Design, synthesis and biological activity of novel HIV ... - UJ IR

DESIGN, SYNTHESIS AND

BIOLOGICAL ACTIVITY OF NOVEL

HIV INTEGRASE INHIBITORS

by

TELISHA TRAUT

Thesis in fulfilment for the

degree

PHILOSOPHIAE DOCTOR

in

CHEMISTRY

in the

FACULTY OF SCIENCE

of the

UNIVERSITY OF JOHANNESBURG

Promoter: Prof. D. B. G. Williams

Co-promoter: Dr R. Hewer

May 2012

Foreword Page II

TABLE OF CONTENTS

LISTS OF EQUATIONS, FIGURES, SCHEMES AND TABLES VI

ABBREVIATIONS XI

SYNOPSIS XV

ACKNOWLEDGEMENTS XVII

SCIENTIFIC CONTRIBUTIONS XVIII

1.1 Conference proceedings XVIII

CHAPTER 1: LITERATURE SURVEY 20

1.1 Introduction 20

1.2 Drug design and discovery 21 1.2.1. A historical perspective on drug discovery 21 1.2.2 The influence of pharmacognosy on modern drug design and discovery 21 1.2.3 Chemical and biological space 24 1.2.4 The drug development pipeline 26

1.3 HIV/AIDS 27 1.3.1 HIV Morphology, life cycle and antiretroviral treatment strategies 29

1.3.1.1 Viral entry inhibitors 31 1.3.1.2 Capsid disassembly 31 1.3.1.3 Inhibitors of reverse transcriptase polymerase activity 32 1.3.1.4 Disruptors of protein-protein and protein-nucleic acid interactions 34 1.3.1.5 Integrase inhibitors 36 1.3.1.6 Silencing of viral mRNA 39 1.3.1.7 Aspartyl protease inhibitors 40 1.3.1.8 Maturation inhibitors and cellular restriction factors 41 1.3.1.9 Viral latency 43

1.3.2 Global response to the HIV/AIDS pandemic 44

1.4 Rationale of the study 45

1.5 Hypothesis 46

1.6 Objectives of the study 46

1.7 References 47

CHAPTER 2: IN SILICO METHODS 48

2.1 Introduction 48

Foreword Page III

2.2 Construction of the HIV-1 IN monomer 50 2.2.1 Preparing the catalytic core domain 51 2.2.2 Preparing the C-terminal domain 54 2.2.3 Preparing the N-terminal domain 54 2.2.4 Comparison between the HIV-1 IN monomer model and a monomer of the PFV IN crystal structure 55

2.3 Construction of the HIV-1 IN strand-transfer complex model 59 2.3.1 Dimerisation model of HIV-1 IN 59 2.3.2 Inclusion of host cofactor LEDGF/p75 60 2.3.3 Inclusion of viral DNA 62 2.3.4 Tetramerisation of HIV-1 IN around host DNA 65

2.4 In silico screening: Hit finding from database mining 70 2.4.1 Preparation of the receptor sites 71 2.4.2 Preparation of the ZINC compound database 71 2.4.3 Docking of the screening library 72

2.5 Conclusion 77

2.6 References 79

CHAPTER 3: CHEMICAL SYNTHESIS 80

3.1 Introduction and background 80

3.2 Synthesis of substituted 1-aminoalkyl-3-hydroxy-3-pyrrolin-2-ones 81 3.2.1 Retrosynthetic analysis 81 3.2.2 Synthesis of the pyruvate starting material 8 83 3.2.3 Synthesis of the pyrrolidinone target compound family 86 3.2.4 Purification 88 3.2.5 Difficulties encountered and solved during the synthesis 89

3.3 Characterisation 89 3.3.1 Nuclear magnetic resonance spectroscopy (NMR) 90 3.3.2 Mass spectrometry (MS) 95 3.3.4 Infrared spectroscopy (IR) 98 3.3.5 X-ray crystallography 99

3.3.5.1 Single X-ray crystal analysis of the synthesised pyrrolidinone compounds 99 3.3.5.2 Single X-ray crystal analysis of the synthesised pyrrolidinone HCl-salts 106 3.3.5.3 Additional crystal structures 111

3.4 Conclusion 114

3.5 References 116

CHAPTER 4: BIOLOGICAL EVALUATION 117

4.1 Introduction 117

4.2 Biological evaluation 118 4.2.1 Direct enzyme assays 118 4.2.2 Structure-activity relationships (SAR) 121 4.2.3 Evaluation of systemic and cellular toxicity 126 4.2.4 Aqueous solubility (LogS) 131 4.2.5 Role of the pH and pKa on solubility and salt formation 132 4.2.6 Membrane permeability 136 4.2.7 Plasma protein binding 138 4.2.8 Compound compliance to Lipinski’s Rule of Five 140

Foreword Page IV

4.3 Conclusion 143

4.4 References 145

CHAPTER 5: EXPERIMENTAL METHODS 146

5.1 General methods 146 5.1.1 Solvents and reagents 146 5.1.2 Spectroscopic data and methods 146

5.1.2.1 Nuclear magnetic resonance (NMR) 146 5.1.2.2 Mass spectrometry (MS) 146 5.1.2.3 Infrared spectrometry (IR) 147 5.1.2.4 Melting points (M.p.) 147 5.1.2.5 X-ray crystallography 147 5.1.2.6 Elemental analysis (EA) 148 5.1.2.7 Absorbance measurements in biological assays 148 5.1.2.8 pH measurements 148 5.1.2.9 Centrifugation 148 5.1.2.10 Microscopy 149

5.2 In silico methods 149 5.2.1 Modelled structure of the three-domain HIV-1 IN monomer 149 5.2.2 Model of the HIV-1 IN dimer with cognate DNA 151

5.2.2.1 Constructing the HIV-1 IN dimer 151 5.2.2.2 Model structure and positioning of the viral DNA 151

5.2.3 Preparation of the PFV IN crystal structure 152 5.2.4 Construction of the HIV-1 IN strand-transfer complex model 152

5.3 Chemical synthesis 154 5.3.1 Synthesis of the pyruvate ester starting material 154 5.3.2 Synthesis of the substituted 1-aminoalkyl-3-hydroxy-3-pyrrolin-2-ones 155 5.3.3 Salt formation of the substituted 1-aminoalkyl-3-hydroxy-3-pyrrolin-2-ones 201

5.4 Biological evaluation 211 5.4.1 Solubility studies 212 5.4.2 Cell viability assays 213 5.4.3 Antiviral activity assays 213 5.4.4 Membrane permeability 215

5.5 References 217

APPENDIX A: SINGLE CRYSTAL DATA 218

A.1 Pyrrolidinone 11.1 218



A.4 Pyrrolidinone 13.3a 232

A.5 Pyrrolidinone 13.3b 240

A.6 Pyrrolidinone 12.2-HCl 243



Foreword Page V


A.10 A co-crystallised set of salts: The hemi-HCl and hemi-oxalic acid forms of morpholine 259

A.11 2,4-Dimethoxybenzaldehyde starting material 260

APPENDIX B: BIOLOGICAL DATA 263

B.1 Graphical representations of the pI and pKa values of the pyrrolidinone compounds 263

Foreword Page VI

LISTS OF EQUATIONS, FIGURES, SCHEMES AND TABLES

CHAPTER 1:

FIGURES: FIGURE 1.1 THE RELATIONSHIP BETWEEN BIOLOGICAL SPACE AND CHEMICAL SPACE, INCLUDING DISCRETE “POCKETS”

FOR DIFFERENT BIOLOGICAL TARGETS. (FIGURE ADAPTED FROM THE ORIGINAL IMAGE IN REFERENCE 52.) ...... 25 FIGURE 1.2 THE STAGES OF DEVELOPMENT OF AN “IDEALISED” DRUG CANDIDATE BEING DEVELOPED FOR SYSTEMIC

USE. (FIGURE COMPILED FROM DATA IN REFERENCES 68 AND 66) ..................................................................... 27 FIGURE 1.3 THE GLOBAL PREVALENCE OF HIV IN 2009, AS REPORTED IN THE 2010 WHO UNAIDS REPORT. ................ 28 FIGURE 1.4 ANNOTATED SCHEMATIC REPRESENTATION OF THE HIV-1 LIFE-CYCLE. (THIS FIGURE WAS PREPARED USING

CHEMBIODRAW VERSION 12.0 SOFTWARE.) ....................................................................................................... 29 FIGURE 1.5 THE STRUCTURE OF THE TRIM5 RESTRICTION FACTOR FAMILY, ILLUSTRATING A) THE DOMAIN STRUCTURE

OF TRIM5Α, AND B) THE OVERALL SECONDARY STRUCTURE OF THE CLOSELY RELATED TRIM-CYP, HIGHLIGHTING THE C-TERMINAL CAPSID BINDING DOMAIN. (THE FIGURE WAS ADAPTED FROM THAT REPRESENTED IN REFERENCE 104). ................................................................................................................................................. 32

FIGURE 1.6 SCHEMATIC REPRESENTATION OF HIV-1 RT, WITH THE RNASEH FUNCTIONALITY HIGHLIGHTED IN ORANGE AND INDICATED BY THE ARROW. ......................................................................................................................... 35

FIGURE 1.7 SCHEMATIC REPRESENTATION OF THE DOMAIN STRUCTURE OF LEDGF/P75. ............................................ 35 FIGURE 1.8 SCHEMATIC ILLUSTRATIONS OF THE A) IMMATURE AND B) MATURE HIV-1 VIRIONS; WITH C) A SCHEMATIC

REPRESENTATION OF THE GAG POLYPEPTIDE, ILLUSTRATING THE FIVE PROCESSING SITES AND THE POSITION OF THE UNCLEAVED STRUCTURAL PROTEINS.109 (THE FIGURE WAS ADAPTED FROM THAT REPRESENTED IN REFERENCE 109). ................................................................................................................................................. 41

TABLES: TABLE 1.1 FDA APPROVED HIV-1 INHIBITORS ............................................................................................................... 30 TABLE 1.2 CHARACTERISTICS OF THE TWO CLASSES OF RT INHIBITORS: N(T)RTIS AND NNRTIS .................................... 33 TABLE 1.3 EXAMPLES OF HIV-1 INIS IN CLINICAL TRIALS.201 .......................................................................................... 39 TABLE 1.4 HIV-1 PR INHIBITORS IN PRECLINICAL DEVELOPMENT.231 ............................................................................. 40

CHAPTER 2:

FIGURES: FIGURE 2.1 THE POSITIONING OF TWO DIVALENT METAL IONS BETWEEN THE CHELATING OXYGEN ATOMS OF THE

CATALYTIC TRIAD RESIDUES: (A) IN THE HIV-1 IN ACTIVE SITE; (B) IN THE TN5 TRANSPOSASE TEMPLATE, SHOWING MN2+ AS THE DIVALENT METAL; AND (C) IN THE PFV IN ACTIVE SITE. THESE FIGURES WERE CREATED USING TRIPOSTM SYBYL8.0 RENDERING IN SGI RGB. ............................................................................................. 52

FIGURE 2.2 A) RESOLVED LOOP STRUCTURES REPRESENTED IN VARIOUS CONFORMATIONS AROUND THE HIV-1 IN ACTIVE SITE (1BL3 = YELLOW, 1B9F = PURPLE, 1BIS = GREEN, 2ITG = RED); B) THE MISSING LOOP REGION IN THE CCD OF HIV-1 IN (1QS4; RESIDUES 141-149) REBUILT USING TEMPLATE 1B9F. THE DARK BLUE STRAND REPRESENTS THE MODEL (BASED ON 1QS4), WHILE THE CYAN STRAND REPRESENTS THE TEMPLATE STRAND (BASED ON 1B9F32). THESE FIGURES WERE CREATED USING TRIPOSTM SYBYL8.0 RENDERING IN SGI RGB. .......... 53

FIGURE 2.3 RELATIVE ORIENTATION OF THE THREE SUBDOMAINS IN: A) THE HIV-1 IN MONOMER MODEL AND B) THE PFV IN MONOMER CRYSTAL STRUCTURE. THE DISTANCES BETWEEN CENTROIDS DEFINED FOR EACH SUBDOMAIN AND THE ANGLE FORMED BETWEEN THE FLEXIBLE LINKERS ARE INDICATED IN EACH CASE. THESE FIGURES WERE CREATED USING TRIPOSTM SYBYL8.0 RENDERING IN SGI RGB. ..................................................... 57

FIGURE 2.4 SECONDARY STRUCTURE ALIGNMENT OF THE PFV IN (BLUE CHAIN) AND THE HIV-1 IN (YELLOW CHAIN) SUB-DOMAINS: A) THE NTD; B) THE CTD; AND C) THE CCD. THESE FIGURES WERE CREATED USING

ACCELRYS DISCOVERY STUDIOTM

RENDERING. ...................................................................................... 58 FIGURE 2.5 REPRESENTATION OF THE HIV-1 IN DIMER STRUCTURE GENERATED, ILLUSTRATING THE DISTANCE

BETWEEN THE ACTIVE SITES IN THE RESPECTIVE MONOMERS. THIS FIGURE WAS CREATED USING TRIPOSTM SYBYL8.0 RENDERING IN SGI RGB. ....................................................................................................................... 60

Foreword Page VII

FIGURE 2.6 THE PROTEIN INTERFACE OF DIMERIC HIV-1 IN AND LEDGF/P75 IS SHOWN, ILLUSTRATING THE STRONG ATTRACTIVE INTERACTIONS ANCHORING LEDGF/P75 TO MONOMERS A AND B. THIS FIGURE WAS CREATED USING TRIPOSTM SYBYL8.0 RENDERING IN SGI RGB. ............................................................................................. 61

FIGURE 2.7 A) SCHEMATIC REPRESENTATION OF THE NUCLEOTIDE SEQUENCE AT THE U5 LTR OF VDNA AND THE NUMBERING SCHEME USED TO IDENTIFY SPECIFIC NUCLEOTIDES; AND B) REPRESENTATION OF THE INTERACTION OF VDNA WITH HIV-1 IN ACTIVE SITE RESIDUES AFTER 3’-END PROCESSING. ALL RELEVANT RESIDUES AND NUCLEOTIDES ARE REPRESENTED IN CAPPED STICK FORMAT: THE CATALYTIC TRIAD (DARK BLUE); THE UNPAIRED 5’-END RESIDUES OF VDNA (COLOURED BY ATOM TYPE); NUCLEOTIDES IN POSITIONS 1 AND 2 (RED); NUCLEOTIDES IN POSITIONS 3 AND 4 (GREEN); AND NUCLEOTIDES 5 TO 8 (PURPLE). THIS FIGURE WAS CREATED USING TRIPOSTM SYBYL8.0 RENDERING IN SGI RGB. ............................................................................. 64

FIGURE 2.8 ILLUSTRATING A POSSIBLE MULTIMERISATION SCENARIO BETWEEN TWO HIV-1 IN DIMERS THROUGH THEIR ASSOCIATION WITH A SINGLE VDNA CHAIN, IN THE ABSENCE OF HOST DNA. ONLY THE ENDS OF THE VDNA CHAIN (WHITE RIBBONS WITH COLOURED BASE-PAIRING) ARE SHOWN WHERE BOUND TO A HIV-1 IN DIMER (RENDERED IN PINK/ORANGE AND TURQUOISE/GREEN, RESPECTIVELY); THE POSITION OF THE LEDGF/P75 IDB (YELLOW AND MAGENTA RIBBONS) AND BOTH ACTIVE SITES IN EACH HIV-1 IN DIMER ARE INDICATED, INCLUDING TWO BOUND METAL IONS (GREEN SPHERES) AND A BOUND INHIBITOR STRUCTURE IN EACH ACTIVE SITE. THE PLACEMENT OF THE INHIBITOR STRUCTURE WAS MADE FOR ILLUSTRATIVE PURPOSES ONLY. THIS FIGURE WAS CREATED USING TRIPOSTM SYBYL8.0 RENDERING IN SGI RGB. ...................................... 66

FIGURE 2.9 A) FRONTAL VIEW, AND B) TOP VIEW OF THE HIV-1 IN DIMER-OF-DIMERS ASSOCIATED AROUND A 27 BASE-PAIR HOST DNA FRAGMENT. THE COMPLEX ILLUSTRATES THE ASSOCIATION OF THE BIOLOGICALLY ACTIVE HIV-1 IN TETRAMER WITH THE LEDGF/P75 IDB; THE BINDING POSITION OF THE U5 AND U3 ENDS OF VDNA; AND THE POSITION OF THE CATALYTIC ACTIVE SITES FOR FULL-SITE STRAND TRANSFER 5 BASE PAIRS (~15 Å) APART. THESE FIGURES WERE CREATED USING TRIPOSTM SYBYL8.0 RENDERING IN SGI RGB. .......................................... 69

FIGURE 2.10 THE 3D BINDING POSE PREDICTED FOR COMPOUND 1 IN THE HIV-1 IN ACTIVE SITE AND THE 2D INTERACTIONS FORMED BETWEEN 1 AND ACTIVE SITE RESIDUES. THESE FIGURES WERE CREATED USING ACCELRYS DISCOVERY STUDIOTM RENDERING. ..................................................................................................... 73

FIGURE 2.11 THE 3D BINDING POSE PREDICTED FOR COMPOUND 3 IN THE HIV-1 IN ACTIVE SITE AND THE 2D INTERACTIONS FORMED BETWEEN 3 AND ACTIVE SITE RESIDUES. THESE FIGURES WERE CREATED USING ACCELRYS DISCOVERY STUDIOTM RENDERING. ..................................................................................................... 76

SCHEMES: SCHEME 2.1 SIMPLIFIED CARTOON IMAGE ILLUSTRATING THE CAPTURE OF HOST DNA BY THE STRAND TRANSFER

COMPLEX. THIS FIGURE WAS CREATED USING CHEMBIODRAW ULTRATM VERSION 12.0. .................................... 67 SCHEME 2.2 SEQUENCE OF THE PALINDROMIC INSERTION SITE: SHOWING POSSIBLE INTERACTIONS OF HOST DNA

NUCLEOTIDES WITH HIV-1 IN ACTIVE SITE RESIDUES. (RESIDUES OF THE INTERACTING HIV-1 IN DIMERS ARE INDICATED IN RED AND PURPLE RESPECTIVELY, WHILE THE ARROWS INDICATE THE PUTATIVE CLEAVAGE SITES 5 BASE PAIRS APART.) ............................................................................................................................................. 70

TABLES: TABLE 2.1 SEQUENCE SIMILARITY PERCENTAGES AND BACKBONE RMSD VALUES BETWEEN THE MODEL (WITH

COMPLETED LOOP SEQUENCE BASED ON 1B9F) AND THE LOOP CONFORMATIONS PRESENT IN 1BIS CHAIN B, 2ITG AND 1BL3 CHAIN C. (STRUCTURES WERE ALIGNED BASED ON CCD RESIDUES 56 – 80) ............................... 54

TABLE 2.2 LIPINSKI’S RO5 FOR THE DISCOVERY OF ORALLY BIO-AVAILABLE PHARMACEUTICALS AND THE SET OF PARAMETERS USED IN THE CREATION OF THE ZINC SCREENING LIBRARY. .......................................................... 71

TABLE 2.3 MOLECULAR DOCKING SOFTWARE AND SOURCES. ...................................................................................... 72 TABLE 2.4 DOCKING SCORES AND STRAND TRANSFER INHIBITION OF SOME PYRROLIDINONE DERIVATIVES AND

KNOWN HIV-1 IN INHIBITORS. ............................................................................................................................. 74 TABLE 2.5 CENTRES FOR THE INCORPORATION OF VARIANCE IDENTIFIED IN THE PYRROLIDINONE MOLECULAR

STRUCTURE. ......................................................................................................................................................... 76

CHAPTER 3:

FIGURES: FIGURE 3.1 THE CHEMICAL STRUCTURES OF PARENT COMPOUND 1 (ZINC02602549), IDENTIFIED AS POTENTIAL HIV-1

IN INHIBITOR THOUGH IN SILICO DATABASE SCREENING; AND COMMERCIALLY AVAILABLE ANALOGUES 2-7,

Foreword Page VIII

WITH POTENTIAL FOR HIV-1 IN STRAND TRANSFER INHIBITION AS CONFIRMED IN EXPERIMENTAL IN VITRO ASSAYS. ................................................................................................................................................................ 80

FIGURE 3.2 A SERIES OF SPECTRA FOR A TWO-SPIN SYSTEM: THE FREQUENCY OF SPIN 1 (Ν0,1) IS KEPT CONSTANT WHILE THAT OF SPIN 2 (Ν0,2) IS MOVED CLOSER TO SPIN 1, RESULTING IN MORE STRONGLY COUPLED SPECTRA ILLUSTRATING THE “ROOFING” EFFECT. LINES 1–3 SHOW VARIOUS FORMS OF A SECOND-ORDER AB SCALAR SPIN-SPIN COUPLING SYSTEM, WHILE LINE 4 SHOWS AN EXAMPLE OF A FIRST-ORDER AX SCALAR SPIN-SPIN COUPLING SYSTEM. ............................................................................................................................................. 91

FIGURE 3.3 1H NMR SPECTRA OF COMPOUND 11.4 DETERMINED AT NEUTRAL PH, SHOWING THE FULL SPECTRUM INDICATING THE SINGLET RESONANCE ARISING FROM THE CHIRAL PROTON FORMED UPON SUCCESSFUL SYNTHESIS (“*”). .................................................................................................................................................. 92

FIGURE 3.4 1H NMR SPECTRA SHOWING THE RATIO OF FIRST- TO SECOND-ORDER SPIN-SPIN SYSTEMS IN SAMPLES OF: A) THE PROTONATED PYRROLIDINONE COMPOUND, 11.6, DETERMINED AT PH 3; AND B) THE FREE BASE FORM OF PYRROLIDINONE COMPOUND 11.6, DETERMINED AT PH 7. ........................................................................... 93

FIGURE 3.5 PYRROLIDINONE COMPOUND 11.4, ILLUSTRATING A) THE OPEN CHAIN FREE-BASE FORM; AND B) THE CYCLIC STRUCTURE PROPOSED TO FORM UPON PROTONATION; C) 1H NMR SPECTRA OF COMPOUND 11.4 DETERMINED AT PH 7: DISTINGUISHING BETWEEN PEAKS OF EQUIVALENT (DASHED LINES) AND NON-EQUIVALENT (SOLID LINES) PROTONS IN THE PROPYL CHAIN (H21AB VS. H21A AND H21B; H22AB VS. H22A AND H22B; AND H23AB VS. H23A AND H23B). ................................................................................................................ 95

FIGURE 3.6 A) SCHEMATIC OF AN ESI INTERFACE AND; B) SCHEMATIC OF THE MECHANISM OF ION FORMATION. ..... 97 FIGURE 3.7 A) TYPICAL HR-ESI+ SPECTRUM OBTAINED FOR THE PYRROLIDINONE COMPOUNDS; B) HR-ESI+ SPECTRUM

WITH CHARACTERISTIC ISOTOPIC DISTRIBUTION PATTERN OBTAINED FOR CHLORIDE-CONTAINING PYRROLIDINONE COMPOUNDS. ........................................................................................................................... 98

FIGURE 3.8 ILLUSTRATING THE CONJUGATED DOUBLE BOND CHARACTER OF THE PYRROLIDINONE COMPOUNDS. .... 99 FIGURE 3.9 ORTEP DIAGRAM AND SCHEMATIC REPRESENTATION OF A MOLECULE OF 11.1 IN THE ASYMMETRIC UNIT.

.......................................................................................................................................................................... 100 FIGURE 3.10 CRYSTAL LATTICE OF 11.1 SHOWING THE CONTACTS FORMED WITHIN THE CRYSTAL. .......................... 100 FIGURE 3.11 ORTEP DIAGRAM AND SCHEMATIC REPRESENTATION OF ONE MOLECULE OF 11.2 IN THE ASYMMETRIC

UNIT. ................................................................................................................................................................. 101 FIGURE 3.12 CRYSTAL LATTICE OF 11.2 SHOWING THE CONTACTS FORMED WITHIN THE CRYSTAL. .......................... 102 FIGURE 3.13 ORTEP DIAGRAM AND SCHEMATIC REPRESENTATION OF ONE MOLECULE OF 11.5 IN THE ASYMMETRIC

UNIT. ................................................................................................................................................................. 103 FIGURE 3.14 CRYSTAL LATTICE OF 11.5 SHOWING THE CONTACTS FORMED WITHIN THE CRYSTAL. .......................... 103 FIGURE 3.15 ORTEP DIAGRAM AND SCHEMATIC REPRESENTATION OF TWO MOLECULES OF 13.3A IN THE

ASYMMETRIC UNIT. ........................................................................................................................................... 104 FIGURE 3.16 CRYSTAL LATTICE OF 13.3A SHOWING THE CONTACTS FORMED WITHIN THE CRYSTAL. ........................ 104 FIGURE 3.17 ORTEP DIAGRAM AND SCHEMATIC REPRESENTATION OF COMPOUND 13.3B SHOWING: A) ONE OF THE

EIGHT MOLECULES IN THE ASYMMETRIC UNIT; AND B) THE CRYSTAL LATTICE AND THE CONTACTS FORMED WITHIN THE CRYSTAL. ....................................................................................................................................... 105

FIGURE 3.18 ORTEP DIAGRAM AND SCHEMATIC REPRESENTATION OF ONE MOLECULE OF 12.2-HCL AND ONE CHLORIDE COUNTER ION IN THE ASYMMETRIC UNIT. ....................................................................................... 106

FIGURE 3.19 CRYSTAL LATTICE OF 12.2-HCL SHOWING: A) THE HYDROGEN BONDS; AND B) OTHER INTER- AND INTRAMOLECULAR CONTACTS FORMED WITHIN THE CRYSTAL LATTICE. ........................................................... 107

FIGURE 3.20 ORTEP DIAGRAM AND SCHEMATIC REPRESENTATION OF THE ASYMMETRIC UNIT FORMED FOR COMPOUND 12.3-HCL. ...................................................................................................................................... 107

FIGURE 3.21 CRYSTAL LATTICE OF 12.3-HCL SHOWING: A) THE HYDROGEN BONDS; AND B) OTHER INTER- AND INTRAMOLECULAR CONTACTS FORMED WITHIN THE CRYSTAL LATTICE. ........................................................... 108

FIGURE 3.22 ORTEP DIAGRAM AND SCHEMATIC REPRESENTATION OF THE ASYMMETRIC UNIT FORMED FOR COMPOUND 13.2-HCL. ...................................................................................................................................... 109

FIGURE 3.23 CRYSTAL LATTICE OF 13.2-HCL SHOWING: A) THE HYDROGEN BONDS; B) Π--- Π STACKING OF THE AROMATIC FEATURES IN THE MOLECULE; C) INTERACTIONS WITH CHLORIDE ATOMS; AND D) OTHER INTER- AND INTRAMOLECULAR CONTACTS FORMED WITHIN THE CRYSTAL LATTICE. ........................................................... 109

FIGURE 3.24 ORTEP DIAGRAM AND SCHEMATIC REPRESENTATION OF TWO MOLECULES OF 13.5-HCL IN THE ASYMMETRIC UNIT. ........................................................................................................................................... 110

FIGURE 3.25 CRYSTAL LATTICE OF 13.5-HCL SHOWING THE CONTACTS FORMED WITHIN THE CRYSTAL. ................... 110 FIGURE 3.26 ORTEP DIAGRAM AND SCHEMATIC REPRESENTATION OF THE 2,4-DIMETHOXYBENZALDEHYDE STARTING

MATERIAL SHOWING: A) THE FOUR MOLECULES IN THE ASYMMETRIC UNIT; AND B) THE CRYSTAL LATTICE AND THE CONTACTS FORMED WITHIN THE CRYSTAL. ................................................................................................ 111

FIGURE 3.27 ORTEP VIEW OF THE CO-CRYSTALLISED SET OF SALTS FORMED FROM THE PROTONATED AMINE STARTING MATERIAL, OXALIC ACID (FROM THE DIOXANE SOLVENT) AND THE CHLORIDE COUNTER ION (FROM THE ADDED HCL). ............................................................................................................................................... 112

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FIGURE 3.28 PACKING DIAGRAM OF THE HYBRID SALT STRUCTURE. .......................................................................... 112 FIGURE 3.29 INTERMOLECULAR INTERACTIONS IN THE HYBRID SALT STRUCTURE. .................................................... 113 FIGURE 3.30 CRYSTAL LATTICE OF THE HYBRID SALT STRUCTURE SHOWING INTERMOLECULAR CH…CL, NH…CL AND

NH…O INTERACTIONS FORMED WITHIN THE CRYSTAL. ..................................................................................... 114 FIGURE 3.31 CRYSTAL LATTICE OF THE HYBRID SALT STRUCTURE SHOWING INTERMOLECULAR CH…O, NH…O AND

OH…O INTERACTIONS FORMED WITHIN THE CRYSTAL. ..................................................................................... 114

SCHEMES: SCHEME 3.1 RETROSYNTHETIC REACTION PATHWAY PROPOSED FOR THE PYRROLIDINONE FAMILY OF COMPOUNDS.

............................................................................................................................................................................ 82 SCHEME 3.2 ILLUSTRATING THE APPLICATION OF Β-KETO ESTER AND Β-DIKETONE INTERMEDIATES IN ORGANIC

SYNTHESIS: A RANGE OF END-PRODUCTS CAN BE ACHIEVED FROM TRANSFORMATION OF THE CLASSIC CLAISEN CONDENSATION PRODUCTS., ............................................................................................................................... 83

SCHEME 3.3 SCHEMATIC REPRESENTATION OF THE CLASSIC CLAISEN CONDENSATION REACTION. ............................. 84 SCHEME 3.4 SCHEMATIC REPRESENTATION OF THE MIXED / CROSSED CLAISEN CONDENSATION REACTION. ............. 84 SCHEME 3.5 SCHEMATIC REPRESENTATION OF THE INTRAMOLECULAR DIECKMANN CONDENSATION REACTION. ..... 84 SCHEME 3.6 THE MECHANISM OF THE CLAISEN CONDENSATION REACTION. ............................................................... 85 SCHEME 3.7 SYNTHESIS OF STARTING MATERIAL 8 (PYRUVATE ESTER). ....................................................................... 86 SCHEME 3.8 GENERAL METHOD FOR THE SYNTHESIS OF THE PYRROLIDINONE DERIVATIVES. ..................................... 86 SCHEME 3.9 PROPOSED MECHANISMS OF THE CONDENSATION AND CYCLISATION REACTIONS THAT RESULT IN THE

FINAL PYRROLIDINONE PRODUCTS (ILLUSTRATED USING COMPOUND 11.1 AS AN EXAMPLE). ........................... 88

TABLES: TABLE 3.1 HYDROGEN BONDS, SYMMETRY OPERATORS AND Π-RING INTERACTIONS OBSERVED IN CRYSTALS OF

PYRROLIDINONE 11.2. ....................................................................................................................................... 101 TABLE 3.2 HYDROGEN BONDS, SYMMETRY OPERATORS AND Π-RING INTERACTIONS OBSERVED IN CRYSTALS OF

PYRROLIDINONE 13.3A. ..................................................................................................................................... 104

CHAPTER 4:

EQUATIONS: EQUATION 4.1 ............................................................................................................................................................ 136

FIGURES: FIGURE 4.1 THE 3D BINDING POSE PREDICTED FOR 11.6 IN THE HIV-1 IN ACTIVE SITE AND THE 2D INTERACTIONS

FORMED BETWEEN 11.6 AND ACTIVE SITE RESIDUES. THESE FIGURES WERE CREATED USING ACCELRYS DISCOVERY STUDIOTM RENDERING. ................................................................................................................... 123

FIGURE 4.2 THE 3D BINDING POSE PREDICTED FOR RAL IN THE HIV-1 IN ACTIVE SITE AND THE 2D INTERACTIONS FORMED BETWEEN RAL AND ACTIVE SITE RESIDUES. THESE FIGURES WERE CREATED USING ACCELRYS DISCOVERY STUDIOTM RENDERING. ................................................................................................................... 123

FIGURE 4.3 THE 3D BINDING POSE PREDICTED FOR 15.2 IN THE HIV-1 IN ACTIVE SITE AND THE 2D INTERACTIONS FORMED BETWEEN 15.2 AND ACTIVE SITE RESIDUES. THESE FIGURES WERE CREATED USING ACCELRYS DISCOVERY STUDIOTM RENDERING. ................................................................................................................... 126

FIGURE 4.4 AQUEOUS SOLUBILITY: ALIGNMENT OF THE PREDICTED AND EXPERIMENTALLY DETERMINED VALUES FOR A) THE LOGS FORM (ONE-TAILED DISTRIBUTION OF A TWO-SAMPLE UNEQUAL VARIANCE STUDENT T-TEST; P-VALUE = 1.37E-11); AND B) THE SOLUBILITY IN NATURAL NUMBERS (ONE-TAILED DISTRIBUTION OF A TWO-SAMPLE UNEQUAL VARIANCE STUDENT T-TEST; P-VALUE = 6.04E-10). ............................................................. 132

FIGURE 4.5 DISTRIBUTION OF THE MOLECULAR PROPERTIES OF THE SYNTHESISED PYRROLIDINONE COMPOUNDS AND SEVERAL KNOWN HIV-1 IN INHIBITORS BASED ON THE CALCULATED PARTITION COEFFICIENT (ALOGP) AND THE MOLECULAR WEIGHT (MW). ............................................................................................................................. 142

SCHEMES: SCHEME 4.1 BIOLOGICAL PROFILES AND HIV-1 IN INHIBITION (INI) ACTIVITIES OF THE SERIES 11 COMPOUNDS,

COMPARED TO COMPOUND 3. .......................................................................................................................... 122

Foreword Page X

SCHEME 4.2 BIOLOGICAL PROFILES AND HIV-1 IN INHIBITION ACTIVITIES OF THE SERIES 15 COMPOUNDS, COMPARED TO COMPOUND 3. ............................................................................................................................................. 125

TABLES: TABLE 4.1 TOXICITY AND ACTIVITY OF THE SYNTHESISED PYRROLIDINONE COMPOUNDS. ......................................... 120 TABLE 4.2 TOPKATTM PREDICTIONS FOR THE PYRROLIDINONE COMPOUNDS. ............................................................ 130 TABLE 4.3 CALCULATED PKA AND PI VALUES OF THE SYNTHESISED PYRROLIDINONE COMPOUNDS. ......................... 135 TABLE 4.4 THE MEMBRANE PERMEABILITY POTENTIAL OF THE PYRROLIDINONE COMPOUNDS. ............................... 137 TABLE 4.5 ATTRIBUTES PREDICTED FOR THE SYNTHESISED PYRROLIDINONE COMPOUNDS. ...................................... 139 TABLE 4.6 COMPOUND COMPLIANCE TO LIPINSKI’S RO5............................................................................................ 141

CHAPTER 5:

EQUATIONS: EQUATION 5.1 AQUEOUS SOLUBILITY (IN µM) ........................................................................................................... 213 EQUATION 5.2 PERMEABILITY (IN CM/S) .................................................................................................................... 215 EQUATION 5.3 MASS RETENTION (%) ......................................................................................................................... 215

Foreword Page XI

ABBREVIATIONS

°C Degrees Celsius

µL Microlitre

2D Two dimensional

3’-OH 3’-Terminal hydroxyl group

3D Three-dimensional

Ǻ Ǻngstrøm, 10-10

AcN Acetonitrile

ADMET Adsorption, distribution, metabolism, excretion, toxicity

AIDS Acquired immunodeficiency syndrome

APOBEC3 or A3 Apolipoprotein B mRNA-editing enzyme 3

ATR Attenuated total reflectance

AZT 3’-Azidothymidine, zidovudine

BBB Blood-brain barrier

bp Base pairs

BST-2 Bone marrow stromal cell antigen 2

CA Capsid protein

CC50 The concentration of test compound responsible for 50% reduction in cell viability

CCD Catalytic core domain

CD4 Plasma membrane-bound receptor protein located on suitable host cells

CDCl3 Deuterated chloroform

CDER Centre for Drug Evaluation and Research

CI Chemical Ionisation

CLogP or ALogP Calculated LogP

cm-1 Wave numbers

CO Side chain carbonyl

CON Lactam carbonyl

CTD C-terminal domain

CXCR4 and CCR5 Co- or fusion receptors for viral entry

CYP450 Cytochrome P450

DCM Dichloromethane

DC-SIGN Dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin

DDE Asp-Asp-Glu; catalytic triad residues of HIV-1 IN

dDNA Donor DNA

dMeOH Deuterated methanol, CD3OD

DMSO Dimethyl sulfoxide

DNA Deoxyribonucleic acid

dNTPs Deoxynucleoside triphosphates

DOS Diversity-oriented synthesis

DTP Developmental Therapeutics Program

DTP Developmental toxicity potential

EA Elemental analysis

EI Electron Impact

Foreword Page XII

ELISA Enzyme-linked immunosorbent assay

EPA Environmental Protection Agency

FCS Foetal calf serum

FDA Food and Drug Administration

g Grams

gp120 Viral surface glycoprotein 120

gp41 Viral glycoprotein 41

GPI Glycosyl-phosphatidylinositol

HAART Highly active antiretroviral therapy

H-bonds Hydrogen bonds

HHCC His-His-Cys-Cys; Conserved zinc-binding residues in HIV-1 IN

HIA Human intestinal absorption

HIV Human immunodeficiency virus

HIV-1 Human immunodeficiency virus type 1

HIV-2 Human immunodeficiency virus type 2

HR TOF-ESI MS High resolution time-of-flight electron spray ionisation mass spectral analysis

HR2 Heptad repeat 2

HTS High-throughput-screening

Hz Hertz

IBD Integrase binding domain

IC50 The concentration of test compound responsible for a 50% inhibition of normal enzyme function.

IN Integrase

INIs HIV-1 integrase inhibitors

IR Infrared spectroscopy

kDa kiloDalton

kV kiloVolts

L Litre

L-CA L-chicoric acid

LEDGF/p75 Lens-epithelium derived growth factor p75

LogS Aqueous solubility

LTR Long terminal repeat

MA Matrix protein

MeOH Methanol

MHz Megahertz

miRNA-RISC MicroRNA RNA-induced silencing complexes

mL Millilitre

mM Millimolar

mmHg Millimetres of mercury

mmol Millimoles

mol Moles

mRNA Messenger RNA

MS Mass spectral

MW Molecular Weight

N(t)RTIs Nucleoside/nucleotide reverse transcriptase inhibitors

NaOMe Sodium methoxide

NC Nucleocapsid proteins

NCEs New chemical entities

NCI National Cancer Institute

Foreword Page XIII

ncRNA Non-coding RNAs

NCEs New chemical entities

NIH National Institutes of Health

NLS Nuclear localisation signal

nm Nanometre

NMR Nuclear magnetic resonance

NNRTIs Non-nucleoside reverse transcriptase inhibitors

NTD N-terminal domain

OPS Optimum prediction space

ORTEP Oak Ridge Thermal Ellipsoid Plot Program

PAGE Polyacrylamide gel electrophoresis

PAMPA Parallel artificial membrane permeability assay

PBS Phosphate buffered saline

PC Personal computer

PDB Protein data bank

PFV Prototype foamy virus

PFV IN Prototype foamy virus integrase

PHAs Polyhydroxylated aromatic compounds

PIC Pre-integration complex

PPB Plasma protein binding

ppm Parts per million

PR Protease

PSA Polar surface area

PVDF Polyvinylidene fluoride

PWWP Pro-Trp-Trp-Pro

QSTR Quantitative structure-toxicity relationship

RAL Raltegravir

RCSB Research Collaboratory for Structural Bioinformatics

RMSD Root mean square deviation

RNA Ribonucleic acid

RNAi RNA-interference

Ro5 Rule of Five

RT Reverse transcriptase

SAR Structure activity relationship

SH3 SRC Homology 3 domain

SI Selectivity indexes

SP1 and SP2 Spacer peptides 1 and 2

SSC Saline-sodium citrate

T-20 Enfuvirtide

TAR Viral trans-activation responsive

Tat Virus-encoded arginine-rich RNA-binding protein

TBS Tris buffered saline

tDNA Target DNA

TM Transmembrane

TMS Tetramethyl silane

TOF-ESI MS Time of flight electron spray ionisation mass spectral analysis

TRIM5 Tripartite motif 5

UNAIDS United Nations Program on HIV/AIDS

US United States (of America)

Foreword Page XIV

USD United States dollar

UV-Vis Ultraviolet-visible region

V Volts

vDNA Viral DNA

VHF Very-high-frequency

Vif Viral infectivity factor

Vpu Viral protein U

WHO World Health Organisation

WOE Weight-of-evidence

Foreword Page XV

SYNOPSIS

Despite nearly three decades of intensive research, the HIV/AIDS pandemic remains a major challenge to

modern medicine. The discovery and development of antiretroviral agents acting against various essential

viral processes and enzymatic targets have greatly enhanced the quality of life for infected individuals, but

no cure or preventative vaccine is available as yet and HIV infection is currently considered irreversible.

Furthermore, the emergence of viral resistance to every class and type of antiretroviral treatment agent

necessitates the continued discovery of antiretroviral agents with novel mechanisms of action. The first

antiretroviral agent targeting the retroviral integrase enzyme (InsentressTM, Raltegravir) received regulatory

approval from the United States Food and Drug Administration during 2007, validating HIV-1 integrase as a

therapeutic target and providing a much-needed second- or third-line treatment option for treatment

experienced patients. This enzyme was selected as a target for the current work.

As limited data were available on the primary and secondary structure of the biologically relevant HIV-1

integrase enzyme, a first step in the present work was the construction of monomeric, dimeric and

tetrameric models of the enzyme with biologically relevant catalytic centres incorporating both viral and

host co-factors and DNA. The models were constructed to identify potential inhibitors of the strand-

transfer reaction of HIV-1 integrase and were based on observations and interactions reported in the

literature and on crystal structure data of HIV-1 integrase sub-domains and related structures available in

the Protein Data Bank. The monomeric model was used as the macromolecular target in docking studies

with “drug-like” compound databases, identifying the pyrrolidinone compound class as an in silico hit

candidate for further development.

Initial activity screening of a number of commercially available pyrrolidinone analogues against

recombinant HIV-1 subtype B integrase in direct enzyme assays confirmed the predicted potential for

strand transfer inhibition of the compound class, and provided initial support in the further development of

this compound class as inhibitors of HIV-1 integrase that target the strand-transfer step. Retrosynthetic

analysis of the pyrrolidinone hit candidates provided a facile one-pot, three-component synthetic pathway

from readily available starting materials, which generally gave the proposed products cleanly and in

acceptable yields. A range of closely related analogues were designed and synthesised. The analogues

making up this series generally differed by only one functional group, in order to enable initial structure-

activity relationship investigations during later stages of the project.

Foreword Page XVI

The synthesised pyrrolidinone analogues were screened through a range of direct and cell-based in vitro

assays to determine the toxicity and strand-transfer activity of each. In general, the pyrrolidinone

compounds proved well-tolerated in PM1 cell culture, with clear potential to further develop the strand-

transfer inhibition of the compound family in second- and further-generation optimisation stages.

Furthermore, the aqueous solubility and membrane permeability of each compound were determined in

vitro, providing initial biological profiles of each test compound. As no in vivo testing was performed with

any of the compounds during this first round of drug discovery and optimisation, several computational

models were employed to extrapolate the in vitro and structural data to possible in vivo scenarios.

Two pyrrolidinone analogues (11.6 and 15.2) were identified as low micro-molar strand-transfer inhibitors

of wildtype-equivalent HIV-1 integrase, with low toxicity in cell culture and favourable solubility and

permeability profiles. Resistance screening of these two compounds against four mutant HIV-1 integrases

(Q148H; Q148H/G140S; N155H and N155H/E92Q) has shown some promise, with compound 15.2 retaining

a measure of activity against the Raltegravir-resistant N155-mutants. These hit candidates will form the

basis of structure-activity relationship optimisations in second- and further generation design stages.

Foreword Page XVII

ACKNOWLEDGEMENTS

I gratefully acknowledge the following institutions and people for their valuable input and contributions

throughout this project:

Funding:

• The Council for Mineral Technology, Mintek

• Project AuTEK, a joint venture between Mintek and Harmony Gold, Ltd.

• South African Department of Science and Technology (DST):

Research and Professional Development Grant (Fellow 2008-2010)

Advancement of Academic Qualifications Grant (Recipient 2011)

• South African National Research Foundation (NRF)

People:

• My project supervisor, Prof. D. Bradley G. Williams

• My co-supervisor, Dr Raymond Hewer

• My friends and colleagues from the Biomedical group (Advanced Materials Division, Mintek, South

Africa) for the sharing of knowledge and valuable discussions (and for putting up with my wild

enthusiasm for crystals and colours)

• Dr Erik Kriel for solving and refining all of the single crystal X-ray data

• Dr Salerwe Mosebi for doing the activity screening of the purchased analogues

• Qasim M. Fish for doing the activity screening against the HIV-1 IN mutants

• My family for never failing to ask when I’ll be finished

• My friends for continuously reminding me that no problem looks quite as insurmountable with a

glass of good red wine in hand. And for never failing to ask when I’ll be finished…

• Nigel, for never asking when I’ll be finished…!

Foreword Page XVIII

SCIENTIFIC CONTRIBUTIONS

1.1 Conference proceedings

2011

• TITLE: “Pyrrole-Carbaldehyde Inhibitors of HIV-1 Integrase: A Medicinal Chemistry Approach.” T.

Traut, R. Hewer, J. Coates, D.B.G. Williams, 5th SA AIDS conference, Durban, South Africa, June 2011.

• TITLE: “The Design and Synthesis of Pyrolle-Carbaldehydes as HIV-1 Integrase Strand-Transfer

Inhibitors.” T. Traut, D. B. G. Williams, J. Coates, R. Hewer, 24th

International Conference on

Antiviral Research (ICAR) 2011, Sofia, Bulgaria, 8-11 May 2011.

• TITLE: “The Design and Synthesis of a Pyrrole-Carbaldehyde Hit Family as HIV-1 Integrase Inhibitors.”

T. Traut, R, Hewer, J. Coates and D. B. G. Williams, MedChem Europe 2011, Munich, Germany, 28-

29 March 2011.

• TITLE: “The Design and Synthesis of a Pyrrole-Carbaldehyde Hit Family as HIV-1 Integrase Inhibitors.”

T. Traut, R, Hewer, J. Coates and D. B. G. Williams, SACI2011, 40th

South African Chemical Institute

Convention, Witwatersrand University, Johannesburg, January 2011.

2010

• TITLE: “Die ontwerp en sintese van 'n belowende reeks pirool-karbaldehiede as potensiële MIV-1

integrase inhibeerders.” T. Traut, R. HEWER, J. COATES, D.B.G. WILLIAMS., SAAWK Symposia, Pretoria,

RSA, 4-5 November 2010.

• TITLE: “In Silico Models of HIV-1 Integrase: Application in the Design and Discovery of Antiretroviral

Agents.” T. Traut, R. HEWER, J. COATES, D.B.G. WILLIAMS, Keystone Symposium: Computer Aided Drug

Design and Chemistry in Drug Discovery, Canada, 19-26 April 2010.

“Science is not a sacred cow. Science is a horse. Don't worship it. Feed it.”

Abba (Aubrey) Eben

AFFIDAVIT: MASTER’S AND DOCTORAL STUDENTS

TO WHOM IT MAY CONCERN

This serves to confirm that I, Telisha Traut (Full Name(s) and Surname), ID Number:

8101250003082, Student number: 920100805 enrolled for the Qualification: PhD Organic

Chemistry, Faculty of Science of the University of Johannesburg, herewith declare that my

academic work is in line with the Plagiarism Policy of the University of Johannesburg with which I

am familiar.

I further declare that the work presented in the thesis (minor dissertation/dissertation/thesis) is

authentic and original unless clearly indicated otherwise and in such instances full reference to

the source is acknowledged and I do not pretend to receive any credit for such acknowledged

quotations, and that there is no copyright infringement in my work.

I declare that no unethical research practices were used or material gained through dishonesty.

I understand that plagiarism is a serious offence and that should I contravene the Plagiarism

Policy notwithstanding signing this affidavit, I may be found guilty of a serious criminal offence

(perjury) that would amongst other consequences compel the UJ to inform all other tertiary

institutions of the offence and to issue a corresponding certificate of reprehensible academic

conduct to whomever request such a certificate from the institution.

Signed at Johannesburg _______________ on this _______ of ____________________2012

Signature__________________________________ Print name_________________________

STAMP COMMISSIONER OF OATHS

Affidavit certified by a Commissioner of Oaths

This affidavit conforms with the requirements of the JUSTICES OF THE PEACE AND COMMISSIONERS OF OATHS

ACT 16 OF 1963 and the applicable Regulations published in the GG GNR 1258 of 21 July 1972; GN 903 of 10 July

1998; GN 109 of 2 February 2001 as amended.

Chapter 1: Literature Survey Page 20

CHAPTER 1: LITERATURE SURVEY

1.1 Introduction

Since the earliest stages of humankind’s development, man has attempted to find ways to alleviate

the symptoms and sources of illness.1 Historically, these attempts were mainly fuelled through

serendipitous discovery and ranged from ingestion or application, to infusions, concoctions, and

tinctures of all manner of plant, animal and mineral material. In some instances, these early

treatments were surprisingly successful: the ancient Chinese healers were famous for their healing

elixirs composed mainly of herbal concoctions. As an example, consider the ancient practise of

chewing the root, leaves and stem of Dichroa febrifuga to alleviate fevers associated with malarial

infections.2 Centuries later, the active ingredient was identified as an alkaloid, febrifugine.

3

Although the clinical antimalarial use of febrigugine in an isolated form was precluded due to

severe liver toxicity, several analogues have been identified with more promising toxicity

profiles.4,5

Just as often, however, these early treatments caused more problems than they cured:

for example, during the Qin Dynasty in China (221-207 BC), mercury was erroneously considered

a life-giving mineral and the belief that ingesting mercury would confer life and longevity caused

the painful death of at least one ancient noble, the Emperor Ying Zheng.6,7

These early trial and

error attempts at altering a disease state were founded mostly on superstition and assumption, with

no clear concept of the workings of the human body and bore little resemblance to the modern-day

concept of medicinal chemistry. Through radical developments of procedures and technology in

various fields, notably that of computational and organic chemistry, biology and medicine, the

field of medicinal chemistry has evolved and finally emerged as a discrete scientific discipline.8

The fundamental principles of medicinal chemistry are based on the relationship between structure

and activity:9 Certain chemical structures or functional groups are responsible for certain

biological responses and the contribution of the various functional groups in a chemical structure

can be determined in an additive manner to determine the behaviour of the molecule in a biological

environment.


1.2 Drug design and discovery

1.2.1. A historical perspective on drug discovery

One of the first reports of a possible relationship between chemical structure and biological

activity was published in 1869.10

The authors speculated on the possibility of enhancing the muscle

relaxant properties of a drug through the incorporation of a quaternary ammonium group into the

chemical structure. Although this hypothesis was later disproved,11

the report introduced a

revolution in the way that the scientific community regarded drug action. The first notion of a drug

receptor was independently introduced by John N. Langley and Paul Ehrlich in 1878 and further

developed during the early part of the 20th

century12,13

(for more information on the lives and work

of these two researchers, the reader is referred to two excellent reviews by Maehle14

and Prüll,15

respectively). Drawing on his own and Langley’s previous work, as well as the elegant “lock-and-

key” model explaining enzyme-substrate binding proposed by Emil Fischer,16,17

Ehrlich

formulated the receptor hypothesis during the early years of the 20th

century,18

to explain the

biological action of chemical structures. The realisation of the importance of drug-receptor binding

to drug action is illustrated in Ehrlich’s famous postulate: corpora non agunt nisi fixata, translated

as “compounds do not act unless bound”.19

In addition, Ehrlich’s postulates allowed formation of

the earliest theories on bio-activation, the therapeutic index and drug resistance.15

Further

important developments in the field of drug-receptor binding include amongst many others, the

work of Linus C. Pauling20

(on the application of the key-lock model to enzyme transition states);

Daniel E. Koshland21

(extending the key-lock theory to arrive at the induced fit theory) and

Corwin H. Hansch22

(on the importance of lipophilicity in medicinal chemistry). Lastly, Ehrlich

also introduced the concept of a “magic bullet”,23

describing compounds with selective toxicity

towards disease states that would cause minimal harm to the host organism. Building on Ehrlich’s

“magic bullet” concept, Seymour S. Cohen introduced a novel strategy for the selective targeting

of infectious diseases in 1977,24

suggesting the formation of multidisciplinary research teams

focussed on the selective inhibition of uniquely parasitic enzymes and proteins that are crucial to

the survival and function of the infectious parasite.

1.2.2 The influence of pharmacognosy on modern drug design and discovery

Although many of the pioneers in the early days of pharmacology and medicinal chemistry had

access to chemistry laboratories capable of performing synthesis, synthetic research was limited by

the techniques, equipment and social ideas of the time8 (the reader is referred to an account on the

discovery of anaesthesia to illustrate some of the social hurdles blocking scientific progress25

). The


techniques used were much more sophisticated than in ancient times and the research was

generally guided by sound scientific principles instead of superstition, but much of the research

into drugs and biologically active molecules during the 19th

and early-20th

century still depended

on compounds directly derived from natural sources, as the depth of chemical diversity and degree

of complexity found in these natural products defied complete synthesis.

The term “pharmacognosy” was coined during the early years of the 19th

century (between 1811

and 1815) to describe the study of drugs from plant, animal or mineral origin in their crude or

unprepared form.26

Although most of the pharmocognostic studies performed during the 19th

century and the beginning of the 20th

century focussed on botanical specimens in whole or

powdered form, rapid development and interest in the areas of microbes and marine organisms

have greatly expanded the research field, and by extension, the potential sources of novel

biologically active molecular species. More recently, different branches of pharmacognosy have

developed as a consequence of the multi-disciplinary nature of the field (including medical

ethnobotany; ethnopharmacology; phytotherapy; phytochemistry; zoopharmacognosy and marine

pharmacognosy).26

It has been suggested that, although the ancient materia medica have

undoubtedly been the source of a variety of valuable pharmacologically active substances, the

influence these natural products has had on modern drug research may have been disproportionate

to the actual therapeutic benefit derived from their use.1 Furthermore, it is important to recognise

the fact that most pharmacologically active substances derived from plant and/or animal origin

originally evolved as mechanisms to protect the plant or animal from predators (i.e. as

poisons),27,28

and administration of these “poisons” to human patients may be associated with high

toxicity. In response, herbal remedies distributed by herbal nostrums contain the active ingredients

in very low concentrations only,29,30

while homeopathic remedies containing natural products have

been diluted to the extent where the concentration of active ingredient is virtually negligible.31

Nonetheless, the large global market that currently exists for natural product applications in the

health sector illustrates the importance that is still attached to traditional medicine and treatments

derived from natural sources. In addition to the large international demand for Ayurvedic

(traditional Indian medicine) and traditional Eastern medicines (annual revenues estimated at US$

5 billion in Western Europe during 2003-2004; US$ 14 billion in China during 2005),32

traditional

medicine plays an important role in many developing countries in Africa and Asia, where up to

80% of the local population may depend on natural products for primary health care.32

In many

instances, the demand for these health products from natural sources far outweighs the readily


available supply, leading to the exhaustion of the natural resources and prompting the production

of synthetically manufactured alternatives where possible.33

During the period 1981-2002, a total of 1031 new chemical entities (NCEs) were introduced into

the market.34

Of these, only ~5% were complete natural products (50/1031 compounds); ~23%

were semi-synthetic derivations from natural products (240/1031 compounds); and ~14% were

synthetic compounds based on natural product pharmacophores (144/1031 compounds). While

~43% of the total number of NCEs introduced were completely chemically synthesised products, a

quarter of these compounds (~25%; 97/386 compounds) were classified as natural product mimics.

This clearly illustrates the importance of natural products, or natural product scaffolds, as sources

of knowledge in the design of synthetic drugs.34

As mentioned previously, natural products exhibit a greater structural diversity and degree of

complexity than synthetic drugs. Additionally, substantial differences can be distinguished in the

structural composition of natural products and synthetic drugs.35

Natural products often contain a

greater ratio of oxygen to nitrogen hetero-atoms, and have a significant number of stereogenic

centres.35

Natural products are often not orally bioavailable, but have been found to address some

biological targets not accessible to synthetic drugs.34

Furthermore, several scaffolds identified from

natural product sources have been classified as “privileged” structures, as they have proved

capable of binding multiple classes of protein targets, and have subsequently found widespread use

in the design of synthetic drugs.36

In contrast, synthetic drugs are often based on nitrogen-rich

hetero-aromatic scaffolds small enough to bind in the active site of enzymes through

complementarities in shape and binding-affinity. The presence of stereogenic centres is greatly

reduced or they are completely absent, and the drugs are generally designed to be orally

bioavailable.35

Many synthetic drug design approaches rely on scaffolds or pharmacophores derived from

biologically active natural products. In these instances, the structural elements (or motif) most

likely to be responsible for the observed biological activity of the natural product is identified and

only the smallest fragment that still retains the original biological activity is synthesised. This

approach greatly reduces the time and financial resources required during the drug design and

synthesis phase, as the structural motif that results in a particular biological response is already

known. Furthermore, the emerging field of diversity-oriented synthesis (DOS) is currently a major

area of interest, as it effectively attempts to bridge the gap between natural product chemistry and


synthetic drug design: DOS libraries are designed to incorporate the structural diversity of natural

products while retaining the ease of preparation characteristic of synthetic drugs.37

These libraries

are not designed against one specific receptor, but rather to provide selective probes for a variety

of different receptors.

1.2.3 Chemical and biological space

Chemical space, shortly defined as the set of all known molecular structures, derived from any

combination of available atoms, is incalculably vast.38,39,40

Currently, advanced high-throughput-

screening (HTS) techniques allow for the screening of libraries containing 105-10

6

compounds.41,42,43

Although this is a significant achievement in itself and illustrates the revolution

in technology that has taken place during the 20th

century, the number is still virtually negligible

when compared to all of chemical space. This realisation prompted pharmaceutical companies and

other drug discovery initiatives to increase their compound output through technologies such as

DOS (discussed previously), combinatorial chemistry, parallel synthesis and ultra-high throughput

in vitro screening, in the hopes of finding more leads that could be developed into drugs.44,45,46

Despite the exponential increase in compound numbers synthesised and screened for biological

activity, the number of drugs reaching the market remained constant,47

highlighting the need for

more efficient compound selection criteria. Molecules were pre-selected for biological screening

based on certain pre-defined criteria: one of the generally applied criteria evaluates the “drug-

likeness” of the molecule, i.e the potential for biological activity based on similarities between the

molecular structures of the target compound and that of known bioactive molecules.48,49

Drug-like

space has been suggested to occupy a distinct space within the continuum of chemical space and

has been estimated to include between 1018

and 10200

molecules, depending on the selection

criteria employed during the calculation.50,51,52

Drug-like space possesses absorption, distribution,

metabolism and excretion (ADME) properties similar to that of orally administered drugs and can

generally be described by Lipinski’s Rule of Five (Ro5).39

In its original form, the Ro5 considers

four basic parameters of pharmaceutical agents developed for oral administration and defines an

optimal range for each of these parameters (ranges defined in multiples of 5): the molecular weight

(≤500 g/mol); the calculated LogP (≤5); the number of hydrogen bond donors (≤5); and the

number of hydrogen bond acceptors (≤10). Importantly, although the current definition of drug-

like space (or ADME space) has proved to be a useful tool in the identification of inhibitors of

certain biological targets, not all drug-like molecules have biological activity. Additionally, not all

biologically active molecules fall within the current parameters of drug-like space. Instead,

compounds that bind to the same protein target classes have been found to cluster together in

Chapter 1: Literature Survey

discrete regions of chemical space, defined by particular che

referred to as

biological space,

biological space

different biological target spaces

Figure 1.

“pockets” for different biological targets.

One of the most important aspects of modern

libraries that target the biologically relevant areas in chemical space.

composition of these libraries directly influences the success of drug screening programs, and

several approaches have been proposed in the design of efficien

that “general purpose” libraries for screening against multiple protein targets should meet the

following basic requirements:

the library should exhibit a

structurally related hit clusters; 4)

compounds; and 5) the compounds listed in the library must be chemically tractable. Ad

quality control and manual curation of compound libraries and databases are crucial to avoid

wasting time and resources on drug screening programs using fundamentally flawed or biased

databases.



referred to as biological space

biological space, drug

biological space forming di


1.1 The relationship between biological space and chemical spac







following basic requirements:

the library should exhibit a





databases.56,57



biological space

drug-like space

forming discrete “pockets” within chemical space, the pockets representative of


The relationship between biological space and chemical spac







following basic requirements:55

the library should exhibit a high degree of chemical diversity; 3)






or biologically

like space and chemical space is illustrated graphically in Figure 1.1, with

screte “pockets” within chemical space, the pockets representative of

different biological target spaces that in some instances coincide with


“pockets” for different biological targets. (Figure adapted from the original image in reference






55 1) the library should be rich in putative bioactive compounds; 2)

high degree of chemical diversity; 3)

structurally related hit clusters; 4) it should be free of artefact





biologically-relevant chemical space

and chemical space is illustrated graphically in Figure 1.1, with


that in some instances coincide with


(Figure adapted from the original image in reference

One of the most important aspects of modern-day drug research is the compilation of c





1) the library should be rich in putative bioactive compounds; 2)


should be free of artefact




discrete regions of chemical space, defined by particular chemical descriptors

relevant chemical space



that in some instances coincide with



day drug research is the compilation of c







should be free of artefact




mical descriptors

relevant chemical space. The relationship between



that in some instances coincide with drug-like space






several approaches have been proposed in the design of efficient libraries.54



high degree of chemical diversity; 3) it should allow the extraction of

should be free of artefact-causing reactive or unstable




mical descriptors and commonly

The relationship between



like space.

The relationship between biological space and chemical space, including discrete



libraries that target the biologically relevant areas in chemical space.53

The quality and


54 It has been proposed



should allow the extraction of

causing reactive or unstable




Page 25

and commonly




, including discrete

(Figure adapted from the original image in reference 52.)

day drug research is the compilation of compound

The quality and


It has been proposed





compounds; and 5) the compounds listed in the library must be chemically tractable. Additionally,



25

and commonly




, including discrete

)

ompound

The quality and


It has been proposed





ditionally,




The application of structure-based, or structure-assisted drug design approaches have produced a

variety of successful pharmaceutical agents since its relatively recent introduction to the medicinal

chemistry community. Some examples include the design of the influenza neuraminidase drug,

Zanamivir (RelenzaTM

);58 the thymidylate synthase inhibitor Tomudex;59 and the anticancer drug,

imatinib (GlivecTM

), a selective tyrosine kinase inhibitor.60 However, one of the best-known early

successes of structure-based drug design is the development of the human immunodeficiency virus

type 1 (HIV-1) aspartyl protease (PR) inhibitors61

(more details on the virus and the various

inhibitors identified against it, follows in section 1.3 below). The wealth of structural data

available for the retroviral PR enzyme (as well as various enzyme-inhibitor complexes) has

enabled a detailed understanding of the structure and function of the enzyme,62

and has allowed a

comprehensive definition of the biological space occupied by small-molecule inhibitors of this

biological target.63

This in turn enabled researchers (and commercial suppliers) to focus on

particular regions in chemical space and design targeted libraries of compounds with particular

physico-chemical properties known to have biological activity against the target enzyme.64,65

1.2.4 The drug development pipeline

Modern drug discovery and development is typically a multidisciplinary, multiyear and multi-

million dollar process that can be divided into three main stages or components (illustrated in

Figure 1.2), namely: the initial drug discovery stage (design and synthesis of the compounds); the

intermediate preclinical development stage (initial investigations into the biological efficacy,

toxicity, pharmacokinetic and -dynamic profiles, scale-up and possible compound formulation);

and finally the three clinical development phases66

(Phase I involves the monitoring of 20-30

healthy volunteers for the occurrence of possible side-effects and to determine suitable dosages;

Phase II involves the monitoring of 100-300 patient volunteers for compound efficacy and the

possible occurrence of side-effects; Phase III is a large-scale, long-term study of 1000-3000 patient

volunteers and monitors the safety and efficacy profiles of the test compound after continued use).

Successful candidates may receive approval from the relevant approval agencies such as the

United States Food and Drug administration (FDA) or the UK Medicines and Healthcare Products

Regulatory Authority for commercial distribution of the compound.67

It is estimated that, for every

~10 000 compounds investigated in each drug discovery program, ~250 compounds (~2.5%) show

promise in the preclinical development phase.68

Of these, only 5-10 compounds are suitable for

further development and investigation in clinical trials, finally resulting in the approval of one

compound by the FDA (final hit rate of 0.01%).68

The average overall investment required per

marketed drug is a highly controversial topic, with estimates varying greatly between

• Target selection

• Lead finding

• Lead optimisation

• Pharmacological profiling


reports.69,

compounded rate of 12.3% since 1970,

requirements for improved safety and efficacy.

Figure 1.

systemic use.

Despite the massive increase in financial and human capital investment, the number of drugs

gaining FDA approval each year has remained largely constant.

of attrition have been proposed,

processes may be required to arrest the negative trend observed in the approval of new chemical

entities (NCEs

between research within academia and industry.

research from fundamental/basic research towards knowledge transfer and innovation, has

in a closer alignment of goals between academia and industry, effectively forcing a symbiotic

relationship that will be increasingly important for the advancement of drug discovery research.

1.3 HIV/AIDS

The first evidence of a new disease, acq

1981 by a

U.S.A.78

controversy) as a retrovirus, later renamed HIV.

spread rapidly throughout the world, impacting negatively on the lives of millions of people

Drug Discovery

Target selection

Lead finding

Lead optimisation

Pharmacological profiling

2–5 years

~10 000

compounds


,70,71,72 It is estimated that pharmaceutical spending has been growing at an average



Figure 1.2 The stages of development of an “idealised” drug candidate being developed for

systemic use. (Figure compiled from data in references





entities (NCEs).74

An added consideration involves the fading of the traditional distinctions





1.3 HIV/AIDS


1981 by a medical doctor, Dr Michael Gottlieb, at the University of California in Los Angeles,

Less than three years later, the causative agent of this disease was identified (amid much

ersy) as a retrovirus, later renamed HIV.


Discovery

Target selection

Lead optimisation

Pharmacological

Preclinical Development

• Pharmacokinetic evaluation

• Evaluation of short-term toxicology

• Formulation

• Synthesis scale

1.5 y

compounds

250 compounds

Drug

candidate


It is estimated that pharmaceutical spending has been growing at an average



The stages of development of an “idealised” drug candidate being developed for

(Figure compiled from data in references











medical doctor, Dr Michael Gottlieb, at the University of California in Los Angeles,




Preclinical Development

Pharmacokinetic evaluation

Evaluation of term

toxicology

Formulation

Synthesis scale-up

• Pharmacokinetic evaluation

• Tolerability

• The occurance of sidehealthy volunteers

1.5 years

250 compounds

Development

compound


compounded rate of 12.3% since 1970,73

as a consequence of more stringent regulations and the






of attrition have been proposed,73

with some experts suggesting that a revolution in thought and







The first evidence of a new disease, acquired immunodeficiency syndrome (AIDS) was reported in





Clinical Development

Phase I

Pharmacokinetic evaluation

Tolerability

The occurance of side-effects in healthy volunteers

evelopment

compound



requirements for improved safety and efficacy.71








between research within academia and industry.75,76




uired immunodeficiency syndrome (AIDS) was reported in



ersy) as a retrovirus, later renamed HIV.79

During the following three decades,



Pharmacokinetic

The occurance of

healthy volunteers


Phase II

• Small-scale trials in patients to assess efficacy and dosage

• Long-term toxicity studies

5 –

5

compounds




(Figure compiled from data in references 68 and 66


gaining FDA approval each year has remained largely constant.73




76 A recent shift in the focus of academic










Phase II

scale trials in patients to assess efficacy and dosage

term toxicity

•Largecontrolled clinical trials

– 7 years

5-10

compounds




66)


73 Various reasons for the high ra




A recent shift in the focus of academic










Phase III

Large-scale controlled clinical trials

Drug approved for





Various reasons for the high ra













Development

controlled clinical


Phase IV

•Post-marketing surveillance

Continuous

1 compound

rug approved for

marketing

Page 27





Various reasons for the high rate





research from fundamental/basic research towards knowledge transfer and innovation, has resulted


relationship that will be increasingly important for the advancement of drug discovery research.77




During the following three decades, HIV/AIDS



Phase IV

marketing surveillance

Continuous

1 compound

rug approved for

27





te





resulted





HIV/AIDS



globally.

(UNAIDS)

disease, the number of new infections worldwide (global incidence rate) is estimated at 2.6 million

and the mortality rate due to AIDS

Although both the incidence and mortality rates show a significant decrease from previous years

and the number of people living with HIV globally is showing a steady increase,

research into new treatment strategies with alternative mechanisms of action is crucial in order to

continue the positive trends observed.

Figure 1.

Drug development and treatment strategies targeting HIV/AIDS have been largely driven by the

extensive characterisation of the virus and the viral life cycle. HIV is classified as a lentivirus of

the retroviridae

genome that consists of three genes

that are essential for viral replication and survival: reverse transcriptase (RT), inte

the aspartyl protease (PR) mentioned previously (section 1.2.3).

evolved that can be grouped into two strains, HIV

divided into four groups: “major” group ‘M’;

‘O’;84

and the latest identified group ‘P’

Moreover, HIV

their geographical distri

* Note: Subtypes E and I have not been identified as non


globally. The latest data (2010 report) released by the joint United Nation

(UNAIDS)80

reported

the number of new infections worldwide (global incidence rate) is estimated at 2.6 million






1.3 The global prevalence of HIV in 2009, as rep



retroviridae famil







Moreover, HIV-1 group M can be further subdivided into subtypes A to K,

their geographical distri

e: Subtypes E and I have not been identified as non


The latest data (2010 report) released by the joint United Nation

reported that for 2009 an estimated 33.3 million people globally are living with the







The global prevalence of HIV in 2009, as rep



family.81

Members of this family are characterised by a ribonucleic acid (RNA)







1 group M can be further subdivided into subtypes A to K,

their geographical distribution, frequency of occurrence and virulence.



for 2009 an estimated 33.3 million people globally are living with the


and the mortality rate due to AIDS-related complications reached 1.8 million people globally.









genome that consists of three genes gag, pol





and the latest identified group ‘P’85


bution, frequency of occurrence and virulence.





ted complications reached 1.8 million people globally.








pol, and env



evolved that can be grouped into two strains, HIV-1 and HIV

divided into four groups: “major” group ‘M’;82

“no

85 (“pending the identification of further human cases”).



e: Subtypes E and I have not been identified as non-recombinant forms.








The global prevalence of HIV in 2009, as reported in the 2010 WHO UNAIDS report.




env. The pol gene encodes three viral enzymes



1 and HIV-2. The HIV

“non-M, non-

(“pending the identification of further human cases”).



recombinant forms.

The latest data (2010 report) released by the joint United Nations Program on HIV/AIDS







orted in the 2010 WHO UNAIDS report.




gene encodes three viral enzymes


the aspartyl protease (PR) mentioned previously (section 1.2.3). Various genetic variations have

2. The HIV-

-O” group ‘N’;



bution, frequency of occurrence and virulence.82

s Program on HIV/AIDS





and the number of people living with HIV globally is showing a steady increase,80







that are essential for viral replication and survival: reverse transcriptase (RT), integrase (IN) and

Various genetic variations have

-1 strain can be further

O” group ‘N’;83

“outlier” group


1 group M can be further subdivided into subtypes A to K,* varying in terms of

The evolution of new

Page 28






80 continued







grase (IN) and


1 strain can be further

“outlier” group


varying in terms of


28






continued






grase (IN) and


1 strain can be further

“outlier” group


varying in terms of



viral strains and circulating recombinant forms (CRFs) have proved to be major stumbling-blocks

in the development of an effective generalised treatment strategy, as these new viral strains

(emerging through recombination of existing strains or mutation of the viral genome86,87,88

) often

exhibit resistance to the antiretroviral therapies in general use. The various areas currently under

investigation that target and disrupt retroviral replication, including synthetic antiretroviral drugs

(Table 1.1) and various restriction factors, will be shortly discussed in terms of their enzymatic or

protein target, general mode of action and/or point of interference in the viral life-cycle (Figure

1.4). For more information on the routes of transmission89

and the pathogenesis of HIV, the reader

is referred to a review by Pope et al.90

1.3.1 HIV Morphology, life cycle and antiretroviral treatment strategies

Figure 1.4 Annotated schematic representation of the HIV-1 life-cycle. (This figure was prepared

using ChemBioDraw version 12.0 software.)

Host cell

HIV HIV

Host cell

cytoplasm

Host cell

nucleus

Host cell

membrane

CD4 CCR5 /

CXCR4

HIV RNA

and proteins

HIV DNA

RT

IN

PR

Nuclear

membrane

Nuclear

pore Integrated

provirus

Viral

mRNA

Viral

mRNA

Viral

proteins

Viral

assembly

Viral

budding

Viral

maturation

2.) HIV fusion with and

entry into host cell

3.) Uncoating of the viral

capsid and release of viral

particles into the host cell

cytoplasm

4.) Reverse transcription of

vRNA into vDNA by reverse

transcriptase (RT)

5.) Transport of vDNA

into the nucleus

through nuclear pores

6.) Integration of vDNA

into the host genome by

IN

7.) Transcription of

provirus by host cell

machinery

8.) Viral mRNA exits the nucleus

through nuclear pores

9.) Viral mRNA and viral proteins

migrate to and assemble at the inner

surface of the cell membrane

10.) Immature virions bud from the host

cell membrane

11.) Mature, infectious particles are

formed upon activation of PR and

cleavage of the Gag polyprotein

1.) Attachment and

binding of HIV to CD4

and co-receptors


Table 1.1 FDA approved HIV-1 inhibitors†

Brand name Generic Name Manufacturer Name Approval date

N(t)RTIs‡

Combivir lamivudine and zidovudine GlaxoSmithKline 27-Sep-97

Emtriva emtricitabine, FTC Gilead Sciences 02-Jul-03

Epivir lamivudine, 3TC GlaxoSmithKline 17-Nov-95

Epzicom abacavir and lamivudine GlaxoSmithKline 02-Aug-04

Hivid zalcitabine, dideoxycytidine, ddC (no longer

marketed) Hoffmann-La Roche 19-Jun-92

Retrovir zidovudine, azidothymidine, AZT, ZDV GlaxoSmithKline 19-Mar-87

Trizivir abacavir, zidovudine, and lamivudine GlaxoSmithKline 14-Nov-00

Truvada tenofovir disoproxil fumarate and emtricitabine Gilead Sciences, Inc. 02-Aug-04

Videx EC enteric coated didanosine, ddI EC Bristol Myers-Squibb 31-Oct-00

Videx didanosine, dideoxyinosine, ddI Bristol Myers-Squibb 9-Oct-91

Viread tenofovir disoproxil fumarate, TDF Gilead 26-Oct-01

Zerit stavudine, d4T Bristol Myers-Squibb 24-Jun-94

Ziagen abacavir sulfate, ABC GlaxoSmithKline 17-Dec-98

NNRTIs§

Edurant rilpivirine Tibotec Therapeutics 20-May-11

Intelence etravirine Tibotec Therapeutics 18-Jan-08

Rescriptor delavirdine, DLV Pfizer 4-Apr-97

Sustiva efavirenz, EFV Bristol Myers-Squibb 17-Sep-98

Viramune

(Immediate Release) nevirapine, NVP Boehringer Ingelheim 21-Jun-96

Viramune XR (Extended

Release)

nevirapine, NVP

Boehringer Ingelheim 25-Mar-11

Fusion inhibitors

Fuzeon enfuvirtide, T-20 Hoffmann-La Roche & Trimeris 13-Mar-03

Entry Inhibitors

Selzentry maraviroc Pfizer 06-August-07

Integrase inhibitors

Isentress raltegravir Merck & Co., Inc. 12--Oct-07

Protease inhibitors

Agenerase amprenavir, APV GlaxoSmithKline 15-Apr-99

Aptivus tipranavir, TPV Boehringer Ingelheim 22-Jun-05

Crixivan indinavir, IDV, Merck 13-Mar-96

Fortovase saquinavir (no longer marketed) Hoffmann-La Roche 7-Nov-97

Invirase saquinavir mesylate, SQV Hoffmann-La Roche 6-Dec-95

Kaletra lopinavir and ritonavir, LPV/RTV Abbott Laboratories 15-Sep-00

Lexiva Fosamprenavir Calcium, FOS-APV GlaxoSmithKline 20-Oct-03

Norvir ritonavir, RTV Abbott Laboratories 1-Mar-96

Prezista darunavir Tibotec, Inc. 23-Jun-06

Reyataz atazanavir sulfate, ATV Bristol-Myers Squibb 20-Jun-03

Viracept nelfinavir mesylate, NFV Agouron Pharmaceuticals 14-Mar-97

† Data according to the U.S. Food and Drug Administration website;

http://www.fda.gov/forconsumers/byaudience/forpatientadvocates/hivandaidsactivities/ucm118915.htm; Accessed 27 February

2012. ‡ N(t)RTIs: Nucleoside (or nucleotide) reverse transcriptase inhibitors § NNRTIs: Non-nucleoside reverse transcriptase inhibitors


1.3.1.1 Viral entry inhibitors

The first point in the viral life-cycle posing a suitable target for antiretroviral drug intervention is

viral attachment to, and fusion with the host cell. The first step in the process involves the

formation of a stable complex between the viral surface glycoprotein, gp120, and CD4, the plasma

membrane-bound receptor protein located on suitable host cells (monocytes, macrophages, and on

subsets of dendritic and T-helper lymphocytes; also called CD4+ cells).

91,92 Although the formation

of this gp120-CD4 complex is sufficient for successful viral attachment to the host cell, in primate

lentiviruses such as HIV an additional cell surface receptor is required for efficient entry into the

cell.93,94

Two distinct patterns of infectivity (tropisms) exist depending on the identity of the

secondary receptors, CXCR4 and CCR5 (also called co- or fusion receptors).95

Binding of gp120 to the co-receptor (CCR5 or CXCR4) triggers further conformational changes

that result in the exposure of the hydrophobic fusion peptide of viral glycoprotein gp41 and its

subsequent insertion into the plasma membrane of the host cell, resulting in membrane fusion.96

Due to the multi-step nature of the entry process, inhibitors targeting viral entry consist of a

heterogeneous group of compounds that act against different points in the process (CD4 binding,

co-receptor binding or fusion), and as such have diverse mechanisms of action.96

The two

inhibitors currently approved for clinical use (Table 1.1) target two different steps in the entry

process: 1) Maraviroc (FDA approved in 2007) prevents co-receptor binding through interaction

with CCR5 and is the result of structural optimisation through the application of medicinal

chemistry techniques;97

2) Enfuvirtide (T-20; FDA approved in 2003) is a synthetic peptide that

prevents viral fusion with the host cell through competitive inhibition, as it mimics the HR2 region

of the gp41 fusion protein.98

Investigational entry inhibitors include vicriviroc (CCR5 antagonist;

undergoing phase II/III trials), cenicriviroc (CCR5 antagonist; undergoing phase IIb trials)99

and

AMD070 (dual tropic CXCR4 antagonist; undergoing phase I toxicity trials).100

As entry inhibitors

act against one of the earliest points of contact between HIV and host cells, this group of

compounds has attracted considerable interest for their potential use in antiretroviral microbicide

therapies.101,102,103

1.3.1.2 Capsid disassembly

Once the viral envelope has merged with the CD4+ immune cell membranes, the mature viral

capsid (or “core”, containing the viral genome and factors necessary for viral replication) is

released into the host cell. As all of the viral factors required for replication are packaged within


the fullerene

necessary for all further steps in the replication cycle. Although the mechanism of inhibition has

not been unambiguously established, it has been reported that a family of restriction factors present

in primate host cells, the tripar

structure represented in Figure 1.

has been found that binding of TRIM5

transcripts through accelerating the dissociation of the viral capsid, thereby destabilising the viral

core before it can complete essential functions.

Figure 1.

structure of TRIM5

highlighting the C

represented in reference 104)

1.3.1.3 Inhibitors of reverse transcriptase polymerase activity

The first antiretroviral drugs

polymerase function of

viral RNA to viral DNA.

target the polymerase function of the RT enzyme: the nucleoside/nucleotide RT inhibito

(N[t]RTIs) and the non

of inhibitors, as well as the differences between them, are summarised in Table 1.2.

**

Restriction factor TRIM5

monkeys. The factors differ only in the identity of the C

SPRY/B30.2 domain and TRIM

A.


the fullerene-like cone of the HIV







fore it can complete essential functions.

Figure 1.5 The structure of the TRIM5 restriction factor family

structure of TRIM5α

highlighting the C-

represented in reference 104)


The first antiretroviral drugs

polymerase function of

viral RNA to viral DNA.


(N[t]RTIs) and the non


estriction factor TRIM5


SPRY/B30.2 domain and TRIM


like cone of the HIV








The structure of the TRIM5 restriction factor family

structure of TRIM5α,108

and b) the overall secondary structure of the closely related TRIM

-terminal capsid bindi

represented in reference 104).


The first antiretroviral drugs approved

polymerase function of reverse transcriptase

viral RNA to viral DNA. Two classes of antiretroviral drugs with different mechanisms of action


(N[t]RTIs) and the non-nucleoside RT inhibitors (NNRTIs). The characteristics of the two classes


estriction factor TRIM5α is expressed by most primates, while the closely related TRIM


SPRY/B30.2 domain and TRIM-Cyp contains cyclophillin A.

like cone of the HIV-1 capsid,



in primate host cells, the tripartite motif 5 (TRIM5) proteins (the domain and overall secondary

structure represented in Figure 1.5), are able to mediate early blocks to retroviral infection.

has been found that binding of TRIM5α to the capsid surface prevents the accumulation of reverse





terminal capsid bindi


approved for

reverse transcriptase

Two classes of antiretroviral drugs with different mechanisms of action


nucleoside RT inhibitors (NNRTIs). The characteristics of the two classes


is expressed by most primates, while the closely related TRIM


contains cyclophillin A.

1 capsid,104

disassembly (or uncoat



tite motif 5 (TRIM5) proteins (the domain and overall secondary

), are able to mediate early blocks to retroviral infection.

α to the capsid surface prevents the accumulation of reverse


fore it can complete essential functions.106,107



terminal capsid binding domain.


for the treatment of

reverse transcriptase (RT), the enzyme necessary in the transcription of






monkeys. The factors differ only in the identity of the C-terminal capsid binding domain, where TRIM5

contains cyclophillin A.






to the capsid surface prevents the accumulation of reverse




ng domain.109

(The figure was adapted from that


the treatment of HIV

, the enzyme necessary in the transcription of






terminal capsid binding domain, where TRIM5

B.








The structure of the TRIM5 restriction factor family, illustrating a) the domain



HIV-infection







terminal capsid binding domain, where TRIM5

B.

disassembly (or uncoating) of the capsid is







, illustrating a) the domain



infection targeted






is expressed by most primates, while the closely related TRIM-Cyp is expressed by owl

terminal capsid binding domain, where TRIM5α contains a

Page 32

ing) of the capsid is




), are able to mediate early blocks to retroviral infection.105

It




and b) the overall secondary structure of the closely related TRIM-Cyp,**


targeted the DNA



target the polymerase function of the RT enzyme: the nucleoside/nucleotide RT inhibitors,


Cyp is expressed by owl

terminal capsid binding domain, where TRIM5α contains a

32

ing) of the capsid is




It




**


the DNA



rs,



Table 1.2 Characteristics of the two classes of RT inhibitors: N(t)RTIs and NNRTIs110

Characteristic N(t)RTIs NNRTIs

Chemical structure Analogues of the natural nucleoside / nucleotide substrates Chemically diverse; non-nucleoside /

nucleotide

Active form Metabolic conversion to 5’-triphosphates by host-cell

enzymes required No metabolic conversion required

Mechanism of action Incorporate into growing DNA chain, DNA chain-terminators Induce conformational changes in RT,

reducing the catalytic efficiency

Type of inhibition Competitive inhibition Non-competitive

Binding site on RT Catalytic site Allosteric hydrophobic pocket

Spectrum Broad spectrum antiretrovirals HIV-1 specific RT inhibitors

Selectivity Low to moderate Very high

N(t)RTIs are synthetically derived analogues of naturally occurring 2’-deoxy-nucleosides and -

nucleotides usually present within the intra-cellular environment of host cells, and are classified as

viral DNA chain-terminators. The synthetic N(t)RTIs, like their naturally occurring analogues, are

inactive in their parent form and require structural modification before being incorporated into the

growing vDNA chain. Successive rounds of phosphorylation by host cell kinases and

phosphotransferases result in the biologically active deoxynucleoside triphosphates (dNTPs). In

this form, N(t)RTIs are capable of competing with their naturally occurring analogue dNTPs for

incorporation into the viral DNA (vDNA) chain by HIV RT. Synthetic dNTPs / NRTIs lack the 3’-

hydroxyl-group required for the elongation of the vDNA chain: incorporation of the

phosphorylated form of these compounds into the vDNA chain therefore effectively halts viral

replication as genes are incompletely formed.111

3’-Azidothymidine (AZT; zidovudine) was the

first retroviral drug to receive FDA-approval in 1987.112,113

Although AZT is still widely used in

treatment strategies, a further seven N(t)RTIs have been approved for the treatment of HIV-

infection between 1987 and 2003 (Table 1.1). Investigational N(t)RTIs include Festinavir

(undergoing phase II trials), Apricitabine (successfully completed phase II trials), Elvucitabine

(undergoing phase I/II trials), and Racivir (undergoing phase II/III trials). Although NRTIs can be

regarded as the cornerstone of successful antiretroviral treatment, these compounds are subject to

potentially severe limitations in terms of drug-drug interactions,114

the emergence of

resistance,115,116

and the occurrence of adverse effects.117

Unlike the N(t)RTIs, the NNRTI class of inhibitors is highly chemically diverse (currently

consisting of over 50 compound families118

), and inhibit RT in a non-competitive manner through

binding to an allosteric site located approx. 10 Ǻ distant from the enzyme active site. Binding of


NNRTIs induces conformational changes that negatively impacts on the catalytic activity of the

RT enzyme.119

NNRTIs generally have very high specificity for HIV-1 RT,111,120

resulting in high

selectivity indexes (SI; ratio of in vitro cytotoxicity over antiviral activity) for this class of

inhibitors.121

Furthermore, the NNRTIs are biologically active in their parent form and no

metabolic conversion to an active form is required. To date, five NNRTIs have been approved by

the FDA for inclusion in treatment regimens (Table 1.1). Currently, nevirapine is included as the

NNRTI of choice in first-line therapy in resource limited countries, while delavirdine is very rarely

used.110

The next-generation NNRTIs, etravirine and rilpivirine, have demonstrated improved

resistance profiles in treatment-experienced122

and treatment-naïve patients, respectively.

Investigational NNRTIs that may show improved toxicity and resistance profiles to those currently

available include RDEA806 (undergoing phase IIB trials), IDZ899 (undergoing phase II trials) and

Lersivirine (undergoing phase IIB trials).

1.3.1.4 Disruptors of protein-protein and protein-nucleic acid interactions

In addition to the well-established DNA polymerase activity of HIV-1 RT (discussed in section

1.4.1.3), the RT enzyme possesses an RNaseH enzymatic functionality, responsible for the

degradation of the RNA strand within the RNA-DNA duplexes formed during minus-strand DNA

synthesis (Figure 1.6).123

As this RNaseH functionality is essential for successful viral replication

it remains an attractive target for antiretroviral drug intervention.124

Despite the fact that the

RNaseH structure has been well characterised,125,126

the design and development of inhibitors have

been slow, with only a limited number of inhibitors investigated in vitro due to a lack of

therapeutic efficacy, as well as difficulties experienced in cellular uptake, cytotoxicity and/or non-

specific target selection.127,128

The realisation that the RNaseH enzymatic functionality is

dependent on a two-metal ion mechanism, and the identification of the catalytic residues as well as

residues critical for the correct positioning of viral RNA in the RNaseH active site,129,130

have

revived interest in the field. One of the most important inhibitor classes targeting RNaseH

interferes with the metal ion requirement of the enzyme and includes three major groups of

compounds: 1) the N-hydroxyimides;131

2) diketo-acids;132

and 3) hydroxylated tropolones.133

Other compound classes that have shown anti-RNaseH activity include compounds such as the

hydrazones,134

vinylogous ureas,135

napthoquinones,136

and small nucleic acid fragments.137


Figure 1.

orange and indicated by the arrow.

It is well-

is significantly enhanced through association with cellular / host factors.

lens-epithelium derived growth factor p75

flexible tether during the integration process, effectively targeting and linking IN to the host

DNA.139

physiology of higher eukaryotes, including

the nucleus, where it is intimately associated with chromatin

regulation of gene expression through its interaction with the general cellular transcription

apparatus

which are located in the N

association with chromatin: the Pro

signal (NLS);

preferential binding to AT

in the C-terminal half of the protein and interacts specifical

between the conserved domains mainly consists of highly flexible peptide chains without formally

assigned conformations.

Figure 1.

LEDGF/p75

NTD


Figure 1.6 Schematic representation of HIV


-known that the activity of HIV


epithelium derived growth factor p75


LEDGF/p75 is a transcriptional co




apparatus.142

LEDGF/p75 consists of four conserved functional domains



signal (NLS);145

and a dual copy of the AT

preferential binding to AT

terminal half of the protein and interacts specifical



Figure 1.7 Schematic representation of the domain structure of LEDGF

1 - 92

/p75

TD PWWP


Schematic representation of HIV


n that the activity of HIV









which are located in the N-terminal half of the protein and are known to mediate the non



preferential binding to AT-rich DNA)




Schematic representation of the domain structure of LEDGF

NLS

92 148 -

WWP

Schematic representation of HIV


n that the activity of HIV-1 IN, and the subsequent replication efficiency of HIV









terminal half of the protein and are known to mediate the non

association with chromatin: the Pro-Trp-Trp


rich DNA).146,147




NLS

156

Schematic representation of HIV-1 RT, with the RNaseH functionality highlighted

1 IN, and the subsequent replication efficiency of HIV


epithelium derived growth factor p75 (LEDGF/p75


LEDGF/p75 is a transcriptional co-activator that plays an important role in the normal

physiology of higher eukaryotes, including humans. This co





Trp-Pro (PWWP) domain;

and a dual copy of the AT-hook DNA

147 The fourth, integrase binding domain (IBD) is located




AT Hook

178 - 197

1 RT, with the RNaseH functionality highlighted



LEDGF/p75), has been sugge


activator that plays an important role in the normal

humans. This co-activator is predominantly localised in

the nucleus, where it is intimately associated with chromatin in vivo




Pro (PWWP) domain;

hook DNA-binding motif

The fourth, integrase binding domain (IBD) is located

terminal half of the protein and interacts specifically with HIV



347



is significantly enhanced through association with cellular / host factors.138

One of these co

, has been suggested to act as a highly



activator is predominantly localised in

in vivo140,141




Pro (PWWP) domain;143,144

the nuclea

binding motif


ly with HIV-1 IN.


Schematic representation of the domain structure of LEDGF/p75.

347 - 429

IBD



One of these co

sted to act as a highly




and is involved in the


(Figure 1.7


the nuclear localisation

binding motif (so named for their


1 IN.148

The linker regions


.

Page 35

1 RT, with the RNaseH functionality highlighted in

1 IN, and the subsequent replication efficiency of HIV-1,

One of these co-factors,







7), three of

terminal half of the protein and are known to mediate the non-specific

r localisation

(so named for their


The linker regions


530 aa

CTD

35

in

1,

factors,







, three of

specific

r localisation

(so named for their


The linker regions



X-ray crystal structures have illustrated the structural basis for the recognition between the

LEDGF/p75 IBD and the HIV-1 IN catalytic core domain (CCD):149

an inter-helical loop in the

LEDGF/p75 IBD fits into a cleft in the dimer interface formed during the dimerisation of HIV-1

IN,150

with the interaction stabilised through charge-charge interactions mediated through the N-

terminal domain (NTD) of IN, while binding primarily involves the CCD of HIV-1 IN

(specifically through an extensive hydrogen bond network involving essential residue D366151

).152

Further experimental data have confirmed these findings and found the correct binding ratio

between each HIV-1 IN monomer and LEDGF/p75 to be 2:1.153

The structural interaction of

LEDGF/p75 and HIV-1 IN also has a functional implication: knock-down studies have illustrated

the importance of LEDGF/p75 in the correct nuclear localisation of the pre-integration complex

(PIC) and efficient replication of HIV-1,154,155

and making this protein-protein interaction an

attractive target for antiretroviral intervention. A small-scale screen based on yeast and mammalian

two-hybrid assays have identified a benzoic acid derivative, D77, as a nuclear localisation

disruptor. D77 competitively binds to the dimer interface of HIV-1, preventing the interaction

between the LEDGF/p75 IBD and HIV-1 IN and effectively decreasing the replication efficiency

of HIV-1.156

In a larger screen, 700 000 small molecules were evaluated for their potential as

disruptors or inhibitors of the IN-LEDGF/p75 interaction in a luminescent proximity assay

(AlphaScreenTM

), identifying ~90 compounds that selectively inhibited the binding of LEDGF/p75

to IN.157

In an alternative approach, synthetic peptide inhibitors derived from LEDGF/p75 have

shown promise as multimerisation enhancers of HIV-1 IN.158

Upon binding, these peptides

effectively shift the IN multimer from the dimeric DNA-binding form to the non-DNA-binding

tetrameric form, thereby disrupting in vitro integration of viral DNA into the host genome.158

Although complete LEDGF/p75 knock-down in mice have resulted in high embryonic lethality,

disruption of LEDGF/p75 in cell culture is well tolerated.159

Moreover, as the LEDGF/p75

treatment strategies in current development target the interaction between LEDGF/p75 and HIV-1

IN and not the expression of LEDGF/p75 itself,156,157,158

it is likely that inhibitors (or disruptors) of

the IN-LEDGF/p75 interaction may be developed without influencing the necessary interactions

between LEDGF/p75 and its cellular partners.123

1.3.1.5 Integrase inhibitors

HIV-1 IN is essential for the replication of HIV-1 in human cells as it is responsible for the

incorporation of proviral DNA into nuclear DNA, resulting in a transcriptionally active integrated

provirus.160,161

During this integration process, IN catalyses two sequential, spatially distinct,

metal-dependent reactions: 3’-end processing and strand transfer. 3’-End processing is a highly


specific reaction involving the priming of the 3’-end of vDNA through the release of the terminal

dinucleotide from both U3 and U5 ends of the chain, exposing the 3’-hydroxyl group of the highly

conserved dinucleotide at each end.162

This process takes place in the cytoplasm shortly after

reverse transcription.163

In contrast, the strand transfer reaction takes place after translocation of

the PIC into the nucleus; a second trans-esterification reaction targeting the liberated 3’-hydroxyl

group results in the full-site integration of a fully functional copy of the viral genome into the host

cell genome. Although both of these reactions are catalysed by HIV-1 integrase, it has been

proposed that different forms of the enzyme are required for each reaction. Specifically, dimeric

IN has been implicated in the 3’-end processing of blunt-ended vDNA164,165,166

but is unable to

perform the full-site strand transfer reaction in vitro, while a tetrameric organisation of the enzyme

is required for efficient strand transfer in vivo,167,168,169

but which is unable to bind unprocessed

vDNA and catalyse the 3’-end processing reaction.170

The monomeric structure of HIV-1 IN

consists of a 32-kDa unit composed of a polypeptide chain that folds into three independent

functional domains: the NTD (residues 1-55); the CCD (residues 56-209); and the C-terminal

domain (CTD; residues 210-270). Each of these domains is essential for efficient concerted

integration to occur and each has a distinct function during this process. Due to a high

multimerisation potential and protein-protein affinity, the hydrophobic HIV-1 IN is rarely present

in its monomeric form and has been reported in dimeric, tetrameric, octameric, as well as higher

oligomeric states.171,172,173

Until recently,174

limited structural data were available to explain the

overall geometry and function of HIV-1 IN: the hetero-dimeric crystal structures of the CCD/CTD

domains175

and the CCD/NTD domains176

had been solved, but the relative orientations of the

three domains in the enzyme monomer remained unclear. All attempts at crystallisation of the full

monomer (or higher oligomeric states) failed due to enzyme solubility issues and protein

aggregation. This absence of complete experimental structural data gave rise to the development of

several theoretical models of HIV-1 IN,177,178

illustrating possible conformations that the monomer

(and oligomeric forms) may adopt,179,180,181,182,183

and serving as templates in the design of HIV-1

integrase inhibitors (INIs).

Models of HIV-1 IN were mostly constructed through computational or manual super-positioning

of the subunit X-ray crystal structures and in some cases determined for the different subunits with

assistance from the NMR-derived structures.179,180,181,182,183

The first of these full-length models

built from individual domains was reported by Podtelezhnikov.179

In this model, the position of the

domains was based on results obtained from fluorescence depolarisation studies. Importantly, the

linkers between the NTD and CCD (residues 48-55) and the CTD and CCD (residues 210-218)


were not completed and the model did not consider the divalent metal ions or the cofactors that are

required for catalytic activity of HIV-1 IN. The models predicted by De Luca180

and Karki181

were

constructed via super-positioning of two-domain crystal structures. In both of these models, the

domains were fully connected and the divalent metal ions in the enzyme active site were included.

Wijitkosoom183

prepared models of the full-length HIV-1 IN using molecular dynamics to

determine the influence of the two terminal domains (NTD and CTD) on the mobility of the CCD,

as well as the effect of incorporating one divalent metal in the active site. The orientations of the

subunits relative to each other in these monomer models were mostly directed by those observed in

X-ray crystal structures of two-domain fragments (NTD/CCD and CCD/CTD). The complexity of

later models increased greatly, with the inclusion of both divalent metal atoms and the terminal

ends of vDNA bound in the enzyme active site, as well as proposing multimeric and co-factor

bound states of the enzyme complex.184

Additionally, various scenarios were predicted for the

interaction of HIV-1 IN (both complexed to other proteins in the form of the PIC and bound to

vDNA only) with host DNA. For an excellent review and in-depth analysis of the various models

reported for HIV-1 IN, the reader is referred to the book chapter by Liao and Nicklaus.185

The first full-length crystal structure of retroviral integrase, solved for prototype foamy virus

integrase (PFV IN) in the presence of cognate vDNA ends bound to the enzyme active sites174

finally offered a clear insight into the overall structure of the functional enzyme. The solving of

this crystal structure marks an important milestone in the field of antiretroviral drug design

targeting IN. Despite the high degree of structural deviation observed between models generated

for the HIV-1 IN and the crystal structure of PFV IN,185

the tetrameric IN-DNA complex as

reported in the PFV crystal structure will undoubtedly play a pivotal role in drug discovery

programs in the foreseeable future.

Several classes of small molecules have been investigated for their potential as HIV-1 INIs.186

During the early 1990s, topoisomerase II inhibitors were cross-screened against the HIV-1 IN

enzyme in polyacrylamide gel electrophoresis (PAGE) assays,187

leading to the realisation that the

presence of the dihydroxynaphthaquinone moiety was sufficient for inhibition of purified IN. This

study also noted that the keto-enol motif (generally known to chelate divalent metals) were

responsible for a decrease in the enzymatic efficiency of HIV-1 IN. Following from these findings,

investigations were launched into the anti-IN activity of the polyhydroxylated aromatic compounds

(PHAs),188,189,190

as well as non-catechol-containing aromatics such as sulfones and

sulfonamides;191,192

hydrazides;193

and coumarins,194,195

with little success in identifying superior


INIs. Furthermore, several compound classes were identified that could act as potential allosteric

inhibitors of HIV-1 IN and/or DNA binders, including: carbazoles;196

pyrrolidine acetamides;197

naphthamidines;198

and aminobenzimidazoles.198

The first class of compounds that showed

verifiable strand transfer inhibition, subsequently halting the integration reaction, was the diketo

acid class of compounds.199,200

Although many structurally diverse analogues have resulted from

bioisosteric replacement of the carboxylic acid group, rearrangements of the keto-enol

functionality and modification of the fluorobenzyl group, most of the potent HIV-1 INIs comply

with the structural requirements of the diketo acid pharmacophore.201

The discovery of raltegravir

(Isentress; Table 1.1), the only HIV-1 INI currently approved by the FDA, is a good example of

the classical medicinal chemistry approach,202

requiring the consecutive replacement of the diketo

acid moiety with diaryl ketones (resulting in the first HIV-1 INI to enter clinical trials, S-1360),203

dihydroxypyrimidine carboxamides,204

and naphthyridine ketones,205

before finally arriving at the

substituted methylpyrimidinone structure that gave rise to raltegravir.206

Other HIV-1 INIs in

clinical trials (summarised in Table 1.3) include elvitegravir (GS-9137; undergoing phase III

trials),207

GSK-364735 (phase II trials halted)208

and dolutegravir (S/GSK 1349572; undergoing

phase II trials).209

Table 1.3 Examples of HIV-1 INIs in clinical trials.201

Compound Company Structural class Status

Elvitegravir (GS-9137) Gilead Sciences 4-Quinolone-3-carboxylic acid Phase III

GSK-364735 GlaxoSmithKline 1,6-Naphthyridinone carboxamide Halted phase II

Dolutegravir (S/GSK 1349572) Shionogi-GSK Pyrido-pyrazino-oxazine carboxamide Phase III

1.3.1.6 Silencing of viral mRNA

Transcription of the integrated provirus to viral messenger RNA (mRNA) and genomic vRNA

proceeds through the highjacking of host cell mechanisms. It has been proposed that non-coding

RNAs (ncRNA) present in macrophages and T-helper cells may play a role in the “silencing” of

viral mRNA and attenuation of viral replication.210

RNA-interference (RNAi) is proposed to

involve limited and imperfect (“wobbled”) base-pairing between microRNA RNA-induced

silencing complexes (miRNA-RISC) and viral mRNA.211

In most instances the replication

attenuation achieved through the RNAi-activity of the cell is only partial, as the process is

inhibited by the RNAi-suppressor activity of the virus-encoded arginine-rich RNA-binding protein

(Tat),212

and the viral trans-activation responsive (TAR) RNA which acts as an RNAi-decoy.213


1.3.1.7 Aspartyl protease inhibitors

The development of HIV-1 PR inhibitors is regarded as a major success of structure-based drug

design.214,215,216,217,218

Until the advent of the INIs, these inhibitors were considered the most potent

drugs available for the treatment of AIDS.217

The homodimeric HIV-1 PR allows viral maturation

by sequentially cleaving at least 10 asymmetric and nonhomologous sequences in the viral gene

region that code for essential enzymes and proteins (the gag-pol polyproteins), making it a

desirable target for therapy.219,220

The HIV-1 PR inhibitors approved by the FDA (Table 1.1) act as

competitive inhibitors,221

binding at the enzymatic active site and directly competing with the

enzyme’s ability to recognise substrates.222,223

Despite the structural diversity present in this class,

all but one of the initial HIV-1 PR drugs developed act as peptidomimetic transition state

analogues that occupy a similar space in the enzyme’s active site. As these molecules were

designed as peptide analogues, they generally have poor bioavailability and a specific toxicity

profile (including lipodystrophy).224

Additionally, mutations in the HIV-1 PR amino acid sequence

have been shown to cause single- and multidrug resistance to the known peptidomimetic class of

PR inhibitors without substantially altering substrate binding.225,226,227

Subsequently, several non-

peptidic compound classes have been investigated as HIV-1 PR inhibitors through substitution of

the peptidomimetic hydroxyethylene backbone with cyclic urea, 4-hydroxycoumarin, L-mannaric

acid and 4-hydroxy-5,6-dihydro-2-pyrone, respectively.224,228

Inclusion of 4-hydroxy-5,6-dihydro-

2-pyrone as the inhibitor backbone resulted in the identification and subsequent FDA approval of

tipranavir,229

one of the first HIV-1 PR inhibitors to show an alternative resistance profile with

little cross-resistance to the peptidomimetic PR inhibitors. Although the FDA does not currently

report any investigational HIV-1 PR inhibitors,230

several PR inhibitors are currently in preclinical

development (some are represented in Table 1.4).231

Even though the use of PR inhibitors in

antiretroviral treatment strategies has proved particularly successful in decreasing the patient viral

load, it is important to note that severe toxicity and significant drug-drug interactions may be

associated with this class of compounds.232

Table 1.4 HIV-1 PR inhibitors in preclinical development.231

Compound Company Status

SM-309515233

Sumitomo Preclinical

P-1946234

Pharmacor Preclinical

GRL-02031235

NCI/Kumamoto University/ University of Illinois at Chicago Preclinical

GS-8374236

Gilead Preclinical


1.3.1.8 Maturation inhibitors and cellular restriction factor

The Gag polyprotein (transcribed from the integrated provirus through the action of host cell

mechanisms) contains the structural proteins of HIV

cleavage by HIV

nucleocapsid proteins (NC), as well as some spacer peptides (SP1 and SP2) and p6.

step in the assembly of new immature viral particles, the Gag polyprotein migrates to

assembles at the inner surface of the host cell plasma membrane, mainly as a consequence of direct

interactions between MA (at the N

The formation of the immature virions are stabilised through

polypeptide, effectively forming an interaction lattice primarily mediated by CA and SP1 (located

in the central domain of the Gag polyprotein).

of the Gag polyprotein) repo

facilitating the incorporation of the viral genome into the immature virions. The assembly of the

immature virion proceeds in such a way as to orientate the NC/RNA complex towards the

the newly formed virion. Upon the autoproteolysis and subsequent activation of HIV

the late stages of viral assembly, the Gag polyprotein is processed at five different processing sites

(MA-CA; CA

HIV-1 infection)

Figure 1.

schematic representation of the Gag polypeptide

A.

C.

SP2p6





cleavage by HIV-1 PR during viral maturation: the matrix protein (M











ewly formed virion. Upon the autoproteolysis and subsequent activation of HIV


CA; CA-SP1; SP1

1 infection)109

to initiate the maturation of the viral particle (Figure 1.

Figure 1.8 Schematic illustrations of the a) immature and b) mature H


NC

SP2





1 PR during viral maturation: the matrix protein (M













SP1; SP1-NC; NC


Schematic illustrations of the a) immature and b) mature H


NC SP1








interactions between MA (at the N-terminal of the Gag polyprotein) and the plasma membrane.




of the Gag polyprotein) reportedly binds genomic viral RNA with high specificity,





NC; NC-SP2; and SP2




Genomic vRNA

SP1 CA








terminal of the Gag polyprotein) and the plasma membrane.



in the central domain of the Gag polyprotein).238,239

Furthermore, the NC (located at the C

rtedly binds genomic viral RNA with high specificity,





SP2; and SP2-p6; where all five proc



schematic representation of the Gag polypeptide, illustrating the five processing sites and the

MA

CA

Genomic vRNA

NC

CACTD

1.3.1.8 Maturation inhibitors and cellular restriction factors


mechanisms) contains the structural proteins of HIV-1 that are generated through proteolytic














p6; where all five proc



, illustrating the five processing sites and the

B.

CANTD


1 that are generated through proteolytic

1 PR during viral maturation: the matrix protein (MA); capsid protein (CA) and





The formation of the immature virions are stabilised through lateral interactions of the Gag








p6; where all five processing sites are essential for

to initiate the maturation of the viral particle (Figure 1.8).

Schematic illustrations of the a) immature and b) mature HIV




A); capsid protein (CA) and

nucleocapsid proteins (NC), as well as some spacer peptides (SP1 and SP2) and p6.109




lateral interactions of the Gag






ewly formed virion. Upon the autoproteolysis and subsequent activation of HIV-1 PR during


essing sites are essential for

).

IV-1 virions; with c) a


MA

Page 41




109 As a first

step in the assembly of new immature viral particles, the Gag polyprotein migrates to and


terminal of the Gag polyprotein) and the plasma membrane.237



Furthermore, the NC (located at the C-terminal

rtedly binds genomic viral RNA with high specificity,240,241


immature virion proceeds in such a way as to orientate the NC/RNA complex towards the centre of

1 PR during



1 virions; with c) a


41




As a first

and


237



terminal

241


of

1 PR during



1 virions; with c) a



position of the uncleaved structural proteins.109

(The figure was adapted from that represented in

reference 109).

Several non-protease inhibitors of retroviral maturation have been identification, represented by

two classes of compounds. The first class can be represented by Bevirimat242,243

(PA-457 or

betulinic acid; halted phase II trials244

) that reportedly binds to the CA-SP1 processing site and

slows viral maturation.245

The second class binds to the processed CA protein and inhibits capsid

assembly: the first of these CA-binders were the methylphenylurea compounds (including CAP-

1246

) and certain peptide inhibitors (including CA-I247,248

). Although neither of the two maturation

inhibitors identified to date bind strongly enough to be clinically relevant, viral maturation remains

an attractive replication intervention target for further development.

Apolipoprotein B mRNA-editing enzyme 3 (APOBEC3 or A3) genes are uniquely mammalian

genes that incorporate a family of cytidine deaminases believed to play an important role in host

immune responses to retroviral infections. These genes are catalytically active and found in

varying numbers in different mammalian species: for example, one A3 gene has been reported for

mice, while humans reportedly have seven different A3 genes (A3A to A3G).249

Specifically, the

human A3 proteins A3F and A3G have demonstrated significant inhibition of HIV-1,250,251

through

the deamination of multiple cytidine residues to yield uracil residues, thereby effective mutating

the viral genome to death.252

Although uracil is a naturally occurring RNA base, it is often

mistakenly incorporated into DNA chains, forming one of the most frequent DNA lesions.

Although numerous viruses have developed counterstrategies to eliminate these lesions, the

incorporation of uracil may be an innate immune defence system against certain viruses, including

HIV-1.253

During viral assembly at the host cell membrane A3 proteins are packaged into the

newly formed immature virions through association with the viral Gag polyprotein and viral or

cellular RNA,254

to assert the observed antiviral activity during the early phase of viral

replication.255

The viral “accessory” gene viral infectivity factor (Vif), has been reported to

neutralise the antiviral effect of A3 proteins, as it physically prevents their inclusion into the

immature virion particles though a proteasome-mediated degradation of the A3 proteins256

(a

second degradation-independent mechanism has also been reported256

).

Viral protein U (Vpu) is an “accessory” viral protein that is involved in the detachment and

release of newly formed infectious viral particles from the host cell surface.257,258

It has been

reported that the Vpu-dependent release of viral particles vary between different cell lines,259


leading to the discovery of several host factors capable of reducing the efficiency of viral release

upon over-expression.260

Of these, bone marrow stromal cell antigen 1 (BST-2; also called

tetherin, CD317 or HM1.24) is particularly interesting as it is generally well expressed in Vpu-

dependant cell types. BST-2 is a 30-26 kDa type II transmembrane glycoprotein with an N-

terminal transmembrane domain (TM) and a C-terminal glycosyl-phosphatidylinositol (GPI)

anchor.261

It has been proposed that BST-2 effectively tethers otherwise completely detached

virions to the host cell surface,260C

and interferes with Vpu-dependent virion release via a direct

interaction between the respective TM domains of the two proteins.255

Although BST-2 potently

inhibits virion release it is not classified as an attractive target for drug intervention research, as

Vpu is not a strict requirement for viral replication.262

Therefore, although inhibition of Vpu by

BST-2 (or any other compound or protein) may significantly reduce the release of viral particles

and inhibit cell-free infection, viral spread will simply continue in a cell-to-cell manner.257,263

In addition to the well-known cellular restriction factors discussed above (APOBECs, TRIM5α,

BST-2/Tetherin and others), several novel host factors have been identified in recent years to

function as either restriction factors (proteins that counter specific viral proteins) or repressive

factors (inhibitors of the HIV-1 life cycle).264

Of these, TRIM28/KAP1 has been shown to restrict

integration of HIV-1;265

p21(Waf/Cip1/Sdil (p21) has demonstrated an important role during or

after reverse transcription;266

and SAMHD1 has been shown to act prior to integration, possibly

through degrading or otherwise preventing the accumulation of vDNA.267

In addition, the PAF1

complex has demonstrated an important role during the early events from post-entry to integration

of proviral DNA into the host genome;268

SETDB1 plays a role in the methylation of the viral Tat

protein, leading to reduced viral replication; and the HECT domain and RCC1-like domain-

containing protein 5 (HERC5) restricts the early stages of viral assembly at the host membrane.269

1.3.1.9 Viral latency

Despite initial promise and some successes achieved with the intervention approaches discussed

above, complete eradication of HIV-1 infection has not been achieved. This is in large part due to

the persistence of integrated proviral HIV-1 in certain long-lived reservoir cells, such as resting

CD4+ T lymphocytes, macrophages and dendritic cells, even in the presence of intensive treatment

strategies (discussed below).270

The integrated provirus present in these cells is generally

transcriptionally silent while the cells are in a resting state, but upon activation will establish a new

systemic infection in the absence of antiretroviral treatment.271

Dendritic cells have been reported

to capture and internalise extra-cellular virions via the dendritic cell-specific intercellular adhesion


molecule-3-grabbing non-integrin (DC-SIGN) lectin,272

with subsequent in trans transmission of

the captured virions to uninfected T cells. Although the internalised virions remain infectious for

several hours, DC-SIGN does not protect against degradation and the virions are eventually

deactivated.273

In contrast, mature virions encapsulated in the late endosomes of infected

macrophages274

may retain infectivity for weeks,275

effectively creating a medium-term reservoir

for infectious viral particles. While both dendritic and macrophage cells effectively hide infectious

viral particles and contribute to the spread and cell-to-cell transmission of HIV-1, the major long-

term reservoir of HIV-1 infection is resting CD4+ cells that carry an integrated provirus.

270 The

presence of several post-entry blocks against retroviral infection in resting CD4+ cells

276 (including

APOBEC3G277

and TRIM5α105), prompted the realisation that these infected cells derive from

activated CD4+ lymphoblasts that have reverted back to a resting state.

278 Eradication of integrated

provirus from resting CD4+ cells is not yet feasible, although various different strategies have been

attempted. Treatment of latently infected CD4+ populations with interleukin-2 or global T-cell

activators in the presence of intensive antiviral therapy resulted in the enhancement of viral

replication beyond that which can be contained by antiretroviral therapy.279

In a separate study, the

histone-deacetylase inhibitor valproic acid, has demonstrated the ability to induce outgrowth of

HIV-1 from resting CD4+ cells without activation of the cells.

280 When combined with intensive

antiretroviral therapy, such treatments may allow the outgrowth of latent HIV-1 without the

associated activation of the CD4+ cells. A third approach would involve the selective elimination

of infected CD4+ memory cells, however, to date no study has been successful in distinguishing

between infected and non-infected CD4+ memory cells, although some distinction between CD4

+

and CD8+ T cells has been achieved in ex vivo studies.

281 Finally, the various forms of unintegrated

vDNA may be regarded as an additional viral reservoir in certain circumstances, as it has the

capacity to result in pre-integration latency,282

as well as the potential of being rescued at later

stages of the cell cycle, leading to productive infection.283

Certain interactions with host DNA

repair enzymes have been reported to degrade or inactivate unintegrated vDNA, potentially

representing a form of host antiviral defence.284

1.3.2 Global response to the HIV/AIDS pandemic

During the past three decades, the vast intellectual efforts and monetary support poured into the

search for effective antiretroviral agents have resulted in the FDA approval of more than 20

antiretroviral agents currently used in antiretroviral treatment regimes. Several of these were

developed through the structure-based drug design and optimisation approach, discussed

previously.


Although the spread of HIV infection has been slowed in many parts of the world, research into

the development of vaccines and into the discovery of new drugs for the treatment of the many

opportunistic diseases associated with AIDS is a continuous need (for more information on recent

advances in the development of an HIV-1 vaccine, please refer to a review by Girard, et al.285

).

While many successful drugs have been developed, HIV monotherapy has failed due to the

development of resistance to the drugs administered. In an attempt to address this problem,

attention has moved to therapy that uses a combination of three or more potent anti-viral agents

with different mechanisms of action, the so-called highly active antiretroviral therapy

(HAART).286

The continued treatment of HIV-1 with this combination therapy, containing

typically at least two RT inhibitors and one PR inhibitor or more recently two RT inhibitors and an

IN inhibitor, has significantly reduced morbidity and mortality287,288

and is credited with an

approximately three-fold drop in the mortality rate from AIDS since its introduction.289

Despite

this success, the emergence of HIV-1 mutants that resist current drug regimens remains a critical

factor in the clinical failure of antiviral therapy. The relatively rapid appearance of resistant viral

mutants among treated HIV-1 patients can be attributed to the high rate of replication of the virus,

coupled with a high intrinsic rate of mutation due to the infidelity of the HIV-1 reverse

transcriptase. Additionally, the risk of HIV-1 drug resistance increases with poor patient

compliance to prescribed drug usage or when structured treatments are interrupted.290

This

necessitates the continued search for new drugs with alternative modes of action against HIV-1,

which can be used in conjunction with those already available.291

1.4 Rationale of the study

Through millennia of trial and error, the field of medicinal chemistry has evolved into a discrete

science, with modern drug discovery initiatives producing remedies, cures and treatments to most

of the ailments and diseases known to assail humankind. One of the great challenges of medicinal

chemistry in recent years was in dealing with the relatively newly identified HIV/AIDS pandemic.

Vast resources are currently directed at the development of a vaccine against this fast-spreading

disease, and although several potential candidates have been progressing through clinical trials, it

may be any number of years before an effective vaccine will be commercially available. In the

meanwhile, intense research is aimed at the discovery and improvement of different classes of

antiretroviral treatments and medications that will enhance the quality of life for those individuals

already infected, while prevention programmes worldwide attempt to slow the spread of the HIV

infection. This project was designed to reflect the multidisciplinary nature of the drug discovery


field, employing three distinct disciplines (computational modelling, organic chemistry and

biochemistry) in the discovery and development of a range of synthetic compounds with

favourable pharmacokinetic profiles that would exhibit inhibitory potential towards HIV-1 IN, an

enzyme essential to the survival and effective replication of HIV-1.

1.5 Hypothesis

Inhibitors of the HIV-1 IN strand transfer reaction can be designed, synthesised and biochemically

validated through the application of standard medicinal chemistry practices and techniques, to

serve as first generation lead candidates in further rounds of compound optimisation and

development.

1.6 Objectives of the study

In this study, the first generation structure-based design of a new class of HIV-1 INIs will be

investigated. A suitable in silico model of the HIV-1 IN active site will be prepared from existing

structural data. This will be used to identify the docking ability of compounds that exhibit a degree

of inhibitory activity against HIV-1 IN in vitro. Any compound families identified as potential

inhibitors and chosen for further study will be synthesised, purified and characterised using known

organic chemistry techniques. The inhibitory activity and biological profiles predicted for these

compounds will be verified in a range of suitable biochemical assays. In addition, any lead

compound identified from the first generation design will be subjected to structural modification

and optimisation of the structure-activity relationship in iterative design and testing phases. Lastly,

this study will evaluate the initial phases of in vitro testing performed on newly identified

inhibitors, with extrapolations to certain in vivo scenarios; no in vivo testing will be performed

with any compounds identified.


1.7 References

1. Sneader, W., Drug Discovery: A History, John Wiley & Sons Ltd., West Sussex, England,

2005, 3-4.

2. Burns, W. R., Endeavour, 2008, 32, 3, 101-106.

3. Kuehl, F. A., Spencer, C. F., Folkers, K., J. Am. Chem. Soc., 1948, 70, 2091-2093.

4. Zhu, S., Meng, L., Zhang, Q.,Wei, L., Bioorg Med Chem Lett. 2006, 16, 7, 1854–1858.

5. Jiang, S., Zeng, Q., Gettayacamin, M., Tungtaeng, A., Wannaying, S., Lim, A.,

Hansukjariya, P., Okunji, C. O., Zhu, S., Fang, D., Antimicrob. Agents Chemother. 2005,

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2011, 121, 1549-1560.

267. Laguette, N., Sobhian, B., Casartelli, N., Ringeard, M., Chable-Bessia, C., Segeral, E.,

Yatim, A., Emiliani, S., Schwartz, O., Benkirane, M., Nature, 2011, 474, 7353, 654-657.

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269. Woods, M. W., Kelly, J. N., Hattlmann, C. J., Tong, J. G. K., Xu, L. S., Coleman, M. D.,

Quest, G. R., Smiley, J. R., Barr, S. D., Retrovirology, 2011, 8, 1, 95.

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Chapter 2: In Silico Methods Page 48

CHAPTER 2: IN SILICO METHODS

2.1 Introduction

Historically, serendipity has played a major role in drug discovery.1,2

Many examples exist of drugs

that were discovered as fortuitous by-products in the pursuit of entirely different goals (penicillin,3

acetanilide,4 nitrogen mustard,

5 cisplatin,

6,7 warfarin,

8,9 and cyclosporine,

10 to name a few).

Although elements of this so-called “serendipitous discovery” approach still remain, modern drug

discovery has evolved significantly in recent years, with a greater focus being placed on the rational

design of pharmaceutical agents for specific macromolecular targets.11,12

This shift in focus was

aided in no small part by the recent advances in computational capabilities13

and biological assay

development,14

as well as significant improvements in the instrumentation and techniques for the

structural elucidation of proteins.15,16

Through the use of these more advanced technologies, a much

deeper understanding could be gained of the structure and mechanistics of target macromolecules in

their natural environment, and it became possible to design small drug molecules to interact with

and specifically alter the functionality of selected target macromolecules. By definition, medicinal

chemistry is “the discipline concerned with determining the influence of chemical structure on

biological activity”.17

Therefore, the application of medicinal chemistry principles to modern-day

drug design not only requires a complete understanding of the physicochemical properties of the

chemical structure of interest (i.e. the drug) and the mechanisms by which the drug exerts its effect,

but also necessitates a detailed knowledge of the structure and function of the biological target,

whether protein or biological pathway.

Modern-day drug discovery mainly focuses on two broad approaches: the random screening

approach (commonly referred to as the shotgun approach) allows the identification of lead

compounds through the random (often automated) in silico or in vitro screening of compound

libraries against protein receptor targets.18

In contrast, the rational drug design approach involves

the conscious step-by-step construction of a drug-like molecule (or family of related molecules).19,20

The random screening and rational drug design approaches are complementary and are often used in

parallel, and in combination, to design a successful drug.

As mentioned, the evolution of computational methods has greatly aided in the discovery and

design of new “drug-like” molecules.21

One of the most useful computational techniques is three-


dimensional (3D) database searching. 3D databases contain information on the conformational and

functional properties of the molecules contained therein, allowing the identification of lead

compound series based on predicted interactions within the 3D environment of specific receptor

sites (also termed molecular recognition). Two types of 3D database searching have evolved to

allow the identification of enzyme inhibitors.22

Both of these methods make use of a

pharmacophore, a manually or computationally generated set of desirable molecular characteristics

that is grouped, fixed into a specific arrangement in 3D space and finally used as a database probe

to identify only those database molecules that possess all of the desirable elements defined during

the generation of the pharmacophore. The first of these methods was developed to deal with

situations where detailed information of the receptor or active site is not available. It is based on

identifying key similarities in a series of molecules with known activity against the target receptor,

these are subsequently grouped and collectively referred to as a ligand-derived pharmacophore.23

In

this sense, the first method can be regarded as a 3D ligand-based approach to database searching.

The second method is employed when the 3D structure of the target protein or enzyme is known,

either from X-ray crystallography, NMR structure elucidation or homology modelling.23

This

method involves the use of pre-existing structural knowledge of the receptor in the generation of a

receptor-based pharmacophore, and is regarded as a structure-based method for database searching.

Again, these techniques are not mutually exclusive and have been successfully combined to

discover new compound classes targeting, as an example, HIV-1 IN.24

Despite years of intensive research by numerous groups,25

the structure of the biologically-relevant

strand-transfer complex of HIV-1 IN remains elusive, and therefore rational drug design research

centred on the HIV-1 IN enzyme has been either ligand-based, making use of known inhibitor

structures, or based on models of the enzyme constructed from incomplete structural data.26

The

recent elucidation of a crystal structure for the tetrameric strand-transfer complex of the closely

related PFV IN27

has provided much impetus for continued research in this field, providing a clearer

picture of the probable multimeric structure of HIV-1 IN, the positioning and 3’-end processing of

vDNA in the enzyme active site and the insertion of 3’-end processed vDNA into the host genome.

The dimeric model of HIV-1 IN detailed in this chapter was generated based on literature reports

pre-dating the PFV intasome crystal structure published in 2010, and was constructed through the

superpositioning of HIV-1 IN sub-domains that were in the public domain pre-2010. (The

importance and relevance of this information, i.e. the timing of the current work in light of the

publication of the PFV intasome crystal structure, shall become clearer as this Chapter plays out and

as the chemical synthesis and biological testing phases of the project are detailed. In brief, the in


silico work, much of the chemical synthesis and some of the biological testing work had already

been completed by the time the PFV intasome crystal structure was published. The results of the

present study can therefore be weighed against the PFV intasome crystal structure.) All crystal

structures of the HIV-1 IN CCD, CTD and NTD deposited in the protein data bank (PDB; pre-

2010) were retrieved and analysed for quality of resolution, integrity of chain structure and the

presence of co-crystallised elements. Selected data were used to assist in the construction of this

model. The model was constructed to represent a snapshot of the dimeric HIV-1 IN strand transfer

complex before insertion of vDNA into the host genome. The constructed HIV-1 IN monomeric

strand transfer model and the relevant fragments of the PFV IN crystal structure were validated

stereo-chemically using PROCHECK.28

The sequence alignment and the Ramachandran plots after

minimisation are shown for the HIV-1 IN model and the PFV crystal structure (section 5.2.5 of the

experimental methods chapter), and compared to promote a better understanding of the similarities

and differences between the two structures. Upon completion, the model was used in conjunction

with a molecular docking software suite (SYBYLTM

SurflexDock, Tripos Ltd.29

) to predict the

structure of the inter-molecular protein-ligand complex formed between each of the compounds in a

chemically diverse compound database (subset defined from the ZINC 3D compound library30

) and

a specified binding site located within the HIV-1 IN enzyme active site. The same compound

library was subsequently docked into the active site of the PFV enzyme to obtain a comparative

score for each compound in the database. In this way, the structures of potential inhibitors selective

for the HIV-1 IN strand transfer reaction (that retained a high measure of predicted activity against

PFV IN) could be identified.

2.2 Construction of the HIV-1 IN monomer*

The monomeric structure of HIV-1 IN consists of a single 32-kDa unit composed of a polypeptide

chain that folds into three independent functional domains: the NTD (residues 1-55); the CCD

(residues 56-209); and the CTD (residues 210-270). Each of these domains is essential for efficient

concerted integration to occur and each has a distinct function during this process.31

2.2.1 Preparing the catalytic core domain

The CCD (HIV-1 IN: residues 56-209) of retroviral IN contains the absolutely conserved catalytic

triad (HIV-1 IN: D64, D116, E152) motif and the divalent metal ion(s) necessary for the enzymatic

* Important note to the reader: All solvating water molecules were removed during the generation of the HIV-1 IN

model in this work, resulting in a completely desolvated protein model. Although protein preparation in certain modern

software programs may require some rudimentary solvation at all times, backbone RMSD comparisons between the

HIV-1 IN CCD prepared in solvated and vacuum environments respectively, showed no deviation in protein structure

when prepared in a vacuum (calculations performed using Tripos Sybyl8.0).


efficiency of the protein. Mutation of any of these residues significantly reduces or abolishes all

catalytic activities of IN both in vitro32

and in vivo.33,34

The HIV-1 IN CCD structure (PDB code

1QS4)35

selected for use in the model was that of a soluble double mutant IN (F185K, W131E)†

possessing one divalent magnesium cation [Mg2+

(I)] and crystallised with a diketo acid-analogue

inhibitor (5-CITEP) coordinated in the active site, determined at a resolution of 2.10 Å. Tn5

transposase (PDB code 1MM8),36

proposed to have a similar catalytic mechanism to HIV-1 IN, has

been crystallised with two divalent Mn2+

ions [Mn2+

(I) and Mn2+

(II)] coordinated by the invariant

DDE motif and was used as a template to insert the second Mg2+

ion [Mg2+

(II)] into our HIV-1 IN

monomer model. It has been suggested that Mg2+

(II) may only be chelated upon or after vDNA

binding to the HIV-1 IN dimer37

in a manner similar to the stabilising mechanism for metal binding

observed in other nucleotidyltransferases,38,39

as there are insufficient counter-charges in the

unbound enzyme active site to accommodate the simultaneous binding of both divalent metal ions.

In the single-metal HIV-1 IN CCD crystal structure (PDB code 1QS4), the Mg2+

(I) ion is chelated

by oxygen atoms of two of the conserved catalytic triad residues, D64 and D116, while the third of

these invariant active site residues, E152, is located on the α4-helix (Figure 2.1a) with the carboxy-

oxygen atoms orientated away from the metal-binding site in a non-chelating position. To

accommodate the chelation of the Mg2+

(II) ion between D64 and E152 in our two-metal model, the

side-chain conformation of E152 was altered to match the analogous residue E326 of the Tn5

transposase template (Figure 2.1b). Upon insertion of Mg2+

(II) chelated by the oxygen atoms of

D116 and E152, and energy-minimisation of the final structure, the inter-metallic distance in the

double-metal model was calculated to be 4.36 Å with both metal centres held slightly above the

protein surface (Figure 2.1a). The estimated inter-metallic distance was slightly longer than those

observed for Zn2+

and Cd2+

in the avian sarcoma virus active site (3.62 Å and 4.06 Å,

respectively),40

as well as the calculated distance in a model for diketo-acid inhibitor binding to IN

(3.612 Å).41

Although the chelation distances between the carboxy-oxygen atoms of D116 and D64

and Mg2+

(I) were slightly altered to allow for incorporation of Mg2+

(II), no significant movement or

change could be detected in the backbone conformation of the protein upon insertion of the

Mg2+

(II) ion.

The conformation and placement of the catalytic triad and the two divalent metal atoms described

for the model above, was reflected in the recently resolved crystal structure of PFV IN27

(PDB entry

† Note: The soluble double mutant HIV-1 IN used in the generation of the in silico models was structurally identical to

the enzyme used during the in vitro identification of inhibitors to the HIV-1 IN strand transfer reaction (discussed in

section 2.4.3 and in Chapter 4).

Chapter 2:

3L2T). The two divalent magnesium ions in the PFV crystal structure were chelated between the

oxygen atoms o

D185 (analogous to HIV

residues D128 and E221 (analogous to HIV

metallic distance in the PFV crystal structure was measured at 4.27 Å,

correlation to the equivalent distance reported above for our HIV

reader: The author is using ‘our’ in an attempt to r

literature and the present work, to avoid repeating phrases such as ‘the model of the present study’.)

Figure 2.1

catalytic triad residues

Mn2+

as the divalent metal; and (c) in the PFV IN active site. Thes

TriposTM

Sybyl8.0 rendering in SGI RGB.

The two point mutations (F185K, W131E) in the selected HIV

(1QS4) were originally included to render this highly insoluble protein more soluble

increase the possibility of obtaining crystals. In our model, these mutations were manually replaced

with the residues normally present in the wild

and after insertion of the wild

structure of the protein.

Although 1QS4 was mostly well

located next to the

included three unresolved amino acid residues (142, 143 and 144) and is proposed to play an

important role in the binding of vDNA. Conformational dynamics investigations of this area

revealed the potential for large

‡ Note: PDB entry 3L2T used in all comparisons of the generated HIV

structure was deposited in the RCSB Protein Data Bank on 15 December 2009. The structure was accessed and

downloaded from the RCSB server on 1 April 2010. Subsequently, the structure was declared obsolete on 20 October

2010 and superseded by PBD entry 3OYA. The inter

A.

Chapter 2: In Silico Methods


oxygen atoms of the conserved PFV IN catalytic triad: the first, Mg

D185 (analogous to HIV






1 The positioning of two divalent metal ions between the chelating

catalytic triad residues







and after insertion of the wild


Although 1QS4 was mostly well

located next to the α4



revealed the potential for large

Note: PDB entry 3L2T used in all comparisons of the generated HIV


d from the RCSB server on 1 April 2010. Subsequently, the structure was declared obsolete on 20 October


Methods


f the conserved PFV IN catalytic triad: the first, Mg

D185 (analogous to HIV-1 IN residues D64 and D116), while Mg






The positioning of two divalent metal ions between the chelating

catalytic triad residues: (a) in the HIV







and after insertion of the wild-


Although 1QS4 was mostly well-

α4-helix region (residues 149



revealed the potential for large-scale conformational c







1 IN residues D64 and D116), while Mg







in the HIV-1 IN active site







-type residues show

-refined, it contained one highly flexible section (residues 141

helix region (residues 149



scale conformational c




2010 and superseded by PBD entry 3OYA. The inter-

B.




residues D128 and E221 (analogous to HIV-1 IN residues D64 and E152; Figure 2.1c).



reader: The author is using ‘our’ in an attempt to readily lead the reader between references to the



1 IN active site;





with the residues normally present in the wild-type enzyme. A comparison of the structure before

type residues showed no significant alteration of the backbone

refined, it contained one highly flexible section (residues 141

helix region (residues 149-165) near the active site. This fle



scale conformational c




-metallic distance reported for 3OYA is 3.825 Å.




1 IN residues D64 and E152; Figure 2.1c).


correlation to the equivalent distance reported above for our HIV-1 IN model (4.36 Å). (Note to the

eadily lead the reader between references to the



(b) in the Tn5 transposase template, showing


The two point mutations (F185K, W131E) in the selected HIV-1 IN subunit X



type enzyme. A comparison of the structure before

ed no significant alteration of the backbone


165) near the active site. This fle



scale conformational changes (> 20 Å), where the loop structure

Note: PDB entry 3L2T used in all comparisons of the generated HIV-1 IN model structure with the PFV crystal



metallic distance reported for 3OYA is 3.825 Å.


f the conserved PFV IN catalytic triad: the first, Mg2+

(I) between residues D128 and

1 IN residues D64 and D116), while Mg2+

(II) was chelated between


metallic distance in the PFV crystal structure was measured at 4.27 Å,‡ which shows excellent

1 IN model (4.36 Å). (Note to the




the Tn5 transposase template, showing

as the divalent metal; and (c) in the PFV IN active site. These figures were created using

1 IN subunit X






165) near the active site. This fle



hanges (> 20 Å), where the loop structure

1 IN model structure with the PFV crystal




C.

Page





which shows excellent




The positioning of two divalent metal ions between the chelating oxygen atom


e figures were created using

1 IN subunit X-ray crystal structure






165) near the active site. This flexible loop region








Page 52




1 IN residues D64 and E152; Figure 2.1c). The inter-





atoms of the



ray crystal structure

(1QS4) were originally included to render this highly insoluble protein more soluble42

and to




refined, it contained one highly flexible section (residues 141-149)

xible loop region










-




of the



ray crystal structure

and to




149)

xible loop region







Chapter 2:

opens and closes the space around the active site in a gate

residues in positions 140 and 149.

flexibility, although f

decreased or inefficient integration.

IN CCD with fully resolved loop regions (PDB codes: 1B9F,

2ITG47

) illustrated this flexibility. Each of the four additional crystal structures investigated,

represented the loop in a

“open” and “closed” loop conformations respectively, while 1B9F

represented intermediate conformations.

Figure 2.2

active site

in the CCD of HIV

strand represents the model (based on 1QS4), while the cyan stran

(based on 1B9F

Taken together, the d

effectively gave snapshots of the space around the active site in various degrees of “open”

“closed”-ness (illustrated in Figure 2.2a), and allowed the identification of suita

templates for the unresolved loop fragment. The missing residues of 1QS4 were rebuilt to an

intermediate “open” conformation according to the sequence and conformation of the

corresponding residues in 1B9F, yielding the HIV

construction of the three

identity/similarity percentages and backbone

A.




flexibility, although fully functional in terms of vDNA




represented the loop in a



2 a) Resolved loop structures represented in various conformations around the HIV

(1BL3 = yellow, 1B9F = purple, 1BIS = green, 2ITG = red); b) The missing loop region

in the CCD of HIV-1 IN (1QS4; residues 141


(based on 1B9F33

). These figures were created using Tripos

Taken together, the d


ness (illustrated in Figure 2.2a), and allowed the identification of suita




construction of the three


A.

Methods



flexibility, although fully functional in terms of vDNA




represented the loop in a different conformation: 2ITG



Resolved loop structures represented in various conformations around the HIV


1 IN (1QS4; residues 141


These figures were created using Tripos

Taken together, the different loop conformations evidenced by the analysed crystal structures






construction of the three-domain model of the HIV



residues in positions 140 and 149.43

It has been shown that mutants with limited loop / hinge

ully functional in terms of vDNA

decreased or inefficient integration.44

An investigation of additional crystal structures of the HIV



different conformation: 2ITG





1 IN (1QS4; residues 141



ifferent loop conformations evidenced by the analysed crystal structures






domain model of the HIV




ully functional in terms of vDNA




different conformation: 2ITG




1 IN (1QS4; residues 141-149) rebuilt using template 1B9F. The dark blue








corresponding residues in 1B9F, yielding the HIV-1 IN CCD model us

domain model of the HIV

identity/similarity percentages and backbone root mean square deviation

opens and closes the space around the active site in a gate-like manner, dependent upon the hinge


ully functional in terms of vDNA-binding, are impaired for replication due to


IN CCD with fully resolved loop regions (PDB codes: 1B9F,44

1BIS chain B,


different conformation: 2ITG47

and 1BL3 (chain C)




149) rebuilt using template 1B9F. The dark blue


These figures were created using TriposTM







1 IN CCD model us

domain model of the HIV-1 IN monomer (Figure 2.2b). Sequence

oot mean square deviation

B.

like manner, dependent upon the hinge


binding, are impaired for replication due to


1BIS chain B,


and 1BL3 (chain C)

“open” and “closed” loop conformations respectively, while 1B9F33

and 1BIS (chain B)




strand represents the model (based on 1QS4), while the cyan strand represents the template strand







1 IN CCD model used as starting point in the

1 IN monomer (Figure 2.2b). Sequence

oot mean square deviation (RMSD

Page





1BIS chain B,45

1BL3 chain C


and 1BL3 (chain C)46

represented the

and 1BIS (chain B)




d represents the template strand




ness (illustrated in Figure 2.2a), and allowed the identification of suitable rebuilding



ed as starting point in the


RMSD) values between

Page 53




An investigation of additional crystal structures of the HIV-1

1BL3 chain C46

and


represented the

and 1BIS (chain B)45

Resolved loop structures represented in various conformations around the HIV-1 IN






effectively gave snapshots of the space around the active site in various degrees of “open”-ness or

ble rebuilding





values between




1

and


represented the

45

IN





ness or

ble rebuilding





values between


the model (with completed loop sequence based on 1B9F) and the other loop conformations (1BL3,

1BIS and 2ITG) are reported in Table 2.1.

Table 2.1 Sequence similarity percentages and backbone RMSD values between the model (with

completed loop sequence based on 1B9F) and the loop conformations present in 1BIS chain B,

2ITG and 1BL3 chain C. (Structures were aligned based on CCD residues 56 – 80)

% ID§48 RMSD

**

1BIS 97 1.6

2ITG 97 3.8

1BL3 97 1.7

2.2.2 Preparing the C-terminal domain

The CTD (HIV-1 IN: residues 210-270) contains the least conserved sequence of the three

subunits,49

although the overall structure resembles the β-barrel fold of the SRC Homology 3

domain (SH3).50,51

This domain has been shown to bind host DNA strongly but non-specifically49,52

through its numerous basic residues53

and has been implicated in protein-protein interactions

(including interactions with reverse transcriptase).54

The two-domain, hetero-dimerised crystal

structure of HIV-1 IN CCD and CTD (PDB code 1EX4)55

provided the template for inserting the

CTD into our HIV-1 IN monomer model. The CCD of crystal structure 1EX4 was aligned with the

CCD of our monomer model to obtain the position for inserting the CTD into the model. Although

the template CCD was discarded after alignment, the 1EX4 CTD was retained and its features and

coordinates incorporated into our model.

2.2.3 Preparing the N-terminal domain

The third subunit of the three-domain monomer, the NTD (HIV-1 IN: residues 1-55), carries a

conserved HHCC motif formed by residues H12, H16, C40 and C43 in HIV-1 IN56

(and residues

H62, H66, C96 and C99 in PFV IN57

). This motif effectively binds zinc in a 1:1 ratio (see Figure

2.3a),58

proposed to impact on both the mode and affinities of self-association of IN.59,60,61

Mutation

of any residue in this motif has a significant impact on the enzymatic efficiency of IN, as

multimerisation of the enzyme to its active forms is impaired. The two-domain, hetero-dimerised

crystal structure of HIV-1 IN CCD and NTD (PDB code 1K6Y)62

provided the template for

§ Reference structure reflects the Los Alamos consensus B sequence reported in 2008.

** Deviation from reference structure / model is mainly due to variable positioning of flexible loop structure.


inserting the NTD into our HIV-1 IN monomer model. The CCD of crystal structure 1K6Y was

aligned with the CCD of our monomer model to obtain the position for insertion of the NTD into

our model. Although the template CCD was discarded after alignment, the 1K6Y NTD was retained

and its features incorporated into our model. The positions of residues 47-55 of the flexible

connector between the CCD and NTD were not resolved in the 1K6Y crystal structure as it could

not be determined crystallographically. These residues were completed manually in our model and a

random conformation was assigned to the resulting flexible region, whilst keeping fixed the position

of the NTD and CCD (residues 1-46 and 56-270).

2.2.4 Comparison between the HIV-1 IN monomer model and a monomer of the PFV IN crystal

structure

As discussed above, monomeric retroviral IN generally comprises ~290 amino acid residues and is

organised into three distinct functional domains responsible for the various activities and

interactions of the enzyme in vitro and in vivo.31

Although most retroviral INs are closely related in

terms of structure and function, some important differences exist. One of the most significant to the

current work is the fact that PFV IN (392 residues) is substantially longer than HIV-1 IN (288

residues), creating difficulties in the absolute definition of boundaries between the linkers and the

domains.63

It is important to recognise that any direct comparisons between different retroviral IN

structures will be highly dependent on both the definition of the domain boundaries and the rigidity

necessarily imposed on the inherently flexible linkers.

In the current work, the orientation of the CTD and NTD relative to CCD achieved through the

superpositioning and linking of various subdomain structures and reflected in the constructed HIV-1

IN monomer model (Figure 2.3a), was characterised in terms of the distances and angles formed

between centroids defined for each of the three subunits, excluding the flexible linker chains: NTD

(residues 1-42), CCD with chelating E152 (residues 56-198) and CTD (residues 223-270). The

position of the flexible linkers, comprising residues 43-55 and 199-222, were restricted to that

observed in the two-domain crystal structures 1K6Y62

and 1EX4,55

respectively. The PFV monomer

(Figure 2.3b) was characterised in a similar manner as above to enable a direct comparison between

the overall architecture of the HIV-1 IN and PFV IN monomers. Centroid features were defined

based on each of the three subdomains, excluding the flexible linker chains: NTD (residues 8-102),

CCD (residues 121-301) and CTD (residues 313-375). The position and orientation of the flexible

linkers (residues 103-120 and 302-312) were used as represented in the PFV intasome crystal

structure.27

All further comparisons made between the various domains in the three-domain HIV-1


IN and PFV IN structures were made in keeping with the domain boundary definitions set out

above.

The HIV-1 IN NTD comprises a 42 amino acid sequence arranged in three α-helices surrounding a

Zn2+

ion centrally coordinated by the HHCC motif (Figure 2.3a). Although the PFV IN NTD shows

a similarly coordinated Zn2+

ion, this domain is much larger than the HIV-1 IN NTD and consists of

a 94 amino acid sequence arranged in four α-helices and one β-sheet (Figure 2.3b). This finding is

consistent with literature reports of an additional domain consisting of ~50 residues that may

precede the NTD of the PFV IN monomer.63

The difference in domain size becomes immediately

apparent when considering the two protein images in Figure 2.3. As this additional domain will

have a significant impact on all comparisons between the PFV IN and HIV-1 IN models, a second

centroid was defined for the PFV IN NTD, disregarding residues 1-50, to enable a more direct

comparison between the two IN structures. When disregarding the additional pre-NTD domain from

the definition of the PFV IN NTD, the sequence identity and sequence similarity between the NTDs

of the PFV and HIV-1 IN structures were 15% and 33% respectively (Figure 2.4a), indicating a low

degree of sequence conservation. The secondary structure of the PFV and HIV-1 IN NTDs (based

on main-chain atoms) could be aligned with a predicted RMSD value of 3.8 Ǻ, showing similarity

in the overall secondary structure. A similar conclusion can be drawn regarding the CTDs of PFV

IN and the HIV-1 IN model: the secondary structures of the CTDs (based on main-chain atoms)

were aligned to a predicted RMSD value of 3.2 Ǻ, indicating similarity in the domain’s secondary

structure, while the sequence identity and similarity between the CTDs of HIV-1 IN and PFV IN

were only 19% and 34%, respectively (Figure 2.4b). The low sequence similarity and identity

versus the relatively high degree of similarity in secondary protein structure observed for the above-

mentioned sub-domains of PFV IN and HIV-1 IN, suggest that the conserved functions of these

domains (protein multimerisation and the formation of non-specific interactions with proteins and

DNA) are influenced more by the secondary structure than by the nucleotide sequence making up

the chain.

Chapter 2:

Figure 2.3

the PFV IN monomer crystal structure. The distances between centroids defined for each

subdomain and the angle formed between the flexible linkers are indicated in eac

figures were created using Tripos

Some similarities and differences between the CCDs of the PFV and HIV

discussed (section 2.2.1). The CCD of retroviral IN holds the complete catalytic ensemble and is

B.

A.

CTD


3 Relative orientation of the three subdomains in: a) the






NTD

CTD

Methods

Relative orientation of the three subdomains in: a) the






NTD

30 Å




figures were created using TriposTM




26 Å

CTD

NTD







57 Å

CTD

50

34 Å

36˚

Relative orientation of the three subdomains in: a) the HIV






14˚

33 Å

50 Å

HIV-1 IN monomer model and b)




Some similarities and differences between the CCDs of the PFV and HIV-1 INs have already been


CCD

Page

1 IN monomer model and b)


subdomain and the angle formed between the flexible linkers are indicated in each case. These

1 INs have already been


CCD

Page 57



h case. These





h case. These



Chapter 2:

responsible for the catalytic fu

specific interactions and performs a very specific function, one would expect a high degree of

sequence and structural conservation between different IN classes. The sequence identity and

similarity for the respective CCDs were found to be 23% and 39%, respectively, indicating a

slightly higher degree of sequence conservation than observed for the NTDs and CTDs,

respectively, while the secondary structures aligned to the same degree as the other

with a predicted RMSD of 3.4

Figure 2.4

chain) sub

Accelrys Discovery Studio

Although the individual domains in the HIV

significant differences exist in their relative spatial orientation. Firstly, the positions of the NTD and

CTD domains are seemingly inverted, with the length of the f

NTD-CCD linker:

CCD-CTD linker:

Secondly, the angle formed between the positions of the three domains in each monomer va

considerably, with a 36

adopts an arrangement almost approaching planarity, forming a NTD

other models of the HIV

models of Podteleznikov

respectively, as calculated by Wijitkosoom

Thus, although the individual domains of the HIV

the PFV IN monomer (as reported in the recently elucidated crystal structure, 3L2T), signific

differences exist in the overall structure of the molecules. These differences mainly arise as a

consequence of the inherent discrepancy between the flexible nature of proteins (in this case, the

A.


responsible for the catalytic fu



rity for the respective CCDs were found to be 23% and 39%, respectively, indicating a



with a predicted RMSD of 3.4

4 Secondary structure alignment of the PFV IN (blue chain) and the HIV

chain) sub-domains: a) the NTD; b) the CTD; and c) the





CCD linker:

CTD linker:


considerably, with a 36


other models of the HIV

models of Podteleznikov






Methods

responsible for the catalytic function of the enzyme, i.e. integration. As the CCD forms very






with a predicted RMSD of 3.4 Ǻ (Figure 2.4c).

Secondary structure alignment of the PFV IN (blue chain) and the HIV

domains: a) the NTD; b) the CTD; and c) the

Accelrys Discovery StudioTM

rendering.




HIV-1 IN monomer

HIV-1 IN monomer


considerably, with a 36˚ angle observed for the HIV


other models of the HIV-1 IN monomer this angle varies considerably

models of Podteleznikov et al.64

at 76






nction of the enzyme, i.e. integration. As the CCD forms very






Ǻ (Figure 2.4c).



rendering.




1 IN monomer –

1 IN monomer –


˚ angle observed for the HIV


1 IN monomer this angle varies considerably

at 76˚, and De Luca

respectively, as calculated by Wijitkosoom et al.





B.







(Figure 2.4c).



Although the individual domains in the HIV-1 IN and PFV IN structures are quite similar,



– 34 Å; and PFV IN monomer

– 50 Å; and PFV IN monomer


˚ angle observed for the HIV-1 IN monomer, while the PFV IN monomer



, and De Luca et al.

et al.66

).

Thus, although the individual domains of the HIV-1 IN monomer model closely resembles that of











domains: a) the NTD; b) the CTD; and c) the CCD. These figures were created using

1 IN and PFV IN structures are quite similar,


CTD domains are seemingly inverted, with the length of the flexible linkers as follows:

34 Å; and PFV IN monomer

50 Å; and PFV IN monomer


1 IN monomer, while the PFV IN monomer



et al.65

and Wijitkosoom

1 IN monomer model closely resembles that of




C.








These figures were created using



lexible linkers as follows:

34 Å; and PFV IN monomer – 57 Å

50 Å; and PFV IN monomer – 33 Å.



adopts an arrangement almost approaching planarity, forming a NTD-CCD-

1 IN monomer this angle varies considerably (cf. those reported for the

and Wijitkosoom





Page






respectively, while the secondary structures aligned to the same degree as the other sub

Secondary structure alignment of the PFV IN (blue chain) and the HIV-1 IN (yellow




lexible linkers as follows:

57 Å

33 Å.



-CTD angle of 14

. those reported for the

and Wijitkosoom et al.66

at 23° and 28°





Page 58






sub-domains,

1 IN (yellow




Secondly, the angle formed between the positions of the three domains in each monomer varies


CTD angle of 14˚. In


at 23° and 28°


the PFV IN monomer (as reported in the recently elucidated crystal structure, 3L2T), significant








domains,

1 IN (yellow




ries


˚. In


at 23° and 28°


ant




flexible domain linkers) and the rigidity necessarily imposed on these proteins when generating

crystals or workable models thereof.

2.3 Construction of the HIV-1 IN strand-transfer complex model

In an attempt to account for all the steps of the integration process as detailed in Chapter 1, a model

of the HIV-1 IN dimer was constructed to reflect the active site structure after the binding and 3’-

end processing of vDNA. This active site structure represents the active conformation of the

enzyme before the binding of host DNA, and served as the receptor target for structure-based drug

discovery efforts aimed at identifying HIV-1 strand transfer inhibitors.

As this dimeric model cannot account for full-site integration of vDNA into the host genome

(consistent with literature reports67,68,69

), an additional tetrameric (dimer-of-dimers) model of HIV-1

IN with bound vDNA and host cofactor LEDGF/p75 was constructed to illustrate a possible

multimerisation mode of HIV-1 IN around a strand of host DNA (for illustrative purposes only).

2.3.1 Dimerisation model of HIV-1 IN

The dimerisation interface of the HIV-1 IN monomer model was based on that of the dimerised

CCDs in the 1QS4 crystal structure,35

with the respective monomers held in place through strong,

mainly hydrophobic, protein-protein interactions as well as an extensive network of hydrogen bonds

formed between the relatively flat surface areas located on each CCD monomer. The flat

dimerisation interface and the active site of each monomer are located on opposite sides of the

molecule, resulting in a separation of ~40 Å between the active sites in a dimeric HIV-1 IN enzyme

(measured as the distance between the centres of mass defined for each active site based on the

coordinates of the catalytic triad residues, Figure 2.5). For PFV IN, crystal structure 3L2T27

shows a

second co-crystallised PFV CCD that indicates the geometry and mode of interaction between the

two facing monomers of the PFV IN dimer. The dimerisation interface found in the PFV IN crystal

structure closely represents that reflected in the HIV-1 IN model, with the active site faces of the

two monomers in the PFV IN dimer situated on the opposite side of the dimerisation faces, and the

two active sites ~42 Å apart (measured as the distance between the centres of mass defined for each

active site based on the coordinates of the catalytic triad residues, compare Figure 2.5). In both the

HIV-1 IN model and the PFV IN crystal structure the dimeric enzyme form is stabilised by

extensive intermolecular NTD-CCD interactions, as previously reported.70,71

Chapter 2:

Although slight, the

structures of HIV

overall architecture between the enzyme dimers that would translate into any further models o

HIV-1 IN generated from these data. In addition, when considering the substantial difference in

monomer structure imparted by the flexible linkers (as represented in Figure 2.3a and b), it would

be prudent to be aware that the architecture of any fur

from the data could vary considerably from the PFV IN intasome structure as it is reflected in

crystal structure 3L2T.

Figure 2.5

between the active sites in the respective monomers. This figure was created using Tripos


2.3.2 Inclusion of h

Although recombinant HIV

vitro without the need for any additional cellular or viral proteins,

based environments

cellular co

integration process, effectiv


Although slight, the

structures of HIV-1 IN and PFV IN (~40 Å vs. ~42 Å, respectively), indicates a variance in the


1 IN generated from these data. In addition, when considering the substantial difference in




crystal structure 3L2T.

5 Representation of the HIV



2.3.2 Inclusion of host

Although recombinant HIV

without the need for any additional cellular or viral proteins,

based environments is

cellular co-factors, LEDGF/p75, has been suggested to act as a highly flexible tether during the

integration process, effectiv

Methods

Although slight, the ~2 Å difference in distance between the two active sites in the dimeric

1 IN and PFV IN (~40 Å vs. ~42 Å, respectively), indicates a variance in the






crystal structure 3L2T.27

Representation of the HIV



ost cofactor LEDGF/p75

Although recombinant HIV-1 IN can successfully catalyse 3’


is much more complex, involving a host of additional cofactors

factors, LEDGF/p75, has been suggested to act as a highly flexible tether during the

integration process, effectively targeting and linking IN to the host DNA.

difference in distance between the two active sites in the dimeric







Representation of the HIV-1 IN dimer structure generated, illustrating the distance


ofactor LEDGF/p75

1 IN can successfully catalyse 3’


much more complex, involving a host of additional cofactors


ely targeting and linking IN to the host DNA.






be prudent to be aware that the architecture of any further multimeric forms of HIV


1 IN dimer structure generated, illustrating the distance


ofactor LEDGF/p75

1 IN can successfully catalyse 3’










ther multimeric forms of HIV




1 IN can successfully catalyse 3’-end processing and strand transfer

without the need for any additional cellular or viral proteins,72









ther multimeric forms of HIV




end processing and strand transfer

72,73 integration




Page






ther multimeric forms of HIV-1 IN generated




end processing and strand transfer

integration in vivo

much more complex, involving a host of additional cofactors.74

One of these


ely targeting and linking IN to the host DNA.75,76

LEDGF/p75 is

Page 60



overall architecture between the enzyme dimers that would translate into any further models of the



1 IN generated



between the active sites in the respective monomers. This figure was created using TriposTM

end processing and strand transfer in

and in cell-

One of these


LEDGF/p75 is



f the



1 IN generated



TM

in

-

One of these


LEDGF/p75 is

Chapter 2:

predominantly


association with chromatin. These three DNA

NLS79


the C-terminal half of the protein and is specifically responsible for i

with HIV-

flexible peptide chains without formally assigned conformations.

One X-ray crystal structure (PDB code 2B4J

between the LEDGF/p75 IBD and the HIV

factor into the HIV

to interact differently with HIV

stable interaction with monomer A was attributed to the attractive electronic interface (Coulombic

attractions), with no observed hydrogen bonds being present. The acidic residues contributed by

HIV-1 IN monomer A

LEDGF/p75 (e.g. K360, K402 and K364) and

thereby creating counter

interaction of LEDGF/p75 with monomer B can also be partly attributed to hydrophobic

interactions and an attractive electronic interface, the driving force is most likely the extensive

hydrogen-bond network formed between the two protein surfaces.

Figure 2.6

strong attractive interactions anchoring LEDGF/p75 to monomers A and B. This figure was created

using Tripos


predominantly localised




terminal half of the protein and is specifically responsible for i

-1 IN.80

The linker regions between the conserved domains mainly consist of highly


ray crystal structure (PDB code 2B4J


factor into the HIV-1 IN dimer model. The modelling shows LEDGF/p75 (residues G345 to M426)

to interact differently with HIV



1 IN monomer A


thereby creating counter

ction of LEDGF/p75 with monomer B can also be partly attributed to hydrophobic


bond network formed between the two protein surfaces.

6 The protein interface of dimeric HIV


using TriposTM


Methods

localised in the nucleus and consists of four conserved functional domains, three of

which are located in the N-terminal half of the protein and are known to mediate the non








1 IN dimer model. The modelling shows LEDGF/p75 (residues G345 to M426)

to interact differently with HIV-



1 IN monomer A (e.g. D167, Q168, and E170) were offset by the basic residues of


thereby creating counter-charges that assist in the self




The protein interface of dimeric HIV



in the nucleus and consists of four conserved functional domains, three of



and a dual copy of the AT-hook DNA







-1 IN monomer A and



(e.g. D167, Q168, and E170) were offset by the basic residues of


charges that assist in the self




The protein interface of dimeric HIV





association with chromatin. These three DNA-binding domains are the PWWP domain,

hook DNA-binding motif. The fourth domain, IBD, is located in




ray crystal structure (PDB code 2B4J81

) illustrates the structural ba

between the LEDGF/p75 IBD and the HIV-1 IN CCD and was used here to incorporate this co


1 IN monomer A and




LEDGF/p75 (e.g. K360, K402 and K364) and vice versa

charges that assist in the self




The protein interface of dimeric HIV-1 IN and LEDGF/p75 is shown, illustrating the





binding domains are the PWWP domain,

binding motif. The fourth domain, IBD, is located in




illustrates the structural ba

1 IN CCD and was used here to incorporate this co


1 IN monomer A and monomer B, respectively (Figure 2.6). The




vice versa (basic H171 neutralising acidic D366),

charges that assist in the self-assembly of the system. Although the




1 IN and LEDGF/p75 is shown, illustrating the






terminal half of the protein and is specifically responsible for interactions of LEDGF/p75


illustrates the structural basis for the recognition



monomer B, respectively (Figure 2.6). The




(basic H171 neutralising acidic D366),

assembly of the system. Although the






Page





nteractions of LEDGF/p75


sis for the recognition













Page 61


terminal half of the protein and are known to mediate the non-specific

binding domains are the PWWP domain,77,78

the





1 IN CCD and was used here to incorporate this co-













ic

the





-













Hydrogen bonding includes those noted between I365 and Q168 (one H-bond; 2.5 Å), D366 and

E170 (two H-bonds; 1.6 Å and 2.6 Å), D366 and H171 (two H-bonds; 2.1 Å and 2.0 Å) and D366

and T174 (one H-bond; 2.0 Å). A recent study found that residue D366 (which forms a total of 5

hydrogen bonds in our model) is essential for the stable interaction of HIV-1 IN and LEDGF/p75.82

Experimental data have indicated that the correct binding ratio between each HIV-1 IN monomer,

LEDGF/p75 and vDNA is 2:1:1,83

a determination with which our model corresponds.

2.3.3 Inclusion of viral DNA

Several groups have reported the selective recognition and specific binding of the U5 and U3 ends

of linear vDNA strands to the HIV-1 IN active site (involving residues Q148, K156 and

K159)84,85,86

and specifically through residues on the amphipathic α4 helix.87,82

De Luca et al.88

incorporated vDNA into a model of HIV-1 IN through an automated docking algorithm followed by

molecular dynamics simulations, while Wielens et al.89

constructed a model with vDNA placed

according to the crystal structure of the homologous Tn5 transposase90,91

with bound DNA (PDB

code 1MM8).36

Ferro et al.92

recently reported an improved model of HIV-1 IN by using the Tn5

transposase as a template for viral binding into the IN active site containing two divalent metal ions.

In a similar way, the Tn5 template was used to transfer the binding coordinates of the vDNA to

each of the monomers A and B in our dimeric HIV-1 IN model.

A recently reported NMR solution structure of a 17-mer vDNA mimic of the U5 long terminal

repeat (LTR) extremity of HIV-1 DNA (PDB code 1TQR)93

indicates that the six terminal base

pairs of the U5 LTR form a highly conserved nucleotide sequence and that the inherent pre-

organised structure reportedly causes a deviation from regular B-DNA structure. In this structure

(1TQR), the distance between the backbone phosphates of the 3’-terminal G1’T2’ dinucleotide (6.2

Å) was found to be much shorter than the distance between the corresponding bases on the facing

5’-strand (7.0 Å), causing the 3’-oxygen atom of nucleotide A1 (involved in the 3’-end cleavage) to

become more exposed and resulting in a local destabilisation of the third base pair (A1---T1) and a

distortion of the vDNA LTR. A similar destabilisation and partial un-stacking of bases at the LTR

has been reported for avian sarcoma virus IN,94

where it is proposed to enable the cleavage of the 5’

dinucleotide. These characteristics were incorporated into our constructed model by positioning

1TQR in the modelled HIV-1 IN active site according to the coordinates designated by the Tn5

transposase template. The inter-phosphorus distance between the backbone phosphates of the

terminal dinucleotide in the energy-minimised model compared well with those observed in the

NMR solution structure, with the 3’-terminal dinucleotide G1’T2’ and the 5’-terminal dinucleotide

C1’A2’ measured as 6.1 Å (vs. 6.2 Å in the NMR structure) and 7.0 Å (vs. 7.0 Å in the NMR


structure), respectively. In models generated from experimental observations, Alian et al.95

and

Michel et al.96

incorporated evidence that binding of vDNA to one of the monomers in the HIV-1

IN dimer induces asymmetry in the other,97

providing a basis for the 2:1 binding ratio observed

when dimeric IN binds the vDNA LTR end, reflected in our model. The above placement represents

the binding of blunt-ended vDNA to one monomer in the HIV-1 IN dimer and is in good agreement

with reported mutagenesis results and cross-linking of vDNA to HIV-1 IN peptides 49-69, 139-152,

153-167, 247-270, and 271-288.98,99,100,101

Importantly, the inclusion of blunt-ended vDNA into our

model resulted in severe clashing between the terminal 3’-end nucleotides T2’ and G1’ of vDNA

and active site residues within the α4 helix, hinting at a potential driving force for 3’-end

processing. The specific cleavage between nucleotides G1’ and A1 on the 3’-strand of vDNA and

the subsequent release of the terminal G1’T2’ dinucleotide that occurs during 3’-end processing,

were mimicked in our HIV-1 IN model bound to U5-mimic DNA, through manual removal of the

G1’T2’ dinucleotide. The numbering scheme of the U5 vDNA sequence is shown in Figure 2.7a,

while the positioning of the 3’-processed vDNA strand and active site residues of the final strand

transfer model is illustrated in Figure 2.7b.

The released 3’-OH group of nucleotide A1 in the model was directed away from the bulk of the

vDNA chain and towards the metal-bound active site,102

effectively forming a probe that would

extend into the host DNA chain and facilitate strand transfer during the second of the sequential

integration reactions. The 5’-overhang of the vDNA LTR (nucleotides C1’ and A2’) formed several

stabilising interactions with active site residues, all in good agreement with literature reports:103,104

Nucleotide A2’ was positioned within binding distance (defined as 2 Å) of the active site metals and

conserved triad residues D116 and E152 and within a reasonable interaction distance (defined as 3-

4 Å) of residues Y143 and Q148,104

while nucleotide C1’ was within binding distance (2 Å) of

residues Q148103

and triad residue E152 and was located in close proximity to residues 51-64.84

Furthermore, partial H-bond stability between nucleotide base pair A1-T1 in the model was

regained and indications of strong interactions between nucleotide A1 and residues K159 (with

modified side-chain conformation) and K156 could be observed.105

Nucleotide C2 is positioned in

close proximity to the flexible loop regions 49-69 (specifically residue Q62) and 139-152, each

containing a single catalytic triad residue (D64 and E152, respectively).106

Furthermore, nucleotide

T5 has been mapped to interact with the minimal peptide 247-270,99,100

with its facing nucleotide

A5 positioned close to residue E246.107

Chapter 2:

Figure 2.7

numbering scheme used to identify specific nucleotides; and b) Representation of the interaction of

vDNA with HIV

nucleotides are represented in capped stick format: the catalytic

end residues of vDNA (coloured by atom type); nucleotides in positions 1 and 2 (red); nucleotides

in positions 3 and 4 (green); and nucleotides 5 to 8 (purple).

Sybyl8.0 rendering i

The interactions between nucleotide T5 and residues 247

residue E246, mentioned above, could be replicated in our model by setting the backbone


7 a) Schematic representation of the nucleotide sequence at the U5 LTR of vDNA and the


vDNA with HIV-1 IN active site residues after 3’







Methods

a) Schematic representation of the nucleotide sequence at the U5 LTR of vDNA and the


1 IN active site residues after 3’




n SGI RGB.





1 IN active site residues after 3’








1 IN active site residues after 3’-








-end processing. All

nucleotides are represented in capped stick format: the catalytic triad (dark blue); the unpaired 5’


in positions 3 and 4 (green); and nucleotides 5 to 8 (purple). This figure was created using Tripos

The interactions between nucleotide T5 and residues 247-270 and between nucleotide A5 and




end processing. All

triad (dark blue); the unpaired 5’


This figure was created using Tripos

270 and between nucleotide A5 and


Page



relevant residues and

triad (dark blue); the unpaired 5’


This figure was created using Tripos



Page 64




triad (dark blue); the unpaired 5’-


This figure was created using TriposTM






-


TM




conformation of residue S223 to α-helix instead of the random conformation assigned in the

original crystal structure. This slight conformational change caused the CTD to closely approach the

binding site and the bound vDNA, a molecular movement that would enable the formation of the

binding interactions reported in the literature.108,109,110,111

Several previous studies have proposed

that HIV-1 IN may undergo conformational changes during the integration process,108,109,110,111

and

that complexes are more stable after terminal cleavage than those present during earlier steps in the

integration reaction. Experimental results supporting this proposal indicate that residue S230C in

the CTD of HIV-1 IN cross-links most readily with nucleotide A5 of blunt-ended vDNA but after

cleavage this interaction is altered and the cross-link forms most readily with nucleotide A7.107

These observations support the conformational changes predicted by our model that bring into

proximity the CTD, the binding site and the bound vDNA.

Although this discussion only considers the binding of the U5 LTR of vDNA to one dimeric strand

transfer complex of HIV-1 IN, an analogous process would take place at the U3 LTR of vDNA,112

with slight variations where the nucleotide sequence of U3 differs from that of U5. This association

of a single vDNA strand with two HIV-1 IN dimers would facilitate the formation of the dimer-of-

dimers arrangement of HIV-1 IN (illustrated in Figure 2.8) responsible for the collinear integration

of vDNA into host DNA.

2.3.4 Tetramerisation of HIV-1 IN around host DNA

Although the elucidation of the PFV intasome has revealed the most probable association of

tetrameric retroviral IN around a strand of host DNA and has to a large extent made the use of

models based on subdomain structures superfluous, further manipulations of the HIV-1 IN model

were retained for illustrative purposes and will be briefly discussed within a literature context.

Chapter 2:

Figure 2.8

their association with a single vDNA chain, in the absence of host DNA. Only the ends of the

vDNA chain (

dimer (rendered in

IDB (yellow and magenta

including two bound metal ion

The placement of the inhibitor structure was made for illustrative purposes only.

created using Tripos

HIV-1 IN targets host DNA mainly

cofactor (illustrated in Scheme 2.1).

binding of host DNA to the N

first attractive interaction would resu

Further decreases in entropy would be gained upon the formation of more and stronger bonds, and

could be achieved through secondary and tertiary non

AT-hook motifs and NLS sequence in the flexible linker regions of LEDGF/p75. Similarly, binding

of the second HIV

favoured. In addition, these secondary and tertiary interactions woul

distance between host DNA and each of the HIV

and correct positioning of host DNA within the catalytic active site of each dimeric complex.

Simultaneously, it would shorten the d

of linear vDNA and assist in the formation of the dimer


8 Illustrating a possible multimerisation scenario between two HIV


vDNA chain (white ribbons

dimer (rendered in pink/orange

yellow and magenta

including two bound metal ion


created using TriposTM

1 IN targets host DNA mainly


binding of host DNA to the N




ook motifs and NLS sequence in the flexible linker regions of LEDGF/p75. Similarly, binding

of the second HIV-1 IN dimer to the opposing strand of the same host DNA chain would be highly






Methods

Illustrating a possible multimerisation scenario between two HIV


ribbons with coloured base

pink/orange and

yellow and magenta ribbon

including two bound metal ions (


TM Sybyl8.0 rendering in SGI RGB.

1 IN targets host DNA mainly


binding of host DNA to the N-terminal PWWP domain of LEDGF/p75, it is envisioned that this





1 IN dimer to the opposing strand of the same host DNA chain would be highly








with coloured base

and turquoise/

ribbons) and both active sites in each HIV

s (green spheres) and a bound inhibitor structure in each active site.



1 IN targets host DNA mainly through its association with the chromatin

cofactor (illustrated in Scheme 2.1).75,76

Although the initial contact may only involve non

terminal PWWP domain of LEDGF/p75, it is envisioned that this

first attractive interaction would result in a significant decrease in the entropy of association.








Simultaneously, it would shorten the distance between the two HIV




with coloured base-pairing

turquoise/green, respectively); the position of the LEDGF/p75

) and both active sites in each HIV

spheres) and a bound inhibitor structure in each active site.



through its association with the chromatin



lt in a significant decrease in the entropy of association.






distance between host DNA and each of the HIV-1 IN dimers, allowing for the step


istance between the two HIV




pairing) are shown where bound to a HIV

green, respectively); the position of the LEDGF/p75

) and both active sites in each HIV









could be achieved through secondary and tertiary non-specific interactions of host DNA with the




1 IN dimers, allowing for the step


istance between the two HIV

of linear vDNA and assist in the formation of the dimer-of-dimers (tetramer form of HIV



are shown where bound to a HIV


) and both active sites in each HIV-1 IN dimer are indicated,








specific interactions of host DNA with the



favoured. In addition, these secondary and tertiary interactions would effectively shorten the

1 IN dimers, allowing for the step


istance between the two HIV-1 IN dimers bound to each end

dimers (tetramer form of HIV

Page

Illustrating a possible multimerisation scenario between two HIV-1 IN dimers through


are shown where bound to a HIV


1 IN dimer are indicated,


The placement of the inhibitor structure was made for illustrative purposes only. This figure was

through its association with the chromatin-binding LEDGF/p75








d effectively shorten the

1 IN dimers, allowing for the step-wise orientation


1 IN dimers bound to each end

dimers (tetramer form of HIV

Page 66

1 IN dimers through


are shown where bound to a HIV-1 IN




This figure was

binding LEDGF/p75

Although the initial contact may only involve non-specific








wise orientation



dimers (tetramer form of HIV-1 IN

1 IN dimers through


1 IN




This figure was

binding LEDGF/p75

specific








wise orientation



1 IN


responsible for strand transfer). It has been reported that all three of the HIV-1 IN sub-domains

interact with host DNA, with residues in the CCD playing a major role in selecting the non-viral

target sites for integration of 3’-processed vDNA ends.113

Scheme 2.1 Simplified cartoon image illustrating the capture of host DNA by the strand transfer

complex. This figure was created using ChemBioDraw UltraTM

version 12.0.

Integration of vDNA into host DNA occurs in both coding and non-coding regions of the genome,

but it has been reported that in some instances a slight preference is displayed towards

transcriptionally active genomic regions.114

During full-site integration, the insertion sites of vDNA

are located on opposite strands of the host DNA chain, characteristically five base pairs (bp) (~15-

16 Å) apart and are flanked by a 5 bp duplication of genomic sequence.115

Although original

evidence suggested that the integration of vDNA occurs randomly,116

subsequent studies have

pointed towards a non-random integration process,117

with integration sites displaying a preference

for palindromic genome sequences.118

It has been suggested that this symmetrical arrangement in

the target/host DNA sequence may favour the transesterification reaction responsible for cleavage

of the DNA strands during strand transfer, as well as stabilising the tetrameric organisation of HIV-


1 IN as a part of the PIC.119

Simply put, the symmetrical nucleotide sequence that characterises the

insertion sites on host DNA indicates a likely symmetrical arrangement of the catalytic active sites,

5 bp (or approx. 15-16 Å) apart. Furthermore, insertion of vDNA into the host genome has been

found to preferentially occur across the major groove of host DNA that is slightly bent or

unwound.120

DNA approaching this conformation was derived from a 147 bp DNA fragment

(human α-satellite DNA) wrapped around an octameric histone (recombinant Xenopus laevis) (PDB

code 1KX3).121

A sequence specific search highlighted the location of one half of the weakly

symmetrical sequence required for integration (characterised by the sequence …CAGTX…††

where

X represents either A or T). Mutations were introduced to the nine base pairs at the chosen site of

integration to fully resemble the required palindromic sequence, with no further alterations made to

the rest of the structure. The final host DNA molecule used in the construction of the tetrameric

model consisted of a 27 base pair fragment representing highly bent host DNA with the following

sequence introduced across the major groove:

…CAGTTACTG… (+ strand) and …GTCAATGAC… (- strand).

Symmetrical cleavage of the two host DNA strands in this engineered chain, specifically between

nucleotides A and G in the palindromic sequence (…CA / GTTACTG… and …GTCAATG /

AC…), would allow collinear integration of vDNA ends precisely 5 base pairs apart.

A strong sequence requirement has been reported for all but one of the nucleotides in this

palindromic sequence (the base pair at the centre position shows a preference for either A or T

nucleotides), suggesting a strong sequence specific recognition of the first four nucleotides in the

sequence (…CAGTX) by residues located within the HIV-1 IN active site. Manual positioning of

the engineered 27 bp host DNA fragment (discussed above) into the tetramer model of HIV-1 IN

(represented in Figures 2.8a and 2.8b), have identified an alignment that satisfy the distance

requirement between cleavage sites on host DNA (~15-16 Å; 5 base pairs).

†† Note on nucleotide identification: Underlined character (X = A or T) represents the central nucleotide of the

palindromic sequence; character under discussion in text is emphasised in bold font; “/” indicates the strand cleavage

site.

Chapter 2:

Figure 2.9

27 base-pair host DNA fragment. The complex illustrates the association of the biologically active

HIV-1 IN tetramer with the LEDGF/p75 IDB; the binding position of the

vDNA; and the position o

apart. These figures were created using Tripos

A.

B.


9 a) Frontal view, and b) top view of the

pair host DNA fragment. The complex illustrates the association of the biologically active

1 IN tetramer with the LEDGF/p75 IDB; the binding position of the

vDNA; and the position o


Methods

a) Frontal view, and b) top view of the



vDNA; and the position of the catalytic active sites for full


a) Frontal view, and b) top view of the



f the catalytic active sites for full


a) Frontal view, and b) top view of the HIV



f the catalytic active sites for full

These figures were created using TriposTM


HIV-1 IN dimer



f the catalytic active sites for full-site strand transfer 5 base pairs (~15 Å)


1 IN dimer-of-dimers associated around a



site strand transfer 5 base pairs (~15 Å)


Page

dimers associated around a


1 IN tetramer with the LEDGF/p75 IDB; the binding position of the U5 and U3 ends of



Page 69



U5 and U3 ends of




U5 and U3 ends of



Specifically, the alignment highlighted the possible involvement of residues Q148 and V151 during

target site selection, potentially providing stabilising interactions to the GT dinucleotide fragment

opposite the putative cleavage site (…CA / GTTACTG… and …GTCAATG / AC…; Scheme 2.2),

while residue Q146 may aid in the destabilisation of the A---T base pair integrity through

interactions with the adenosine (A) nucleotide (…CA / GTTACTG… and …GTCAATG / AC…)

on the opposing DNA strand. Furthermore, the possibility exists that residue E152 may destabilise

the T---A base pair located directly adjacent the cleavage site (…CA / GTTACTG… and

GTCAATG / AC…) through an interaction with the thymidine (T) nucleotide. Importantly in this

instance, E152 will not be involved in the chelation of the divalent metal atom, but will have to

adopt the original side-chain conformation (discussed previously).

Scheme 2.2 Sequence of the palindromic insertion site: Showing possible interactions of host DNA

nucleotides with HIV-1 IN active site residues. (Residues of the interacting HIV-1 IN dimers are

indicated in red and purple respectively, while the arrows indicate the putative cleavage sites 5 base

pairs apart.)

2.4 In silico screening: Hit finding from database mining

All computational protein manipulations were performed on a dual-core desktop PC with a Red Hat

Enterprise Linux Version 5.0 operating system. All protein manipulations, database preparations

and docking runs were performed using the commercial software package SybylTM

version 8.0,

licensed from TriposTM

(Tripos Inc., St. Louis, MO, USA, 2008). In this work, significant and

meaningful figures will be treated in the following way:

1) For theoretical numerical results (including modelling data), the values will be rounded to

the closest 0.5 to reflect the inherent lack of statistical confidence in the generated numbers

(as a consequence of the inherent uncertainty in the theoretical models used).

2) For experimental in vitro results, the uncertainty will be rounded to the closest 0.5 unit at the

level of the uncertainty if the standard deviation (SD) <0.5 at the uncertainty level; if

however the SD>0.5, the uncertainty will be rounded to the closest whole number.


2.4.1 Preparation of the receptor sites

The HIV-1 IN strand transfer model generated in the first part of this chapter was prepared as one of

the target receptors for the docking of compound databases, while the PFV IN strand transfer

complex as reported in the crystal structure was used as the second target receptor (docking with the

PFV IN strand transfer complex was performed after elucidation of the crystal structure in 2010, to

enable a comparison of the binding mode predicted for potential inhibitors). In an attempt to

simplify the docking process and to shorten the computational processing time, all docking runs

were performed with monomers of either the PFV IN or the HIV-1 IN strand transfer structures.

These monomer structures still contained all of the features of the original tetrameric strand transfer

complexes, including the two divalent magnesium atoms and the 3’-end processed vDNA strands

bound in the enzyme active sites. The catalytic centre and inhibitor binding sites of each target

receptor was defined as a 3 Å sphere around the conserved catalytic triad (HIV-1 IN: D64, D116

and E152; and PFV IN: D128, D185 and E221), including the two divalent magnesium atoms, as

well as several nucleotides of the bound vDNA (see Figure 2.7 for a representation of the active site

of the HIV-1 IN model).

2.4.2 Preparation of the ZINC compound database

The ZINC database30

(containing 6,220,335 unique compounds in subset #13: “Clean Drug-like” on

2009/04/02) was accessed through the on-line interface (http://zinc.docking.org/) and filtered

according to a modified version of Lipinski’s Rule of Five (Ro5). As discussed, the Ro5 provides

guidelines for the design of compounds with good oral bio-availability and affords a good starting

point for most drug design projects; however, in this instance certain parameters of the Ro5 were

modified in an attempt to identify only the most “drug-like” compounds. Specifically, the CLogP

parameter was adjusted to filter out compounds with median to low lipophilicity; the molecular

weight parameter was adjusted to filter out molecules smaller than 100 g/mol; and molecules with a

single positive or negative charge were allowed (outlined in Table 2.2). The compound subset was

generated and downloaded on 2 April 2009 from ZINC version 9 containing a total of 732 unique

molecules.

Table 2.2 Lipinski’s Ro5 for the discovery of orally bio-available pharmaceuticals and the set of

parameters used in the creation of the ZINC screening library.

Lipinski’s Ro5 Modified Parameters

CLogP ≤ 5 3 ≤ CLogP ≤ 4

Hydrogen bond donors ≤ 5 1 ≤ # Hydrogen bond donors ≤ 10

Hydrogen bond acceptors ≤10 1 ≤ # Hydrogen bond acceptors ≤10


Molecular weight (g/mol) ≤ 500 100 ≤ Molecular weight (g/mol) ≤ 500

-1 ≤ Net Charge ≤ +1

The resulting library was manually inspected to ensure the chemical viability and correct chemical

structure of all molecules in the subset. Furthermore, all hydrogen atoms were added and the

compound library was prepared to reflect the correct 3D compound structure; AMBER7_FF99 atom

types were assigned to all atoms; Gasteiger-Huckel charges were added to the ligand atoms; all β-

factors were replaced with charges; and pre- and post-minimisation were performed using the

MMFF94s force field without initial optimisation. Finally, the compound library generated for

screening against HIV-1 IN consisted of 732 structurally diverse, commercially available, “drug-

like” molecules.

2.4.3 Docking of the screening library

A number of virtual screening techniques, and programs, have been developed to predict the

geometries of biomolecular ligand-receptor complexes and to discover new ligand structures as

leads for drug design. Most of the available software programs were developed to identify ligand-

or receptor-based pharmacophores and to perform 3D database searching. Some of the most widely

used pharmacophore identification software programs are summarised in Table 2.3.

Table 2.3 Molecular docking software and sources.122

Software Program Source Information

Surflex-Dock Tripos, Inc. (Prof. Ajay N. Jain): http://www.tripos.com/

FlexX BioSolveIT GmbH: www.biosolveit.de/FlexX/

LigandFit Accelrys, Inc.: www.accelrys.com/

GOLD Cambridge Crystallographic Data Centre: www.ccdc.cam.ac.uk/

DOCK University of California (Irwin Kuntz): www.dock.compbio.ucsf.edu/

AUTODOCK The Scripps Research Institute (David Goodsell): www.scripps.edu/

Glide Schrödinger Inc.: www.schrodinger.com

All of the docking programs listed in Table 2.3 also incorporate a scoring function. As each

compound is docked into the receptor site, a user-defined number of possible compound

conformations or “poses” are generated. The scoring function then rates each of these poses as a

function of the “goodness-of-fit” within the active site, dependent on the number and strength of

predicted interactions, as well as the presence of complementary geometric features to that of the

active site. Each pose is assigned a score, enabling the identification of compounds predicted to

have a high binding affinity for the active site.

Chapter 2:

The ZINC screening library was docked

elucidation of the crystal structure, into the

software program (Table 2.3)

bound in the active sites

interactions between the active site and the docking library.

ten conformational poses were specified for each compound in the screening library, to ensure a

thorough exploration of each docking site within reasonable processing times.

score obtained for the 732 molecules in the prepared ZINC compound library was 5.66 (range from

1.33 to 10.48), supporting the “drug

poses generated for the top

analysed and the predicted interactions with the receptor site summarised; illustrated in Figure 2.10.

Figure 2.10

interactions formed between 1 and active site residues. These figures were created using Accelrys

Discovery Studio

Pyrrolidinone ZINC02602549 (co

receptor (docking score of 10.5), and retained an acceptably high score against the PFV IN target

receptor (docking score 7.5). The docking scores obtained for known inhibitors of HIV

both receptor targets are included in Table 2.4 to enable a direct comparison of the results.

The pyrrolidinone compound class

agents and has shown favourable pharmacokinetic profiles


ZINC screening library was docked


software program (Table 2.3)

bound in the active sites



ough exploration of each docking site within reasonable processing times.



poses generated for the top


10 The 3D binding pose predicted for compound 1 in the HIV


Discovery StudioTM

rendering.







Methods

ZINC screening library was docked


software program (Table 2.3), considering the metal atoms and

bound in the active sites. No further restrict






poses generated for the top-scoring compound candidates (HIV


The 3D binding pose predicted for compound 1 in the HIV


rendering.







ZINC screening library was docked firstly


, considering the metal atoms and

No further restrict





1.33 to 10.48), supporting the “drug-like” nature of the compounds

scoring compound candidates (HIV




Pyrrolidinone ZINC02602549 (compound 1




The pyrrolidinone compound class has been tested against a variety of bacterial and disease


firstly into the HIV

elucidation of the crystal structure, into the PFV IN target receptor site

, considering the metal atoms and

No further restrictions were placed on the structure in terms of preferred





like” nature of the compounds

scoring compound candidates (HIV




1) scored exceptionally high against the HIV




has been tested against a variety of bacterial and disease


into the HIV-1 IN target receptor

target receptor site

, considering the metal atoms and the pre

ions were placed on the structure in terms of preferred

interactions between the active site and the docking library. A total of five pre




like” nature of the compounds

scoring compound candidates (HIV-




scored exceptionally high against the HIV






1 IN target receptor

target receptor site using the Surflex

the pre-processed 3’


A total of five pre




like” nature of the compounds in the library.

-1 IN target receptor only) were


The 3D binding pose predicted for compound 1 in the HIV-1 IN active site and the 2D


scored exceptionally high against the HIV





for use as orally administered

Page

1 IN target receptor site

using the Surflex

processed 3’-end of v


A total of five pre-docking poses and


ough exploration of each docking site within reasonable processing times. The average docking


in the library. The docking

1 IN target receptor only) were


1 IN active site and the 2D


scored exceptionally high against the HIV-


receptor (docking score 7.5). The docking scores obtained for known inhibitors of HIV-1 IN aga




Page 73

site and, upon

using the Surflex-DockTM

end of vDNA


docking poses and


The average docking


The docking





-1 IN target


1 IN against


has been tested against a variety of bacterial and disease-causing


and, upon

TM

DNA


docking poses and


The average docking


The docking




1 IN target


inst

causing



pharmaceutical agents.123,124,125,126

A set of six commercially available analogues was sourced from

suppliers and tested in a proof-of-concept assay against the HIV-1 IN enzyme in a direct enzyme-

linked immunosorbent assay (ELISA) (2-7; Table 2.4). At the time, none of the vendors contacted

had readily available stock of compound 1, and sourcing this compound would have entailed

resynthesis (escalating the associated costs and delaying delivery time).

Table 2.4 Docking scores and strand transfer inhibition of some pyrrolidinone derivatives and

known HIV-1 IN inhibitors.

Compound structure Compound name Strand transfer

inhibition % (HIV-1 IN)‡‡

Docking score:

HIV-1 IN§§

Docking score:

PFV IN§§

In silico hit:

1

4-[(1-benzofuran-2-

yl)carbonyl]-5-[4-

(dimethylamino)phenyl]-3-

hydroxy-1-[3-(imidazol-1-

yl)propyl]-5H-pyrrol-2-one

ND* 10.5 7.5

Purchased analogues:

2

1-(3-(1H-imidazol-1-yl)propyl)-

4-(benzofuran-2-carbonyl)-3-

hydroxy-5-phenyl-1H-pyrrol-2-

(5H)-one

8 10.5 7.0

3



hydroxy-5-(p-tolyl)-1H-pyrrol-

2-(5H)-one

55 9.5 7.5

4


4-(4-chlorobenzoyl)-5-(4-

(dimethylamino)phenyl)-3-

hydroxy-1H-pyrrol-2-(5H)-one

25 8.0 6.0

‡‡

In vitro % inhibition of recombinant HIV-1 IN; performed at 10 µM compound concentration with 3’-end processed

vDNA; performed by Dr. S. Mosebi (Biomed group, AMD, Mintek, South Africa); values rounded to the nearest whole

number. §§

Values rounded off to the nearest 0.5 to reflect the lack of statistical confidence in the generated numbers.

O O

NHO

O N

N


Table 2.4 continued.

5


(4-(dimethylamino)phenyl)-3-

hydroxy-1-(3-

morpholinopropyl)-1H-pyrrol-

2-(5H)-one

28 9.0 7.5

6


hydroxy-5-(2-methoxyphenyl)-

1-(3-morpholinopropyl)-1H-

pyrrol-2(5H)-one

1 ND ND

7

3-hydroxy-4-(7-

methoxybenzofuran-2-

carbonyl)-5-(4-

methoxyphenyl)-1-(3-

morpholinopropyl)-1H-pyrrol-

2(5H)-one

25 ND ND

Known Inhibitors of HIV-1 IN:

Raltegravir

N-(2-(4-((4-

fluorobenzyl)carbamoyl)-5-

hydroxy-1-methyl-6-oxo-1,6-

dihydropyrimidin-2-yl)propan-

2-yl)-5-methyl-1,3,4-

oxadiazole-2-carboxamide

84 6.5 7.0

* ND = Not determined

An activity cut-off value of 50% inhibition at 10 µM compound concentration was established for

use throughout the project, as an attempt to focus research efforts on the most promising candidates.

One of the commercially available analogues [compound 3; 1-(3-(1H-imidazol-1-yl)propyl)-4-

(benzofuran-2-carbonyl)-3-hydroxy-5-(p-tolyl)-1H-pyrrol-2(5H)-one] showed promising inhibition

of the HIV-1 IN enzyme (55% inhibition as measured against a positive and negative control),

warranting further investigation and providing a basis for continued research into the anti-integrase

activity of the pyrrolidinone compound family (Figure 2.11a and b). Using structure-based drug

design and medicinal chemistry principles, a family of derivatives was designed based on the parent

compound identified through in silico screening, compound 1, and the lead candidate from

experimental screening of commercially available analogues, compound 3. The derivatisation was

performed not only in an attempt to optimise binding to the HIV-1 IN active site, but also to enable

Chapter 2:

the study of structure

variance were identified in the compound scaffold and are illustrated in Table 2.

the contribution an

positions of variance.

Figure 2.11

interactions formed between

Discovery Studio

Table 2.5 Centres for the incorporation of variance identified in the pyrrolidinone molecular

structure.

1 analogue used in synthesis:

Methyl-(2Z)-

hydroxy


the study of structure


the contribution and effect of different functional groups that were investigated in each of the

positions of variance.

11 The 3D binding pose predicted for compound 3 in the HIV


Discovery StudioTM

rendering.

Centres for the incorporation of variance identified in the pyrrolidinone molecular

Arom1


-4-(1-benzofuran

hydroxy-4-oxobut-2-enoate

Methods

the study of structure-activity relationships during later stages of the project. Three centres of


d effect of different functional groups that were investigated in each of the



rendering.


O O

HO

O


benzofuran-2-yl)-2-

enoate

activity relationships during later stages of the project. Three centres of




3 and active site residues. These figures were created using Accelrys


N

ON

N

6 analogues synthesised:

Aromatic vs. aliphatic

Steric influence (size)

Electronic influence (N/O)

Chain length of aliphatic linker







N

R2






-







HO

Arom









The 3D binding pose predicted for compound 3 in the HIV-1 IN active site and the 2D



NO

O

O

m1

R

Cn

R2


Presence vs. absence


Electronic Influence (Heteroatom)

Position of substituents

Additional substitutions

Page


variance were identified in the compound scaffold and are illustrated in Table 2.5. Also illustrated is





R3

2

R3







Page 76


. Also illustrated is












. Also illustrated is





A total of 450 structurally related pyrrolidinone analogues were generated from the original

structures of 1 and 3. Each of these analogues was analysed for chemical tractability, feasibility and

the presence of undesirable functional groups, and then re-docked into the HIV-1 IN strand transfer

monomer. The potential for keto-enol tautomerisation and the presence of one chiral centre in each

of the molecules were considered, and different versions of each molecule were generated to

account for both the keto and enol forms, as well as both the (R) and (S) conformations at the chiral

centre. Notably, a slight preference for the R-enantiomer was evident from the docking results of

the pyrrolidinone analogues (38% preference for the S-enantiomer vs. 62% preference for the R-

enantiomer). Finally, the 50 top-scoring analogue structures were earmarked for chemical synthesis

(discussed in Chapter 3).

2.5 Conclusion

Prior to the elucidation of the PFV intasome crystal structure in 2010, structural data on the

biologically relevant forms of retroviral integrase was severely limited and rational drug discovery

efforts targeting HIV-1 IN were largely based on incomplete models generated from partial

structural data. Although many compounds were identified that showed some inhibition of the 3’-

end processing or strand transfer reactions catalysed by HIV-1 IN, only one HIV-1 IN inhibitor

gained FDA approval (RaltegravirTM

or Isentress®

developed by Merck; approved by the FDA in

2007). The solvation of the biologically relevant tetrameric structure of retroviral integrase provided

a major breakthrough in this research field, finally enabling rational drug design targeting the

integrase enzyme. It is worth noting that even though the structure determined was that of the

related PFV integrase, HIV-1 IN is proposed to function in a similar manner, allowing the

generation of more accurate and biologically relevant HIV-1 IN models.

In a literature-based approach (focussed on reports published during 2009 and earlier), a series of

models was generated of HIV-1 IN, depicting the monomeric, dimeric and tetrameric forms of the

enzyme. Specifically, the monomeric enzyme form described here was constructed through

superpositioning of sub-domain crystal structures in a manner similar to that reported by

Podteleznikov et al.,64

De Luca et al.,65

and Wijitkosoom et al.,66

while the inclusion of viral DNA

was based on reports by Alian et al.95

and Michel et al.96

At each step in the construction, the HIV-1

IN model was compared to the corresponding PFV IN fragment to promote a more complete

understanding of the differences (and similarities) between the two structures.

Although the sequence similarity between PFV IN and HIV-1 IN was low, the overall monomeric

structures showed some similarity, attributed to a high degree of structural similarity between the


three subdomains, with the position and arrangement of the flexible subdomain linkers contributing

to the variance observed. The effect of the linkers became progressively more pronounced as the

models increased in size and complexity (monomer < dimer << tetramer). As a consequence, the

tetramer model structure of HIV-1 IN generated in a manner similar to literature reports differed

significantly to the tetrameric structure of the PFV intasome as observed in the crystal structure.

Additionally, although both structures incorporated the requisite two divalent magnesium ions, the

binding position observed for vDNA in the PFV IN crystal structure was not predicted by the HIV-1

IN model. As such, there was a slight difference in the active site structure of the two enzymes

which would conceivably translate into any drug design efforts.

A 3D screening library containing 732 chemically diverse molecules were defined from the ZINC

compound database, according to a modified version of Lipinski’s Ro5. The inter-molecular

protein-ligand complexes formed between each of the molecules in the screening library and a

specified binding site located within the HIV-1 IN and PFV IN active sites were predicted with the

use of a molecular modelling software suite program. In this way, the structures of potential

inhibitors selective for the HIV-1 IN strand transfer reaction could be predicted that still retained an

acceptably high score against PFV IN. Pyrrolidinone compound 1 scored exceptionally highly

against the HIV-1 IN active site (docking score 10.5) and retained an acceptable score against the

PFV IN active site (docking score 7.5). Furthermore, both docking scores compared favourably

with scores obtained for several known inhibitors of HIV-1 IN, docked against the same active sites

in an identical procedure. Subsequently, six analogues of the identified structure were purchased

from commercial sources and tested in a proof-of-concept ELISA against recombinant HIV-1 IN

enzyme. One of these analogues, compound 3, showed promising inhibition of HIV-1 IN catalysed

strand transfer (>50% enzyme inhibition at 10 µM compound concentration), and supported further

research into the pyrrolidinone class of compounds. Finally, a total of 450 derivatives were

designed based on the hit structures identified from the in silico and initial in vitro screening

(compounds 1 and 3, respectively), incorporating a variety of functional groups and steric

influences to enable a comprehensive probing of the HIV-1 IN active site. From these derivatives,

~50 were earmarked for chemical synthesis (discussed in Chapter 3).


2.6 References

1. Ban, T. A., Dialogues Clin. Neurosci., 2006, 8, 3, 335-344.

2. Roberts, R. M., Serendipity – Accidental Discoveries in Science, John Wiley & Sons, New

York, 1989.

3. Fleming, A., J. Roy. Inst. Pub. Health Hyg., 1945, 8, 36-49

4. Cahn, A., Hepp, P., Centralblatt Klin. Med., 1886, 7, 561-564.

5 Golman, A. G., Rall, T. W., Nies, A. S., Taylor, P., (eds), Goodman and Gilman’s The

Pharmacological Basis of Therapeutics, 1990, Pergamon Press, New York, p. 1389.

6. Rosenberg, B., Van Camp, L., Trosko, J., Mansour, V., Nature, 1969, 222, 385-386.

7. Rosenberg, B., Van Camp, L., Krigas, T., Nature, 1965, 205, 698-699.

8. Holmes, R. W., Love, J., J. Am. Med. Assoc., 1952, 148, 11, 935-937.

9. Hirsh, J., Fuster, V., Ansell, J., Halperin, J. L., J. Am. Coll. Cardiol.,2003, 41, 9, 1633-

1652.

10. Mueller, W., Herrmann, B., N. Engl. J. Med.,1979, 301, 555.

11. Silverman, R. B., The Organic Chemistry of Drug Design and Drug Action, 2nd Edition,

2004, Elsevier Academic Press, Burlington, USA, p.2-3.

12. Kubinyi, H., J. Recept. Signal Transduct. Res., 1999, 19, 15-39.

13. Thomas, G., Fundamentals of Medicinal Chemistry, 2003, John Wiley & Sons Ltd., West

Sussex, England, p.95.

14. Wu, G., Assay Development: Fundamentals and Practices, 2010, John Wiley & Sons, Inc.,

New Jersey, USA, Preface, p.xiii.

15. Usón, I., Sheldrick, G. M., Curr. Opin. Struct. Biol.,1999, 9, 643-648.

16. Glazer, A. M. in: Ladd, M. F. C., Palmer, R. A., Structure Determination by X-ray

Crystallography, 4th Edition, 2003, Kluwer Academic / Plenum Publishers, New York,

USA, p.v-vii, 618-677.

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Chapter 3: Chemical Synthesis Page 80

CHAPTER 3: CHEMICAL SYNTHESIS

3.1 Introduction and background

In silico screening (“docking”, discussed in Chapter 2) against a model of the HIV-1 IN enzyme

identified 4-[(1-benzofuran-2-yl)carbonyl]-5-[4-(dimethylamino)phenyl]-3-hydroxy-1-[3-(imidazol-

1-yl)propyl]-5H-pyrrol-2-one (code ZINC02602549; Figure 3.1, parent compound 1) as a potential

inhibitor that may selectively target the strand transfer process. Additionally, commercially

available analogues (compounds 2-7; Figure 3.1) of compound 1 have confirmed the proposed

inhibition potential in direct enzyme assays (discussed in Chapter 2).

O O

NHO

O N

O

O O

7

Figure 3.1 The chemical structures of parent compound 1 (ZINC02602549), identified as potential

HIV-1 IN inhibitor though in silico database screening; and commercially available analogues 2-7,

with potential for HIV-1 IN strand transfer inhibition as confirmed in experimental in vitro assays.

An investigation of available literature showed that pyrrolidinone compounds of this type have been

extensively tested against a variety of bacterial and other disease-causing agents and have shown


favourable pharmacokinetic profiles for use as orally administered pharmaceutical agents.1

Chemical synthesis of a structurally related family was undertaken to confirm the predicted

inhibitory activity in experimental assays (discussed in Chapter 4) and to form an idea of the

pharmacophore responsible for any observed activity (for use in second and later generation

design). Additionally, any data derived from these compounds would also facilitate the study of

structure-activity relationships during later stages of the project.

3.2 Synthesis of substituted 1-aminoalkyl-3-hydroxy-3-pyrrolin-2-ones

3.2.1 Retrosynthetic analysis

In 1990 the Nobel Prize in Chemistry was awarded to Professor E. J. Corey for his revolutionary

work in the “development of the theory and methodology of organic synthesis”.2 Although his

influence can be seen in many areas of modern organic chemistry (including novel synthetic

reagents and reactions), arguably the most important of his contributions was the introduction of his

theory on the retrosynthetic planning of organic reactions. Application (and further development) of

this theory over the ensuing five decades has had far-reaching effects on the way that modern

organic reactions are planned. Instead of the synthetic planning phase being strongly dependent on

an assumed starting point (pre-1957), it became possible to design a synthesis pathway through

performing an iterative series of deconstruction steps, starting with the final target molecule and

ending with a range of commercially available starting materials. As computational capabilities

became more advanced, databases and software programs were developed that enabled the

automation of the retrosynthetic analysis process (for example IGOR,3,4

CONAN,5 THERESA,

6

RETROSYN7 and Route Designer

8 to name a few).

The reasons for performing a retrosynthetic analysis of parent compound 1 were two-fold: it would

facilitate the derivatisation of the compound family, while confirming the chemical feasibility of

any derivatives designed. Although several possible reaction pathways leading to compound 1 were

identified, only the best option (and the route eventually followed) will be outlined below.


Scheme 3.1 Retrosynthetic reaction pathway proposed for the pyrrolidinone family of compounds.

Compound 1 could be disconnected as illustrated in Scheme 3.1 to yield three commercially

available starting materials. Three disconnections of the pyrrolidinone ring system, specifically

disconnecting the delocalised electron-rich system on the left-hand side of the molecule from the

dimethylamino-benzylic group, as well as breaking the bonds on both sides of the pyrrolidinone-N,

resulted in synthons that corresponded to readily available commercial starting materials: pyruvate

esters (starting material 8), diamines (starting materials 9.1-9.6) and aryl-aldehydes (starting

materials 10.1-10.9). A further retrosynthetic step was introduced to enable the synthesis of 8 from

two less complex starting materials (starting materials 8.1 and 8.2). There are several advantages to

this reaction pathway: a one-step reaction yields the required pyruvate starting material 8 following

which the condensation and cyclisation of the pyrrolidinone product can be performed in a one-pot

multicomponent reaction using equimolar amounts of the three starting materials (8, 9.1-9.6 and

10.1-10.9); no additional catalysts (acids, bases, etc.) or special conditions are required for the

reaction to take place; and importantly, the intermediate product formed in each step of the multi-

step reaction will act as starting material for the subsequent reaction, effectively eliminating the

need to isolate higher-energy intermediate products and driving the reaction irreversibly forward

towards the most thermodynamically stable product (according to Le Chatelier’s principle).

A library of 900 analogues of parent compound 1 was designed based on commercially available

starting materials of the three classes of compounds identified in the retrosynthetic analysis (design

discussed in Chapter 2). Re-docking this in silico library to the original protein model provided a


docking score for each of the designed analogues, effectively ranking the respective compounds in

terms of predicted HIV-1 IN binding affinity. Finally, a set of approximately 50 of the most

promising designed analogues was earmarked for chemical synthesis to validate the molecular

modelling predictions.

3.2.2 Synthesis of the pyruvate starting material 8

The Claisen condensation reaction was developed by Rainer Ludwig Claisen (1851–1930) in 1881

and is classified as a nucleophilic acyl substitution reaction. This carbon-carbon bond-forming

reaction takes place between two carbonyl-containing reagents in the presence of a strong base,

where at least one of the reagents is an enolisable ester and the base used is usually an alkoxide. The

products of this exothermic reaction, known as β-keto esters or β-diketones, are important

intermediates in a variety of chemical syntheses and open the way to a range of different end-

products (see Scheme 3.2).

Scheme 3.2 Illustrating the application of β-keto ester and β-diketone intermediates in organic

synthesis: a range of end-products can be achieved from transformation of the classic Claisen

condensation products.9,10

Additionally, β-keto esters and β-diketones have found numerous applications as end-products in

various fields as a result of their highly electrophilic nature and their ability to chelate metal

systems.11

Modifications of the classic Claisen procedure have given rise to a spectrum of variations

that include the mixed (or crossed) Claisen condensation and the Dieckmann condensation

reactions.


While the classic Claisen condensation describes reactions involving only one enolisable ester

(Scheme 3.3), the mixed Claisen condensation describes a similar reaction with two different

reagents, one of these being an enolisable ester, while the other is a non-enolisable carbonyl-

containing compound (Scheme 3.4). The third variation, the Dieckmann condensation, describes

one molecule with two ester moieties that can react intra-molecularly to form a cyclic β-keto ester

(Scheme 3.5). As is commonly observed in cyclic systems, the formation of 5- or 6-membered rings

is favoured over smaller or larger rings due to the lesser amount of ring strain experienced in the

system.

Scheme 3.3 Schematic representation of the classic Claisen condensation reaction.

Scheme 3.4 Schematic representation of the mixed / crossed Claisen condensation reaction.

Scheme 3.5 Schematic representation of the intramolecular Dieckmann condensation reaction.

Although these reactions take place with different reagents, the mechanism in all Claisen reactions

remains the same (Scheme 3.6). Upon addition of a strong base (usually an alkoxide) the acidic α-

hydrogen of the enolisable ester is deprotonated, resulting in a resonance-stabilised ester enolate

ion. The strongly nucleophilic enolate ion attacks the second carbonyl carbon in a nucleophilic

substitution process resulting in a hemi-acetal intermediate. The unstable intermediate collapses,

reforming the carbonyl and resulting in a loss of the leaving group, leading to the β-keto ester

product. Addition of a mild acid solution neutralises the basic alkoxide and allows for recovery of

the protonated β-keto ester product.


Scheme 3.6 The mechanism of the Claisen condensation reaction.12

The crossed Claisen reaction was employed in the synthesis of the pyruvate ester starting material

common to all of the synthesised compounds in this project, methyl (2Z)-4-(1-benzofuran-2-yl)-2-

hydroxy-4-oxobut-2-enoate (starting material 8). During the synthesis of this starting material, 2-

acetyl benzofuran (starting material 8.1) and dimethyl oxalate (starting material 8.2) were reacted in

the presence of sodium methoxide base to give the required product (Scheme 3.7). Specifically, an

equimolar solution of 8.1 and 8.2 in dry methanol (light yellow solution) was added to a dry

solution of sodium methoxide (colourless solution) over a period of 1 hour, then stirred at room

temperature for 4–6 hours and stirred overnight at room temperature. Oxalates are generally very

reactive under the conditions used in the Claisen condensation reaction, as each of the two carbonyl

groups on the molecule makes the other more electrophilic and therefore more susceptible to attack

by the nucleophilic enolate ion produced upon addition of the base.13

The non-aqueous methoxide

base in methanol solvent was used in an attempt to avoid the alkaline hydrolysis of the ester bond

that may occur when stronger bases such as sodium hydroxide in aqueous medium are employed.

During the overnight reaction, a light pink precipitate formed in the reaction vessel that gradually

intensified in colour, indicative of the deprotonated enolate form of the pyruvate ester. To obtain

and isolate the neutral, protonated product 8, 15% aqueous sulfuric acid was added to the mixture,

resulting in an immediate colour change of the precipitate (pink to bright yellow). Extraction with a

biphasic water/dichloromethane (DCM) system and evaporation of the organic solvent afforded

high yields of the powdered pyruvate ester product (yellow), which was subsequently recrystallised

from warm methanol. As the product obtained was of high purity, it could be used in subsequent

synthesis steps without the need for further purification.


The synthesised pyruvate ester starting material 8 was characterised via 1H and

13C{

1H} NMR

analysis, as well as high resolution time-of-flight electron spray ionisation mass spectral analysis

(HR TOF-ESI MS). All characterisation data point towards successful completion of the reaction

and are reported in Chapter 5. All other starting materials could be sourced directly from

commercial suppliers and were used without further modification or purification.

Scheme 3.7 Synthesis of starting material 8 (pyruvate ester).

3.2.3 Synthesis of the pyrrolidinone target compound family

R1 = Benzofuran-2-yl; R

2 = 1-Propyl-imidazole (11.1-11.6), 4-Propyl-morpholine (12.1-12.6), Propyl-N’,N’-dimethyl-

3-amine (13.1-13.6), 3-Propylamine (14.1-14.6), 2-Ethyleneamine (15.1-15.9), 5-Aminopentane (16.1-16.9); R3 = H

(11.1; 12.1; 13.1; 15.1; 16.1), 2-Chloro (11.2; 12.2; 13.2; 14.2; 15.2; 16.2), 4-Fluoro (11.3; 12.3; 13.3; 15.3; 16.3), 4-

Methoxy (11.4; 12.4; 13.4; 14.4; 15.4; 16.4), 3-Methoxy (11.5; 12.5; 13.5; 15.5; 16.5), 4-Hydroxy-3-methoxy (14.6;

15.6; 16.6), 2,4-Dimethoxy (14.7; 15.7; 16.7), 2,4-Dihydroxy (14.8; 15.8; 16.8), 4-tert-Butyl (11.9; 12.9; 13.9; 14.9;

15.9; 16.9).

Scheme 3.8 General method for the synthesis of the pyrrolidinone derivatives.

The synthesis of the proposed family of compounds was based on literature reports of similar

compounds.14

In following the facile synthetic procedure outlined by the authors and illustrated in

synthetic Scheme 3.8, equimolar amounts of starting materials 8, 9 (9.1-9.6) and 10 (10.1-10.9)

were dissolved in 1,4-dioxane and stirred at room temperature until the desired pyrrolidinone

product precipitated from the solution. In some instances, the reaction proceeded to completion

within minutes, but mostly the reactions required stirring at room temperature overnight, during

which time the product precipitated from the mixture. Such a long reaction time would be

undesirable in an industrial setup and attempts were made to optimise and shorten the reaction time

and to increase the yield achieved with each reaction. An immediate improvement in reaction time

O O

OH O

R1

+ H 2 N R2

Cn +

R 3

O O

H O N

O R

1

R3

R 2

C n

*


and yield of product could be observed when the solvent volume used in each reaction was reduced

to ca. 5 mL. As product formation in the lesser volume started virtually immediately upon addition

of the last reagent and (for most reactions) proceeded to completion within minutes, no further

optimisation in terms of reaction time was performed for the purposes of this project.

Product formation is dependent on a relatively complex cascade of events, one possibility of which

is illustrated in Scheme 3.9 using the synthesis of 11.1 as an example.

In a first step, the primary amine of 9.1 reacts with the aldehyde of 10.1 to give a Schiff-base

intermediate product, releasing a water molecule in the process. Normally, the equilibrium of this

Schiff-base reaction lies to the left, favouring the reactants and inhibiting product formation

(Scheme 3.9, Step 1). According to Le Chatelier’s principle, the position of the reaction equilibrium

can be altered by changing the reaction conditions, allowing optimisation for product formation.15,16

Applied to the Schiff-base reaction, the equilibrium can be manipulated to favour product formation

by continuously removing one of the products as the reaction proceeds. Usually, the water formed

during the reaction is removed as an azeotrope. In this specific instance, however, the Schiff-base is

removed from the system through participation in a subsequent reaction (nucleophilic attack on 8;

Scheme 3.9, step 2), forcing the equilibrium towards the right, favouring product formation.

Specifically, the lone pair electrons on the basic Schiff-base intermediate deprotonates the acidic

CH2-group of the tricarbonyl compound, leaving an activated nucleophile (carbanion) and an

activated electrophile (iminium ion). The carbanion attacks the activated iminium ion, generating a

carbon-carbon bond and resulting in the formation of a secondary amine. It is possible that this

happens as a (semi) concerted reaction via a six-membered ring transition state. The molecule

subsequently undergoes an intramolecular cyclisation when the secondary amine adds to the ester-

carbonyl, forming the pyrrolidinone ring and resulting in the release of the methanol leaving group.


Step 1:

Step 2:

Scheme 3.9 Proposed mechanisms of the condensation and cyclisation reactions that result in the

final pyrrolidinone products (illustrated using compound 11.1 as an example).

3.2.4 Purification

One of the major advantages of the synthetic route chosen for the preparation of the pyrrolidinone

compounds was the facile purification of the prepared products. All starting materials used and by-

products formed were fully soluble in the chosen dioxane solvent, but the products formed during

the course of the reaction were not soluble and therefore precipitated out of the solution. In most

cases, the pure product could be obtained through a simple centrifugation step to collect the

precipitate, washing with diethyl ether or n-hexane and in vacuo drying of the product.


Recrystallisation from ethanol resulted in several samples providing crystals suitable for single

crystal X-ray analysis. The characteristics and properties of these crystals will be discussed in a

subsequent section (section 3.3.5).

3.2.5 Difficulties encountered and solved during the synthesis

Although the products formed easily and the isolation and purification of these products were, for

the most part, relatively uncomplicated, attempts at characterisation and further evaluation of some

compounds were hindered by solubility problems. These solubility issues were not only

experienced in aqueous medium, but the dried product powders were insoluble (or minimally

soluble) in all general laboratory solvents, including dimethyl sulfoxide (DMSO), alcohols,

chlorinated solvents, etc. In a proof-of-concept trial to overcome this obstacle, small amounts of the

product powders were suspended in a range of different laboratory solvents. These suspensions

were then gradually heated (with stirring) until the suspended powder in question dissolved.

Although several of the products could be dissolved (and recrystallised) from absolute ethanol at 70

°C, this high-temperature method was not ideally suited for the characterisation of the products. In a

second approach, small amounts of the product powders were suspended and stirred in a range of

laboratory solvents as before, but in a slight modification of the above method, aqueous

hydrochloric acid (HCl) was added to each suspension at room temperature. It was observed that

upon addition of HCl to a suspension of the product powders in methanol, nearly all of the products

tested gave fully homogeneous solutions suitable for solution-based characterisation (NMR and

MS). The few exceptions notably included compounds that carried the tert-butyl substituent, but

even in these instances, the compounds were sufficiently soluble to allow full characterisation.

In several instances, a powdered form of the pyrrolidinone hydrochloride salt could be isolated from

the acidified methanol solution. As these powders were prepared from pure solutions of the free

pyrrolidinones in methanol acidified with aqueous HCl, further purification was not required. The

powders were washed with n-hexane and/or diethyl ether to remove residual HCl, and subsequently

dried in vacuo. The full characterisation data for each isolated pyrrolidinone hydrochloride salt is

included in Chapter 5.

3.3 Characterisation

As discussed previously, the initial characterisation of members of the pyrrolidinone family of

compounds proved problematic due to the insoluble nature of some compounds in most laboratory


solvents. Through protonation of these compounds by addition of HCl, the solubility problems

could mostly be overcome and as such it became possible to obtain solution-based characterisation

data, including interpretable NMR and MS spectra for all synthesised compounds.

3.3.1 Nuclear magnetic resonance spectroscopy (NMR)

The magnetic resonance of hydrogen nuclei was first demonstrated in 1946 by two independent

researchers, Edward Mills Purcell (1912-1997) and Felix Bloch (1905-1983). Their original

hypothesis rested on two known facts of their time: 1. electromagnetic radiation is composed of

both an electric and a magnetic component, and 2. electronic spectroscopy results from the

interaction of an electric moment with the electric component of electromagnetic radiation. Purcell

and Bloch reasoned that in a similar manner, the interaction of a short-wave, very-high-frequency

(VHF) magnetic moment with the magnetic component of electromagnetic radiation should give

rise to magnetic spectroscopy. The experimental validation of their hypothesis paved the way for

the discovery in the 1950s that the magnetic moment is highly sensitive to changes in the electronic

and chemical environment under study. Differences in the chemical (and therefore also in the

electronic) environment of hydrogen atoms cause a shift in the frequency of the magnetic moment,

so that hydrogen resonances are characteristic of the chemical group they are bonded to.

Furthermore, it was found that magnetic moments of one magnetically active atom or functional

group could couple with the magnetic moments of neighbouring magnetically active atoms in the

same molecule, whether directly bound or separated by several bonds, giving rise to characteristic

splitting patterns (multiplets: doublets, triplets, quartets, etc.) for different functional group

resonances. The simplest form of scalar spin-spin coupling is described by the first order spin

system (Figure 3.2, line 4). In these systems the chemical shift between the coupled functional

groups (∆νA – ∆νX) is significantly larger than the coupling constant (JAX): in general, if a certain

nucleus A is coupled to n identical nuclei X (each being magnetically active), A will show up as n +

1 lines in the NMR spectrum and the separation between the lines will be equal to the coupling

constant between nuclei A and X. The number of lines in a first order spin system, also called an AX

system, is found by a binomial expansion known as the Pascal Triangle.17,18,19

In more complicated systems, where the chemical shift between the coupled functional groups

approaches the coupling constant (∆ν ~ J; Figure 3.2, lines 1–3), more transitions of a similar

energy will be present and the spectrum will show more lines than predicted by a simple first order

analysis. In these instances, a certain nucleus A couples to a nucleus B with very similar energy

(each being magnetically active) and gives rise to a second order spin system, or AB system. Often

seen in ring-structures, AB systems result from cases of magnetic non-equivalence, where two


nuclei will have the same chemical environment and the same chemical shift, but with different

couplings. A third form of scalar spin-spin coupling systems is the ABX system, in which two nuclei

(A and B) have comparable chemical shifts while the third (X) is very different. ABX spin systems

are common in tri-substituted aromatic systems.

Figure 3.2 A series of spectra for a two-spin system: The frequency of spin 1 (ν0,1) is kept constant

while that of spin 2 (ν0,2) is moved closer to spin 1, resulting in more strongly coupled spectra

illustrating the “roofing” effect. Lines 1–3 show various forms of a second-order AB scalar spin-

spin coupling system, while Line 4 shows an example of a first-order AX scalar spin-spin coupling

system.20

The NMR spectra of the synthesised compounds in pyrrolidinone form were determined in dimethyl

sulfoxide (DMSO) or acidified deuterated methanol (dMeOH), while those of the HCl-salts were

determined in dMeOH, with tetramethyl silane (TMS) and the residual solvent peaks as reference

peaks in all spectra. The 1H and

13C NMR spectra of all starting materials were determined and used

as a guide to determine the completion of each reaction. Specifically, for each synthesised

compound, a singlet resonance appears in both the 1H and

13C NMR spectra (in the regions 5.5–6.5

ppm and 65.0–70.1 ppm, respectively) indicative of the presence of a chiral proton formed on the

pyrrolidinone ring upon successful completion of the reaction (marked with “*” in Scheme 3.8 and

Figure 3.3). This position contains the only chiral centre in the synthesised molecules and is a

common feature throughout the series. Aromatic resonances resulting from the benzo-furanyl,

Line 1:

Line 2:

Line 3:

Line 4:


carbonyl and phenyl substituents appear within the expected regions for all compounds (7.0–8.0

ppm in the 1H NMR and 110–180 ppm in the

13C NMR spectra).

Figure 3.3 1H NMR spectra of compound 11.4 determined at neutral pH, showing the full spectrum

indicating the singlet resonance arising from the chiral proton formed upon successful synthesis

(“*”).

The proton signal from the enol-alcohol, another moiety common throughout the compound series,

resonates between 9.5 and 10.0 ppm, but in most instances this signal shows significant broadening

and is not resolved in the 1H NMR spectra. In contrast, the imine-H on the imidazole-ring shows up

as a sharp peak at ~9.0 ppm (as illustrated for compound 11.4 in Figure 3.3). Solution NMR spectra

of most of the pyrrolidinone compounds determined at neutral pH showed interesting complexity in

the aliphatic region, arising from the simultaneous existence of two different spin systems (first-

order AX and second-order AB spin systems) within each sample, representing two different

protonation states. In solution, each of the pyrrolidinone compounds is proposed to form both the

free base and the protonated species, each giving rise to one of the observed spin systems. The

existence of two protonation states also gave rise to a duplication of the aromatic imine-H peak of

the imidazole ring (as illustrated in Figure 3.3). As the complexity of the spectra hindered the

interpretation thereof, attempts were made to simplify the spectra. To this end, NMR spectra for

each compound were recorded in deuterated methanol acidified to pH ~3.0 with 2M HCl in an

effort to force the compounds to adopt the protonated state and to facilitate the allocation of spin

system to protonation state. Analysis and comparison of the spectra at pH 7 and pH 3 allowed

several conclusions to be drawn based on some assumptions: firstly, the concentration of the

protonated compound species will increase at lower pH values, while the concentration of the free

form would decrease; secondly, the two protonation states are each represented by either the AX or

the AB spin systems observed in the aliphatic regions of the 1H NMR spectra; and thirdly, a change

in the concentration ratio of the two protonation states would result in a concomitant change in the

peak ratio of the two spin systems. Upon acidification, the spectra showed a significant increase in

*


the concentration of AB spin system peaks, thereby identifying these as representative of the

protonated compound species (Figure 3.4a and b).

A

B

Figure 3.4 1H NMR spectra showing the ratio of first- to second-order spin-spin systems in samples

of: a) the protonated pyrrolidinone compound, 11.6, determined at pH 3; and b) the free base form

of pyrrolidinone compound 11.6, determined at pH 7.

Although acidification increased the ratio of the AB spin system peaks relative to AX spin system

peaks, and by extension the ratio of protonated to free compound form in a manner highly specific

to each compound under study, complete conversion to the fully protonated state could not be

achieved at pH 3. As such, where possible the characterisation data recorded for the pyrrolidinone

compounds include characteristic peaks for both the major and minor product species at pH 3, as

well as the ratio between the two species under acidic conditions. In addition to the free and

protonated forms discussed above, a third protonation state can be identified for some of the

pyrrolidinone compounds. This third species is present in very low concentrations and most likely

represents the zwitterionic form of the compounds (data not shown).

A cursory investigation of the pyrrolidinone compound structure was performed to explain the

presence of the two different spin systems observed for each compound. The first-order AX spin

system and specifically the couplings and peak multiplicity for the alkyl groups on the aliphatic

bridge is relatively straight-forward and easily predicted from the structure of the free base.

However, the presence of the second-order AB system, later assigned to the protonated compound

form was not anticipated from the initial structure analysis and warranted further investigation.

The presence of strong coupling resulting in an AB spin system would indicate non-equivalence of

the protons within the –CH2– alkyl groups included in the propyl carbon chain, most likely due to a


loss of rotational freedom. A constrained conformation enforced on the alkyl protons upon

formation of a cyclic-system through interaction of the lactam-carbonyl with the protonated

terminal amine on the carbon chain may explain this observation. Furthermore, as discussed

previously, the pyrrolidinone-ring contains a stereogenic proton (marked with “*” in Scheme 3.8

and Figure 3.3). As such, the N-CH2 protons (H21a and H21b) are prochiral which, with limited or

restricted rotation about the N-C bond will give rise to the observed AB system. The structures of

the open chain (free base) and cyclic (protonated) forms of the pyrrolidinone compounds are

illustrated in Figures 3.5a and b, respectively.

Each of the alkyl groups in the chain seems affected to a different extent: using compound 11.4 as

an example (Figure 3.5c), it can be shown that the two protons of the alkyl group immediately

adjacent to the nitrogen of the pyrrolidinone-ring (H21A and H21B) showed the greatest degree of

non-equivalence, with a chemical shift separation of 0.63 ppm (252.2 Hz) between the multiplets of

H21A and H21B. In contrast, the chemical shift separation between the multiplets of H22A and H22B

was measured at 0.11 ppm (44.4 Hz) and similarly for H23A and H23B at 0.08 ppm (32.5 Hz),

indicating that protons situated further away from the pyrrolidinone-ring were less affected by the

formation of the constrained cyclic-system. Additionally, the appearance of both first-order and

second-order characteristics within the same 1H NMR spectrum, and the demonstrated influence of

pH on the ratio between the species, may point towards the transient nature of the cyclic system,

with the ratio of first- to second-order spin character in each spectrum indicative of the

concentration of freely rotating vs. cyclic forms.

For the hydrochloride salts of the pyrrolidinone compounds, this second-order AB spin system is

greatly minimised with respect to that observed for the acidified pyrrolidinone compounds, and

resembles that of the free pyrrolidinone base (illustrated in Figure 3.4b).

Due to the fact that the two neighbouring protons on the imidazole-ring of compounds 11.1 – 11.6

are nearly chemically equivalent (H-25 and H-26 in Figure 3.5A and B below), the ortho H-H

coupling between these two protons and the meta H-H coupling between both of these protons and

the imino-H (H-24 in Figure 3.5A and B) of the imidazole-ring is extremely small. While in some

instances (notably 11.1) the peaks for these protons appear as triplets (overlapping doublet-of-

doublets structure), in most instances this apparent triplet peak structure could not be well-resolved

and the peaks for these three protons (H-24, H-25 and H-26 in Figure 3.5A and B) appear as broad

singlets (compounds 11.2 – 11.6).


O

N

N

O

HO N

O

O

21

22 23

24

25

26

N

HO

O

NN

H

21

22

23

O

O

O

24

25 26

Figure 3.5 Pyrrolidinone compound 11.4, illustrating a) the open chain free-base form; and b) the

cyclic structure proposed to form upon protonation; c) 1H NMR spectra of compound 11.4

determined at pH 7: Distinguishing between peaks of equivalent (dashed lines) and non-equivalent

(solid lines) protons in the propyl chain (H21AB vs. H21A and H21B; H22AB vs. H22A and H22B; and

H23AB vs. H23A and H23B).

3.3.2 Mass spectrometry (MS)

Electron Impact (EI) and Chemical Ionisation (CI) are widely used methods for generating ions for

mass spectrometry. EI is a direct technique, based on the bombardment of vapour phase sample

molecules with high-energy electrons (generally 70 eV), often resulting in substantial fragmentation

of molecules subjected to this characterisation technique. In contrast, CI relies on the ionisation of a

reagent gas introduced into the source. The sample molecules collide with the ionised reagent gas

molecules and undergo secondary ionisation, either by proton transfer from the ionised gas reagent

producing a [M+1]+ ion or by electrophilic addition of the reagent gas to the sample molecule (peak

weight depends on reagent gas used). This “softer” technique generally has a greater sensitivity than

EI (towards the generation of molecular ion peaks), but less information is obtained regarding the

molecular structure by virtue of less fragmentation of the ions. This means that CIMS is not a very

useful technique in structure elucidation or peak matching: it is mainly used for the detection of

molecular weights.21

H23A : H23B H21A H21B H22A : H22B

A. B.

C.

H23AB H21AB H22AB


A representative test batch of the synthesised compounds was submitted for mass spectral (MS)

analysis. Both EI and CI were employed in the analysis of the pyrrolidinone compounds and the

pyrrolidinone HCl-salts in an initial analysis. Low resolution CIMS of the pyrrolidinone compounds

11.3 and 11.4 gave a base peak at 126 without further fragmentation, corresponding to the [M+1]+

peak of the liberated amine starting material (1H-Imidazole-1-propanamine). In contrast, the low

resolution EIMS spectra of 11.1 and 11.5 showed excessive fragmentation with no formation of a

molecular ion. The HCl-salts were analysed via low resolution CIMS only, and did not ionise, so no

useable spectra could be obtained for any of the compounds in the test batch. As the correct

distribution and fragmentation patterns could not be obtained using EIMS or the “softer” CIMS,

alternative methods of analysis were investigated.

Time of flight electron spray ionisation mass spectral analysis (TOF-ESI MS) provided an

alternative technique in the characterisation of the pyrrolidinone compounds and the corresponding

HCl-salt derivatives. The compounds in powder form were submitted for analysis via TOF-ESI MS.

During a standard application of ESI, each of the samples to be tested was dissolved in a polar,

volatile solvent (1:1 mixture of 0.1% formic acid in water and acetonitrile) and 1 µL of each

solution was pumped through a narrow capillary or needle at a high flow rate. A voltage of 3.5 kV

was applied to the tip of the capillary, generating the strong electric field necessary for the

detachment of charged droplets from the tip of the eluent column in the capillary. Under these high

voltage conditions, the detached droplets are so small that they are hardly affected by the Tyndall

effect (droplet size approaches the wavelengths of visible light; 400-750 nm).22 As the detached

droplets are carried along their horizontal trajectory towards the low voltage endplate of the

spectrometer, a flow of inert gas (N2) at elevated temperature (350 °C) causes the evaporation of

solvent from the charged droplet, thereby reducing the size and increasing the charge density to a

point where the Rayleigh limit is reached (droplet size smaller than the wavelength of visible light;

< 400 nm).22

At this point, the Coulombic repulsion forces within the droplet start to exceed the

surface tension and the droplets subdivide. This iterative process finally results in a state where each

of the minuscule droplets contains only one molecule of the sample. Once all of the solvent has

evaporated, a net charge remains on the sample molecule, enabling the detection of the molecular

ion. The ESI interface and the mechanism of droplet (and molecular ion) formation are

schematically represented in Figures 3.6a and b.


Figure 3.6 a) Schematic of an ESI interface and; b) Schematic of the mechanism of ion

formation.23

High resolution ESI+ spectra could be obtained for the majority of the compounds, clearly showing

the isotopic distribution pattern specific to, and expected for, each sample. For the most part, this

pattern consisted of three peaks representing molecular ions formed of different isotopic forms of

the test compound due to the incorporation of one of the naturally occurring isotopes of carbon (12

C

– 98.9% natural abundance; 13

C – 1.1% natural abundance) and nitrogen (14

N – 99.7% natural

abundance; 15

N – 0.3% natural abundance), effectively giving a distribution pattern of [M+H]+,

[M+H+1]+ and [M+H+2]+ with regards to the calculated molecular weight of the test compound

(Figure 3.7a). Notable exceptions included compounds containing the chloro-benzaldehyde moiety.

Instead, these compounds showed an effective distribution pattern of [M+H]+, [M+H+2]

+ and

[M+H+3]+. The distribution pattern of the chloride-containing molecular ions formed during the

analysis reflect the additional incorporation of two stable, naturally occurring isotopes of chloride,

35Cl (75.8% natural abundance) and 37Cl (24.2% natural abundance) (Figure 3.7b). This

characteristic isotope distribution pattern confirmed the presence of chloride in the analysed sample,

and can therefore be regarded as further proof that the correct product formed.


Figure 3.7 a) Typical HR-ESI+ spectrum obtained for the pyrrolidinone compounds; b) HR-ESI+

spectrum with characteristic isotopic distribution pattern obtained for chloride-containing

pyrrolidinone compounds.

3.3.4 Infrared spectroscopy (IR)

Before the advent of more modern characterisation tools such as NMR and MS, infrared

spectroscopy (IR) was used with great success in the identification of unknown compounds, with a

particular emphasis on functional group analysis. In fact, IR is still regarded is an invaluable tool in

structure determination, especially when used in combination with NMR and MS, as the data

obtained from these different characterisation methods are perfectly complementary. Whereas NMR

and MS provide ways of identifying specific functional groups through a consideration of the

interactions and environments of the atoms involved, IR allows the identification of these functional

groups by detecting the stretching and bending of bonds rather than any specific property of the

atoms themselves. The appearance of characteristic bands indicate the presence of certain functional

groups and give useful structural information through simple inspection and reference to

generalised charts of characteristic group frequencies.

As other analytical methods (NMR, MS and X-ray crystallography) were employed to determine

and confirm the structure of the compounds synthesised, IR was mainly used to confirm the

presence of the lactam carbonyl (CON), formed upon ring closure and present in all of the

synthesised compounds (signals typically manifest at 1650–1700 cm-1

). The keto-enol tautomer

system formed between the side chain carbonyl (CO) and the enol-alcohol (OH) on the

pyrrolidinone-ring (signals typically manifest at 1660–1670 cm-1 and 3200–3500 cm-1, respectively)

were also diagnostic.24

It has been proposed25

that increasing aromatisation and/or extending

A. B.

Compound 13.6 Compound 13.2


conjugated double-bond systems effectively stabilises the overall molecular structure, thereby

reducing the absorption of infrared radiation energy and effectively shifting the absorption bands to

lower wavenumbers. This effect can be observed in the synthesised pyrrolidinone compounds,

where the conjugated character of the system is extended through the formation of a keto-enol

system (viz. Figure 3.8), resulting in a downward shift in the positions of the side chain CO and

enol-OH bands (for the side chain CO: ~1600 cm-1 instead of 1660–1670 cm-1, and for the enol-OH:

~3120–3140 cm-1

instead of 3200–3500 cm-1

). In some instances the enol-OH band was obscured

by the formation of a wide trough formed between 3000–3500 cm-1

that effectively concealed the

bands usually present in this area (enol-OH, aliphatic CH, aromatic CH, NH, NH2). The

transmission band of the lactam-CO was observed in the range of 1675–1710 cm-1

, well correlated

with literature values.24

Figure 3.8 Illustrating the conjugated double bond character of the pyrrolidinone compounds.

3.3.5 X-ray crystallography

The most efficient and certain way to establish the structure of a specific compound is to crystallise

it and perform X-ray crystallography on the single crystals obtained. In this way, a representation of

the chemical structure can be obtained, including exact bonding distances and bond-angles. The

structural data for all crystals reported in this manuscript are included in Chapter 5 (complete data

tables in Appendix A).

3.3.5.1 Single X-ray crystal analysis of the synthesised pyrrolidinone compounds

Several of the synthesised products formed crystals suitable for single-crystal analysis and were

characterised on a Bruker APEXII diffractometer using a molybdenum Kα radiation source,

confirming the formation of the expected product in all cases.

Pyrrolidinone 11.1:

Product 11.1 (Figure 3.9) crystallised with two disordered solvent molecules (DMSO) in the

asymmetric unit. The compound crystallised in a monoclinic crystal system, with P21/n space group

symmetry. Most bond lengths and angles were within expected ranges, confirming the successful


synthesis of the proposed structure. Notably, the C-OH bond length of the ring-bound enol-alcohol

at 1.263(4) Å was shorter than would be expected for a single bond and is comparable to that of the

lactam- and side chain carbonyls at 1.224(4) Å and 1.244(4) Å, respectively. This, together with the

contribution of the alkene within the pyrrolidinone ring, extends the aromatic character of the

benzo-furanyl side chain further into the molecule and results in nearly one half of the molecule

presenting a planar and relatively rigid conformation. The delocalisation of electrons throughout the

planar part of the molecule favours the formation of hydrophobic interactions and may assist with

metal chelation within the active site of the HIV-1 IN enzyme. Another noteworthy feature was the

protonation of the imidazole imine-N. The fully aromatised nature of the imidazole ring (bond

lengths between 1.326–1.388 Å) and the subsequent delocalisation of electron density may activate

the ring, resulting in a partial deprotonation of the enol OH by the terminal N-atom of the

imidazole-ring (forming a zwitterion), thereby effectively generating the substantial hydrogen-

bonding observed between the proton and the two oxygen atoms of the pyrrolidinone ring

(illustrated in Figure 3.10). It is important to note that this protonation does not impact the

aromatisation or planar structure of the imidazole ring.

N

N

O

HO N

O

O

Figure 3.9 ORTEP diagram and schematic representation of a molecule of 11.1 in the asymmetric

unit.

Figure 3.10 Crystal lattice of 11.1 showing the contacts formed within the crystal.


Pyrrolidinone 11.2:

Product 11.2 (Figure 3.11) also crystallised with two solvent molecules in the asymmetric unit, but

in this case the solvent molecules were well ordered. The compound crystallised in a monoclinic

crystal system with space group symmetry P21/c, similar to compound 11.1. Most bonds and angles

were within expected ranges, confirming the successful synthesis of the proposed structure. In a

similar manner as observed for crystal 11.1, the aromaticity and planar character of the benzo-

furanyl side chain was extended into the pyrrolidinone-ring through inclusion of the enol-alcohol

and contraction of the C-OH bond to a length of 1.277(2) Å. Consequently, this bond is much

shorter than would be expected for a single bond and compare well to the lactam- and side chain

carbonyl bonds at 1.237(2) Å and 1.249(2) Å, respectively. The protonation of the imidazole ring

seen for crystal 11.1 is not observed in this instance. The crystal lattice is stabilised by a series of

intermolecular interactions involving both the pyrrolidinone and the two co-crystallised DMSO

molecules. For details on each interaction and a representation of the crystal lattice, refer to Table

3.1 and Figure 3.12, respectively.

Cl

N

N

O

HO N

O

O

Figure 3.11 ORTEP diagram and schematic representation of one molecule of 11.2 in the

asymmetric unit.

Table 3.1 Hydrogen bonds, symmetry operators and π-ring interactions observed in crystals of

pyrrolidinone 11.2.

Hydrogen bonds (Å� and ˚)

D-H…A D(D-

H)

D(H…A) D(D…A) <(DHA) Symmetry operators

i. O3-H3b…N3 0.84 2.44 2.872(2) 148 1-x, 1-y, 2-z

ii. C2-H2…O5 0.95 2.48 3.323(3) 148 1-x, -½+y, 3/2-z

iii. C3-H3…O6 0.95 2.51 2.353(3) 148 1-x, -½+y, 3/2-z

iv. C20-H20a…O6 0.99 2.44 3.232(3) 137 -x, 1-y, 1-z

v. C24-H24…O2 0.95 2.34 3.220(2) 154 1-x, ½+y, 3/2-z

vi. C24-H24…O2 0.98 2.56 3.514(3) 166 1-x, 1-y, 2-z

vii. C24-H24…O2 0.98 2.58 3.321(3) 132 x, y, -1+z


A nalysis of C-H…Cg (π-ring) interactions (H…Cg < 3.0 Å – Gamma < 30.0˚)

X-H…Cg H…Cg C-H…Cg C…Cg Symmetry operators

i. C19-H19…Cg2 2.69 99 2.989(2) x, y, z

ii. C22-H22a…Cg4 2.88 117 3.447(2) x, ½-y, ½+z

iii. C26-H26b…Cg5 2.81 138 3.603(3) x, ½-y, ½+z


Pyrrolidinone 11.5:

The crystal structure representation of product 11.5 (Figure 3.13) again shows the incorporation of

two solvent molecules. This structure did not resolve well with the molybdenum Kα radiation

source (Rint, wR2 and R values too high) and future work will include a recollection of data using a

copper radiation source. Even though a thorough analysis of the structure is not feasible at this

point, the bond distances and angles could be used to confirm the proposed structure. As with the

previous structures, the C-OH bond of the enol-alcohol was shortened to 1.265(5) Å to allow

incorporation into the aromatic planar framework of the benzo-furanyl side chain. Partial

protonation of the imidazole-imine enabled the formation of additional hydrogen bonds, thereby

further stabilising the crystal structure. The monoclinic crystal lattice with space group P21/c, is

stabilised by a series of intermolecular interactions involving both the pyrrolidinone and the two co-

crystallised DMSO molecules. For a representation of the crystal lattice, refer to Figure 3.14.


O

N

N

O

HO N

O

O

Figure 3.13 ORTEP diagram and schematic representation of one molecule of 11.5 in the

asymmetric unit.


Pyrrolidinone 13.3:

Two different single crystal systems suitable for analysis formed for compound 13.3: the first

crystal isolated was the desired product 13.3a, present as the major product; the second product

[1,5-di(benzofuran-2-yl)-3-(4-fluorophenyl)pentane-1,5-dione, 13.3b] was only observed during

crystallisation (no trace of this compound could be found in NMR or MS analysis of the purified

product powder) and formed from the condensation of two molecules of the pyruvate ester starting

material (8) and one molecule of the 4-fluorobenzaldehyde starting material (10.3).

Compound 13.3a crystallised in a monoclinic crystal system with space group P21/c, similar to the

pyrrolidinone crystals discussed previously. However, in contrast to the crystal structures of

products 11.1, 11.2 and 11.5, the crystal structure of product 13.3a (Figure 3.15) shows two

independent zwitter-ionic product molecules in the asymmetric unit with no incorporation of

solvent molecules. Interestingly, the crystal does not show the enol-alcohol present in the previous

crystal structures. Instead, both oxygen atoms on the pyrrolidinone ring are in the keto form,

facilitating the formation of additional hydrogen bonds and stabilising the crystal lattice. The


extended conjugation and planar structure observed for the benzo-furanyl side chain and half of the

pyrrolidinone-ring is conserved in this structure. The zwitter-ionic character is complete by

protonation of the terminal tertiary amine. The formation of zwitter-ions is a common occurrence in

amino acids, where it aids in crystallisation by stabilising the overall crystal packing and unit cell

structure.26 A series of intra- and intermolecular interactions involving both of the molecules in the

asymmetric unit cell lead to lattice stability in the crystal. For details on each interaction and a

representation of the crystal lattice, refer to Table 3.2 and Figure 3.16, respectively.

F

N

O

HO N

O

O

Figure 3.15 ORTEP diagram and schematic representation of two molecules of 13.3a in the

asymmetric unit.

Figure 3.16 Crystal lattice of 13.3a showing the contacts formed within the crystal.

Table 3.2 Hydrogen bonds, symmetry operators and π-ring interactions observed in crystals of

pyrrolidinone 13.3a.

Hydrogen bonds (Å� and ˚)

D-H…A D(D-H) D(H…A) D(D…A) <(DHA) Symmetry operators

i. O2-H2a…N4 0.84 1.87 2.8541(19) 154 x, y, z


ii. O6-H6a…N2 0.84 1.85 2.646(2) 158 1+x, y, z

iii. C8-H8b…O6 0.99 2.54 3.414(2) 148 1+x, y, z

iv. C11-H11a…O5 0.98 2.47 3.082(3) 121 1+x, y, z

v. C21-H21…F1 0.95 2.44 3.341(2) 159 x, y, z

vi. C26-H26…O3 0.95 2.53 3.374(2) 149 1-x, 1-y, 2-z

vii. C35-H35a…O4 0.98 2.38 3.196(3) 137 1-x, ½+y, 3/2-z

A nalysis of Cg…Cg (π-ring) interactions

Cg…Cg Cg…Cg Symmetry operators

i. Cg2…Cg3 3.8331(11) x, y, z

ii. Cg3…Cg7 3.9939(11) 1-x, 1-y, 2-z

iii. Cg7…Cg8 3.8301(11) x, y, z

A nalysis of C-H…Cg (π-ring) interactions (H…Cg < 3.0 Å – Gamma < 30.0˚)

X-H…Cg H…Cg C-H…Cg C…Cg Symmetry operators

i. C2-H2…Cg2 2.75 98 2.028(2) x, y, z

ii. C10-H10a…Cg6 2.65 125 3.311(2) 1-x, -½+y, 3/2-z

iii. C29-H2b…Cg2 2.89 125 3.517(2) 1-x, -½+y, 3/2-z

iv. C30-H30…Cg7 2.75 98 3.027(2) x, y, z

v. C31-H31…Cg32.85 142 3.6954(19) 1-x, 1-y, 2-z

In contrast, compound 13.3b formed monoclinic crystals with a C2/c space group and eight

molecules in the asymmetric unit (Figure 3.17a). The crystal lattice was stabilised through the

formation of numerous short intermolecular interactions, resulting in layers reminiscent of

corrugated sheets (Figure 3.17b).

Figure 3.17 ORTEP diagram and schematic representation of compound 13.3b showing: a) one of

A. B.


the eight molecules in the asymmetric unit; and b) the crystal lattice and the contacts formed within

the crystal.

3.3.5.2 Single X-ray crystal analysis of the synthesised pyrrolidinone HCl-salts

Pyrrolidinone 12.2-HCl:

Pyrrolidinone hydrochloride salt 12.2-HCl (Figure 3.18) crystallised in an orthorhombic crystal

system with a Pna21 space group and four molecules in the asymmetric unit (Z=4), including a

protonated product molecule and a chloride counter ion. Importantly, in contrast to the free

pyrrolidinone compounds, the enol-alcohol regained some measure of single-bond character and at

1.328(2) Å, is significantly longer than the lactam- and side chain carbonyls at 1.225(3) Å and

1.226(2) Å, respectively. The chloride counter ion effectively formed a hydrogen-bonded bridge

between the protonated amine (3.074 Å) and the enol functionalities (2.941 Å), and contributed

greatly to the overall stability of the crystal lattice (Figure 3.19). Additionally, the protonated amine

formed a second hydrogen bond with the lactam-carbonyl (3.052 Ǻ), providing additional

stabilisation to the crystal lattice. Furthermore, the crystal lattice was stabilised through a network

of intra- and intermolecular interactions involving both of the molecules in the asymmetric unit cell.

For a representation of the crystal lattice, refer to Figure 3.19.

HN

O

HO N

O

O

ClO

Cl

Figure 3.18 ORTEP diagram and schematic representation of one molecule of 12.2-HCl and one

chloride counter ion in the asymmetric unit.


Figure 3.19 Crystal lattice of 12.2-HCl showing: a) the hydrogen bonds; and b) other inter- and

intramolecular contacts formed within the crystal lattice.


Pyrrolidinone hydrochloride salt 12.3-HCl (Figure 3.20) crystallised in a monoclinic crystal system

with a Pn space group. The unit cell formed with two protonated product molecules in the

asymmetric unit. The charges were balanced by two chloride counter ions. As observed for

compound 12.2-HCl, the enol-alcohol regained some single-bond character (1.341(4) Å), when

compared to the lactam- and side chain carbonyls (1.236(5) Å and 1.239(5) Å, respectively). Two

hydrogen bonds formed within the crystal lattice: the chloride counter ion formed a hydrogen-bond

to the protonated amine (3.018 Å); while the morpholine-ring oxygen formed a hydrogen bond to

the enol functionality (2.688 Å) (Figure 3.21a). Furthermore, the crystal lattice was stabilised

through a network of intra- and intermolecular interactions involving both of the molecules in the

asymmetric unit cell (Figure 3.21b).

HN

O

HO N

O

O

ClO

F

Figure 3.20 ORTEP diagram and schematic representation of the asymmetric unit formed for

A. B.


compound 12.3-HCl.

Figure 3.21 Crystal lattice of 12.3-HCl showing: a) the hydrogen bonds; and b) other inter- and



Pyrrolidinone hydrochloride salt 13.2-HCl (Figure 3.22) crystallised in a triclinic crystal system

with a P-1 space group. The unit cell formed with two protonated product molecules in the

asymmetric unit, and the charges were balanced by two chloride counter ions. As observed in

crystals of the other pyrrolidinone salts, the enol-alcohol regained a measure of single-bond

character (1.3107(17) Å), when compared to the lactam- and side chain carbonyls (1.2250(18) Å

and 1.2653(17) Å, respectively). Three hydrogen bonds formed within the crystal lattice (Figure

3.23a): the expected hydrogen-bond formed between the chloride counter ion and the protonated

amine (3.037 Å); while a stabilising hydrogen-bond network formed between the side chain

carbonyl groups and the enol-oxygen atoms of the two product molecules in the unit. Specifically,

the side chain carbonyl formed an intramolecular hydrogen bond to the enol functionality (2.617 Å),

while an intermolecular hydrogen bond formed between the enol-alcohols of the two product

molecules (2.896 Å). The formation of the intermolecular hydrogen bond may indicate a partial

protonation of the enol-alcohol, in line with the formation of zwitterionic species observed in other

molecule crystals. An extensive network of additional intra- and intermolecular interactions

involving both of the molecules in the asymmetric unit cell provided further stabilisation to the

crystal lattice (Figure 3.23b-d).

A. B.


HN

O

HO N

O

O

Cl

Cl

Figure 3.22 ORTEP diagram and schematic representation of the asymmetric unit formed for

compound 13.2-HCl.

Figure 3.23 Crystal lattice of 13.2-HCl showing: a) the hydrogen bonds; b) π--- π stacking of the

aromatic features in the molecule; c) interactions with chloride atoms; and d) other inter- and


A. B.

C. D.



Pyrrolidinone hydrochloride salt 13.5-HCl (Figure 3.24) crystallised in a triclinic crystal system

with a P-1 space group. The unit cell formed with one protonated product molecule and one

chloride counter ion, with the incorporation of one water molecule. Importantly, in contrast to the

free pyrrolidinone compounds, the enol-alcohol regained some measure of single-bond character

and at 1.3237(15) Å, is significantly longer than the lactam- and side chain carbonyls at 1.2332(14)

Å and 1.2297(16) Å, respectively. The chloride counter ion and the incorporated H2O molecule

both formed numerous intermolecular hydrogen bonds with one another and with the protonated

pyrrolidinone molecule, and contributed greatly to the overall stability of the crystal lattice.

Furthermore, the crystal lattice is stabilised through a series of intramolecular CH…Cl, NH…Cl,

OH…Cl, CO…H, OH…O and C-H…π interactions involving all three of the molecules in the

asymmetric unit cell. For a representation of the crystal lattice, refer to Figure 3.25.

NH

O

HO N

O

O

O

Cl

Figure 3.24 ORTEP diagram and schematic representation of two molecules of 13.5-HCl in the

asymmetric unit.

Figure 3.25 Crystal lattice of 13.5-HCl showing the contacts formed within the crystal.


3.3.5.3 Additional crystal structures

2,4-Dimethoxybenzaldehyde starting material:

One of the starting materials used in the synthesis of compounds 14.7, 15.7 and 16.7 (2,4-

dimethoxybenzaldehyde) crystallised out of the 1,4-dioxane solvent to form monoclinic crystals

with a P21/c space group (Figure 3.26a). The unit cell formed with four molecules in the

asymmetric unit. The crystal lattice was stabilised through the formation of numerous short

intermolecular interactions, resulting in layers reminiscent of corrugated sheets (Figure 3.26b).

Figure 3.26 ORTEP diagram and schematic representation of the 2,4-dimethoxybenzaldehyde

starting material showing: a) the four molecules in the asymmetric unit; and b) the crystal lattice

and the contacts formed within the crystal.

A co-crystallised set of salts – The hemi-HCl and hemi-oxalic acid forms of morpholine:

During the synthesis of the water-soluble hydrochloride salts of the pyrrolidinone carbaldehyde

compounds, crystals grown from 12.6-HCl showed an interesting hybrid structure (Figure 3.27),

possibly formed as a by-product during the synthetic reaction. This structure was formed between

the protonated amine starting material (propylamino-morpholine), residual dioxane solvent

converted to oxalic acid and chloride counterions generated from the dilute HCl solution added to

the system. Interestingly, although all of the crystals formed in this sample holder showed the same

hybrid structure, NMR and MS characterisation of the same sample in powder form before

crystallisation indicated the target pyrrolidinone salt (12.6-HCl) as the major product, with no sign

of either the hybrid structure or significant amounts of the amine starting material being present in

the sample. Therefore, the abundance of hybrid crystals formed in the sample would indicate that

the hybrid structure is the most thermodynamically stable; in solution these crystals may have

formed at the expense of the energetically less favourable pyrrolidinone salt.

A. B.


Figure 3.27 ORTEP view of the co-crystallised set of salts formed from the protonated amine

starting material, oxalic acid (from the dioxane solvent) and the chloride counter ion (from the

added HCl).

The crystal formed in a monoclinic crystal system with a C2/c space group and eight molecules

within the unit cell. As can be seen in the crystal packing diagram (Figure 3.28), the structure is

ordered into sheets with distinct layers of protonated di-amine molecules, bridged by a layer of

oxalic acid molecules on one side and by a layer of chloride counter ions on the other side.

Neighbouring di-amine layers have opposite directionality, with individual molecules in each layer

positioned in a head-to-tail manner. This gives rise to a rippled planar surface reminiscent of a sheet

of corrugated metal. This effect is further enhanced when considering the network of interactions

formed in the crystal lattice (Figure 3.29).

Figure 3.28 Packing diagram of the hybrid salt structure.

Each respective layer that makes up the hybrid crystal lattice is firmly positioned by an extensive

network of inter-molecular and inter-layer interactions. The di-amine layers are formed through

direct H-bonding of the electronegative oxygen atom in the morpholine ring “head” to one of the


electropositive protons of the protonated amine “tail” of each respective di-amine molecule in the

layer. Although the morpholine “heads” of opposing di-amine molecules in the respective layers are

placed in close proximity within the crystal lattice, no direct interactions between these layers can

be observed. Instead, the oxalic acid and chloride counter ion layers act as bridges between the di-

amine layers, with the chloride counter ion layer anchoring the “head” region of individual di-amine

molecules in one layer to the “tail” region of the equivalent molecules in the opposite layer.

Additionally, the chloride counter ion layers provide a further stabilising effect to the di-amine

layers as these ions connect the “heads” of individual di-amine molecules in the layer to the

protonated “tails” of the preceding di-amine molecule in the same layer (in effect showing head-to-

tail interactions between layers as well as enabling indirect inter-molecular head-to-tail interactions

within each layer).

Figure 3.29 Intermolecular interactions in the hybrid salt structure.

The indirect secondary, inter-molecular stabilising interactions discussed above are not observed

within the oxalic acid layers. While these layers form bridges between subsequent di-amine layers

and are necessary in the construction of the crystal lattice, the oxalic acid molecules do not seem to

contribute greatly to the stability of each respective di-amine layer. In fact, the oxalic acid

molecules anchor themselves to the di-amine layers through intra-molecular interactions to the

hydrophobic propyl chain (“body”) of each di-amine molecule exclusively. For representations of

the crystal lattice, refer to Figures 3.30 and 3.31, respectively.


Figure 3.30 Crystal lattice of the hybrid salt structure showing intermolecular CH…Cl, NH…Cl

and NH…O interactions formed within the crystal.

Figure 3.31 Crystal lattice of the hybrid salt structure showing intermolecular CH…O, NH…O and

OH…O interactions formed within the crystal.

3.4 Conclusion

A family of closely related compounds, designed through derivatisation of the in silico hit

compound 1 identified through database screening (discussed in Chapter 2), was synthesised. The

retrosynthetic analysis identified three possible classes of starting materials: aldehydes, primary

amines and pyruvate ester moieties. The aldehyde and amine starting materials were readily

available through commercial sources, but the pyruvate ester moiety was synthesised. High yields

of this starting material could be obtained through use of the Claisen reaction in anhydrous media.

The synthesis of the pyruvate ester moiety was performed based on published literature reports,

with few modifications.

The synthesis of the pyrrolidinone target compounds proceeded well without any major

complications. In most instances, the product precipitated out of solution and could be collected by


centrifugation, yielding moderate returns of the pyrrolidinone product in powder form. Some of

these products could be recrystallised from ethanol. Due to the insoluble nature of some of the

synthesised compounds in nearly all generally available laboratory solvents, characterisation proved

a challenge. Several of the products formed single crystals suitable for X-ray analysis and

confirmed the correct formation of the proposed products in all cases, but limited solution-based

NMR or MS data could be obtained in these instances. In an effort to overcome this obstacle, and in

line with literature, the products with low solubility profiles were protonated and/or converted to

their corresponding hydrochloride salts (isolated in pure powder form). This conversion rendered

the compounds water-soluble and enabled their characterisation via 1H and

13C NMR, HR TOF-ESI

MS, and IR analysis. None of the compounds synthesised were analysed via elemental analysis as

our laboratory did not have access to a facility offering this service.

In a serendipitous discovery, a by-product formed during the synthesis of compound 12.6-HCl

provided crystals of an interesting hybrid structure formed by reaction of the diamine with two

different acids, namely HCl and oxalic acid. The hybrid structure shows layers of the protonated di-

amine starting material generated during the reaction, stabilised by direct head-to-tail inter-

molecular interactions and bridged by layers of chloride counter-ions or solvent-derived oxalic acid

molecules, respectively. These bridge layers anchor each new di-amine layer to the preceding layer

through inter- or intra-molecular interactions (chloride or oxalic acid layers, respectively).

Chapter 3: Chemical Synthesis

3.5 References

1. a) Gein, V. L., Shumilovskikh, E. V., Andreichikov, Y. S., et al., Pharm. Chem. J., 1991,

25, 12, 884-887; b) Gein, V. L., Popov, A. V., Kolla, V. E., et al., Pharm. Chem. J., 1993,

27, 5, 343-346; c) Gein, V. L., Voronina, E. V., Ryumina, T. E., et al., Pharm. Chem. J.,

1996, 30, 2, 95-96; d) Gein, V. L., Yushkov, V. V., Splina, T. A., Pharm. Chem. J., 2008,

42, 5, 255-257; e) Gein, V. L., Kasimova, N. N., Moiseev, A. L., et al., Pharm. Chem. J.,

2007, 41, 9, 476-479; f) Gein, V. L., Yushkov, V. V., Kasimova, N. N., et al., Pharm.

Chem. J., 2007, 41, 5, 256-263.

2. Corey, E. J., Angew. Chem. Int. Ed. Engl., 1991, 30, 5, 455-465.

3. Bauer, J., Herges, R., Fontain, E., Ugi, I., Chimia, 1985, 39, 43–58.

4. Bauer, J., Tetrahedron Comp. Methodol., 1989, 2, 269-280.

5. Barberis, F., Barone, R., Arbelot, M., et al., J. Chem. Inf. Comput. Sci., 1995, 35, 467-471.

6. Schwab, C. H., Bienfait, B., Gasteiger, J., Chem. Central J., 2008, 2 (Suppl 1), P46.

7. Blurock, E. S., J. Chem. Inf. Model., 1990, 30, 505-510.

8. Law, J., Zsoldos, Z., Simon, A., et al., J. Chem. Inf. Model., 2009, 49, 593-602.

9. Brückner, R., Wender, P. A., Organic Mechanisms: Reactions, Stereochemistry and

Synthesis, 3rd Edition, 2010, Springer-Verlag, Heidelberg, Germany, pp. 544-545.

10. Reusch, W., Virtual Textbook of Organic Chemistry, 1999 (most recent revision 6/2010);

http://www2.chemistry.msu.edu/faculty/reusch/VirtTxtJml/crbacid3.htm#react7

11. Moszner, N., Zeuner, F., Salz, U., et al., Polymer Bull., 1994, 33, 43-49; Al-Niaimi, N. S.,

Al-Saadi, B. M., J. Inorg. Nucl. Chem., 1973, 35, 12, 4207-4216.

12. McMurry, J., Organic Chemistry Enhanced Edition, 7th Edition, Volume 2, 2010,

Brooks/Cole, Cengage Learning Inc., Canada, pp. 888-890.

13. Clayden, J., Greeves, N., Warren, S., Wothers, P., Organic Chemistry, Oxford University

Press, 2001, pp.728.

14. a) Gein, V. L., Yushkov, V. V., Kasimova, N. N., et al., Pharm. Chem. J., 2007, 41, 7, 367-

371; b) Gein, V. L., Kasimova, N. N., Potemkin, K. D., Russ. J. Gen. Chem., 2002, 72, 7,

1150-1151; c) Gein, V. L., Gein, L. F., Porseva, N. Yu., et al., Pharm. Chem. J., 1997, 31,

5, 251-254; d) Gein, V. L., Popov, A. V., Kolla, V. É., et al., Khim.-Farm. Zh., 1993, 27, 5,

42-45; e) Gein, V. L., Shumilovskikh, E. V., Andreichikov, Yu. S., Khim.-Farm. Zh., 1991,

25, 12, 37-40.

Chapter 3: Chemical Synthesis

15. Bettelheim, F. A., Brown, W. H., Campbell, M. K., Farrell, S. O., Introduction to General,

Organic and Biochemistry, 9th Edition, 2010, Brooks/Cole, Cengage Learning Inc.,

Canada, pp. 229-230.

16. Le Chatelier, H., Recherches Experimentales Et Theoriques Sur Les Equilibres Chimique,

1888, Kessinger Publishing LLC., Whitefish, USA.

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19. Shaler, T. A., J. Chem. Educ., 1991, 68, 10, 853-854.

20. Figure adapted from: Keeler, J., Chapter 2, “NMR and energy levels”, © James Keeler,

2002 & 2004. Downloaded from the internet on 7 November 2011; http://www-

keeler.ch.cam.ac.uk/lectures/Irvine/chapter2.pdf.

21. Silverstein, R. M., Webster, F. X., Kiemle, D. J., Spectrometric Identification of Organic

Compounds, 7th Edition, 2005, John Wiley & Sons, Inc., USA, pp.127 – 176.

22. Yamashita, M., Fenn, J. B., J. Phys. Chem., 1984, 88, 4451-4459.

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Research Council, on 7 November 2011:

http://www.bris.ac.uk/nerclsmsf/techniques/hplcms.html.

24. Pretsch, E., Clerc, T., Seibl, J., Simon, W., Tables of Spectral Data for Structure

Determination of Organic Compounds, 2nd Edition, 1989, Translated from the German by

Biemann, K., Springer-Verlag Inc., Heidelberg, Germany, pp. B35 – B60, B240, I85, I132,

I135-I152.

25. Jianxin, X., Shanqing, C., Chinese Sci. Bul., 1998, 43, 12, 1048-1050.

26. Kwon, O. Y., Kim, S. Y., No, K. T., Kang, Y. K., Jhon, M. S., and Scheraga, H. A., J.

Phys. Chem., 1996, 100, 17670-17677.

Chapter 4: Biological Evaluation Page 117

CHAPTER 4: BIOLOGICAL EVALUATION

4.1 Introduction

Compounds similar and related to the pyrrolidinone class of compounds synthesised in this work

have been tested against a range of bacterial and fungal infections in mice and rodents,1 showing

minimal adverse effects towards the in vivo systems tested. However, to the author’s knowledge,

this class of compounds has not been tested for any potential antiretroviral activity, and in

particular, HIV-1 IN activity. The in silico identification of the compound class as potential HIV-1

IN inhibitors, the design of suitable compound analogues, and the synthesis, characterisation and

structures of all compounds tested are discussed in other chapters of this manuscript. This chapter

deals exclusively with the biological aspects of the project and as such only structures relevant to

the compounds discussed in the text are reported. It is important to keep in mind that, structurally,

the test compounds have only a few points of variance: 1) the character of the R2-group varies in

terms of the amine functionality (hetero-aromatic vs. hetero-aliphatic ring vs. bulky aliphatic non-

ring vs. free amine); 2) the character of the R3-group varies as a consequence of the character and

position of substitutions on the aromatic ring; and 3) the length of the aliphatic side-chain linking R2

to the pyrrolidinone ring. Any one, or a combination of these three structural factors, should explain

any difference observed in biological activity or effect.

A range of experimental and predictive computational assays were employed in an attempt to gain

an insight into the inhibitory activity, as well as generating an initial ADMET profile for each of the

pyrrolidinone test compounds. The in vitro biological evaluation described in this work details only

the early phases of first-generation compound development. In the interest of creating an

extrapolated understanding of the behaviour that the pyrrolidinone compounds may display under

later-stage in vitro and in vivo conditions, a range of predictive in silico models were included in the

initial biological evaluation. At this early stage in the investigation, evaluation of the synthesised

compounds through the chosen range of in silico models will serve to red-flag any areas that may

pose hurdles to the future development of each compound synthesised. It is important to note,

however, that any model, regardless of type, is limited by the fact that it describes and represents a

closed system,2,3

based on a limited amount of pre-determined data. As such, it cannot reliably

predict or describe any point that falls outside of its prediction space. Additionally, models are not

universally applicable, and therefore great care should be exercised to ensure the relevance of any

particular model to both the test dataset and the application. Considering the aforementioned


limitations of in silico models, the biological profiles predicted for hit candidates of second- and

later-generation compound development phases will be validated in suitable experimental assays.

4.2 Biological evaluation

All theoretical predictions of molecular properties, ligand-receptor interactions and potential

toxicity effects were performed using the Discovery StudioTM

software suite (Accelrys, version 2.5)

and the Marvin Beans software suite (ChemAxon, version 5.2). Analysis and manipulation of the

raw results data were performed using Microsoft Excel 2010 and Origin6.1 (OriginLab Corporation,

version 6.1). Statistical analyses were performed using Microsoft Excel 2010. In this work,

significant and meaningful figures were treated in the following way:

1) For experimental in vitro results, the uncertainty was rounded to the closest 0.5 unit at the

level of the uncertainty if the standard deviation (SD) <0.5 at the uncertainty level; if

however the SD>0.5, the uncertainty was rounded to the closest whole number.

2) For theoretical numerical results (including modelling data), the values were rounded to the

closest 0.5 to reflect the inherent lack of statistical confidence in the generated numbers (as a

consequence of the inherent uncertainty in the theoretical models used).

4.2.1 Direct enzyme assays

For any compound to have clinical usefulness as a drug, it has to be sufficiently active against the

intended target receptor, at biologically relevant concentrations. In an attempt to place the research

and development focus on compounds with the best potential of eventually becoming a drug,

pharmaceutical companies and research groups have established an activity cut-off value.4,5

For

example, the Developmental Therapeutics Program (DTP) conducted by the National Cancer

Institute (NCI) / National Institutes of Health (NIH) in the US, routinely start the first phase of the

NCI-60 DTP Human Tumour Cell Line Screen with an activity evaluation at a single dose of 10 µM

of each compound (according to the process outlined on the organisation’s website,

http://dtp.nci.nih.gov/branches/btb/ivclsp.html; accessed on 17 January 2012). This value serves as

a benchmark during the activity screening of compounds, where compounds with an activity below

that of the benchmark will not undergo further evaluation. This value is not uniform throughout the

industry, but a benchmark compound concentration of 10 µM (highest concentration) is generally

considered as a good starting point for activity screening. Each of the compounds synthesised in this

project was tested against a soluble double-mutant (F185H/C280S) of recombinant HIV-1 subtype


B IN enzyme* in a cell-free assay to determine its inhibitory effect on strand-transfer activity (and

therefore its potential for further development) at a final, single-dose concentration of 10 µM and

the results are summarised in Table 4.1. The addition of pre-processed vDNA ensured that only

inhibitors of the strand-transfer reaction gave a positive response. It is important to note that the

assay does not exclude compounds with dual activity (for example, dual strand-transfer / 3’-end

processing inhibitors), but all compounds identified as inhibitors in this assay must posses some

degree of inhibitory activity against strand-transfer. Although there is some debate as to whether L-

chicoric acid (L-CA) can be classified as a 3’-end processing or strand transfer inhibitor,6,7,8

in the

present work this compound gave reproduceable inhibition of HIV-1 IN strand transfer and as such,

it was included as an assay control in the single-dose screen. Raltegravir (RAL; the only HIV-1 IN

inhibitor currently approved by the FDA), was included as a second control compound in all in vitro

screens. The test compounds showed variable potential as HIV-1 IN inhibitors across the series,

with eight compounds (including the L-CA reference) showing promising inhibition of recombinant

HIV-1 subtype B IN at the test concentration (>50% inhibition at 10 µM compound concentration;

results summarised in Table 4.1), warranting more in-depth activity screening. One additional

compound, 11.3, showed 49% inhibition at the test concentration and was included in further

studies, effectively bringing the number of compounds earmarked for five-dose activity screening

and IC50 determination, to ten.

The IC50 values determined for RAL, L-CA and the eight pyrrolidinone compounds are summarised

in Table 4.1. In the present study, RAL and L-CA gave reproducible IC50 values of 0.55 (±0.05) µM

and 2 (±1.5) µM, respectively. These results are higher than comparative IC50 values found in

literature reports,9,10,11

but were consistent with results routinely determined in our laboratory. For

the most part, a good correlation could be seen between the single- and five-dose results obtained

for all compounds, however, the inhibitory activity observed in the single-dose screen of compound

14.5 could not be reproduced in the dose-response screen, most likely due to compound degradation

during storage (product powder showed a change in colour from brown to black). Two of the

pyrrolidinone compounds, 11.6 and 15.2 exhibited promising activity in this first-generation

screening [IC50 values of 9 (±4) µM and 6 (±2) µM, and SI values of 11 and 15, respectively] and

are currently undergoing activity screening against four additional drug resistant HIV-1 subtype B

IN mutants (Q148H; Q148H/G140S; N155H and N155H/E92Q).† Initial results have shown

promise, with compound 15.2 retaining a measure of activity against the RAL-resistant N155-

mutants.

* The F185H/C280S double-mutant HIV-1 IN demonstrated activity equivalent to wild-type in ELISA-based assays (data not

shown). † Testing against resistant mutants performed by Mr. Q. M. Fish (Biomedical group, AMD, Mintek, South Africa).


Table 4.1 Toxicity and activity of the synthesised pyrrolidinone compounds.

Cytotoxicity (µM)‡ ST activity (µM)

§ SI value

**

Code CC50 in PM1; (±Std. Dev.) P-value††

% Inhibition @ 10µM; (±Std. Dev.) P-value††

IC50 in direct ELISA; (±Std. Dev.) CC50 / IC50

RAL ≥100 (±0.0) - 84 - - 0.55 (±0.05) ≥182

L-CA ≥100 (±0.0) 0.115 94 (±0.1) 0.120 2 (±1.5) ≥54

11.1 34 (±1.0) 0.000 21 (±3.0) 0.030

11.2 10.0 (±0.2) 0.000 23 (±6.5) 0.017

11.3 18 (±3.5) 0.000 49 (±9.0) 0.033 24 (±1.5) 1

11.4 30 (±5.5) 0.001 35 (±3.0) 0.039

11.5 16 (±1.0) 0.000 58 (±9.0) 0.063 18 (±2.0) 1

11.6 98 (±2.0) 0.071 68 (±4.5) 0.188 9 (±4.0) 11

12.1 97 (±1.0) 0.002 33 (±1.5) 0.044

12.2 63 (±10.5) 0.003 2 (±1.5) 0.025

12.3 99 (±2.0) 0.211 22 (±3.0) 0.029

12.4 85 (±3.0) 0.005 44 (±4.0) 0.046

12.5 98 (±2.0) 0.067 29 (±4.5) 0.026

12.6 55 (±2.5) 0.000 15 (±4.0) 0.022

13.1 59 (±5.5) 0.003 18 (±5.0) 0.021

13.2 98 (±3.0) 0.067 39 (±5.0) 0.032

13.3 15 (±3.0) 0.000 2 (±2.5) 0.020

13.4 98 (±3.0) 0.223 10 (±4.5) 0.013

13.5 ≥100 (±0.0) 0.211 17 (±9.0) 0.007

13.6 99 (±1.5) 0.152 26 (±1.5) 0.037

14.1 98 (±2.0) 0.068 27 (±8.0) 0.012

14.2 95 (±2.0) 0.020 42 (±9.5) 0.021

14.3 97 (±1.0) 0.002 29 (±3.0) 0.035

14.4 92 (±11.0) 0.243 35 (±4.5) 0.030

14.5 66 (±7.0) 0.007 58 (±16.0) 0.090 0 (±1.0)

14.6 100 (±1.0) 0.105 22 (±6.5) 0.014

15.1 97 (±2.5) 0.075 57 (±5.0) 0.076 29 (±4.5) 3

15.2 96 (±4.0) 0.085 67 (±10.0) 0.172 7 (±1.5) 15

15.3 66 (±8.50) 0.002 54 (±18.0) 0.043 13 (±1.5) 5

15.4 60 (±7.5) 0.040 56 (±12.5) 0.103 21 (±5.0) 3

15.5 55 (±1.0) 0.002 32 (±3.0) 0.033

15.6 31 (±1.0) 0.000 16 (±3.5) 0.022

15.7 41 (±3.0) 0.000 11 (±1.5) 0.027

15.8 45 (±4.0) 0.001 0 (±0.2) 0.027

15.9 37 (±2.0) 0.000 24 (±4.0) 0.022

16.1 ≥100 (±0. 0) 0.211 13 (±5.0) 0.016

16.2 ≥100 (±0.0) 0.211 30 (±8.0) 0.014

16.3 84 (±13.5) 0.085 15 (±3.0) 0.023

16.4 ≥100 (±0.0) 0.211 35 (±7.0) 0.020

16.5 93 (±11.5) 0.202 31 (±4.0) 0.032

16.6 ≥100 (±0.0) 0.211 43 (±4.0) 0.038

16.7 92 (±8.0) 0.105 41 (±5.5) 0.030

16.8 ≥100 (±0.0) 0.211 45 (±2.0) 0.056

16.9 62 (±8.5) 0.050 44 (±7.0) 0.029

‡ CC50 = the concentration of test compound (µM) responsible for 50% reduction in cell viability. § IC50 = the concentration of test compound (µM) responsible for a 50% inhibition of normal enzyme function. ** SI = Selectivity index; the ratio of CC50 / IC50. †† Independent T-test with non-equivalent variance performed against RAL; p-values < 0.05 designates a significant difference.


Although further testing is needed to confirm the initial results, preliminary testing indicate the

possibility of the pyrrolidinone compounds inhibiting HIV-1 IN strand transfer via an alternative

mechanism than RAL.

4.2.2 Structure-activity relationships (SAR)

In an initial investigation into the possible relationship between the molecular structure of first-

generation lead compounds 11.6 and 15.2, and the inhibitory effect these compounds exert on the

HIV-1 IN enzyme, the binding positions predicted for each compound in the HIV-1 IN active site

were examined (Accelrys, Discovery StudioTM

, Flexible Docking application).

For each compound, the number and type of interactions predicted to form between the enzyme

active site and the ligand’s functional groups were quantified, and any complementarities in their

respective geometries that may improve the binding of ligand to active site were noted.

Additionally, certain trends in terms of functional group substitutions and their effect on the

observed biological activity were noted, using the HIV-1 IN inhibition of purchased analogue 3

(54% as measured in the single-dose direct enzyme assay) as a starting point. Specifically, six

different functional groups in various ring-positions were introduced for the series 11 compounds

and nine for the series 15 compounds, respectively. The following parameters were investigated for

each compound series: 1) presence vs. absence of functional groups; 2) the steric influence (due to

size); 3) the electronic influence (due to heteroatom presence); 4) the position of the substituents;

and 5) the effect of additional substituents.

In an evaluation of the compounds 11.1-11.6 (Scheme 4.1), several conclusions could be drawn

regarding the effect of functional group identity and position. The molecular structures of

compounds in this series were closely aligned to the molecular structures of the in silico hit

compound 1 and purchased analogue 3, with the identity and position of the R3-phenyl ring

substituents as the only point of variance across the series.


Scheme 4.1 Biological profiles and HIV-1 IN inhibition (INI) activities of the series 11 compounds,

compared to compound 3.

A decrease in HIV-1 INI activity resulted for compounds 11.1 (21%) and 11.2 (23%), with R3-

substitutions either completely absent (11.1) or in the ortho-position (11.2). Some measure of INI

activity was restored when substitutions were introduced into the para- and meta-positions on the

phenyl-ring (11.3, 11.4 and 11.5), with a preference for bulky, hydrophobic functional groups

substituted into the para-position (compound 11.6). An examination of the binding pose predicted

for 11.6 in the HIV-1 IN active site, reveals the potential for the formation of hydrogen bonds to

active site residues, as well as to the 3’-end overhang of processed vDNA (Figure 4.1).

Furthermore, the presence of the bulky tert-butyl-group on the para-position of the phenyl ring

effectively forces compound 11.6 to adopt a conformation where the lactam-enol is in a position to

chelate one of the metal ions in the active site, in a manner similar to that predicted for RAL (Figure

4.2). The adopted conformation is further stabilised by the insertion of the imidazole-group into a

Chapter 4: Biological Evaluation

partially hydrophilic groove formed between active site residues and the terminal nucleotides of the

bound vDNA. Confirmation of the single

evidence in

inhibition.

Figure 4.1

interactions formed between 11.6 and active


Figure 4.2

interactions formed between RAL and active site re


For compounds

the molecular structures of the

compounds. Specifically, the R




evidence in support of compound

1 The 3D binding pose predicted for 11.6 in the HIV



2 The 3D binding pose predicted for RAL in the HIV



For compounds 15.1-

the molecular structures of the

compounds. Specifically, the R




support of compound

The 3D binding pose predicted for 11.6 in the HIV



rendering.

The 3D binding pose predicted for RAL in the HIV



rendering.

-15.9 (Scheme 4.2), the general backbone structure varied significantly from

the molecular structures of the in silico

compounds. Specifically, the R2-



bound vDNA. Confirmation of the single-dose activity results in a five

support of compound 11.6 having some potential for HIV



rendering.



rendering.

(Scheme 4.2), the general backbone structure varied significantly from

in silico hit compound

-group of the series


dose activity results in a five

having some potential for HIV


interactions formed between 11.6 and active site residues. These figures were created using




hit compound

group of the series 15


dose activity results in a five



site residues. These figures were created using


interactions formed between RAL and active site residues. These figures were created using


hit compound 1, purchased analogue

compounds consisted of a primary amine in


dose activity results in a five-dose screen provided further


The 3D binding pose predicted for 11.6 in the HIV-1 IN active site and the 2D


The 3D binding pose predicted for RAL in the HIV-1 IN active site and the 2D

sidues. These figures were created using


, purchased analogue


Page


dose screen provided further

having some potential for HIV-1 IN strand






, purchased analogue 3 and the series


Page 123



1 IN strand-transfer






nd the series 11




transfer






11



contrast to the aromatic imidazole-functionality present in compounds 1, 3 and the compounds in

series 11. Also, the aliphatic chain in the series 15 compounds was reduced by one –CH2– from the

propyl-chain present in 1, 3 and 11.1-11.6, to an ethyl-bridge between the pyrrolidinone-N and the

primary amine of the R3-group. Furthermore, the identity and position of the R

3-phenyl ring

substituents was a third point of variance across the series.

A good measure of HIV-1 IN inhibitory activity could be observed for compound 15.1 (57% INI

activity), with no substituent on the phenyl ring, as well as for compounds 15.3 (54% INI activity)

and 15.4 (56% INI activity), with single-substitutions in the para-position of the phenyl-ring.

However, an enhancement of INI activity could be observed upon substitution of a small,

electronegative functional group (Cl-atom) into the ortho-position of the phenyl ring for compound

15.2 (67% INI activity).

In contrast, single-substitutions in the meta-position (15.5; 32% INI activity), bulky hydrophobic

substitutions in the para-position (15.9; 24% INI activity) and the presence of additional

substitutions on the phenyl-ring (15.6, 15.7 and 15.8) resulted in a reduction of the HIV-1 IN

inhibitory activity (16%, 11% and 0% INI activity, respectively).

A closer examination of the binding pose predicted for compound 15.2 in the HIV-1 IN active site,

reveals the potential for the formation of five hydrogen bonds to active site residues and the 3’-end

overhang of the processed vDNA bound in the active site (Figure 4.3). It is worth noting that the

binding position predicted for 15.2 differs significantly from that observed for compound 11.6. In

this instance, the lactam-carbonyl of compound 15.2 was shifted away from the active-site bound

magnesium ion, limiting the potential for metal chelation. Due to this shift, the chloro-substituted

phenyl-ring is ideally positioned to nestle into a partially hydrophilic groove located between the 3’-

end of bound vDNA and active site residues (specifically residues E152 and K156). The presence of

a primary amine linked to the pyrrolidinone-ring through a flexible ethyl-bridge, enables the

formation of a labile six-membered ring through interaction of the lone-pair electrons on the

primary amine with the electron-deficient carboxyl-group on the pyrrolidinone-ring. This ring may

provide sufficient stability and rigidity to lock compound 15.2 in a conformation favourable to

interactions with the enzyme active site. Confirmation of the single-dose activity results in a five-

dose screen provided further evidence in support of compound 15.2 having some potential for HIV-

1 IN strand-transfer inhibition.


Scheme 4.2 Biological profiles and HIV-1 IN inhibition activities of the series 15 compounds,

compared to compound 3.


Figure 4.3



The fact that all

form stabilising interactions with the terminal adenosine nucleotide on the 3’

pre-processed vDNA strand bound in the IN active site, correlates well with th

action proposed for strand transfer inhibitors.

moderate activity against HIV

models generated in Chapter 2), there is clear s

of these compounds against HIV

structure and the biological response to the optimised structures will be required in second

later-generation d

compound series will be undertaken by a multidisciplinary team, thereby s

the iterative phases of design, synthesis and biological evaluation.

4.2.3 Evaluation of systemic and cellular toxicity

Literature reports on biological evaluations of similar structures,

bacteriostatic properties (

and the toxicity profiles of the pyrrolidinone compound class in r

literature has highlighted the relatively low toxicity associated with this class of compounds

As the current work does not include any

evaluation of the pyrroli

results from this study with literature reports cannot be confidently made. Instead, the


3 The 3D binding pose predicted for



The fact that all three of the compounds discussed above (


processed vDNA strand bound in the IN active site, correlates well with th


moderate activity against HIV




generation design and development stages. It is envisioned that further development of this


iterative phases of design, synthesis and biological evaluation.

valuation of systemic and cellular toxicity


bacteriostatic properties (




evaluation of the pyrroli



The 3D binding pose predicted for

interactions formed between 15.2


rendering.

three of the compounds discussed above (




moderate activity against HIV-1 IN (providing some measure of validity to the




esign and development stages. It is envisioned that further development of this





bacteriostatic properties (assays performed against




evaluation of the pyrrolidinone compounds in a range of



The 3D binding pose predicted for

15.2 and active site residues. These figures were created using

rendering.





1 IN (providing some measure of validity to the


of these compounds against HIV-1 IN. Much refinement and optimisation of both the molecular







assays performed against



As the current work does not include any in vivo

dinone compounds in a range of


The 3D binding pose predicted for 15.2

and active site residues. These figures were created using




action proposed for strand transfer inhibitors.12

Although compounds


models generated in Chapter 2), there is clear scope for development and improvement of the SAR

1 IN. Much refinement and optimisation of both the molecular







assays performed against Escherichia



in vivo studies and is only concerned with the biological

dinone compounds in a range of


15.2 in the HIV


three of the compounds discussed above (11.6, 15.2



Although compounds


cope for development and improvement of the SAR







scherichia coli



studies and is only concerned with the biological

dinone compounds in a range of in vitro


in the HIV-1 IN active site and the 2D


15.2 and RAL) were predicted to



Although compounds 11.6






compound series will be undertaken by a multidisciplinary team, thereby significantly accelerating

Literature reports on biological evaluations of similar structures,1 have mainly focussed on the

coli and Staphylococcus

and the toxicity profiles of the pyrrolidinone compound class in rodents (mice and rats). The



assays, direct comparisons of the


Page



and RAL) were predicted to

form stabilising interactions with the terminal adenosine nucleotide on the 3’-end overhang of the

processed vDNA strand bound in the IN active site, correlates well with the mechanism of

and 15.2 have shown

1 IN (providing some measure of validity to the in silico





ignificantly accelerating

have mainly focussed on the

taphylococcus

odents (mice and rats). The





Page 126




end overhang of the

e mechanism of

have shown

in silico active site



structure and the biological response to the optimised structures will be required in second- and




taphylococcus aureus),


literature has highlighted the relatively low toxicity associated with this class of compounds in vivo.



results from this study with literature reports cannot be confidently made. Instead, the in vitro




end overhang of the

e mechanism of

have shown

active site



and




),


.



in vitro


results obtained in this work can be seen as complementary to the in vivo results reported in the

literature.

In an attempt to determine whether the synthesised pyrrolidinones caused any adverse effects to

human blood cells, the in vitro cytotoxicity of each compound was evaluated in the healthy human

lymphocyte cell line PM1.13

The endpoint was determined by means of the CellTiter Aqueous One

Solution (Promega, USA).14,15

The cytotoxic effects of the compounds on the cell-line under study

were evaluated alongside a positive control of mock-treated cells and a cell-free negative control

under identical conditions. The CC50 values obtained for each compound is reported in Table 4.1,

and reflect the concentration of each compound that resulted in a loss of 50% of the cell viability.

In general, the test compounds showed variable toxicity across the series, with clear potential for

improvement in future generation compound design. Specifically, treatment of the cells with

compounds 11.1-11.5 (with imidazole as the R2-group) resulted in low CC50 values (~10 µM).

Importantly however, most of the test compounds showed good toxicity profiles with CC50 values

in the non-toxic to moderately toxic range (CC50 >50µM) supporting further development of more

in-depth ADMET profiles.

An important consideration in the design of orally-administered pharmaceuticals is the first-pass

effect. A complete first-pass effect describes molecules in an in vivo setting that have been

completely inactivated through metabolic transformations during the first pass through the liver,

before reaching the intended active site and exerting any of the intended pharmaceutical activity.16

Due to the extensive interaction between the liver tissues and the orally-administered compound,

potential for organ and especially hepatotoxicity needs to be evaluated during the early stages of

discovery and/or development. Using a computationally derived model for the prediction of dose-

dependent hepatotoxicity,17

all of the synthesised pyrrolidinone compounds were evaluated as

potential hepatotoxins. Each compound was scored in terms of its similarity to a previously

compiled database of known hepatotoxins and classified as either a toxin (score of one, with

increasing confidence as the probability value approaches 1) or a non-toxin (score of 0, with

increasing confidence as the probability value approaches 0), using a SAR technique that employs

recursive partitioning.

With the exception of compounds 13.6 and 16.9, all of the synthesised pyrrolidinones were

classified as potential hepatotoxins (score of 1.0); however the confidence of the predictions was

not high in all cases (probability values approach 0.5 instead of either 1.0 to identify a toxin or 0.0


to identify a non-toxin; see items marked with “*” in Table 4.2). Further, more in-depth in vitro

assays will be necessary to confirm these predictions.

The TOPKAT theoretical toxicity prediction application (Accelrys, Discovery StudioTM

) is

effectively a collection of cross-validated quantitative structure-toxicity relationship (QSTR)

models, for the in silico assessment of a range of specific toxicological endpoints from a 2D

molecular structure. Although the model would give a numerical result for any compound

evaluated, the prediction results should be assessed to determine whether the model is applicable,

the result is meaningful and the prediction is acceptable.18

For the QSTR models in the TOPKAT

application, this is accomplished through a rigorous post-prediction analysis: Firstly, a fragment-

comparison is done to ensure all fragments in the query molecule have been considered during the

model development (i.e. all fragments are covered in the model’s training set); secondly, a

univariate analysis is performed on a set of descriptors generated for each query molecule based on

the electronic attributes of the specific molecular structure, and compared to the descriptors

contained in the training set; thirdly, a multivariate analysis is performed to ensure the query

structure is within the optimum prediction space (OPS) of the model; lastly, the confidence of the

predicted result can be assessed through a similarity analysis. For each query compound, the

training set is searched for compounds similar in terms of the types of descriptors generated from

the molecular structure, as well as the values of these descriptors. Thus, if a query compound falls

within the OPS of the model and it is considered identical or very similar to compounds in the

training set for which the model gave a correct prediction (predicted value = experimental value),

then there is a high probability of the predicted result being correct (high confidence in the

predicted result).

Each of the synthesised pyrrolidinone compounds was screened against three QSTR models in the

TOPKAT application (Table 4.2): the weight-of-evidence (WOE) rodent carcinogenicity model; the

Ames mutagenicity model; and the developmental toxicity potential (DTP) model. For each

compound, the applicability of the model was assessed based on whether it could accurately predict

the toxicity potential of similar compounds in the training set found within the same OPS as the test

compound (included in Table 4.2).

In the WOE carcinogenicity model, the carcinogenic potential of each test compound was evaluated

against the US FDA Centre for Drug Evaluation and Research (CDER) WOE protocol and scored

as a carcinogen (values approaching 1.00) if it is rated as 1) a multiple-site carcinogen in at least

one sex / species combination (male or female / rat or mouse), or 2) a single-site carcinogen in at


least two sex / species combinations. The model could only be confidently applied to 50% of the

synthesised compounds, and in all cases a high probability of carcinogenic potential was predicted.

For the remaining 50%, the molecular descriptors defined from the compound structure fell outside

the OPS limits of the model, reducing the confidence in the computed probability and making the

quality of the predicted results unknowable.

The Ames mutagenicity model is based on the US Environmental Protection Agency’s (EPA)

GeneTox protocol, where tests are performed against five strains of Salmonella typhimurium, with

and without S9 activation and quantified using the Histidine Reversion Assay. Each test compound

was assessed and scored as a non-mutagen if there was a high probability of it producing a negative

response in the Histidine Reversion assay, with or without S9 activation, against all five Salmonella

strains (computed probability of mutagenicity approaching 0.0). The model could be confidently

applied to only two of the synthesised compounds, 12.6 and 13.1. In both instances, the compounds

were scored as non-mutagens. For all the other compounds, the molecular descriptors defined from

the compound structure fell outside the OPS limits of the model, reducing the confidence in the

computed probability and making the quality of the predicted results unknowable.

The DTP model was developed from 374-open literature references and includes only rat oral data.

Developmental toxicity (DT) was evidenced by reduced foetal growth, foetal death, resorption, and

both external and visceral teratology. The test compounds were scored based on the probability of

each to induce either no evidence of DT (computed probability between 0.00 and 0.30), or any

evidence of DT (computed probability between 0.70 and 1.00; no degree of DTP severity can be

assigned to the probability). The model could be applied to all of the synthesised compounds with a

high degree of confidence (all compounds fell within the OPS of the model). With the exception of

compounds 15.3 and 15.8, all of the synthesised compounds were predicted to cause some degree of

developmental toxicity.


Table 4.2 TOPKATTM

predictions for the pyrrolidinone compounds.

Hepatotoxicity prediction Carcinogenicity Mutagenicity DTP model

Compound

codes

(0 = Non-toxin;

1 = Toxin) Probability Weight of evidence (WOE)

Acceptability

of model Ames test

Acceptability

of model

Acceptability

of model

11.1 1 0.95 1.00 Y 0.00 N (3) 0.96 Y

11.2 1 0.96 1.00 N (3) 0.00 N (3) 1.00 Y

11.3 1 0.98 1.00 N (3) 0.00 N (3) 0.89 Y

11.4 1 0.97 1.00 Y 0.00 N (3) 1.00 Y

11.5 1 0.96 1.00 Y 0.00 N (3) 1.00 Y

11.6 1 0.70* 1.00 Y 0.00 N (3) 0.96 Y

12.1 1 0.68* 1.00 Y 0.00 N (3) 0.97 Y

12.2 1 0.68* 1.00 N (3) 0.00 N (3) 1.00 Y

12.3 1 0.62* 1.00 N (3) 0.00 N (3) 0.92 Y

12.4 1 0.70* 1.00 Y 0.00 N (3) 1.00 Y

12.5 1 0.70* 1.00 Y 0.00 N (3) 1.00 Y

12.6 1 0.59* 1.00 Y 0.00 Y 0.97 Y

13.1 1 0.76* 1.00 Y 0.00 Y 0.96 Y

13.2 1 0.88 1.00 N (3) 0.00 N (3) 1.00 Y

13.3 1 0.80 1.00 N (3) 0.00 N (3) 0.90 Y

13.4 1 0.76* 1.00 Y 0.00 N (3) 1.00 Y

13.5 1 0.88 1.00 Y 0.00 N (3) 1.00 Y

13.6 0 0.28 1.00 Y 0.00 N (3) 0.96 Y

14.1 1 0.97 1.00 N (3) 0.00 N (3) 1.00 Y

14.2 1 0.97 1.00 Y 0.00 N (3) 0.99 Y

14.3 1 0.96 1.00 N (3) 0.00 N (3) 1.00 Y

14.4 1 0.72* 1.00 N (3) 0.00 N (3) 1.00 Y

14.5 1 0.98 1.00 N (3) 0.00 N (3) 0.89 Y

14.6 1 0.58* 1.00 Y 0.00 N (3) 0.92 Y

15.1 1 0.97 1.00 Y 0.00 N (3) 0.77 Y

15.2 1 0.96 1.00 N (3) 0.00 N (3) 1.00 Y

15.3 1 0.98 1.00 N (3) 0.00 N (3) 0.50 Y

15.4 1 0.93 1.00 Y 0.00 N (3) 0.98 Y

15.5 1 0.93 1.00 N (3) 0.00 N (3) 0.98 Y

15.6 1 0.93 1.00 N (3) 0.00 N (3) 1.00 Y

15.7 1 0.84 1.00 N (3) 0.00 N (3) 1.00 Y

15.8 1 0.92 1.00 N (3) 0.00 N (3) 0.70 Y

15.9 1 0.66* 1.00 Y 0.00 N (3) 0.79 Y

16.1 1 0.97 1.00 Y 0.00 N (3) 0.96 Y

16.2 1 0.96 1.00 N (3) 0.00 N (3) 1.00 Y

16.3 1 0.95 1.00 N (3) 0.00 N (3) 0.89 Y

16.4 1 0.94 1.00 Y 0.00 N (3) 1.00 Y

16.5 1 0.95 1.00 Y 0.00 N (3) 1.00 Y

16.6 1 0.94 1.00 N (3) 0.00 N (3) 1.00 Y

16.7 1 0.91 1.00 N (3) 0.00 N (3) 1.00 Y

16.8 1 0.97 0.99 N (3) 0.00 N (3) 0.96 Y

16.9 0 0.37 1.00 Y 0.00 N (3) 0.96 Y

Y = Yes; N = No; (3) = Model not suitable for assessment of compound due to compound falling outside of the model’s OPS.


4.2.4 Aqueous solubility (LogS)

Advances in combinatorial chemistry and high-throughput compound screening during recent years

have resulted in a substantial increase in the number of development candidates identified; however,

this increase does not reflect in the number of drugs attaining FDA approval. As a result, the

traditional gap between the research and development phases have shrunk considerably, with

pharmaceutical companies now including the profiling of drug candidates based on their ADMET

properties as a routine practise early in the discovery phase.19

One important requirement for all

good drug candidates is a suitable solubility profile in aqueous / biologically relevant medium.20

Specifically, adequate solubility in the surrounding media helps to ensure successful uptake of the

test compound and effective distribution within the test system.

The aqueous solubility of each of the synthesised pyrrolidinone compounds was determined both

theoretically21

(Accelrys Discovery StudioTM

, ADMET Descriptors application) and

experimentally: a good agreement between both the log and natural values of the predicted and

experimental results could be established (Figure 4.4a: T-test p-value of the log form = 1.37E-11;

and Figure 4.4b: T-test p-value of the natural numbers = 6.04E-10). Notably, for certain compounds

with a methoxy group on the 3-position of the phenyl ring (specifically compounds 12.4, 14.2, 15.4,

16.4 and 15.6), the experimentally determined aqueous solubility was slightly lower than predicted.

For experimental determination of the aqueous solubility, the well-established 96-well format

MultiScreen Solubility FilterplateTM

protocol was used and the concentration of compound

dissolved in phosphate buffered saline (PBS, pH 7.4) measured via UV-Vis spectrometry against a

control (chloramphenicol with known aqueous solubility). The results (illustrated in log-form in

Figure 4.4) indicate moderate to good solubility in aqueous medium for the majority of compounds

tested (40–70% compound dissolution). However, compounds 15.3, 15.8 and most of the

compounds in series 16 showed excellent aqueous solubility (70 – 100% compound dissolution)

comparable to that of the reference compound, chloramphenicol. In contrast, compounds 11.6, 12.6,

14.6 and 16.9 showed a decreased aqueous solubility in comparison to the corresponding

compounds with an un-substituted (or minimally substituted) phenyl-ring, most likely due to the

presence of the large hydrophobic tert-butyl substituent. Nevertheless, all compounds showed

acceptable solubility profiles and were deemed sufficiently soluble in aqueous medium to support

further investigations to be conducted in biological environments.


Figure 4.4 Aqueous solubility: Alignment of the predicted and experimentally determined values

for a) the logS form (one-tailed distribution of a two-sample unequal variance student T-test; p-

value = 1.37E-11); and b) the solubility in natural numbers (one-tailed distribution of a two-sample

unequal variance student T-test; p-value = 6.04E-10).

4.2.5 Role of the pH and pKa on solubility and salt formation

The pKa values of the synthesised pyrrolidinone compounds in an aqueous environment were

calculated in an effort to determine the effect that changes in pH may have on the compound

ionisation state (results represented in Table 4.3). It should be kept in mind that due to the levelling

effect of water,22

the effective pKa range in aqueous medium is from -1.74 to 15.74. This range will

be sufficient to identify the strength of weak acids and weak bases in situ as these compounds

generally have pKa values in the approximate range -2 to 12,23

but strong acids and bases (for

-7

-6

-5

-4

-3

-2

-1

0

0 100 200 300 400 500

So

lub

ilit

y (

Mo

l/L)

MW (g/mol)

LogS (experimental; Mol/L) LogS (calculated; Mol/L)

0

0.0001

0.0002

0.0003

0.0004

0.0005

0.0006

0.0007

0.0008

0 100 200 300 400 500

So

lub

ilit

y (

Mo

l/L)

MW (g/mol)

Solubility (experimental; Mol/L) Solubility (calculated; Mol/L)

A.

B.


example, HCl) will completely dissociate, severely limiting the direct determination of the pKa of

these compounds in water. Instead, the pKa values of strong acids and bases are determined in

alternative solvents with more suitable pKa ranges and the data is then extrapolated to give an

approximate pKa in water.

Each of the pyrrolidinone compounds synthesised in this project contains at least one ionisable

hydroxyl-group (enol on the pyrrolidinone-ring common to all compounds) and one ionisable

amine-group (substituent R2). As such, the synthesised pyrrolidinones are amphoteric molecules

that should be able to act as either an acid (calculated pKa = -2 to 7) or a base (calculated pKa = 7

to 12) depending of the pH of the environment. Additionally, each molecule should therefore have

at least two pKa values (to account for the acidic, neutral and basic forms at varying pH values).

Table 4.3 reports the pKa values predicted for the major species formed for each pyrrolidinone

compound, denoted by pKa1, pKa2, and in some instances pKa3. In line with their amphoteric

nature, several species were predicted for each compound dependent on the pH (extrinsic factor) as

well as the number and identity of ionisable functional groups on each molecule (intrinsic factor).

The identities of the predicted species dominant in specific pH ranges were generally conserved

throughout the six compound series synthesised (11.1-11.6, 12.1-12.6, 13.1-13.6, 14.1-14.6, 15.1-

15.9 and 16.1-16.9).‡‡

Also represented in Table 4.3, is the absorption probability of each

compound as determined using a model of human intestinal absorption (HIA) after oral

administration24


, ADMET Descriptors application). The HIA model

evaluates each compound in terms of the calculated 2D polar surface area (PSA) and the calculated

LogP, and defines well-absorbed compounds as those absorbed at least 90% in the human

bloodstream.

For compounds with R2 = propyl-imidazole (compounds 11.1-11.6), a total of five species were

predicted in different protonation states and in differing concentrations depending on the pH. As

these compounds are predicted to predominantly form charged species, they would most likely

experience difficulties in crossing lipid membranes through passive diffusion.

The preferential formation of zwitterions over the neutral species predicted for compounds with R2

= propyl-morpholine (compounds 12.1-12.6), may affect the absorption and permeability of these

compounds due to impaired passive diffusion.

‡‡ Representative figures of the pI and the pKa distribution patterns predicted for each compound series are included in Appendix B.


For compounds 13.1-13.6 (R2 = propyl-dimethylamine), the zwitterionic compound form was

absent, and the neutral species was predicted to form in the pH range between ~5.0 and ~8.6.

Absorption of these neutral compounds is therefore likely to proceed through passive diffusion.

For compounds 14.1-14.6 (R2 = propylamine), the zwitterionic compound form was mostly absent,

and the neutral species was predicted to form in the pH range between ~4.8 and ~9.6. Notably, the

presence of additional ionisable groups resulted in the formation of additional species (specifically,

view the pKa and pI spectra of compounds 14.3 and 14.5 in Appendix B). Absorption of the neutral

species of compounds 14.1-14.6 is likely to proceed through passive diffusion.

The species predicted for compounds 15.1-15.9 and 16.1-16.9 closely resembled those observed for

compounds 14.1-14.6. For these compound series, the neutral species were predicted to form

between pH ~ 4.8 to ~8.8 for compounds 15.1-15.9 and between pH ~5.0 to ~10.0 for 16.1-16.9

respectively, indicating a good possibility of absorption through passive diffusion.

One of the most common ways to circumvent solubility problems is to convert the insoluble product

to a salt.25

Recognising the need to consider the effect of salt formation on the compounds

synthesised in this project, a cursory investigation into the HCl salt formation of the compounds

was performed.26

It is a generally accepted rule-of-thumb that stable and efficient salt formation

requires at least a three unit difference between the pKa values of the test compound (acting as the

base) and that of the added acid.27,28

This difference in the pH values will typically ensure that

ionisation is complete and that only one ionisation state is formed. (See Table 4.3 for pKa values of

the pyrrolidinone compounds and hydrochloric acid.) To this end, suspensions of the pyrrolidinone

compounds in methanol were treated with HCl and the resulting precipitates isolated, dried and

characterised. The pure product powders of 12 pyrrolidinone hydrochloride salts could be obtained

(these being 11.5, 11.6, 12.1, 12.2, 12.3, 12.5, 12.6, 13.1, 13.2, 13.3, 13.5 and 13.6), confirming the

ease with which these salts could be prepared, should the need arise. The preparation of these salts

is discussed in more detail in Chapter 3 (section 3.2.5), while information on the characterisation of

each compound is outlined in Chapter 5.


Table 4.3 Calculated pKa and pI values of the synthesised pyrrolidinone compounds.

Compound

codes

pI pKa1 pKa2 pKa3 HIA model prediction

(0 = Good; 1 = Moderate; 2 = Poor)

HCl -7.0

11.1 4.8 4.8 5.2 0

11.2 5.0 5.0 14.0 0

11.3 4.6 4.8 0

11.4 4.8 4.8 5.2 0

11.5 4.8 4.8 5.2 0

11.6 4.9 4.8 5.4 0

12.1 5.7 5.2 6.2 0

12.2 5.5 5.0 6.2 14.0 0

12.3 5.5 5.0 6.2 0

12.4 5.6 5.0 6.2 0

12.5 5.6 5.0 6.2 0

12.6 5.7 5.4 6.2 0

13.1 6.9 5.2 8.6 0

13.2 6.7 5.0 8.6 14.0 0

13.3 6.7 5.0 8.6 0

13.4 6.8 5.2 8.6 0

13.5 6.8 5.2 8.6 0

13.6 7.0 5.4 8.6 0

14.1 7.2 4.8 9.6 14.0 0

14.2 7.3 5.0 9.6 0

14.3 7.1 5.0 9.6 10.0 1

14.4 7.2 5.0 9.6 14.0 0

14.5 6.7 5.0 9.0 9.6 1

14.6 7.4 5.4 9.6 0

15.1 7.0 5.2 9.0 0

15.2 6.8 4.8 9.0 14.0 0

15.3 6.8 4.8 9.0 0

15.4 7.0 5.0 9.0 0

15.5 7.0 5.0 9.0 0

15.6 6.8 5.0 9.0 10.0 1

15.7 6.9 5.0 9.0 13.8 0

15.8 6.5 4.8 8.8 10.8 1

15.9 7.1 5.2 9.0 0

16.1 7.6 5.2 10.0 0

16.2 7.4 5.0 10.0 14.0 0

16.3 7.4 5.0 10.0 0

16.4 7.5 5.2 10.0 0

16.5 7.5 5.2 10.0 0

16.6 7.2 5.0 9.8 1

16.7 7.4 5.0 10.0 14.0 0

16.8 6.7 5.0 9.0 10.0 2

16.9 7.7 5.4 10.2 1


4.2.6 Membrane permeability

Passive diffusion through a lipid layer (such as the human intestinal layer) generally takes place

across a concentration gradient according to Fick’s Law:29

dM / dt = A•Peff (Clumen ref

– Cblood ref

)

Equation 4.1

where A is the surface area of the membrane, Peff is an effective permeability coefficient and the

reference concentrations are on the two opposite sides of the intestinal mucosa; Clumen ref

and Cblood

ref. It is usually assumed that the reference blood concentration (C

blood ref) is negligible in comparison

with the lumen concentration (Clumen ref

).30

Additionally, the polarity of chemical compounds has

been shown to have a significant effect on the compound permeability,31

with small non-polar

molecules showing enhanced potential for membrane permeation. As polarity depends largely on

the ionisation state of a particular compound,32

it follows that good membrane permeation requires

a test compound in a non-ionised or neutral state.33

Furthermore, charged molecules may be

influenced by electric field potentials (such as membrane potentials).34

The permeability of a representative number of the synthesised pyrrolidinone compounds was

determined in the parallel artificial membrane permeability assay (PAMPA) model, and the

percentage membrane retention through the phospholipid layer of the polyvinylidene fluoride

(PVDF) membrane was calculated according to the manufacturer’s instructions (Table 4.4). The

direct relationship between the lipophilicity of a compound and its membrane binding potential has

been well-established.33

More lipophilic molecules tend to have a higher binding affinity to the

phospholipid membrane layers, and therefore show higher membrane retention. Conversely,

molecules with higher aqueous solubility (more hydrophilic molecules) show a lower tendency to

bind to the lipid-rich membrane layers, and should therefore display lower membrane retention. The

above-mentioned relationships between parameters could not be conclusively established in the

current dataset.

Also represented in Table 4.4, is the predicted blood-brain penetration of each compound as

determined in a linear regression model35


, ADMET Descriptors

application). This robust model evaluates the potential of test molecules to cross the blood-brain

barrier (BBB) as a ratio of concentrations: specifically, each potential is represented by the base 10

logarithm of (compound concentration in the brain) / (compound concentration in the blood).


All of the compounds tested against the model showed a low to moderate BBB penetration, except

for compound 13.6. In total, the molecular structure of 13.6 contains five methyl groups (three on

the tert-butyl substituent of the phenyl ring and an additional two on the amino-propyl nitrogen),

effectively rendering it sufficiently lipophilic (ALogP = 4.58; Table 4.6) to interact with and cross

the BBB.

Table 4.4 The membrane permeability potential of the pyrrolidinone compounds.

Compound

Codes

Permeability Retention LogS BBB model prediction

Pe (cm/s) Std. dev. (µM fraction) Std. dev. % (1 = High; 2 = Medium; 3 = Low)

11.1 -4.04E-06 ±(7.80E-06) 0.21 ±(0.10) 21 2.71 3

11.2 -4.03E-06 ±(1.78E-05) 0.63 ±(0.04) 63 2.38 3

11.3 -4.99E-06 ±(1.04E-05) 0.40 ±(0.14) 40 2.18 3

11.4 4.61E-06 ±(2.03E-05) 0.5 ±(0.26) 50 2.32 3

11.5 1.11E-07 ±(1.37E-05) 0.34 ±(0.02) 34 2.46 3

11.6 1.67E-05 - 0.83 ±(0.34) 83 2.43 2

12.1 -4.99E-06 ±(1.05E-05) 0.33 ±(0.24) 33 2.09 3

12.2 3.10E-06 ±(1.23E-05) 0.22 ±(0.09) 22 2.33 2

12.3 6.90E-07 ±(1.04E-05) 0.32 ±(0.07) 32 2.20 3

12.4 -1.05E-06 ±(1.13E-05) 0.41 ±(0.21) 41 2.31 3

12.5 3.89E-07 ±(6.57E-06) -0.01 ±(0.15) -1 2.22 3

12.6 1.15E-05 ±(2.46E-05) 0.62 ±(0.14) 62 2.44 2

13.1 -3.51E-06 ±(8.10E-06) 0.26 ±(0.14) 26 1.99 2

13.2 -2.37E-06 ±(8.74E-06) 0.33 ±(0.14) 33 2.38 2

13.3 -3.61E-07 ±(8.01E-06) 0.21 ±(0.14) 21 2.12 2

13.4 8.90E-07 ±(7.52E-06) -0.72 ±(0.41) -72 2.47 2

13.5 -2.96E-06 ±(6.44E-06) 0.03 ±(0.24) 3 2.82 2

13.6 -5.57E-05 - 1.07 ±(0.12) 107 2.48 1

14.1 1.03E-05 ±(2.49E-05) 0.50 ±(0.06) 50 2.38 3

14.2 1.01E-05 ±(2.47E-05) 0.31 ±(0.22) 31 2.36 3

14.3 -8.04E-05 - 0.29 ±(0.97) 29 2.19 *

14.4 -5.71E-05 - 0.84 ±(0.17) 84 2.36 *

14.5 1.82E-05 ±(5.49E-05) 0.10 ±(0.56) 10 2.43 *

14.6 4.26E-06 ±(2.06E-05) 0.60 ±(0.03) 60 2.48 *

15.1 1.75E-06 ±(1.21E-05) 0.35 ±(0.09) 35 2.05 3

15.2 3.80E-06 ±(1.54E-05) 0.33 ±(0.07) 33 2.41 3

15.3 -1.28E-05 ±(2.25E-05) 0.78 ±(0.09) 79 2.39 3

15.4 -1.09E-05 ±(1.87E-05) 0.70 ±(0.12) 70 2.56 3

15.5 -7.60E-06 ±(1.58E-05) 0.62 ±(0.11) 62 2.29 3

15.6 3.42E-05 - 0.71 ±(0.63) 71 2.53 *

15.7 -2.62E-06 - 1.85 ±(0.57) 185 2.33 *

15.8 -3.98E-05 - 0.76 ±(0.25) 76 2.43 *


4.2.7 Plasma protein binding

An increase in aromatic ring count has been implicated in an increase in the plasma protein

sequestration of pharmaceutical compounds.36

Furthermore, plasma protein binding (PPB) has been

reported to adversely affect the biological activity of therapeutic agents (and potentially the antiviral

activity of the synthesised pyrrolidinones), as it effectively reduces the concentration of compound

available to exert an effect on the target site.37

Nonetheless, numerous examples exist of successful

drugs that exhibit a high degree (>95%) of PPB16

(some examples include: the anti-depressant drug

Amitriptyline; Clofibrate used to treat high cholesterol; Diazepam used in the treatment of anxiety

disorders, etc.).

The extent to which each of the synthesised pyrrolidinone compounds binds plasma protein was

predicted using a plasma protein binding model38


, ADMET

Descriptor application) and the results represented in Table 4.5. Most of the synthesised compounds

showed a high (>95%) potential for PPB, with the exception of compounds 12.1, 12.2, 12.3 that

showed a low PPB potential (<90%); and 13.6 and 16.9 that showed a moderate PPB potential

(>90%).

Additionally, the potential of each synthesised pyrrolidinone to inhibit CYP2D6 was predicted

using a computational model39


, ADMET Descriptors application).

CYP2D6 is a member of the cytochrome P450 (CYP450) family and plays a very important role in

the metabolism and clearance of xenobiotics from the biological system,40

therefore inhibition of

this protein in an in vivo setting would prolong the drug-life and enhance the bioavailability of the

test compound by decreasing the drug clearance rate. Most of the synthesised pyrrolidinones were

predicted to have some inhibition potential towards CYP2D6 (Table 4.5), with the notable

exception of compounds that contained the hydrophobic tert-butyl substituent on the phenyl ring

(12.6, 13.6, 14.6, 15.9 and 16.9). Further exceptions included several compounds with a di-

substituted phenyl ring (14.3, 14.4, 14.5, 15.6, 15.8, 16.7 and 16.8).


Table 4.5 Attributes predicted for the synthesised pyrrolidinone compounds.

PPB model prediction CYP2D6 inhibition

(0 = <90%; 1 = >90%; 2 = >95%) (0 = Non-inhibitor; 1 = Inhibitor) Probability

11.1 2 1 0.69*

11.2 2 1 0.85

11.3 2 1 0.65*

11.4 2 1 0.70*

11.5 2 1 0.69*

11.6 2 1 0.65*

12.1 0 1 0.62*

12.2 0 1 0.77

12.3 0 1 0.65

12.4 2 1 0.67

12.5 2 1 0.67

12.6 2 0 0.44

13.1 2 1 0.79

13.2 2 1 0.72

13.3 2 1 0.78

13.4 2 1 0.61

13.5 2 1 0.77

13.6 1 0 0.38

14.1 2 0 0.46

14.2 2 1 0.59

14.3 2 0 0.44

14.4 2 0 0.44

14.5 2 0 0.48

14.6 2 0 0.38

15.1 2 1 0.57

15.2 2 1 0.51

15.3 2 1 0.54

15.4 2 1 0.60

15.5 2 1 0.53

15.6 2 0 0.44

15.7 2 1 0.51

15.8 2 0 0.44

15.9 2 0 0.33

16.1 2 1 0.88

16.2 2 1 0.59

16.3 2 1 0.69

16.4 2 1 0.70

16.5 2 1 0.69

16.6 2 1 0.56

16.7 2 0 0.44

16.8 2 0 0.44

16.9 1 0 0.42


4.2.8 Compound compliance to Lipinski’s Rule of Five

One of the best known and most widely accepted concepts in designing orally administered

pharmaceuticals is the Ro5 proposed by Chris Lipinski and co-workers in 1997.20,41

This simple

concept can be used to predict the likelihood of a molecule having absorption problems due to poor

solubility and/or permeability. In its original form, the Ro5 considered four basic parameters of

pharmaceutical agents developed for oral administration and defined an optimal range for each of

these parameters (ranges defined in multiples of 5): the molecular weight (≤500 g/mol); the

calculated LogP (≤5); the number of hydrogen bond donors (≤5); and the number of hydrogen bond

acceptors (≤10). The rule further states that compounds found to violate more than two of these

parameters were likely to have poor absorption and would most likely be ill-suited to oral

administration. Later additions to the Ro5 included criteria related to the number of rotatable bonds

(≤10) and the PSA (≤140 Å2).

42 Because of its simplicity and easy implementation, the Ro5 rapidly

gained popularity in the drug discovery community and soon variations were developed for use in

fragment-based drug design (for example the Rule of Three, Ro3).43

During the past decade, the

Ro5 has surpassed its original rule-of-thumb status, and has become an indispensable tool in the

medicinal chemist’s arsenal. However, it has been proposed that the almost indiscriminate use of

the Ro5 may disproportionately bias current drug discovery efforts and hamper rather than help the

discovery of new pharmaceutical agents.44

Although the original screening library compiled from the ZINC database (discussed in Chapter 2)

was filtered according to the Ro5 and therefore fully compliant to all of the requirements, one of the

analogues derived from the in silico lead and subsequently synthesised (discussed in Chapter 3)

exceeded the molecular weight parameter stipulated in the Ro5. Specifically, the molecular weight

of synthesised analogue 12.6 (MW = 502.6 g/mol) exceeded the required 500 g/mol. Compound

12.6 would therefore receive a Lipinski score of 3/4 in contrast to the other compounds, all of which

scored a perfect 4/4 (see Table 4.6). In spite of this minor violation, compound 12.6 was still

included in all further studies. Although a direct relationship between molecular weight and

aqueous solubility has been established for certain types and classes of compounds,45

many

additional factors may play a role in the dissolution of compounds in aqueous medium.


Table 4.6 Compound compliance to Lipinski’s Ro5.

Original Lipinski Ro5 Lipinski Score Additions to the Ro5 Final drug-score

MW ALogP Donors Acceptors Rotatable Bonds PSA

(≤500) (≤5) (≤5) (≤10) (≤10) (≤140 Å2)

Test Compounds:

11.1 427.4 2.0 2 5 4/4 7 87.5 6/6

11.2 461.9 2.7 2 5 4/4 7 87.5 6/6

11.3 445.4 2.2 2 5 4/4 7 87.5 6/6

11.4 457.5 2.0 2 6 4/4 8 96.4 6/6

11.5 457.5 2.0 2 6 4/4 8 96.4 6/6

11.6 483.6 3.4 2 5 4/4 8 87.5 6/6

12.1 446.5 2.9 1 5 4/4 7 83.6 6/6

12.2 480.9 3.5 1 5 4/4 7 83.6 6/6

12.3 464.5 3.1 1 5 4/4 7 83.6 6/6

12.4 476.5 2.9 1 6 4/4 8 92.5 6/6

12.5 476.5 2.9 1 6 4/4 8 92.5 6/6

12.6 502.6 4.3 1 5 3/4 8 83.6 5/6

13.1 404.5 3.2 1 4 4/4 7 74.7 6/6

13.2 438.9 3.8 1 4 4/4 7 74.7 6/6

13.3 422.4 3.4 1 4 4/4 7 74.7 6/6

13.4 434.5 3.2 1 5 4/4 8 83.6 6/6

13.5 434.5 3.2 1 5 4/4 8 83.6 6/6

13.6 460.6 4.6 1 4 4/4 8 74.7 6/6

14.1 410.8 2.9 2 4 4/4 6 97.9 6/6

14.2 406.4 2.2 2 5 4/4 7 106.8 6/6

14.3 422.4 2.0 3 6 4/4 7 127.6 6/6

14.4 436.5 2.2 2 6 4/4 8 115.7 6/6

14.5 408.4 1.7 4 6 4/4 6 139.5 6/6

14.6 432.5 3.6 2 4 4/4 7 97.9 6/6

15.1 362.4 2.2 2 4 4/4 5 97.9 6/6

15.2 396.8 2.8 2 4 4/4 5 97.9 6/6

15.3 380.4 2.4 2 4 4/4 5 97.9 6/6

15.4 392.4 2.1 2 5 4/4 6 106.8 6/6

15.5 392.4 2.1 2 5 4/4 6 106.8 6/6

15.6 408.4 1.9 3 6 4/4 6 127.6 6/6

15.7 422.4 2.1 2 6 4/4 7 115.7 6/6

15.8 394.4 1.7 4 6 4/4 5 139.5 6/6

15.9 418.5 3.6 2 4 4/4 6 97.9 6/6

16.1 404.5 3.2 2 4 4/4 8 97.9 6/6

16.2 438.9 3.9 2 4 4/4 8 97.9 6/6

16.3 422.4 3.5 2 4 4/4 8 97.9 6/6

16.4 434.5 3.2 2 5 4/4 9 106.8 6/6

16.5 434.5 3.2 2 5 4/4 9 106.8 6/6

16.6 450.5 3.0 3 6 4/4 9 127.6 6/6

16.7 464.5 3.2 2 6 4/4 10 115.7 6/6

16.8 436.5 2.8 4 6 4/4 8 139.5 6/6

16.9 460.6 4.6 2 4 4/4 9 97.9 6/6


Table 4.6 continued.

Known INIs:

5CITEP 289.7 2.1 3 5 4/4 3 102.0 6/6

Elvitegravir 447.9 4.7 2 6 4/4 7 88.5 6/6

L-708906 404.4 4.4 2 6 4/4 9 94.1 6/6

L-731988 289.3 2.7 2 4 4/4 5 81.6 6/6

L-870810 432.5 0.9 2 6 4/4 4 111.5 6/6

L-870812 427.4 0.2 2 6 4/4 5 114.8 6/6

MA-DKA 249.2 1.8 4 6 4/4 5 110.6 6/6

MK-2048 461.9 2.4 2 5 4/4 4 108.9 6/6

Raltegravir 444.4 -0.3 3 7 4/4 6 148.1 6/6

S-1360 313.3 2.5 2 4 4/4 5 88.2 6/6

The distribution of molecular properties of the pyrrolidinone compounds can be graphically

compared to that of a series of known HIV-1 IN inhibitors (Figure 4.5), by plotting the calculated

LogP (ALogP) against the molecular weight (MW) and imposing the upper Lipinski limit of each

parameter onto the pharmacological space represented in the graph.46

Figure 4.5 Distribution of the molecular properties of the synthesised pyrrolidinone compounds and

several known HIV-1 IN inhibitors based on the calculated partition coefficient (ALogP) and the

molecular weight (MW).

-0.5

1.5

3.5

5.5

0 100 200 300 400 500

ALo

gP

MW (g/mol)

Lipinski upper limit: ALogP = 5 Lipinski upper limit: MW = 500 g/mol

Pyrrole-carbaldehydes Known INIs


4.3 Conclusion

A range of synthesised pyrrolidinone compounds, predicted to have anti-retroviral activity through

inhibition of the strand-transfer reaction of HIV-1 IN, were evaluated in a range of biological and

computational assays to generate activity and initial ADMET profiles for each compound.

Biological assays (inhibition potential in direct enzyme assays and whole-cell cytotoxicity assays)

have identified two pyrrolidinone compounds (11.6 and 15.2) with promising SI values, warranting

further, more in-depth analysis of the biological profiles and mechanism of action of the

pyrrolidinone class of compounds, and in particular of compounds 11.6 and 15.2. An initial SAR

evaluation of the series 11 compounds (based on the inhibition of a soluble HIV-1 IN double-

mutant with wild-type activity) indicated the preference for a bulky, hydrophobic substituent in the

para-position of the phenyl-ring (substituent-R3), when an aromatic R

2-amino group (imidazole) is

linked to the pyrrolidinone-ring through a flexible hydrophobic propyl-chain. In contrast, when the

R2-group is a primary amine and the alkyl chain is shortened to an ethyl-bridge as for the series 15

compounds,; the preferred conformation includes a small, electronegative substituent capable of

forming hydrogen-bonds in the ortho-position of the phenyl-ring. In this instance, the presence of

additional substituents all but abolishes the HIV-1 INI activity observed for similar compounds.

Furthermore, screening of the most promising compounds (11.6, 15.2 and 15.3) against four drug

resistant HIV-1 IN mutants (single- and double mutants: Q148H; Q148H/G140S; N155H and

N155H/E92Q) is underway to determine whether the compounds retain their observed activity

against INI-resistant enzymes. Promising initial results indicate a possibility of the pyrrolidinone

compounds having a different mechanism of action to RAL, as some inhibitory activity is retained

in the RAL-resistant mutants.

Most of the pyrrolidinone compounds were predicted to have a high propensity for binding to

plasma proteins and metabolic enzymes (CYP2D6), and at least some potential for adverse effects

(carcinogenic, mutagenic, DT and/or hepatotoxic). Accordingly, if the predictions are correlated

through experimentally observed toxicity characteristics, second- and further-generation compound

design will attempt to minimise the occurrence of various toxicophores in the compound family

while retaining the pharmacophore responsible for the observed biological activity.

The facile conversion of the free pyrrolidinone compounds to the corresponding HCl-salts has

demonstrated an effective solution to any potential solubility problems. Furthermore, a predictive

investigation into the character of the pyrrolidinone compounds at different pH environments have

enabled a clearer understanding of how these compounds may behave in a biological system.


In conclusion, the in silico identification of an inhibitor class predicted to have activity against HIV-

1 IN strand transfer, has resulted in compounds with inhibitory activity against HIV-1 IN strand

transfer in vitro, providing a measure of validation to the computational models generated in this

work. Although much optimisation and refinement needs to be done for second- and later-

generation compound design and testing (in silico and in vitro), the promising activity and generally

acceptable biological ADMET profiles observed for the pyrrolidinone compounds have provided a

good starting point in the development of this compound class as inhibitors of HIV-1 IN strand

transfer.


4.4 References

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9. Temesgen, Z., Siraj, D. S., Ther. Clin. Risk Man., 2008, 4, 493-500.

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18. Kulkarni, S. A., Zhu, J., SAR QSAR Environ. Res., 2008, 19, 1-2, 39-54.

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2010, 398, 1, 227-238.

20. Lipinski, C. A., Lombardo, F., Dominy, B. W., Feeney, P. J., Adv. Drug Deliv. Rev., 1997,

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Pharm. Res.,1992, 9, 1243-1251..

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Chapter 5: Experimental Methods Page 146

CHAPTER 5: EXPERIMENTAL METHODS

5.1 General methods

5.1.1 Solvents and reagents

All solvents used were chemically pure or pre-purified.1 All solvents were freshly distilled before

use. All other reagents were analytically and synthetically pure.

5.1.2 Spectroscopic data and methods

5.1.2.1 Nuclear magnetic resonance (NMR)

All 1H,

13C{

1H} and

15N{

1H} NMR spectra were recorded on a Bruker Avance Ultrashield-400

spectrometer. In all cases deuterated solvents were used for measurements and the resulting spectra

referenced to the residual non-deuterated solvent peaks and TMS. The peak multiplicities are

abbreviated as follows:

s–singlet; d–doublet; dd–doublet of doublets; t–triplet; dt–doublet of triplets; p–pentet; dp–doublet

of pentets; and m–multiplet. The coupling constant (J) is calculated in Hertz and reported to the

nearest 0.1 Hz.2

In general, no peak splitting due to C-H coupling is expected for 13

C{H} spectra. Therefore, all

peaks in the 13

C{H} spectra of the compounds reported in this chapter appears as singlets and are

not explicitly reported, except for those compounds where a fluorine atom forms a part of the

molecule (compounds 11.3, 12.3, 13.3, 14.3, 15.3 and 16.3). For these compounds, C-F coupling

occurs and the carbons on the phenyl ring bearing the F-substituent present as doublet signals in the

13C{H} spectra of the relevant compounds (explicitly reported).

5.1.2.2 Mass spectrometry (MS)

TOF-ESI+ and TOF-ESI- mass spectral analysis was performed on a Waters API Q-TOF Ultima

spectrometer at the Central Analytical Facility, University of Stellenbosch, South Africa. In all

instances, milligram quantities of the samples were dissolved in a 1:1 mixture of 0.1% formic acid

in water and acetonitrile; 1-3 µl of sample solution were injected via a Waters UPLC instrument;

the capillary and cone voltages were set to 3.5 kV and 35 V, respectively; the source and

desolvation temperature were set to 100 °C and 350 °C, respectively; nitrogen desolvation gas was


pumped into the system at a flow rate of 350 L/hour, while the cone gas flow rate was set to 50

L/hour.

5.1.2.3 Infrared spectrometry (IR)

Infrared spectroscopy (IR) was performed on a Perkin-Elmer 881 spectrometer fitted with an

attenuated total reflectance (ATR) accessory and a diamond ATR crystal. Only peaks of the

characteristic functional groups are reported in wavenumber (cm-1

) and referenced to generalised

charts of characteristic group frequencies.3 All samples were measured in powder form and pressed

into a pellet directly onto the ATR diamond.

5.1.2.4 Melting points (M.p.)

Melting points were collected using a Stuart SMP3 melting point apparatus and scanning was done

between 30 °C and 300 °C at 10 °/minute. The melting point ranges have been reported to the

nearest 0.5 °C.

5.1.2.5 X-ray crystallography

Crystal evaluation and data collection were performed on a Bruker APEXII diffractometer with Mo

Kα (λ = 0.71073 Å) radiation and diffractometer to crystal distance of 4.00 cm, at the Department

of Chemistry, University of Johannesburg, South Africa. The initial cell matrix was obtained from

three series of scans at different starting angles. Each series consisted of 12 frames collected at

intervals of 0.5º in a 6º range with the exposure time of 10 seconds per frame. The reflections were

successfully indexed by an automated indexing routine built in the APEXII program suite.4 The

final cell constants were calculated from a set of 6460 strong reflections from the actual data

collection.

The data were collected by using the full sphere data collection routine to survey the reciprocal

space to the extent of a full sphere to a resolution of 0.75 Å. Data were harvested by collecting

frames at intervals of 0.5º scans in ω and φ with exposure times of 20 seconds per frame. These

highly redundant datasets were corrected for Lorentz and polarisation effects. The absorption

correction was based on fitting a function to the empirical transmission surface as sampled by

multiple equivalent measurements.4

The systematic absences in the diffraction data were uniquely consistent for the space group P21/c

that yielded chemically reasonable and computationally stable results of refinement.5 A successful


solution by the direct methods of SHELXS975 provided all non-hydrogen atoms from the E-map.

All non-hydrogen atoms were refined with anisotropic displacement coefficients. All hydrogen

atoms except those on the solvent water molecules were included in the structure factor calculation

at idealised positions and were allowed to ride on the neighbouring atoms with relative isotropic

displacement coefficients.

The final least-squares refinement of parameters against data resulted in residuals R (based on F2

for I≥2σ) and wR (based on F2 for all data) values unique to each crystal. The final difference

Fourier map was featureless. The molecular diagrams are drawn with 50% probability ellipsoids.6

5.1.2.6 Elemental analysis (EA)

Due to the unavailability of elemental analysis services at the time of completion of this project, no

elemental analysis data are included for any of the synthesised compounds. Instead, the correct

formation of the expected products were confirmed by single crystal analysis (section 5.1.2.5) and

the purity of all compounds submitted for biological screening were confirmed via HR TOF-ESI

mass spectrometry (section 5.1.2.2).

5.1.2.7 Absorbance measurements in biological assays

All absorbance measurements of biological samples performed during this project was recorded on

an X-MarkTM

Microplate Spectrophotometer (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Further manipulations of the raw data were performed using Microsoft Excel 2007 and 2010

software.

5.1.2.8 pH measurements

All pH measurements obtained during this project were performed on a Consort C830 pH meter.

The pH meter was calibrated with known standards (pH 4.0 and pH 7.0) before each measurement

to ensure conformity between data sets.

5.1.2.9 Centrifugation

The centrifuge employed for cell isolation and purification in this project was the Eppendorf 5810R.


5.1.2.10 Microscopy

An Olympus CKX41 microscope with a CC-12 digital colour camera using AnalySIS® FIVE

software was employed for the visualisation of all cell matter. All cell counts were performed on a

CountessTM

automated cell counter (Invitrogen Corporation, Carlsbad, CA, USA).

5.2 In silico methods

Theoretical adverse effect and ADMET predictions were performed using the Accelrys Discovery

StudioTM

software package (licensed from Accelrys Inc., San Diego, CA, 2010, USA) while

chemical structure visualisations and modifications were performed using ChemBioDraw Ultra

version 12.0 (CambridgeSoft, Cambridge, MA, USA, 2010) and MarvinSketch version 5.2.4

(ChemAxon Kft., Budapest, Hungary, 2009). All statistical data and graphs were calculated and

created in Microsoft Excel 2010. Biological CC50 and IC50 data were calculated using Origin

version 6.1 (OriginLab Corp. Northampton, MA, 2000, USA).

All computational protein manipulations were performed on a dual-core desktop PC with a Red Hat

Enterprise Linux Version 5.0 operating system. All protein visualisations, manipulations and

docking runs were performed using the commercial software packages SybylTM

version 8.0,

licensed from TriposTM

(Tripos Inc., St. Louis, MO, USA, 2008), and Accelrys Discovery StudioTM

version 2.5 licensed from Accelrys (Accelrys Inc., San Diego, CA, 2010, USA).

5.2.1 Modelled structure of the three-domain HIV-1 IN monomer

The full-length HIV-1 IN monomer was obtained by assembling structures available in the

Research Collaboratory for Structural Bioinformatics (RCSB) PDB7 using Sybyl 8.0.

The HIV-1 IN three-domain model was built starting from a crystal structure of the CCD only (PDB

code 1QS4).8 This crystal structure consists of three identical chains, each containing one Mg

2+ ion

coordinated by two of the catalytic triad residues, D64 and D116. In each chain, there is an

unresolved loop region situated in close proximity to the active site (residues 141-144).

Additionally, Chain A contained 5-CITEP, a bound inhibitor of the diketo-acid class that was

manually discarded in the construction of our model. All water molecules and Chains B and C were

discarded. The missing loop region was built onto the existing chain with the Biopolymer Building

function of Sybyl 8.0 in accordance with the HIV Sequence Compendium 2008.9 The conformation

was assigned based on the conformation of the homologous region in the crystal structure of the

inhibitor-free CCD of HIV-1 IN (PDB code 1B9F).10

In the original crystal structure (1QS4), two


mutations (F185K and W131E) were introduced into the protein to facilitate crystallisation. These

mutations were manually replaced in Sybyl 8.0 with the naturally occurring residues. In order to

place the second Mg2+

ion [Mg2+

(II)], the two-metal crystal structure of Tn5 transposase (PDB code

1MM8) was structurally aligned with the CCD of HIV-1 IN via the conserved catalytic triad

residues. The coordinates of the second divalent metal in the Tn5 active site were transferred to the

HIV-1 IN active site and the Mg2+

ion inserted.

The protein structure of the CCD was prepared via the Structure Preparation Tool encoded in Sybyl

8.0. All hydrogen atoms were added. The force field AMBER7FF99 was used to assign partial

charges to the protein. The side-chain amides of all asparagine and glutamine residues and the

protonation state and orientation of all histidine residues were optimised for H-bonding potential.

Finally, the entire molecule was energy-minimised using the MMFF94s force field without initial

optimisation, using the Powell method11

in a maximum of 5000 iterations to a termination gradient

of 0.5 kcal/(molÅ).

The effect of solvation on the protein model structure was investigated by preparing a second

version of the CCD from 1QS4 in an identical manner as described above; except that all water

molecules within 3 Å of any Chain A residue and bound Mg2+

ion were retained. Also, the missing

loop region between residues 141 and 144 was not repaired but used as it appeared in the original

crystal structure. Upon completion of the energy-minimisation process, the structures of the

solvated and non-solvated models of the CCD were compared via a backbone RMSD analysis.

The CTD of the IN model was derived from the two-domain, hetero-dimerised crystal structure of

HIV-1 IN CTD and CCD (PDB code 1EX4).12

All water molecules and Chain B were discarded.

This structure was well resolved with no unresolved regions or bound cofactors. The protein was

prepared with the Biopolymer Protein Preparation Tool function of Sybyl 8.0 as described above.

The missing side-chain of residue K211 was completed and orientated to reduce protein “bumping”.

Finally, the biomolecule was energy-minimised using the AMBER7FF99 force field as described

before.

To obtain the orientation between the CCD and CTD, our CCD model was aligned with residues

56-209 (CCD) of 1EX4, following which these residues were discarded. The remaining residues

210-270 (CTD) of 1EX4 were merged into the molecular area of our CCD model and the two

chains joined by forming a peptide bond between residues 209 and 210. The result was a two-

domain 56-270 monomer, energy-minimised with the MMFF94s force field.


The tetrameric two-domain crystal structure of HIV-1 IN CCD and NTD was extracted from the

PDB (PDB code 1K6Y).13

The crystal structure contains one Zn2+

ion chelated in the HHCC region

of each NTD. It is well resolved except for a missing loop region between residues 47-55. All water

molecules were deleted, as were Chains B, C and D with the associated metal ions. The missing

loop region between residues 47 and 55 was completed by building the correct sequence9 onto the

C’-terminal of residue 46 and assigning a random conformation in accordance to that observed in

the NMR structure of dimeric HIV-1 IN NTD (PDB code 1WJD).14

Chain A and the chelated Zn2+

were prepared using the Biopolymer Protein Preparation Tool as described previously and the

molecule was energy-minimised using the AMBER7FF99 force field. The CCD of Chain A (PDB

code 1K6Y) was aligned to that of our two-domain CCD-CTD model and residues 56-209 of 1K6Y

were removed. Residues 1-55 of 1K6Y were merged into the same molecular area as our two-

domain model and finally the chains were joined by forming a peptide bond between residues 55

and 56. The entire biomolecule was energy-minimised with the MMFF94s force field as described

previously.

5.2.2 Model of the HIV-1 IN dimer with cognate DNA

5.2.2.1 Constructing the HIV-1 IN dimer

The CCD dimer (interacting Chains A and B) of the original, unaltered HIV-1 IN crystal structure

(1QS4) was used as a template to construct the IN dimer model. Two molecules of the constructed

three-domain monomer of the present study were aligned with Chains A and B of 1QS4

respectively, the monomers merged into the same molecular area and the template deleted to give

the HIV-1 IN dimer AB.

5.2.2.2 Model structure and positioning of the viral DNA

The interaction of vDNA with the HIV-1 IN dimer model was adapted from the crystal structure of

the homologous Tn5 IN enzyme (PDB code 1MM8).15

One molecule of template 1MM8 was

aligned with one of the HIV-1 IN monomers in the AB dimer through the absolutely conserved

catalytic triad (D64, D116 and E152 of HIV-1 IN were aligned with D97, D188 and E326 of Tn5

IN, respectively) to act as a template for the insertion of the vDNA LTR. The sequence and

structure of the U5 LTR of HIV-1 was reported in an NMR solution structure (PDB code 1TQR).16

This NMR structure consists of a blunt-ended 17-mer DNA chain with the binding or cleaving site

at the 3’-end distinguishable by a loss of regular base stacking and a distorted minor groove. The 3’-

end of the U5 LTR vDNA chain of 1TQR was aligned with the 3’-end of the DNA substrate in the


1MM8 templates, the DNA chain merged into the same molecular area as the HIV-1 IN dimer

model, and the template was deleted. Finally, the terminal two nucleotides on the 3’-end of the

vDNA were removed manually to imitate 3’-end processing.

5.2.3 Preparation of the PFV IN crystal structure

The newly elucidated crystal structure of PFV integrase used in this study (PDB code 3L2T)17

contains an active site with the invariant catalytic triad motif (DDE) coordinating two divalent

magnesium ions. Additionally, one molecule of the INI, MK0518 (RAL), is held in the active site

through chelation to the divalent metals. A pre-processed 17/19-mer mimic of the U5 vDNA end is

bound in the active site. The crystal also shows the dimeric interface of the PFV IN, with a second

co-crystallised PFV IN CCD interacting with the first monomer.

All water molecules in the crystal structure as well as the co-crystallised inhibitor were discarded.

All hydrogen atoms were added. The structure was well resolved with no unresolved regions. The

protein was prepared with the Biopolymer Protein Preparation Tool function of Sybyl 8.0 as

described previously. The force field AMBER7FF99 was used to assign partial charges to the

protein. The side-chain amides of all asparagine and glutamine residues and the protonation state

and orientation of all histidine residues were optimised for H-bonding potential. Finally, the entire

molecule was energy-minimised with the AMBER7FF99 force field without initial optimisation

according to the Powell method11

in a maximum of 5000 iterations to a termination gradient of 0.5

kcal/(molÅ).

5.2.4 Construction of the HIV-1 IN strand-transfer complex model

A crystal structure of the HIV-1 IN CCD in complex with the IBD of LEDGF/p75 has been solved

and is available through the PDB (PDB code 2B4J).18

This crystal structure consists of two units of

dimerised CCDs, which are complexed to LEDGF/p75 in a 1:1 ratio. Interacting Chains A (IN

CCD) and C (LEDGF/p75) of 2B4J were retained and refined with the Biopolymer Protein

Preparation Tool and energy-minimised with the AMBER7FF99 force field as described previously.

The CCD (Chain A) of this molecule was aligned with the CCD in one of the three-domain HIV-1

IN monomers in the dimeric model. All residues of Chain C (LEDGF/p75) were merged into the

same molecular area as the three-domain model, resulting in a model for the LEDGF/p75-bound,

full-length dimer of HIV-1 IN.


The tetrameric form of HIV-1 IN responsible for the strand transfer reaction was constructed based

on the tetrameric crystal structure of 1K6Y13

(previously used for the positioning of the NTD in the

HIV-1 IN monomer). One molecule of the three-domain, LEDGF-bound HIV-1 IN dimer model in

association with viral DNA was aligned with each of the dimers in the 1K6Y template. All residues

were merged into the same molecular area to give a representation of the HIV-1 IN tetramer. Two

of the four LEDGF/p75 molecules (Chains A and C) caused steric clashes within the tetrameric

strand transfer model and were removed. The resulting model of the HIV-1 IN consisting of a

homo-tetrameric IN with four active sites (of which two are catalytic) containing two divalent

magnesium ions each, complexed to two pre-processed U5 ends of vDNA and two molecules of

LEDGF/p75 was minimised with the MMFF94s force field without initial optimisation, using the

Powell method in a maximum of 5000 iterations to a termination gradient of 0.5 kcal/(mol*A).

The tetrameric form of HIV-1 IN inserts 3’-processed viral cDNA into highly bent host DNA and

the integration takes place across a major groove.19,20

A plausible model of host DNA that

approaches this particular conformation was found in the Protein Data Base (PDB code 1KX3).21

This crystal structure consists of the nucleosome core particle, and includes a 147 bp DNA fragment

(derived from human α-satellite DNA) wrapped around the octameric histone (derived from

recombinant Xenopus laevis).

As only the conformation of the DNA chain was of interest in this case, all of the histone amino

acids, the coordinated metal atoms and all water molecules were discarded. A sequence specific

analysis of the DNA fragment was performed and point mutations were introduced into the DNA

sequence of both + and - DNA strands to complete the palindromic sequence of host DNA observed

at the site of integration (refer to Scheme 2.2). The resulting molecule of target DNA was manually

positioned in the hydrophobic groove formed between the active sites of opposing CCDs in the AB

and CD dimers of the HIV-1 IN tetramer. The placement was optimised to satisfy all experimentally

observed interactions. The points of possible interaction between protein residues and host DNA

nucleotides were analysed and key interactions identified. The logistics and possible mechanism of

vDNA integration into the palindromic sites on target DNA was evaluated and finally a possible

mode of dimer-dimer association around the target DNA was proposed.


5.3 Chemical synthesis

5.3.1 Synthesis of the pyruvate ester starting material

5.3.1.1 Methyl (2Z)-4-(1-benzofuran-2-yl)-2-hydroxy-4-oxobut-2-enoate (8)

The reaction was carried out according to literature procedures.22

Using flamed-out glassware and

under inert gas, 10 mmol (0.54 g) NaOMe was added to 20 mL dry MeOH and dissolved by

stirring. The solution was cooled to room temperature and the flask immersed in tap water. An

equimolar mixture of 2-acetyl benzofuran (starting material 8.1; 10 mmol, 1.60 g) and dimethyl

oxalate (starting material 8.2; 10 mmol, 1.46 g) in 10 mL dry MeOH was prepared in a dropping

funnel. The equimolar mixture was run into the NaOMe solution over a period of 1 hour, with

continuous stirring. Stirring was continued at room temperature for 4 hours and the mixture left

overnight at room temperature. The following day, the flask was cooled in an ice bath and ice cold

15% H2SO4 (approx. 10 mL) rapidly run into the mixture through the dropping funnel. The mixture

was stirred for 5 minutes and the product extracted with a biphasic system (DCM/H2O). The solvent

was removed in vacuo and the product purified through recrystallisation from warm MeOH to give

the title compound 8 as dark yellow crystals (2.127 g; 8.67 mmol; 87% yield).

Keto form:

1H NMR: (400 MHz, CDCl3) δH 7.59 (1H, d, J = 8.0 Hz, H-5); 7.47 (1H, d, J = 8.0 Hz,

H-2); 7.40 (1H, d, J = 1.2 Hz, H-7); 7.34 (1H, dt, J = 7.7 and 1.3 Hz, H-3),

7.20 (dt, J = 7.5 and 1.2 Hz, H-4); 3.80 (2H, s, H-10); 2.50 (3H, s, H-13)

13C NMR: (75 MHz, CDCl3) δC 188.8 (2C, C-11 and C-9); 157.9 (1C, C-12); 155.7 (1C,

C-1); 152.5 (1C, C-8); 128.3 (1C, C-6); 127.0 (1C, C-3); 123.9 (1C, C-4);

123.3 (1C, C-5); 113.2 (1C, C-7); 112.4 (1C, C-2); 53.6 (1C, C-10); 26.4 (3C,

C-13)

HR TOF-ESI+: m/z 247.0609 ([M+H]+, 100%); 248.0640 ([M+H+1 {isotope}]

+, 14%),

249.0653 ([M+H+2 {isotope}]+, 2%)

Calculated for [C13H10O5 + H]: 247.0606; found: 247.0609.

Melting point: 116–117.5 °C


5.3.2 Synthesis of the substituted 1-aminoalkyl-3-hydroxy-3-pyrrolin-2-ones

With some exceptions (notably 14.3, 14.4, 14.5, 15.5, 15.6, 15.7, 15.8, 15.9, 16.2, 16.4, 16.5, 16.6,

16.7, 16.8 and 16.9) the synthesised pyrrolidinone compounds were rendered sufficiently soluble

for solution-based NMR characterisation through acidification of the deuterated solvent (MeOD).

However, the compounds showed a tendency to form multiple products in solution. In a number of

instances, the various product forms could be distinguished and the peak assignments completed for

each product formed (major and minor products). However, the large number of product forms

present in solutions of compound series with an amino R2-terminal (series 14.1-14.6, 15.1-15.9 and

16.1-16.9) resulted in significant overlapping of the product peaks. In several instances, the

assignment of peaks could not be done with any degree of certainty; for these compounds, only the

1H and

13C NMR signals of the chiral-CH formed during ring formation are reported, in addition to

the IR, HR TOF-ESI+, melting points and crystal structure data (where applicable) stated for each

compound.

5.3.2.1 1-(3-(1H-imidazol-1-yl)propyl)-4-(benzofuran-2-carbonyl)-3-hydroxy-5-phenyl-1H-pyrrol-

2(5H)-one (11.1)

The reaction was carried out according to literature procedures.23

0.200 g (0.816 mmol) of 8 was

dissolved in 10 mL 1,4-dioxane. Separately, 1 molar equivalent aminopropyl-imidazole (0.816

mmol; 0.097 mL) and 1 molar equivalent of benzaldehyde (0.816 mmol; 0.087 mL) was dissolved

in 10 mL 1,4-dioxane. The solution of 8 was added to the second starting material solution and the

mixture stirred at room temperature for 4 hours. The precipitate formed was filtered off and dried to

give the title compound 11.1 as a yellow solid (0.710 mmol; 0.305 g; 87% yield).

Yield: 87%.

1H NMR: (400 MHz, CD3OD) δH 9.00 (1H, s, H-23); 7.87 (1H, d, J = 0.8 Hz, H-7);

7.73 (1H, dt, J = 7.7 and 0.9 Hz, H-5); 7.69 (1H, t, J = 1.8 Hz, H-25); 7.60

(1H, dq, J = 8.4 and 0.8 Hz, H-2); 7.56 (1H, t, J = 1.8 Hz, H-24); 7.49 (1H,


dt, J = 7.3 and 1.3 Hz, H-3); 7.39 (2H, d, J = 7.2 Hz, H-16 and H-18); 7.33-

7.23 (4H, m, H-4, H-15, H-17 and H-19); 5.89 (1H, s, H-13); 4.46 (2H, t, J =

7.4 Hz, H-22, minor product); 4.31 (1H, dt, J = 14.1 and 6.8 Hz, H-22A of the

ABX system, major product); 4.23 (1H, dt, J = 14.1 and 7.0 Hz, H-22B of the

ABX system, major product); 3.63 (1H, t, J = 7.3 Hz, H-20A of ABX system,

major product); 3.05 (2H, t, J = 7.7 Hz, H-20, minor product); 2.97 (1H, dt, J

= 14.5 and 6.1 Hz, H-20B of ABX system, major product); 2.34 (2H, p, J =

7.6 Hz, H-21, minor product); 2.14 (1H, dp, J = 12.8 and 6.6 Hz, H-21A of the

ABX system, major product); 2.03 (1H, dp, J = 14.3 and 6.9 Hz, H-21B of the

ABX system, major product).

13C NMR: (100 MHz, CDCl3) δC 177.7 (1C, C-11); 166.6 (1C, C-9); 159.4 (1C, C-12);

157.0 (1C, C-1); 152.7 (1C, C-8); 137.0 (1C, C-23); 136.7 (1C, C-14); 130.1

(3C, broad s, C-15, C-17 and C-19); 129.9 (1C, C-3); 129.2 (2C, C-16 and C-

18); 128.3 (1C, C-6); 125.3 (1C, C-4); 124.7 (1C, C-5); 123.3 (1C, C-25);

121.1 (1C, C-24); 119.8 (1C, C-10); 116.8 (1C, C-7); 113.1 (1C, C-2); 63.0

(1C, C-13); 48.0 (1C, C-22); 38.8 (1C, C-20); 29.6 (1C, C-21).

15N NMR: (40 MHz, MeOD) δN 182.5 (1N, s, N-3); 172.4 (1N, s, N-2); 138.4 (1N, s, N-

1).

IR: νmax / cm-1

3151, 1683, 1597.


+, 20%),

430.1741 ([M+H+2 {isotope}]+, 2%)

Calculated for [C25H21N3O4 + H]: 428.1610; found: 428.1627.

Melting point: 170–171.5 °C.

Single-crystal analysis:

Empirical formula C25H17N3O4S4

Formula weight 551.66

Temperature 293(2) K

Wavelength 0.71073 Å

Crystal system Monoclinic

Space group P21/n

Unit cell dimensions

a = 9.3499(8) Å

b = 17.4394(14) Å

c = 17.9530(15) Å

α = 90°

β = 102.189(2)°

γ = 90°

Volume 2861.4(4) Å3


Z 4

Density (calculated) 1.281 Mg/m3

Crystal size 0.18 x 0.19 x 0.25 mm3

Theta range for data collection 2.28 to 28.44°

Completeness to theta = 28.44˚ 99.6%

Refinement method Full-matrix least-squares on F2

Goodness-of-fit on F2 1.037

Final R-indices [I>2sigma(I)] R1 = 0.0923, wR2 = 0.2537

5.3.2.2 1-(3-(1H-imidazol-1-yl)propyl)-4-(benzofuran-2-carbonyl)-5-(2-chlorophenyl)-3-hydroxy-

1H-pyrrol-2(5H)-one (11.2)

The reaction was carried out according to literature procedures23

as described for section 5.3.2.1,

with 1.0 molar equivalents of aminopropyl-imidazole (0.816 mmol; 0.097 mL) and 2-

chlorobenzaldehyde (0.816 mmol; 0.092 mL) dissolved in 10 mL 1,4-dioxane added to 0.200 g

(0.816 mmol) of 8 dissolved in 10 mL 1,4-dioxane. Title compound 11.2 was isolated as a yellow

solid (0.734 mmol; 0.345 g; 90% yield).

Yield: 90%.


7.70 (1H, d, J = 7.6 Hz, H-5); 7.69-7.68 (1H, m, H-25); 7.61-7.56 (2H, m, H-

17 and H-24); 7.54 (1H, dd, J = 8.4 and 0.8 Hz, H-2); 7.49-7.44 (2H, m, H-16

and H-19); 7.38 (1H, t, J = 4.6 Hz, H-3); 7.34-7.31 (1H, m, H-18); 7.28 (1H,

t, J = 8.0 Hz, H-4); 6.41 (1H, s, H-13); 4.38 (2H, t, J = 7.4 Hz, H-22, minor

product); 4.26 (1H, dt, J = 14.0 and 6.7 Hz, H-22A of the ABX system, major

product); 4.16 (1H, dt, J = 14.0 and 6.9 Hz, H-22B of the ABX system, major

product); 3.50 (1H, dt, J = 14.5 and 7.2 Hz, H-20A of the ABX system, major


product); 2.95 (1H, dt, J = 12.9 and 6.7 Hz, H-20B of the ABX system, major

product); 2.90 (2H, t, J = 7.2 Hz, H-20, minor product); 2.25 (2H, p, J = 7.5

Hz, H-21, minor product); 2.08 (1H, dp, J = 13.4 and 7.2 Hz, H-21A of the

ABX system, major product); 1.97 (1H, dp, J = 14.1 and 6.7 Hz, H-21B of the



157.1 (1C, C-1); 152.4 (1C, C-8); 136.6 (1C, C-14); 136.4 (1C, C-23); 135.2

(1C, C-15); 131.1 (1C, C-16); 130.7 (1C, C-3); 130.0 (1C, C-19); 129.3 (1C,

C-17); 128.4 (1C, C-6); 127.9 (1C, C-18); 125.4 (1C, C-4); 124.7 (1C, C-5);

123.4 (1C, C-25); 121.2 (1C, C-24); 118.4 (1C, C-10); 116.7 (1C, C-7); 113.2

(1C, C-2); 58.6 (1C, C-13); 39.2 (1C, C-22); 37.7 (1C, C-20); 30.1 (1C, C-21)


1)

IR: νmax / cm-1

3146, 1682, 1599


+, 30%),

465.1295 ([M+H+3 {isotope}]+, 5%)

Calculated for [C25H20ClN3O4 + H]: 462.1221; found: 462.1216

Melting point: 132.5–133 °C


Empirical formula C29H32ClN3O6S2





Space group P21/c


a = 8.5726(17) Å

b = 24.547(5) Å

c = 14.313(3) Å

α = 90°

β = 95.128(5)°

γ = 90°

Volume 2999.7(10) Å3

Z 4






Refinement method Semi-empirical from equivalents

Goodness-of-fit on F2 Full-matrix least-squares on F2

Final R-indices [I>2sigma(I)] 1.025

R1 = 0.0447, wR2 = 0.1115

5.3.2.3 1-(3-(1H-imidazol-1-yl)propyl)-4-(benzofuran-2-carbonyl)-5-(4-fluorophenyl)-3-hydroxy-



as described for section 5.3.2.1.

0.200 g (0.816 mmol) of 8 was dissolved in 5 mL of 1,4-dioxane. One molar equivalent of

aminopropyl-imidazole (0.816 mmol; 0.097 mL) was added to the dioxane mixture, resulting in a

light yellow precipitate forming. One molar equivalent of 4-fluorobenzaldehyde (0.816 mmol;

0.088 mL) was added to the mixture and stirred at room temperature for 5 minutes. The solvent was

removed in vacuo and title compound 11.3 was isolated as a yellow powder (0.410 mmol; 0.183 g;

50% yield). No further purification was needed.

Yield: 50%

1H NMR: (400 MHz, CD3OD) δH 9.05 (1H, s, H-23); 7.89 (1H, s, H-7); 7.74-7.72 (2H,

m, H-5 and H-25); 7.61-7.57 (2H, m, H-2 and H-24); 7.49 (1H, t, J = 14.8 Hz,

H-3); 7.45-7.42 (2H, m, H-15 and H-19); 7.30 (1H, t, J = 7.4 Hz, H-4); 7.03

(2H, t, J = 8.6 Hz, H-16 and H-18); 5.91 (1H, s, H-13); 4.47 (2H, t, J = 7.3

Hz, H-22, minor product); 4.34 (1H, dt, J = 13.7 and 6.8 Hz, H-22A of the

ABX system, major product); 4.26 (1H, dt, J = 13.8 and 7.0 Hz, H-22B of the

ABX system, major product); 3.64 (1H, dt, J = 14.5 and 7.2 Hz, H-20A of the

ABX system, major product); 3.06 (2H, t, J = 7.6 Hz, H-20, minor product);

2.94 (1H, dt, J = 14.5 and 6.4 Hz, H-20B of the ABX system, major product);

2.35 (2H, p, J = 7.5 Hz, H-21, minor product); 2.18 (1H, p, J = 6.6 Hz, H-21A


of the ABX system, major product); 2.06 (1H, p, J = 7.9 Hz, H-21B of the


13C NMR: (100 MHz, CDCl3) δC 177.6 (1C, C-11); 166.5 (1C, C-9); 164.2 (1C, d, JC,F =

246.5 Hz, C-17); 159.5 (1C, C-12); 157.0 (1C, C-1); 152.6 (1C, C-8); 136.7

(1C, C-23); 133.1 (1C, d, JC,F = 2.9 Hz, C-14); 131.4 (2C, d, JC,F value did

not resolve well, H-15 and H-19); 130.0 (1C, C-3); 128.3 (1C, C-6); 125.4

(1C, C-4); 124.8 (1C, C-5); 123.4 (1C, C-25); 121.2 (1C, C-24); 119.6 (1C,

C-10); 117.0 (3C, C-16, C-18 and C-7); 113.2 (1C, C-2); 62.2 (1C, C-13);

48.0 (1C, C-22, major product); 47.6 (1C, C-22, minor product); 38.8 (1C, C-

20, major product); 37.7 (1C, C-20, minor product); 29.7 (1C, C-21, major

product); 29.2 (1C, C-21, minor product).

15N NMR: (40 MHz, MeOD) δN 172.8 (1N, s, N-3), 182.5 (1N, s, N-2)

IR: νmax / cm-1

3144, 1680, 1601


+, 20%),

462.1282 ([M+H+2 {isotope}]+, 2%)

Calculated for [C25H20FN3O4 + H]: 446.1516; found: 446.1518.


5.3.2.4 1-(3-(1H-imidazol-1-yl)propyl)-4-(benzofuran-2-carbonyl)-3-hydroxy-5-(4-

methoxyphenyl)-1H-pyrrol-2(5H)-one (11.4)





yellow precipitate forming. One molar equivalent of 4-methoxybenzaldehyde (p-anisaldehyde;

0.816 mmol; 0.099 mL) was added to the mixture and stirred at room temperature for 5 minutes.

The solvent was removed in vacuo and title compound 11.4 was isolated as a yellow powder (0.473

mmol; 0.217 g; 58% yield). No further purification was needed.


Yield: 58%

1H NMR: (400 MHz, CD3OD) δH 9.03 (1H, s, H-24); 7.84 (1H, s, H-7); 7.72-7.70 (2H,

m, H-5 and H-26); 7.61-7.56 (2H, m, H-2 and H-25); 7.47 (1H, dt, J = 7.8

and 1.2 Hz, H-3); 7.30-7.26 (3H, m, H-16, H-19 and H-4); 6.82 (2H, d, J =

8.8 Hz, H-15 and H-20); 5.85 (1H, s, H-13); 4.47 (2H, t, J = 7.4 Hz, H-23,

minor product); 4.32 (1H, dt, J = 14.0 and 6.8 Hz, H-23A of the ABX system,

major product); 4.24 (1H, dt, J = 14.0 and 7.0 Hz, H-23B of the ABX system,

major product); 3.66 (3H, s, H-18 [methyl-group]); 3.60 (1H, dt, J = 14.5 and

7.4 Hz, H-21A of the ABX system, major product); 3.06 (2H, t, J = 7.7 Hz, H-

21, minor product); 2.97 (1H, dt, J = 14.5 and 6.0 Hz, H-21B of the ABX

system, major product); 2.35 (2H, p, J = 7.5 Hz, H-22, minor product); 2.14

(1H, p, J = 7.0 Hz, H-22A of the ABX system, major product); 2.03 (1H, p, J

= 6.8 Hz, H-22B of the ABX system, major product).

13C NMR: (100 MHz, CDCl3) δC 177.7 (1C, C-11); 166.4 (1C, C-9); 161.5 (1C, d, J =

246.5 Hz, C-17); 159.8 (1C, C-12); 157.0 (1C, C-1); 152.6 (1C, C-8); 136.7

(1C, C-24); 130.5 (2C, C-16 and C-19); 129.9 (1C, C-3); 128.5 (1C, C-14);

128.3 (1C, C-6); 125.3 (1C, C-4); 124.8 (1C, C-5); 123.4 (1C, C-26); 121.2

(1C, C-25); 119.8 (1C, C-10); 116.9 (1C, C-7); 115.5-115.4 (2C, m, C-15 and

C-20); 113.2 (1C, C-2); 62.5 (1C, C-13); 55.9 (1C, C-18); 48.0 (1C, C-23,

major product); 47.6 (1C, C-23, minor product); 38.7 (1C, C-21, major

product); 37.7 (1C, C-21, minor product); 29.7 (1C, C-22, major product);

29.2 (1C, C-22, minor product).


1)

IR: νmax / cm-1

3142, 1678, 1599


+, 22%),

460.1841 ([M+H+2 {isotope}]+, 3%)


Melting point: 139–140 °C





as described for section 5.3.2.1,

with 1 molar equivalents of aminopropyl-imidazole (0.816 mmol; 0.097 mL) and 3-methoxy

benzaldehyde (m-anisaldehyde; 0.816 mmol; 0.111 mL) dissolved in 10 mL 1,4-dioxane added to

0.200 g (0.816 mmol) of 8 dissolved in 10 mL 1,4-dioxane. Title compound 11.5 was isolated as a

yellow solid (0.682 mmol; 0.313 g; 84% yield).

Yield: 84%


7.65 (1H, d, J = 8.0 Hz, H-5); 7.60 (1H, s, H-26); 7.52-50 (1H, m, H-2); 7.47

(1H, t, J = 1.4 Hz, H-25); 7.40 (1H, dt, J = 7.8 and 1.2 Hz, H-3); 7.22 (1H, dt,

J = 7.5 and 0.6 Hz, H-4); 7.12 (1H, t, J = 8.0 Hz, H-16); 6.86 (1H, s, H-20);

6.82 (1H, d, J = 7.6 Hz, H-15); 6.71 (1H, dd, J = 8.2 and 2.2 Hz, H-17); 5.80

(1H, s, H-13); 4.37 (2H, t, J = 7.4 Hz, H-23, minor product); 4.23 (1H, dt, J =

14.1 and 6.8 Hz, H-23A of the ABX system, major product); 4.15 (1H, dt, J =

14.0 and 7.0 Hz, H-23B of the ABX system, major product); 3.61 (3H, s, H-

19 [methyl-group]); 3.54 (1H, dt, J = 14.1 and 7.2 Hz, H-21A of the ABX

system, major product); 2.94 (1H, dt, J = 13.3 and 6.8 Hz, H-21B of the ABX

system, major product); 2.89 (2H, t, J = 6.1 Hz, H-21, minor product); 2.26

(2H, p, J = 5.7 Hz, H-22, minor product); 2.06 (1H, dp, J = 13.3 and 7.0 Hz,

H-22A of the ABX system, major product); 1.95 (1H, dp, J = 14.1 and 7.2 Hz,

H-22B of the ABX system, major product).


159.4 (1C, C-12); 157.0 (1C, C-1); 152.7 (1C, C-8); 138.5 (1C, C-24); 136.7

(1C, C-14); 131.3 (1C, C-16); 129.9 (1C, C-3); 128.3 (1C, C-6); 125.3 (1C,

C-4); 124.7 (1C, C-5); 123.3 (2C, C-15 and C-26); 121.2 (1C, C-25); 119.7

(1C, C-10); 116.8 (1C, C-7); 115.6 (1C, C-17); 115.2 (1C, C-20); 113.2 (1C,


C-2); 63.0 (1C, C-13); 56.2 (1C, C-19); 48.1 (1C, C-23); 38.9 (1C, C-21);

29.6 (1C, C-22).


1)

IR: νmax / cm-1

3140, 1680, 1601


+, 20%),

460.1851 ([M+H+2 {isotope}]+, 2%)

Calculated for [C26H23N3O5 + H]: 458.1716; found: 458.1721



Empirical formula C30H36N3O7S2





Space group P21/c


a = 10.297(3) Å

b = 17.004(5) Å

c = 18.512(5) Å

α = 90°

β = 108.601(15)°

γ = 90°

Volume 3072.0(15) Å3

Z 4








R1 = 0.1064, wR2 = 0.2047

5.3.2.6 1-(3-(1H-imidazol-1-yl)propyl)-4-(benzofuran-2-carbonyl)-5-(4-(tert-butyl)phenyl)-3-

hydroxy-1H-pyrrol-2(5H)-one (11.6)






white precipitate forming. One molar equivalent of tert-butylbenzaldehyde (0.816 mmol; 0.137 mL)

was added to the mixture and stirred at room temperature for 5 minutes. The solvent was removed

in vacuo and title compound 11.6 was isolated as a yellow powder (0.571 mmol; 0.276 g; 70%

yield). No further purification was needed.

Yield: 70%

1H NMR: (400 MHz, CD3OD) δH 8.96 (1H, s, H-27); 7.85 (1H, s, H-7); 7.69 (1H, d, J =

8.0 Hz, H-5); 7.63 (1H, s, H-29); 7.56-7.54 (2H, m, H-2 and H-28); 7.47 (1H,

t, J = 7.8 Hz, H-3); 7.33-7.26 (5H, m, H-4, H-15, H-16, H-22 and H-23); 5.85

(1H, s, H-13); 4.47 (2H, t, J = 7.4 Hz, H-26, minor product); 4.33 (1H, dt, J =

14.6 and 7.1 Hz, H-26A of the ABX system, major product); 4.23 (1H, dt, J =

13.2 and 6.3 Hz, H-26B of the ABX system, major product); 3.62 (1H, dt, J =

14.7 and 7.1 Hz, H-24A of the ABX system, major product); 3.09 (2H, t, J =

7.5 Hz, H-24, minor product); 2.99 (1H, dt, J = 14.4 and 6.4 Hz, H-24B of the

ABX system, major product); 2.37 (2H, p, J = 7.6 Hz, H-25, minor product);

2.16-2.0 (2H, m, H-25, major product); 1.15 (9H, s, H-19, H-20 and H-21).


155.1 (1C, C-1); 151.6 (1C, C-8); 150.6 (1C, C-17); 134.6 (1C, C-27); 131.8

(1C, C-14); 128.2 (1C, C-3); 127.1 (2C, C-15 and C-23); 126.3 (1C, C-6);

125.3 (2C, C-16 and C-22); 123.5 (1C, C-4); 122.9 (1C, C-5); 121.4 (1C, C-

29); 119.4 (1C, C-28); 118.1 (1C, C-10); 115.4 (1C, C-7); 111.3 (1C, C-2);


60.9 (1C, C-13); 46.2 (1C, C-26); 37.1 (1C, C-24); 33.6 (3C, C-19, C-20 and

C-21); 29.9 (1C, C-25); 27.7 (1C, C-18).

15N NMR: (40 MHz, MeOD) δN 182,2 (1N, s, N-3); 173.1 (1N, s, N-2)

IR: νmax / cm-1

3121, 1680, 1603


+, 24%),

486.2369 ([M+H+2 {isotope}]+, 2%)



5.3.2.7 4-(benzofuran-2-carbonyl)-3-hydroxy-1-(3-morpholinopropyl)-5-phenyl-1H-pyrrol-2(5H)-

one (12.1)



0.200 g (0.816 mmol) of 8 was dissolved in 5 mL of 1,4-dioxane. One molar equivalent of 4-

aminopropyl-morpholine (0.816 mmol; 0.119 mL) was added to the dioxane mixture, resulting in a

yellow precipitate forming. One molar equivalent of benzaldehyde (0.816 mmol; 0.083 mL) was

added to the mixture and stirred at room temperature for 5 minutes. The solvent was removed in

vacuo and title compound 12.1 was isolated as a yellow powder (0.400 mmol; 0.179 g; 49% yield).

No further purification was needed.

Yield: 49%

1H NMR: (400 MHz, CD3OD) δH 7.77 (1H, s, H-7); 7.64 (1H, d, J = 8.0 Hz, H-5); 7.53

(1H, d, J = 8.4 Hz, H-2); 7.40 (1H, t, J = 7.8 Hz, H-3); 7.34 (2H, d, J = 7.6

Hz, H-16 and H-18); 7.25-7.14 (4H, m, H-15, H-17, H-19 and H-4); 5.89

(1H, s, H-13); 3.93 (4H, d, J = 12.0 Hz, H-23 and H-26); 3.79 (4H, d, J =

12.4 Hz, H-24 and H-25); 3.66-3.31 (2H, m, H-20); 3.16-2.92 (2H, m, H-22);

2.18-1.86 (2H, m, H-21).



157.2 (1C, C-1); 152.1 (1C, C-8); 136.6 (1C, C-14); 130.4 (3C, C-15, C-17

and C-19); 130.1 (1C, C-3); 129.4 (2C, C-16 and C-18); 128.3 (1C, C-6);

125.4 (1C, C-4); 124.9 (1C, C-5); 119.4 (1C, C-10); 117.4 (1C, C-7); 113.5

(1C, C-2); 65.1 (2C, C-24 and C-25); 63.0 (2C, C-23 and C-26); 56.0 (1C, C-

13); 53.6 (1C, C-22); 39.5 (1C, C-20); 23.7 (1C, C-21).

15N NMR: (40 MHz, MeOD) δN 140.3 (1N, s, N-1), 31.8 (1N, s, N-2)

IR: νmax / cm-1

3121, 1694, 1599


+, 20%),

449.2064 ([M+H+2 {isotope}]+, 3%)



5.3.2.8 4-(benzofuran-2-carbonyl)-5-(2-chlorophenyl)-3-hydroxy-1-(3-morpholinopropyl)-1H-

pyrrol-2(5H)-one (12.2)

Cl

N

O

HO N

O

O

O

12

3

4

56 7

8

9 10

11

12

13

14

15

1617

18

19

20

21 22

23

2425

26

Exact Mass: 480.1452Molecular Weight: 480.9401

N-1

N-2





yellow precipitate forming. One molar equivalent of 2-chlorobenzaldehyde (0.816 mmol; 0.092

mL) was added to the mixture and stirred at room temperature for 5 minutes. The solvent was


74% yield) that crystallised as 12.2-HCl from the acidified solution during characterisation. No

further purification was needed.

Yield: 74%

1H,

13C and

15N NMR: No solution-based characterisation data could be obtained as product

salt 12.2-HCl crystallised from the acidified solution. The resulting


product was insoluble in a range of common NMR solvents (MeOD,

DMSO, D2O and CDCl3).

IR: νmax / cm-1

3119, 1686, 1605


+,

29%), 484.1630 ([M+H+3 {isotope}]+, 5%)

Calculated for [C26H25ClN2O5 + H]: 481.1530; found: 481.1543.


5.3.2.9 4-(Benzofuran-2-carbonyl)-5-(4-fluorophenyl)-3-hydroxy-1-(3-morpholinopropyl)-1H-






yellow precipitate forming. One molar equivalent of 4-fluorobenzaldehyde (0.816 mmol; 0.088 mL)




Yield: 62%


(1H, d, J = 8.4 Hz, H-2); 7.43-7.36 (3H, m, H-3, H-15 and H-19); 7.23 (1H, t,

J = 7.4 Hz, H-4); 6.96 (2H, t, J = 8.6 Hz, H-16 and H-18); 5.90 (1H, s, H-13);

3.95-3.76 (4H, m, H-24 and H-25); 3.68-3.36 (2H, m, H-22); 3.26-2.88 (6H,

m, H-20, H-23 and H-26); 2.18-1.93 (2H, m, H-21).


164.2 (1C, d, JC,F = 248.6 Hz, C-17); 157.3 (1C, C-1); 151.7 (1C, C-8); 132.3

(1C, C-14); 131.4 (2C, d, JC,F = 8.6 Hz, C-15 and C-19); 130.3 (1C, C-3);


128.2 (1C, C-6); 125.6 (1C, C-4); 124.8 (1C, C-5); 119.0 (1C, C-10); 117.4

(2C, d, JC,F = 21.8 Hz, C-16 and C-18); 117.4 (1C, C-7); 113.7 (1C, C-2);

68.4 (2C, C-24 and C-25); 65.1 (2C, C-23 and C-26); 61.7 (1C, C-13); 53.8

(1C, C-22); 39.0 (1C, C-20); 23.6 (1C, C-21).

15N NMR: (40 MHz, MeOD) δN 140.6 (1N, s, N-1).

IR: νmax / cm-1

3119, 1688, 1603


+, 21%),

467.1973 ([M+H+2 {isotope}]+, 3%)

Calculated for [C26H25FN2O5 + H]: 465.1826; found: 465.1834


5.3.2.10 4-(benzofuran-2-carbonyl)-3-hydroxy-5-(4-methoxyphenyl)-1-(3-morpholinopropyl)-1H-






yellow precipitate forming. One molar equivalent of 4-methoxybenzaldehyde (0.816 mmol; 0.099




Yield: 54%



Hz, H-16 and H-19); 7.20 (1H, t, J = 7.6 Hz, H-4); 6.75 (2H, d, J = 8.8 Hz, H-

15 and H-20); 5.84 (1H, s, H-13); 3.93 (4H, d, J = 12.4 Hz, H-25 and H-26);


3.78 (4H, t, J = 12.2 Hz, H-24 and H-27); 3.58 (3H, s, H-18); 3.36 (2H, t, J =

14.6 Hz, H-23); 3.09-2.92 (2H, m, H-21); 1.99-1.91 (2H, m, H-22).


160.1 (1C, C-12); 157.1 (1C, C-1); 152.5 (1C, C-8); 130.7 (2C, C-16 and C-

19); 129.9 (1C, C-3); 128.4 (1C, C-14); 128.3 (1C, C-6); 125.3 (1C, C-4);

124.7 (1C, C-5); 119.9 (1C, C-10); 116.8 (1C, C-7); 115.5 (2C, C-15 and C-

20); 113.2 (1C, C-2); 65.0 (2C, C-25 and C-26); 62.6 (1C, C-13); 59.0 (2C,

C-24 and C-27); 53.5 (1C, C-23); 52.9 (1C, C-18); 39.1 (1C, C-21); 23.6 (1C,

C-22).

15N NMR: (40 MHz, MeOD) δN 140.3 (1N, s, N-1)

IR: νmax / cm-1

1690, 1601


+, 22%),

479.2191 ([M+H+2 {isotope}]+, 3%)



5.3.2.11 4-(benzofuran-2-carbonyl)-3-hydroxy-5-(3-methoxyphenyl)-1-(3-morpholinopropyl)-1H-






yellow precipitate forming. One molar equivalent of 3-methoxybenzaldehyde (0.816 mmol; 0.099




Yield: 52%



(1H, dd, J = 8.4 and 0.4 Hz, H-2); 7.32 (1H, dt, J = 7.9 and 1.0 Hz, H-3); 7.13

(1H, dt, J = 7.6 and 0.8 Hz, H-4); 6.85 (1H, s, H-15); 6.64 (1H, d, J = 7.2 Hz,

H-19); 6.57 (2H, d, J = 8.0 Hz, H-18 and H-20); 5.77 (1H, s, H-13); 3.93

(4H, d, J = 12.4 Hz, H-25 and H-26); 3.70 (4H, d, J = 12.6 Hz, H-24 and H-

27); 3.59 (3H, s, H-17); 3.57-3.48 (2H, m, H-23); 3.30-2.88 (2H, m, H-21);

1.97-1.78 (2H, m, H-22).


157.1 (1C, C-1); 152.7 (1C, C-8); 149.3 (1C, C-16); 148.1 (1C, C-14); 129.9

(1C, C-3); 128.3 (1C, C-6); 125.3 (1C, C-4); 124.7 (1C, C-5); 121.9 (1C, C-

19); 120.0 (1C, C-10); 116.7 (3C, C-7, C-18 and C-20); 113.3 (2C, C-2 and

C-15); 65.0 (2C, C-24 and C-27); 63.0 (2C, C-25 and C-26); 55.8 (1C, C-13);

53.5 (1C, C-23); 52.8 (1C, C-17); 39.2 (1C, C-21); 23.6 (1C, C-22).

15N NMR: (40 MHz, MeOD) δN 139.7 (s, 1N).

IR: νmax / cm-1

1684, 1609


+, 31%),

479.2193 ([M+H+2 {isotope}]+, 4%)


Melting point: 271.5–272.5 °C

5.3.2.12 4-(benzofuran-2-carbonyl)-5-(4-(tert-butyl)phenyl)-3-hydroxy-1-(3-morpholinopropyl)-






yellow precipitate forming. One molar equivalent of tert-butylbenzaldehyde (0.816 mmol; 0.137





Yield: 71%


(1H, d, J = 8.4 Hz, H-2); 7.26 (1H, dt, J = 7.8 and 1.0 Hz, H-3); 7.11-7.09

(4H, m, H-15, H-16, H-22 and H-23); 7.07 (1H, t, J = 7.5 Hz, H-4); 5.78 (1H,

s, H-13); 3.84-3.73 (4H, m, H-27 and H-30); 3.54-3.45 (4H, m, H-28 and H-

29); 3.25-3.17 (2H, m, H-26); 3.0-2.78 (2H, m, H-24); 1.96-1.80 (2H, m, H-

25); 0.99 (9H, s, H-19, H-20 and H-21).


157.3 (1C, C-12); 153.5 (1C, C-1); 152.0 (1C, C-8); 133.2 (1C, C-14); 130.2

(1C, C-3); 129.1 (2C, C-15 and C-23); 128.3 (1C, C-6); 127.4 (2C, C-16 and

C-22); 125.5 (1C, C-4); 124.9 (1C, C-5); 119.5 (1C, C-10); 117.6 (1C, C-7);

113.6 (1C, C-2); 68.3 (2C, C-28 and C-29); 65.1 (2C, C-27 and C-30); 62.7

(1C, C-13); 53.6 (1C, C-26); 39.4 (1C, C-24); 35.8 (3C, C-19, C-20 and C-

21); 32.4 (1C, C-25); 23.7 (1C, C-18)

15N NMR: (40 MHz, MeOD) δN 141.4 (1N, s, N-1)

IR: νmax / cm-1

1690, 1609


+, 32%),

505.2735 ([M+H+2 {isotope}]+, 4%)



5.3.2.13 4-(benzofuran-2-carbonyl)-1-(3-(dimethylamino)propyl)-3-hydroxy-5-phenyl-1H-pyrrol-

2(5H)-one (13.1)




0.200 g (0.816 mmol) of 8 was dissolved in 5 mL of 1,4-dioxane. One molar equivalent of N1,N1-

dimethyl-1,3-propanediamine (0.816 mmol; 0.103 mL) was added to the dioxane mixture, resulting

in a yellow precipitate forming. One molar equivalent of benzaldehyde (0.816 mmol; 0.083 mL)




Yield: 63%



Hz, H-16 and H-18); 7.23-7.18 (3H, m, H-15, H-17 and H-19); 7.15 (1H, d, J

= 8.0 Hz, H-4); 5.83 (1H, s, H-13); 3.61-3.54 (1H, m, H-20A of the ABX

system); 3.07-2.93 (2H, m, H-22); 2.86-2.80 (1H, m, H-20B of the ABX

system); 2.76 (3H, s, H-23); 2.72 (3H, s, H-24); 1.94-1.72 (2H, m, H-21)


157.0 (1C, C-1); 152.5 (1C, C-8); 137.0 (1C, C-14); 130.2 (3C, C-15, C-17

and C-19); 129.9 (1C, C-3); 129.4 (2C, C-16 and C-18); 128.3 (1C, C-6);

125.3 (1C, C-4); 124.8 (1C, C-5); 119.7 (1C, C-10); 116.9 (1C, C-7); 113.2

(1C, C-2); 63.2 (1C, C-13); 56.3 (1C, C-22); 44.9 (1C, C-23); 43.4 (1C, C-

24); 39.4 (1C, C-20); 24.6 (1C, C-21)

15N NMR: (40 MHz, MeOD) δN 139.6 (1N, s, N-1); 39.2 (1N, s, N-2)

IR: νmax / cm-1

1688, 1611


+, 30%),

407.1844 ([M+H+2 {isotope}]+, 5%)



5.3.2.14 4-(benzofuran-2-carbonyl)-5-(2-chlorophenyl)-1-(3-(dimethylamino)propyl)-3-hydroxy-







in a yellow precipitate forming. One molar equivalent of 2-chlorobenzaldehyde (0.816 mmol; 0.092



62% yield) that crystallised as 13.2-HCl from the acidified solution during characterisation. No

further purification was needed.

Yield: 62%

1H,

13C and

15N NMR: No characterisation data could be obtained as product salt 13.2-HCl

crystallised from the acidified solution. The resulting product was

insoluble in a range of common NMR solvents (MeOD, DMSO, D2O

and CDCl3).

IR: νmax / cm-1

1690, 1609


+,

40%), 442.1432 ([M+H+3 {isotope}]+, 10%)



5.3.2.15 4-(benzofuran-2-carbonyl)-1-(3-(dimethylamino)propyl)-5-(4-fluorophenyl)-3-







in a yellow precipitate forming. One molar equivalent of 4-fluorobenzaldehyde (0.816 mmol; 0.088




Yield: 92%


(1H, d, J = 8.4 Hz, H-2); 7.31-7.27 (3H, m, H-3, H-15 and H-19); 7.09 (1H, t,

J = 7.6 Hz, H-4); 6.56 (2H, t, J = 8.6 Hz, H-16 and H-18); 5.75 (1H, s, H-13);

3.56 (2H, m, H-22); 3.04-2.85 (2H, m, H-20); 2.72 (3H, s, H-24); 2.69 (3H, s,

H-23); 1.85-1.76 (2H, m, H-21).

13C NMR: (100 MHz, CDCl3) δC 177.4 (1C, C-11); 166.6 (1C, C-9); 164.2 (1C, d, JC,F =

246.6 Hz, C-17); 159.8 (1C, C-12); 157.0 (1C, C-1); 152.5 (1C, C-8); 133.1

(1C, d, JC,F = 2.9 Hz, C-14); 131.5 (2C, d, JC,F = 7.7 Hz, C-15 and C-19);

130.0 (1C, C-3); 128.2 (1C, C-6); 125.3 (1C, C-4); 124.8 (1C, C-5); 119.6

(1C, C-10); 117.0-116.8 (3C, m, C-7, C-1 and C-18); 113.2 (1C, C-2); 62.4

(1C, C-13); 56.4 (1C, C-22); 44.0 (1C, C-23); 43.5 (1C, C-24); 39.3 (1C, C-

20); 24.7 (1C, C-21).

15N NMR: (40 MHz, MeOD) δN 139.3 (1N, s, N-1); 39.6 (10N, s, N-2).

IR: νmax / cm-1

1692, 1605


+, 30%),

425.18 ([M+H+2 {isotope}]+, 5%)





13.3a:

Empirical formula C24H23FN2O4





Space group P21/c


a = 13.6407(5) Å

b = 17.1711(7) Å

c = 18.0711(7) Å

α = 90°

β = 95.0980(10)°

γ = 90°

Volume 4216.0(3) Å3

Z 8








R1 = 0.0496 wR2 = 0.1092

13.3b:

Empirical formula C27H19FO4





Space group C2/c


a = 19.754(2) Å

b = 5.8170(6) Å

c = 35.699(4) Å

α = 90°

β = 99.276(4)°

γ = 90°

Volume 4048.6(7) Å3

Z 8








Final R-indices [I>2sigma(I)] R1 = 0.0658 wR2 = 0.1572

5.3.2.16 4-(benzofuran-2-carbonyl)-1-(3-(dimethylamino)propyl)-3-hydroxy-5-(4-methoxyphenyl)-






in a yellow precipitate forming. One molar equivalent of p-methoxybenzaldehyde (0.816 mmol;




Yield: 67%


(1H, d, J = 8.4 Hz, H-2); 7.31 (1H, dt, J = 7.8 and 0.8 Hz, H-3); 7.17 (2H, d, J

= 8.4 Hz, H-16 and H-18); 7.12 (1H, t, J = 7.8 Hz, H-4); 6.67 (2H, d, J = 8.8

Hz, H-15 and H-20); 5.73 (1H, s, H-13); 3.50 (5H, m, H-18 and H-23); 3.01-

2.88 (2H, m, H-21); 2.71 (3H, s, H-24); 2.68 (3H, s, H-25); 1.85-1.69 (2H, m,

H-22).


160.2 (1C, C-12); 157.0 (1C, C-1); 152.5 (1C, C-8); 130.6 (2C, C-16 and C-

19); 129.9 (1C, C-3); 128.5 (1C, C-14); 128.3 (1C, C-6); 125.3 (1C, C-4);

124.7 (1C, C-5); 119.8 (1C, C-10); 116.9 (1C, C-7); 115.5 (2C, C-15 and C-


20); 113.2 (1C, C-2); 68.1 (1C, C-18); 62.8 (1C, C-13); 56.4 (1C, C-23); 44.0

(1C, C-24); 43.4 (1C, C-25); 39.3 (1C, C-21); 24.6 (1C, C-22).


IR: νmax / cm-1

1690, 1611


+, 24%)









in a yellow precipitate forming. One molar equivalent of m-methoxybenzaldehyde (0.816 mmol;




Yield: 69%


(1H, d, J = 8.4 Hz, H-2); 7.34 (1H, t, J = 7.8 Hz, H-3); 7.13 (1H, t, J = 7.4

Hz, H-4); 7.06 (1H, t, J = 8.0 Hz, H-19); 6.84 (1H, s, H-15); 6.79 (1H, d, J =

7.6 Hz, H-20); 6.64 (1H, dd, J = 8.4 and 2.0 Hz, H-18); 5.77 (1H, s, H-13);

3.54-3.50 (5H, m, H-17 and H-23); 3.02-2.88 (2H, m, H-21); 2.71 (3H, s, H-

24); 2.67 (3H, s, H-25); 1.90-1.68 (2H, m, H-22).


159.7 (1C, C-12); 157.0 (1C, C-1); 152.6 (1C, C-8); 138.5 (1C, C-14); 131.4


(1C, C-19); 129.9 (1C, C-3); 128.3 (1C, C-6); 125.4 (1C, C-4); 124.8 (1C, C-

5); 121.1 (1C, C-20); 119.8 (1C, C-10); 116.8 (1C, C-7); 115.3 (2C, C-15 and

C-18); 113.2 (1C, C-2); 68.1 (1C, C-17); 63.2 (1C, C-13); 56.1 (1C, C-23);

44.0 (1C, C-24); 43.3 (1C, C-25); 39.4 (1C, C-21); 24.6 (1C, C-22).


IR: νmax / cm-1

1697, 1605


+, 31%),

437.2032 ([M+H+2 {isotope}]+, 8%)



5.3.2.18 4-(benzofuran-2-carbonyl)-5-(4-(tert-butyl)phenyl)-1-(3-(dimethylamino)propyl)-3-






in a yellow precipitate forming. One molar equivalent of tert-butylbenzaldehyde (0.816 mmol;




Yield: 70%

1H NMR: (400 MHz, CD3OD) δH 7.71 (1H, d, J = 0.8 Hz, H-7); 7.56 (1H, d, J = 7.6

Hz, H-5); 7.43 (1H, d, J = 7.2 Hz, H-2); 7.34 (1H, dt, J = 7.7 and 1.0 Hz, H-

3); 7.19 (4H, s, H-15, H-16, H-22 and H-23); 7.16 (1H, dt, J = 7.6 and 0.8 Hz,

H-4); 5.77 (1H, s, H-13); 3.56 (2H, d, J = 6.8 Hz, H-24); 2.96 (2H, d, J = 6.7


Hz, H-26); 2.73 (3H, s, H-27); 2.68 (3H, s, H-28); 1.85 (2H, dp, J = 35.7 and

6.2 Hz, H-25); 1.08 (9H, s, H-19, H-20 and H-21).


157.1 (1C, C-1); 153.3 (1C, C-8); 152.9 (1C, C-17); 134.0 (1C, C-14); 129.8


C-23); 125.2 (1C, C-4); 124.7 (1C, C-5); 120.1 (1C, C-10); 116.7 (1C, C-7);

113.0 (1C, C-2); 63.1 (1C, C-13); 56.4 (1C, C-26); 43.7 (1C, C-27); 43.3 (1C,

C-28); 39.1 (1C, C-24); 31.6 (3C, C-19, C-20 and C-21); 24.6 (1C, C-25);

24.2 (1C, C-18).

15N NMR: (40 MHz, MeOD) δN 139.8 (1N, s, N-1), 40.2 (1N, s, N-2).

IR: νmax / cm-1

3036, 1690, 1605


+, 34%),

463.2516 ([M+H+2 {isotope}]+, 8%)



5.3.2.19 1-(3-aminopropyl)-4-(benzofuran-2-carbonyl)-5-(2-chlorophenyl)-3-hydroxy-1H-pyrrol-

2(5H)-one (14.1)

NH2

O

HO N

O

OCl

12

3

4

56

7

8

9 10

11

12

13

14

15

16 17

18

19

20

21 22


N-1

N-2



0.200 g (0.816 mmol) of 8 was dissolved in 5 mL of 1,4-dioxane. One molar equivalent of 1,3-

diaminopropane (0.816 mmol; 0.068 mL) was added to the dioxane mixture, resulting in a yellow

precipitate forming. One molar equivalent of 2-chlorobenzaldehyde (0.816 mmol; 0.092 mL) was




Yield: 33%



(1H, d, J = 8.4 Hz, H-2); 7.38-7.34 (2H, m, H-3 and H-15); 7.20-7.11 (2H, m,

H-17 and H-18); 7.08 (1H, dt, J = 7.4 and 1.2 Hz, H-4); 6.99 (1H, dd, J = 7.6

and 1.6 Hz, H-15); 6.32 (1H, s, H-13); 3.70 (2H, m, H-20); 3.04-2.84 (2H, m,

H-22); 1.99=1.71 (2H, m, H-21).


157.1 (1C, C-1); 152.3 (1C, C-8); 136.5 (1C, C-14); 135.0 (1C, C-1); 131.7

(2C, C-17 and C-18); 131.1 (1C, C-15); 130.1 (1C, C-3); 129.4 (1C, C-16);

128.2 (1C, C-6); 125.4 (1C, C-4); 124.8 (1C, C-5); 118.3 (1C, C-10); 116.8

(1C, C-7); 113.2 (1C, C-2); 64.2 (1C, C-20); 58.7 (1C, C-13); 38.4 (1C, C-

22); 27.5 (1C, C-21).


IR: νmax / cm-1

1694, 1638


+, 43%),

414.1152 ([M+H+3 {isotope}]+, 11%)



5.3.2.20 1-(3-aminopropyl)-4-(benzofuran-2-carbonyl)-3-hydroxy-5-(4-methoxyphenyl)-1H-






precipitate forming. One molar equivalent of p-methoxybenzaldehyde (0.816 mmol; 0.099 mL) was





Yield: 36%


7.55-7.50 (1H, m, H-2); 7.44-7.38 (1H, m, H-3); 7.28-7.20 (3H, m, H-4, H-16

and H-19); 6.76 (2H, d, J = 8.8 Hz, H-15 and H-20); 5.81 (1H, s, H-13); 3.60

(3H, s, H-18); 3.31 (2H, t, J = 6.6 Hz, H-21); 2.90 (2H, t, J = 7.6 Hz, H-23);

1.83 (2H, p, J = 7.13 Hz, H-22).


159.6 (1C, C-12); 157.1 (1C, C-1); 152.8 (1C, C-8); 134.6 (1C, C-14); 130.5

(2C, C-16 and C-19); 130.0 (1C, C-3); 128.3 (1C, C-6); 125.4 (1C, C-4);

124.8 (1C, C-5); 120.0 (1C, C-10); 115.6 (2C, C-15 and C-20); 115.5 (1C, C-

7); 113.2 (1C, C-2); 68.1 (1C, C-18); 62.8 (1C, C-13); 38.0 (1C, C-21); 28.1

(1C, C-23); 26.4 (1C, C-22).


IR: νmax / cm-1

3285, 1697, 1640


+, 34%)



5.3.2.21 1-(3-aminopropyl)-4-(benzofuran-2-carbonyl)-3-hydroxy-5-(4-hydroxy-3-






precipitate forming. One molar equivalent of vanillin (0.816 mmol; 0.124 g) was added to the

mixture and stirred at room temperature for 5 minutes. The solvent was removed in vacuo and title


compound 14.3 was isolated as a yellow powder (0.441 mmol; 0.186 g; 54% yield). No further

purification was needed.

Yield: 54%

1H,

13C and

15N NMR: No solution-based characterisation data could be obtained as the

product was not sufficiently soluble in the acidified media.

Additionally, the resulting product was insoluble in a range of

common NMR solvents (MeOD, DMSO, D2O and CDCl3).

IR: νmax / cm-1

3296, 1697, 1647

LR TOF-ESI+: m/z 423.2 ([M+H]+, 20%)


5.3.2.22 1-(3-aminopropyl)-4-(benzofuran-2-carbonyl)-5-(2,4-dimethoxyphenyl)-3-hydroxy-1H-






precipitate forming. One molar equivalent of 2,4-dimethoxybenzaldehyde (0.816 mmol; 0.136 g)




Yield: 50%

1H,

13C and






IR: νmax / cm-1

3294, 1699, 1645

LR TOF-ESI+: m/z 435.2 ([M-H]+, 65%)

LR TOF-ESI-: m/z 437.1 ([M+H]-, 100%)


5.3.2.23 1-(3-aminopropyl)-4-(benzofuran-2-carbonyl)-5-(2,4-dihydroxyphenyl)-3-hydroxy-1H-






precipitate forming. One molar equivalent of 2,4-dihydroxybenzaldehyde (0.816 mmol; 0.113 g)


in vacuo and title compound 14.5 was isolated as a dark brown powder (0.465 mmol; 0.190 g; 57%


Yield: 57%


Hz, H-5); 7.53 (1H, dd, J = 7.6 and 0.8 Hz, H-2); 7.41 (1H, dt, J = 7.8 and 1.2

Hz, H-3); 7.24-7.22 (3H, m, H-4, H-16 and H-18); 6.76 (1H, d, J = 8.8 Hz, H-

19); 5.81 (1H, s, H-13); 3.30 (2H, t, J = 6.0 Hz, H-20); 2.91-2.78 (2H, m, H-

22); 1.90-1.79 (2H, m, H-21).

13C and

15N NMR: No solution-based characterisation data could be obtained as the product was

not sufficiently soluble in the acidified media. Additionally, the resulting

product was either minimally soluble or insoluble in a range of common

NMR solvents (MeOD, DMSO, D2O and CDCl3).

IR: νmax / cm-1

3283, 1647

LR TOF-ESI+: m/z 407.2 ([M-H]+, 65%)



5.3.2.24 1-(3-aminopropyl)-4-(benzofuran-2-carbonyl)-5-(4-(tert-butyl)phenyl)-3-hydroxy-1H-






precipitate forming. One molar equivalent of tert-butylbenzaldehyde (0.816 mmol; 0.137 mL) was




Yield: 63%



Hz, H-3); 7.26-7.18 (5H, m, H-4, H-15, H-16, H-22 and H-23); 5.79 (1H, s,

H-13); 3.61 (2H, d, J = 7.3 Hz, H-24); 2.91 (2H, dt, J = 14.6 and 6.6 Hz, H-

26); 2.03 (2H, p, J = 7.7 Hz, H-25); 1.09 (9H, s, H-19, H-20 and H-21).


157.1 (1C, C-1); 153.3 (1C, C-17); 152.6 (1C, C-8); 133.6 (1C, C-14); 130.0


C-23); 125.3 (1C, C-4); 124.8 (1C, C-5); 120.1 (1C, C-10); 117.0 (1C, C-7);

113.2 (1C, C-2); 68.1 (1C, C-24); 62.8 (1C, C-13); 38.3 (1C, C-26); 35.5 (1C,

C-18); 31.7 (3C, C-19, C-20 and C-21); 27.1 (1C, C-25).


IR: νmax / cm-1

1678, 1647



+, 33%),

435.2224 ([M+H+2 {isotope}]+, 5%)



5.3.2.25 1-(2-aminoethyl)-4-(benzofuran-2-carbonyl)-3-hydroxy-5-phenyl-1H-pyrrol-2(5H)-one

(15.1)




diaminoethane (0.816 mmol; 0.055 mL) was added to the dioxane mixture, resulting in a yellow

precipitate forming. One molar equivalent of benzaldehyde (0.816 mmol; 0.083 mL) was added to

the mixture and stirred at room temperature for 5 minutes. The solvent was removed in vacuo and

title compound 15.1 was isolated as a yellow powder (0.367 mmol; 0.133 g; 45% yield). No further


Yield: 45%

1H NMR: (400 MHz, CD3OD) δH 5.87 (1H, s, H-13).

13C NMR: (100 MHz, CD3OD) δC 177.9 (1C, C-11); 167.5 (1C, C-9); 159.6 (1C, C-12);

157.1 (1C, C-1); 152.6 (1C, C-8); 136.6 (1C, C-14); 130.7-129.7 (4C, m, C-3,

C-15, C-17 and C-19); 129.4 (2C, C-16 and C-18); 128.3 (1C, C-6); 125.4

(1C, C-4); 124.8 (1C, C-5); 120.2 (1C, C10); 117.0 (1C, C-7); 113.2 (1C, C-

2); 68.1 (1C, C-20); 63.4 (1C, C-13); 38.0 (1C, C-21).

15N NMR: (40 MHz, CD3OD) δN 134.6 (1N, s, N-1); 30.8 (1N, s, N-2).

IR: νmax / cm-1

1640


+, 25%),

365.1154 ([M+H+2 {isotope}]+, 11%)




5.3.2.26 1-(2-aminoethyl)-4-(benzofuran-2-carbonyl)-5-(2-chlorophenyl)-3-hydroxy-1H-pyrrol-

2(5H)-one (15.2)









Yield: 42%



159.6 (1C, C-12); 157.2 (1C, C-1); 151.9 (1C, C-8); 136.6 (1C, C-14); 135.0

(1C, C-19); 131.8 (1C, C-16); 130.7 (1C, C-3); 129.3 (1C, C-17); 128.3 (1C,

C-6); 127.9 (1C, C-18); 125.4 (1C, C-4); 124.8 (1C, C-5); 119.1 (1C, C-10);

116.9 (1C, C-7); 113.3 (1C, C-2); 68.1 (1C, C-20); 58.7 (1C, C-13); 38.0 (1C,

C-21).


IR: νmax / cm-1

1640


+, 39%),

400.0965 ([M+H+3 {isotope}]+, 10%)



5.3.2.27 1-(2-aminoethyl)-4-(benzofuran-2-carbonyl)-5-(4-fluorophenyl)-3-hydroxy-1H-pyrrol-

2(5H)-one (15.3)






precipitate forming. One molar equivalent of 4-fluorobenzaldehyde (0.816 mmol; 0.088 mL) was




Yield: 49%


(1H, d, J = 8.8 Hz, H-2); 7.38 (3H, t, J = 7.2 Hz, H-3, H-15 and H-19); 7.19

(1H, t, J = 7.4 Hz, H-4); 7.00-6.92 (2H, m, H-16 and H-18); 5.88 (1H, s, H-

13); 3.82-3.56 (2H, m, H-20); 3.29-3.01 (2H, m, H-21).

13C NMR: (100 MHz, CD3OD) δC 177.6 (1C, C-11); 167.4 (1C, C-9); 164.3 (1C, d, JC,F

= 246.8 Hz, C-17); 159.2 (1C, C-12); 157.0 (1C, C-1); 152.5 (1C, C-8); 132.7

(1C, C-14); 131.6 (2C, d, JC,F = 7.9 Hz, C-15 and C-19); 130.0 (1C, C-3);

128.3 (1C, C-6); 125.4 (1C, C-4); 124.8 (1C, C-5); 120.0 (1C, C-10); 117.0-

116.9 (3C, m, C-7, C-16 and C-18); 113.2 (1C, C-2); 68.1 (1C, C-20); 62.6

(1C, C-13); 39.4 (1C, C-21).


IR: νmax / cm-1

1699, 1636


+, 24%),

383.1302 ([M+H+2 {isotope}]+, 3%)



5.3.2.28 1-(2-aminoethyl)-4-(benzofuran-2-carbonyl)-3-hydroxy-5-(4-methoxyphenyl)-1H-pyrrol-

2(5H)-one (15.4)






precipitate forming. One molar equivalent of p-anisaldehyde (0.816 mmol; 0.099 mL) was added to




Yield: 38%



Hz, H-3); 7.24 (2H, d, J = 8.8 Hz, H-15 and H-20); 7.20 (1H, dt, J = 7.6 and

0.8 Hz, H-4); 6.74 (2H, d, J = 8.8 Hz, H-16 and H-19); 5.83 (1H, s, H-13);

3.78-3.74 (2H, m, H-20); 3.57 (3H, s, H-18); 3.25-2.92 (2H, m, H-21).


159.5 (1C, C-12); 157.1 (1C, C-1); 152.6 (1C, C-8); 130.7 (2C, C-15 and C-

20); 130.0 (1C, C-3); 128.3 (1C, C-6); 128.1 (1C, C-14); 125.3 (1C, C-4);

124.8 (1C, C-5); 120.2 (1C, C-10); 117.0 (1C, C-7); 115.6 (2C, C-16 and C-

19); 113.2 (1C, C-2); 68.2 (1C, C-20); 62.9 (1C, C-13); 55.9 (1C, C-18); 39.4

(1C, C-21).


IR: νmax / cm-1

1699, 1611


+, 27%),

395.1447 ([M+H+2 {isotope}]+, 3%)




5.3.2.29 1-(2-aminoethyl)-4-(benzofuran-2-carbonyl)-3-hydroxy-5-(3-methoxyphenyl)-1H-pyrrol-

2(5H)-one (15.5)

NH2O

HO N

O

O

O

12

3

4

56

7

8

9 10

11

12

13

14

15

16

17

1819

20

21

22


N-1

N-2





precipitate forming. One molar equivalent of m-anisaldehyde (0.816 mmol; 0.099 mL) was added to




Yield: 51%



Hz, H-3); 7.22 (1H, t, J = 8.0 Hz, H-4); 7.18-7.12 (1H, m, H-19); 6.92 (1H, s,

H-15); 6.89-6.84 (2H, m, H-18 and H-20); 5.87 (1H, s, H-13); 3.26 (3H, s, H-

17); H-21 and H-22 could not be resolved.

13C NMR: No solution-based characterisation data could be obtained as the product was




15N NMR: (40 MHz, CD3OD) δN 133.4 (1N, s, N-1).

IR: νmax / cm-1

1711, 1624


+, 21%)



5.3.2.30 1-(2-aminoethyl)-4-(benzofuran-2-carbonyl)-3-hydroxy-5-(4-hydroxy-3-methoxyphenyl)-











Yield: 22%

1H,

13C and





IR: νmax / cm-1

3293, 1653, 1632

HR TOF-ESI-: m/z 407.1263 ([M-H]-, 50%)

Calculated for [C22H20N2O6 – H]: 407.1243; found: 407.1263


5.3.2.31 1-(2-aminoethyl)-4-(benzofuran-2-carbonyl)-5-(2,4-dimethoxyphenyl)-3-hydroxy-1H-







precipitate forming. One molar equivalent of 2,4-dimethoxybenzaldehyde (0.816 mmol; 0.136 g)




Yield: 23%

1H,

13C and





IR: νmax / cm-1

3296, 3213, 1717, 1655

LR TOF-ESI+: m/z 423.1 ([M+H]+, 18%)

LR TOF-ESI-: m/z 421.1 ([M+H]-, 95%)

Melting point: Decomposed at 270 °C

5.3.2.32 1-(2-aminoethyl)-4-(benzofuran-2-carbonyl)-5-(2,4-dihydroxyphenyl)-3-hydroxy-1H-











Yield: 29%

1H,

13C and





IR: νmax / cm-1

3293, 3217, 1699, 1655

LR TOF-ESI+: m/z 393.1 ([M-H]+, 9%)

Melting point: Decomposed at 240 °C

5.3.2.33 1-(2-aminoethyl)-4-(benzofuran-2-carbonyl)-5-(4-(tert-butyl)phenyl)-3-hydroxy-1H-










Yield: 53%


(1H, d, J = 8.0 Hz, H-2); 7.53-7.50 (1H, m, H-3); 7.43-7.39 (3H, m, H-4, H-

15 and H-23); 7.31-7.21 (2H, m, H-16 and H-22); 5.24 (1H, s, H-13); 3.53

(2H, t, J = 6.0 Hz, H-24); 3.08 (2H, t, J = 5.8 Hz, H-25); 1.23 (9H, s, H-19,

H-20 and H-21).

13C and






IR: νmax / cm-1

3295, 3217, 1699, 1645

HR TOF-ESI-: m/z 417.1832 ([M-H]-, 100%); 418.1904 ([M {isotope}]

-, 36%), 419.1919

([M+H {isotope}]-, 8%)

Calculated for [C25H26N2O4 – H]: 417.1814; found: 417.1832


5.3.2.34 1-(7-aminoheptyl)-4-(benzofuran-2-carbonyl)-3-hydroxy-5-phenyl-1H-pyrrol-2(5H)-one

(16.1)

O

HO N

O

O

NH2

12

3

4

56

7

8

9 10

11

12

13

14

15

16 17

18

19

20

21 22

2324


N-1

N-2




diaminopentane (cadaverine; 0.816 mmol; 0.096 mL) was added to the dioxane mixture, resulting in

a yellow precipitate forming. One molar equivalent of benzaldehyde (0.816 mmol; 0.083 mL) was




Yield: 47%



Hz, H-16 and H-18); 7.20 (3H, d, J = 7.2 Hz, H-15, H-17 and H-19); 7.15

(1H, t, J = 7.4 Hz, H-4); 5.84 (1H, s, H-13); 3.64-3.56 (2H, m, H-20); 2.89

(2H, p, J = 6.2 Hz, H-24); 2.06-1.36 (6H, m, H-21, H-22 and H-23).


157.1 (1C, C-1); 152.5 (1C, C-8); 137.0 (1C, C-14); 130.3 (3C, C-15, C-17

and C-19); 130.0 (1C, C-3); 129.3 (2C, C-16 and C-18); 128.3 (1C, C-6);


125.4 (1C, C-4); 124.8 (1C, C-5); 119.7 (1C, C-10); 116.9 (1C, C-7); 113.2

(1C, C-2); 68.2 (1C, C-20); 63.7 (1C, C-13); 51.7 (1C, C-24); 39.0 (1C, C-

23); 31.4 (1C, C-21); 26.2 (1C, C-22).


IR: νmax / cm-1

1674, 1632


+, 26%),

407.1871 ([M+H+2 {isotope}]+, 3%)



5.3.2.35 1-(5-aminopentyl)-4-(benzofuran-2-carbonyl)-5-(2-chlorophenyl)-3-hydroxy-1H-pyrrol-

2(5H)-one (16.2)




diaminopentane (0.816 mmol; 0.096 mL) was added to the dioxane mixture, resulting in a yellow





Yield: 45%

1H,

13C and





IR: νmax / cm-1

1653



+,

38%), 442.1438 ([M+H+3 {isotope}]+, 9%)



5.3.2.36 1-(5-aminopentyl)-4-(benzofuran-2-carbonyl)-5-(4-fluorophenyl)-3-hydroxy-1H-pyrrol-

2(5H)-one (16.3)





precipitate forming. One molar equivalent of 4-fluorobenzaldehyde (0.816 mmol; 0.088 mL) was




Yield: 51%

1H NMR: (400 MHz, CD3OD) δH 7.78-7.73 (1H, m, H-7); 7.64 (1H, t, J = 8.0 Hz, H-5);

7.52-7.48 (1H, m, H-2); 7.41-7.34 (3H, m, H-3, H-16 and H-18); 7.20 (1H, t,

J = 7.6 Hz, H-4); 6.95 (2H, t, J = 8.7 Hz, H-15 and H-19); 5.82 (1H, s, H-

13); 3.64-3.15 (2H, m, H-20); 3.04-2.92 (2H, m, H-24); 1.82-1.45 (6H, m, H-

21, H-22 and H-23).


164.3 (1C, d, JC,F = 246.8 Hz, C-17); 157.1 (1C, C-1); 152.5 (1C, C-8); 132.8

(1C, C-14); 131.5 (2C, C-16 and C-18); 129.9 (1C, C-3); 128.3 (1C, C-6);

125.4 (1C, C-4); 124.8 (1C, C-5); 119.6 (1C, C-10); 117.0 (2C, C-15 and C-


19); 115.8 (1C, C-7); 113.2 (1C, C-2); 62.3 (1C, C-13); 58.9 (1C, C-20); 51.8

(1C, C-24); 38.8 (1C, C-23); 31.7 (1C, C-21); 26.7 (1C, C-22).


IR: νmax / cm-1

1670, 1636

LR TOF-ESI+: m/z 423.2 ([M+H]+, 100%); 424.2 ([M+H+1]

+, 24%)

LR TOF-ESI-: m/z 421.2 ([M-H]-, 100%); 422.2 ([M]

-, 28%)


5.3.2.37 1-(5-aminopentyl)-4-(benzofuran-2-carbonyl)-3-hydroxy-5-(4-methoxyphenyl)-1H-pyrrol-

2(5H)-one (16.4)





precipitate forming. One molar equivalent of p-anisaldehyde (0.816 mmol; 0.099 mL) was added to




Yield: 50%


13C and





IR: νmax / cm-1

1647


+, 30%),

437.1968 ([M+H+2 {isotope}]+, 3%)




5.3.2.38 1-(5-aminopentyl)-4-(benzofuran-2-carbonyl)-3-hydroxy-5-(3-methoxyphenyl)-1H-pyrrol-

2(5H)-one (16.5)





precipitate forming. One molar equivalent of m-anisaldehyde (0.816 mmol; 0.099 mL) was added to




Yield: 54%

1H,

13C and





IR: νmax / cm-1

1649

LR TOF-ESI+: m/z 435.2 ([M+H]+, 100%); 436.2 ([M+H+1]

+, 26%); 437.2

([M+H+2]+, 7%)

LR TOF-ESI-: m/z 433.2 ([M-H]-, 100%); 434.2 ([M]

-, 32%)


5.3.2.39 1-(5-aminopentyl)-4-(benzofuran-2-carbonyl)-3-hydroxy-5-(4-hydroxy-3-











Yield: 35%

1H,

13C and





IR: νmax / cm-1

1638

LR TOF-ESI-: m/z 451.7 ([M+H]-, 26%)


5.3.2.40 1-(5-aminopentyl)-4-(benzofuran-2-carbonyl)-5-(2,4-dimethoxyphenyl)-3-hydroxy-1H-







precipitate forming. One molar equivalent of 2,4-dimethoxybenzaldehyde (0.816 mmol; 0.136 mL)




Yield: 32%

1H,

13C and





IR: νmax / cm-1

1651

LR TOF-ESI+: m/z 465.2 ([M+H]+, 100%); 466.2 ([M+H+1]

+, 30%)

LR TOF-ESI-: m/z 463.2 ([M-H]-, 100%)


5.3.2.41 1-(5-aminopentyl)-4-(benzofuran-2-carbonyl)-5-(2,4-dihydroxyphenyl)-3-hydroxy-1H-











Yield: 29%

1H,

13C and





IR: νmax / cm-1

1638

LR TOF-ESI-: m/z 437.1 ([M+H]-, 28%)


5.3.2.42 1-(5-aminopentyl)-4-(benzofuran-2-carbonyl)-5-(4-(tert-butyl)phenyl)-3-hydroxy-1H-











Yield: 42%

1H,

13C and





IR: νmax / cm-1

3119, 1746, 1611

LR TOF-ESI-: m/z 459.0 ([M-H]-, 51%); 460.0 ([M]

-, 19%); 461.0 ([M+H]

+, 15%)


5.3.3 Salt formation of the substituted 1-aminoalkyl-3-hydroxy-3-pyrrolin-2-ones


methoxyphenyl)-1H-pyrrol-2(5H)-one hydrochloride salt (11.5-HCl)


0.050 g (0.109 mmol) of 11.5 was dissolved in 2.5 mL dry methanol. One drop of 15% HCl was

added to this solution and agitated to mix. Title compound 11.5-HCl precipitated out of the solution

and was isolated as a light yellow powder (0.0899 mmol; 0.044 g; 84% yield).

Yield: 84%


-, 40%); 458.1710

([M+H {isotope}]-, 9%)

Calculated for C26H22N3O5- 456.1559; found 456.1570


5.3.3.2 1-(3-(1H-imidazol-1-yl)propyl)-4-(benzofuran-2-carbonyl)-5-(4-(tert-butyl)phenyl)-3-

hydroxy-1H-pyrrol-2(5H)-one hydrochloride salt (11.6-HCl)



and was isolated as an off-white powder (0.0835 mmol; 0.043 g; 87% yield).

Yield: 87%


+, 34%);

486.2453 ([M+H+2 {isotope}]+, 4%)


Calculated for C29H30N3O4+ 484.2236; found 484.2256.


5.3.3.3 4-(benzofuran-2-carbonyl)-3-hydroxy-1-(3-morpholinopropyl)-5-phenyl-1H-pyrrol-2(5H)-

one hydrochloride salt (12.1-HCl)




Yield: 77%


+, 31%);

449.2128 ([M+H+2 {isotope}]+, 3%)

Calculated for C26H27N2O5+ 447.1920; found 447.1931


5.3.3.4 4-(benzofuran-2-carbonyl)-5-(2-chlorophenyl)-3-hydroxy-1-(3-morpholinopropyl)-1H-

pyrrol-2(5H)-one hydrochloride salt (12.2-HCl)





Yield: 76%


+, 51%);

484.1687 ([M+H+3 {isotope}]+, 10%)

Calculated for C269H26N2O5Cl+ 481.1530; found 481.1540



Empirical formula C26H26Cl2N2O5




Crystal system Orthorhombic

Space group P n a 21


a = 30.505(3) Å

b = 12.7963(14) Å

c = 6.2578(7) Å

α = 90°

β = 90°

γ = 90°

Volume 2442.7(5) Å3

Z 4








5.3.3.5 4-(benzofuran-2-carbonyl)-5-(4-fluorophenyl)-3-hydroxy-1-(3-morpholinopropyl)-1H-






Yield: 79%

HR TOF-ESI+: m/z 465.1838 ([M+H]+, 100); 466.1972 ([M+H+1 {isotope}]

+, 35%);

467.2050 ([M+H+2 {isotope}]+, 3%)

Calculated for C26H26N2O5F+ 465.1826; found 465.1838



Empirical formula C26H26ClFN2O5





Space group P n


a = 5.759(4) Å

b = 20.044(15) Å

c = 10.211(7) Å

α = 90°

β = 100.197(15)°

γ = 90°

Volume 1172.6(15) Å3

Z 2









5.3.3.6 4-(benzofuran-2-carbonyl)-5-(4-(tert-butyl)phenyl)-3-hydroxy-1-(3-morpholinopropyl)-1H-


HN

O

HO N

O

O

ClO





Yield: 77%


-, 89%); 503.2724

([M+H {isotope}]-, 31%)



5.3.3.7 4-(benzofuran-2-carbonyl)-1-(3-(dimethylamino)propyl)-3-hydroxy-5-phenyl-1H-pyrrol-

2(5H)-one hydrochloride salt (13.1-HCl)





Yield: 82%


-, 82%); 405.1950

([M+H {isotope}]-, 22%)



5.3.3.8 4-(benzofuran-2-carbonyl)-5-(2-chlorophenyl)-1-(3-(dimethylamino)propyl)-3-hydroxy-

1H-pyrrol-2(5H)-one hydrochloride salt (13.2-HCl)




Yield: 83%


+, 38%);

442.1413 ([M+H+3 {isotope}]+, 10%)

Calculated for C24H24N2O4Cl+ 439.1425; found 439.1420



Empirical formula C24H24Cl2N2O4





Crystal system Triclinic

Space group P -1


a = 9.8063(12) Å

b = 10.3524(13) Å

c = 11.6777(15) Å

α = 84.013(2)°

β = 84.700(2)°

γ = 72.907(2)°

Volume 1124.6(2) Å3

Z 2








5.3.2.9 4-(benzofuran-2-carbonyl)-1-(3-(dimethylamino)propyl)-5-(4-fluorophenyl)-3-





Yield: 92%


+, 30%);

425.1764 ([M+H+2 {isotope}]+, 5%)

Calculated for C24H24N2O4F+ 423.1720; found 423.1700




1H-pyrrol-2(5H)-one hydrochloride salt (13.5-HCl)



and was isolated as a light yellow powder (0.0943 mmol; 0.044 g; 89% yield).

Yield: 89%


-, 80%); 435.2106

([M+H {isotope}]-, 21%)




Empirical formula C25H29ClN2O6





Space group P -1


a = 6.805(5) Å

b = 10.847(5) Å

c = 16.912(5) Å

α = 74.177(5)°

β = 82.181(5)°

γ = 88.152(5)°

Volume 1189.9(11) Å3

Z 2






Absorption correction Semi-empirical from equivalents



Final R-indices R1 = 0.0385 wR2 = 0.0931

5.3.2.11 4-(benzofuran-2-carbonyl)-5-(4-(tert-butyl)phenyl)-1-(3-(dimethylamino)propyl)-3-





Yield: 83%


-, 89%); 461.2617

([M+H {isotope}]-, 28%)


Melting point: 228.5- 230 °C

5.3.2.12 A co-crystallised set of salts: The hemi-HCl and hemi-oxalic acid forms of morpholine


0.050 g (0.101 mmol) of 12.6 was dissolved in 2.5 mL methanol with trace amounts of 1,4-dioxane.

One drop of 15% HCl was added to this solution and agitated to mix. The title compound

crystallised out of the solution as a by-product.


Empirical formula C9H19ClN2O5





Space group P 2/c


a = 18.949(5) Å

b = 5.685(5) Å

c = 24.783(5) Å

α = 90.000(5)°

β = 109.575(5)°

γ = 90.000(5)°

Volume 2515(2) Å3

Z 8







Final R-indices R1 = 0.0280, wR2 = 0.0785

5.4 Biological evaluation

Aseptic techniques were applied to all biological and biochemical protocols performed with cell

culture. These techniques included the sterilisation of all media and equipment, either by

autoclaving at 121 ˚C for 20 minutes or by spraying with 70% ethanol. This was necessary to avoid

any and all bacterial or fungal contamination. All work with these cell cultures were performed

exclusively in a class II biological safety cabinet with the safety sash down.

The following reagents were obtained through the NIH AIDS Research and Reference Reagent

Program, Division of AIDS, NAID, NIH: PM1 from Dr. Marvin Reitz;24

and pINSD.His.Sol from

Dr. R. Craigie.25


5.4.1 Solubility studies

The aqueous solubility of the described compounds was determined in phosphate buffered saline

(PBS, pH 7.4), using the MultiScreen Solubility Filterplate method as per the manufacturer’s

instructions. Briefly, a 1000 mL volume of 10x PBS stock was prepared by adding 2.0 g KCl (74.55

g/mol; 0.0268 M), 2.40 g KH2PO4 (136.09 g/mol; 0.0176 M), 14.4 g Na2HPO4 (MW = 141.96

g/mol; 0.1014 M) and 80.0 g NaCl (MW = 58.44 g/mol; 1.369 M) to 800 mL ultrapure water. The

solution was mixed to dissolve and the pH adjusted to 7.4 with 1.0 N HCl and q.s. to 1000 mL with

ultrapure water. The final solution was filtered and autoclaved to remove particulates and reduce

bacterial growth. The stock solution was diluted 1:9 to obtain a 1x working concentration in all

assays.

Five standard concentrations (500 µM, 200 µM, 50 µM, 12.5 µM and 3.125 µM) of each compound

were prepared in a 96-well plate via serial dilution, in a 80:20 PBS:acetonitrile (AcN) solution. The

level of DMSO in all standards and samples was maintained at 5% (v/v). The PBS:AcN solution

and DMSO were added to the plate and mixed thoroughly before the addition of compound stock

(dissolved in DMSO). The plate was covered and gently agitated (100–300 rpm) for 30 minutes at

room temperature. 200 µL from each well was transferred to a 96-well disposable UV analysis plate

and scanned at 10 nm increments from 260–500 nm. The absorbance was determined for each well

of the UV analysis plate at each of the 25 different wavelengths of the scan. A standard curve was

constructed for each compound by plotting the absorbance vs. concentration at the optimum

wavelength for each compound (e.g. 290 nm).

Samples were prepared by adding PBS and compound stock (prepared in DMSO) to the wells of a

multiscreen solubility filter plate in triplicate, to yield a compound concentration of 500 µM/well.

The DMSO concentration in each well was maintained at 5%. The plate was covered and gently

agitated (200–300 rpm) for 90 minutes at room temperature. After 90 minutes, the aqueous solution

was vacuum filtered (10" mmHg) from the multiscreen solubility filter plate into a polypropylene

V-bottom plate. 160 µL/well of filtrate from the V-bottom plate was transferred to a 96-well

disposable, UV-Star analysis plate. 40 µL/well of acetonitrile was dispensed to the UV-Star analysis

plate, the plate covered and gently agitated at room temperature for 5 minutes. The absorbance of

each well was determined at the wavelength determined as optimum for each individual compound

(e.g. 290 nm). The final drug concentration in the filtrate was determined by dividing the

absorbance at the optimum wavelength (e.g.290 nm) by the slope of the line from the calibration

curve and multiplying by a dilution factor of 1.25 (as per Equation 5.1).


Aqueous Solubility = A290 nm Filtrate x 1.25

Slope

Equation 5.1 Aqueous solubility (in µM)

5.4.2 Cell viability assays

The cytotoxic effect of the compounds were evaluated in non-infected PM1 cells and determined

via the CellTiter Aqueous One Assay (Promega Corporation, Madison, Winsconsin, USA). Briefly,

a 96-well microtiter plate was seeded with PM1 cells at a low passage number, at a concentration of

1x104

cells per well in a total volume of 100 µL RPMI 1640 media. The media used contained 10%

foetal bovine serum (FBS, Highveld Biological, R.S.A.) and necessary antibiotics. After an

incubation of 1 hour at 37 °C and 5% CO2, the test compounds were added in two-fold serial

dilutions for a total of 5 concentrations (100 µM to 6.25 µM). After an incubation period of 96

hours, 10 µL/well of the CellTiter solution was added; the contents gently mixed and the plates

were incubated under the previously described conditions for 2–4 hours. The plates were read at

time intervals of 2 and 4 hours at an absorbance wavelength of 490 nm on a multiplate reader

(BP800, BioHIT, Finland). CC50 values were determined as the concentration of the test substance

required to reduce cell viability by 50% using Microsoft Excel version 14.06106.5005 (Microsoft

Office Professional Plus 2010, © 2010 Microsoft Corporation) and Origin® version 6.1052

software (OriginLab Corporation, Northampton, MA 01060 USA).

5.4.3 Antiviral activity assays

The antiviral efficacy of the described compounds was evaluated in direct ELISA against

recombinant, wild-type HIV-1 B IN and determined via a colorimetric endpoint measurement

according to the manufacturer’s guidelines.26

Briefly: All buffers, streptavidin-coated multi-well strips and donor DNA (dDNA) stock solution

were brought to room temperature. The assay buffer was prepared to a 1x working concentration

and 2-mercaptoethanol added to a final concentration of 5 mM. The dDNA working concentration

was prepared from stock to a final concentration of 0.15 µM dDNA in assay buffer. 100 µl of the

prepared dDNA working solution was added to each micro-well used in the assay and the strips

incubated at 22 °C for 1 hour. During this incubation period, the IN working solution was prepared

from stock to a final concentration of 1 µM in assay buffer and kept on ice. After the incubation, the

solution was aspirated from the wells and then washed with 1 x PBS buffer (350 µl x 3 washes; no


incubation necessary; plate was dried thoroughly after the third wash). To the negative control wells

was added 100 µl of the assay buffer, while 100 µl of the IN working solution was added to all

other wells. The plate was covered and incubated at 22 °C for 30 minutes after which the solution

was aspirated and the strips washed with 1 x assay buffer (200 µl x 2 washes; 5 minutes incubation

at 22 °C; solution was aspirated with each wash; plate was dried thoroughly after the second wash).

For one-dose experiments:

To each well was added: 89 µl of assay buffer; 1 µl of DMSO to control wells; 1 µl of 1 mM L-CA

or RAL solution in DMSO to compound control wells; 1 µl of 1 mM inhibitor solutions in DMSO

to test wells (to a final compound concentration of 10 µM).

For IC50 determinations:

To each well was added: 89 µl of assay buffer; 1 µl of DMSO to control wells; 1 µl of compound

solutions prepared via two-fold serial dilution (working concentrations: 5.0 mM to 39 µM; final

concentrations: 50 µM–0.39 µM).

The plate was covered and incubated at 37 °C for 30 minutes. During this incubation period, the

target DNA (tDNA) working concentration was prepared from stock to a final concentration of 2.5

µM in assay buffer. The solution was aspirated to mix and covered with foil to protect it from light.

To each well was added 10 µl tDNA working solution and the solution in the wells aspirated to mix.

The plate was covered, protected against light and incubated at 37 °C for 1 hour after which the

solution was aspirated and the wells washed with 2 x saline-sodium citrate (SSC) buffer (300 µl x 3

washes; 10 minutes incubation at 22-25 °C; the plate was dried thoroughly after the third wash).

The anti-FITC-AP antibody working solution was prepared in 1 x tris buffered saline (TBS) buffer

(1:10 000 dilution of stock, 0.05% Tween added) and used at room temperature (store at 4 °C). To

each well was added 200 µl of the prepared antibody solution; the plate was covered and incubated

at 25 °C for 2 hours. After incubation, the solution was aspirated and the wells washed with 1 x

TBS buffer (300 µl x 3 washes; 10 minutes incubation at 22-25 °C; the plate was dried thoroughly

after the third wash). The BluePhos substrate (KPL Inc., Gaithersburg, Maryland, USA) working

solution was prepared by adding equal volumes of Solutions A and B together and aspirating to

mix. This solution was protected from light. To each well was added 100 µl substrate working

solution and the plate was incubated at 37 °C for 1 hour. The absorbance of each well was measured

at 620 nm on an absorbance reader and the percentage inhibition of each compound calculated

against the positive, negative and compound controls. Inhibition data and IC50 curves were

determined using Microsoft Excel version 14.06106.5005 (Microsoft Office Professional Plus 2010,


© 2010 Microsoft Corporation) and Origin® version 6.1052 software (OriginLab Corporation,

Northampton, MA 01060 USA).

5.4.4 Membrane permeability

The membrane permeability of the described compounds was evaluated in vitro, using a pre-coated

PAMPA plate system with a UV-Vis spectroscopic endpoint measurement according to the

manufacturer’s guidelines (BD Biosciences; BD GentestTM

Pre-coated PAMPA Plate System,

Catalogue # 353015; Bedford, MA, USA).

Briefly: All buffers, the pre-coated multi-well plates and compound stock solutions were brought to

room temperature before the start of the assay. Four standard concentrations (200 µM, 50 µM, 12.5

µM and 3.125 µM) of each compound were prepared in a 96-well plate via serial dilution for the

construction of a standard curve: stock solutions in DMSO were diluted with PBS (the DMSO

concentration in each solution was maintained at 5%). Furthermore, 1 mL of a 100 µM working

dilution was prepared of each test compound (DMSO concentration in each sample solution was

maintained at 5%). 300 µl of each test compound solution was added per well in triplicate into the

receiver (donor) plate, while 200 µl of PBS was added to each well of the filter (acceptor) plate.

The pre-coated PAMPA filter plate (with PBS) was slowly lowered onto the receiver plate (with

compound solutions) and the assembly incubated at room temperature for five hours. After

incubation, the plates were carefully separated and the compound concentrations in both plates

determined via UV-Vis spectroscopy (read from 200–1000 nm with 10 nm increments) with the aid

of the standard concentration curves constructed for each compound. The permeability27

and mass

retention of each test compound were calculated using Equations 5.2 and 5.3 below.

Pe = -ln [1-CA(t)/Ceq]

A*(1/VD + 1/VA)*t

Equation 5.2 Permeability (in cm/s)

R = 1-[CD(t)*VD + CA(t)*VA] / (C0 * VD)

Equation 5.3 Mass retention (%)

Where:

C0 = Initial compound concentration in the donor well (mM); CD (t) = Compound concentration in

donor well at time t (mM); CA (t) = Compound concentration in acceptor well at time t (mM); VD =


Donor well volume (300 µl); VA = Acceptor well volume (200µl); Ceq = [CD (t)*VD + CA(t)*VA] /

(VD + VA); A = Filter area (0.3 cm2); t = incubation time (five hours = 18000 seconds).

Chapter 5: Experimental Methods

5.5 References

1. Perrin, D. D., Armarego, W. L. F.,

Oxford,

2. Pavia, D. L., Lampman, G. M., Kriz

Saunders College Publishing,

3. Silverstein, R. M., Webster, F. X., Kiemle, D. J.,

Compounds

4. a.) Bruker

Wisconsin, USA, 2009.; b.) SAINT: Area

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Detector Absorption Correction, Siem

USA, 1996.

5. Sheldrick, G. M.,

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8. Goldgur, Y.; Craigie, R.; Cohen, G. H.; Fujiwara, T.; Yoshinaga, T.; Fujishita, T.;

Sugimoto, H.; Endo, T.; Murai, H.; Davies, D. R.

9. Kuiken, C.; Leitner, T.; Fo

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Appendix A: Single Crystal Data Page 218

APPENDIX A: SINGLE CRYSTAL DATA

A.1 Pyrrolidinone 11.1

Table 1. Crystal data and structure refinement for 11.1. Identification code mo_10bo_tel4_0m

Empirical formula C25 H17 N3 O4 S4





Space group P21/n

Unit cell dimensions a = 9.3499(8) Å α= 90°.

b = 17.4394(14) Å β= 102.189(2)°.

c = 17.9530(15) Å γ = 90°.

Volume 2861.4(4) Å3

Z 4


Absorption coefficient 0.366 mm-1

F(000) 1136


Theta range for data collection 2.28 to 28.44°.

Index ranges -12<=h<=12, -23<=k<=20, -23<=l<=23

Reflections collected 35155

Independent reflections 7195 [R(int) = 0.0357]

Completeness to theta = 28.44° 99.6 %


Data / restraints / parameters 7195 / 3 / 387


Final R indices [I>2sigma(I)] R1 = 0.0923, wR2 = 0.2537

R indices (all data) R1 = 0.1180, wR2 = 0.2803

Largest diff. peak and hole 0.901 and -1.307 e.Å-3

Table 2. Atomic coordinates ( x 104) and equivalent isotropic displacement parameters (Å2x 103)

for 11.1. U(eq) is defined as one third of the trace of the orthogonalised Uij tensor. x y z U(eq)

S(2) 7432(2) 4177(1) 1142(1) 34(1)

C(28) 7220(9) 4133(5) 129(5) 35(2)

O(6) 8200(6) 3463(3) 1477(3) 35(1)

C(29) 8761(10) 4938(5) 1318(6) 40(2)

S(2B) 8547(3) 4428(2) 1047(2) 63(1)

O(6B) 8863(10) 3602(5) 1341(5) 77(2)

C(28B) 9596(13) 4588(7) 375(6) 71(3)

C(29B) 6794(14) 4414(7) 402(7) 73(3)

S(2C) 8160(14) 4281(7) 1278(7) 1(4)

O(6C) 8510(30) 4910(20) 960(20) 0(7)

C(28C) 6290(50) 4330(30) 670(30) 4(9)

S(1) 3924(2) 3363(1) 584(1) 40(1)

O(5) 3421(9) 4108(5) 205(5) 70(2)

C(26) 3181(11) 3348(6) 1440(6) 55(2)

C(27) 2947(13) 2634(8) 65(7) 66(3)

S(1B) 2662(3) 3278(2) 933(2) 61(1)

O(5B) 2466(11) 4125(5) 881(5) 81(2)

C(26B) 896(10) 2866(5) 677(5) 44(2)

C(27B) 3227(18) 3016(10) -136(10) 89(5)


C(27C) 3290(30) 3014(16) 160(16) 10(5)

C(26C) 1620(30) 2853(16) 744(15) 16(5)

S(1C) 3151(11) 3300(5) 695(5) 28(3)

O(5C) 3040(20) 4119(13) 642(13) 22(5)

C(1) 5943(3) 3652(2) 3930(2) 27(1)

C(2) 6197(5) 4225(2) 4483(2) 39(1)

C(3) 6772(4) 4901(2) 4271(2) 37(1)

C(4) 7095(4) 4995(2) 3549(2) 35(1)

C(5) 6852(4) 4412(2) 3019(2) 35(1)

C(6) 6275(3) 3715(2) 3214(2) 26(1)

C(7) 5844(3) 2997(2) 2839(2) 27(1)

C(8) 5282(3) 2564(2) 3334(2) 23(1)

C(9) 4558(3) 1808(2) 3314(2) 24(1)

C(10) 4239(3) 1338(2) 2643(2) 22(1)

C(11) 4709(3) 1338(2) 1957(2) 21(1)

C(12) 3998(3) 649(2) 1504(2) 24(1)

C(13) 3164(3) 690(2) 2649(2) 25(1)

C(14) 1651(3) 965(2) 2715(2) 28(1)

C(15) 988(4) 670(2) 3279(2) 38(1)

C(16) -374(5) 944(3) 3357(3) 49(1)

C(17) -1091(4) 1501(2) 2876(2) 44(1)

C(18) -448(4) 1786(2) 2303(2) 41(1)

C(19) 921(4) 1522(2) 2228(2) 35(1)

C(20) 2287(4) -385(2) 1690(2) 30(1)

C(21) 3072(4) -1109(2) 2037(2) 34(1)

C(22) 4400(4) -1318(2) 1721(2) 34(1)

C(23) 4516(4) -1248(2) 339(2) 28(1)

C(24) 3030(4) -2184(2) -134(2) 35(1)

C(25) 3073(4) -2151(2) 623(2) 39(1)

N(1) 3146(3) 302(2) 1923(1) 26(1)

N(2) 4012(3) -1556(2) 914(2) 29(1)

N(3) 3944(3) -1621(2) -298(2) 29(1)

O(1) 5340(3) 2951(1) 4015(1) 31(1)

O(2) 4156(3) 1601(1) 3901(1) 32(1)

O(3) 5568(2) 1758(1) 1682(1) 26(1)

O(4) 4182(2) 455(1) 876(1) 27(1)

Table 3. Bond lengths [Å] and angles [°] for 11.1. Bond Lengths [Å]

S(2)-O(6) 1.498(6)

S(2)-C(28) 1.789(8)

S(2)-C(29) 1.800(9)

S(2B)-O(6B) 1.542(9)

S(2B)-C(28B) 1.730(12)

S(2B)-C(29B) 1.796(12)

S(2C)-O(6C) 1.32(4)

S(2C)-C(28C) 1.86(5)

S(1)-O(5) 1.496(9)

S(1)-C(27) 1.720(14)

S(1)-C(26) 1.816(10)

S(1B)-O(5B) 1.489(10)

S(1B)-C(26B) 1.769(10)

S(1B)-C(27B) 2.144(18)

C(27C)-S(1C) 1.11(3)

C(27C)-C(26C) 2.08(4)

C(26C)-S(1C) 1.65(3)

S(1C)-O(5C) 1.43(2)

C(1)-O(1) 1.367(4)

C(1)-C(6) 1.390(5)

C(1)-C(2) 1.393(5)

C(2)-C(3) 1.382(5)

C(2)-H(2) 0.9300

C(3)-C(4) 1.401(5)

C(3)-H(3) 0.9300

C(4)-C(5) 1.378(5)

C(4)-H(4) 0.9300

C(5)-C(6) 1.403(5)

C(5)-H(5) 0.9300

C(6)-C(7) 1.438(4)

C(7)-C(8) 1.353(4)

C(7)-H(7) 0.9300

C(8)-O(1) 1.387(4)

C(8)-C(9) 1.479(4)

C(9)-O(2) 1.244(4)

C(9)-C(10) 1.437(4)

C(10)-C(11) 1.391(4)

C(10)-C(13) 1.515(4)

C(11)-O(3) 1.263(4)

C(11)-C(12) 1.522(4)

C(12)-O(4) 1.224(4)

C(12)-N(1) 1.349(4)

C(13)-N(1) 1.466(4)

C(13)-C(14) 1.521(4)

C(13)-H(13) 0.9800

C(14)-C(19) 1.386(5)

C(14)-C(15) 1.393(5)

C(15)-C(16) 1.395(6)

C(15)-H(15) 0.9300

C(16)-C(17) 1.375(7)

C(16)-H(16) 0.9300

C(17)-C(18) 1.389(6)

C(17)-H(17) 0.9300

C(18)-C(19) 1.393(5)

C(18)-H(18) 0.9300

C(19)-H(19) 0.9300


C(20)-N(1) 1.452(4)

C(20)-C(21) 1.526(5)

C(20)-H(20A) 0.9700

C(20)-H(20B) 0.9700

C(21)-C(22) 1.515(5)

C(21)-H(21A) 0.9700

C(21)-H(21B) 0.9700

C(22)-N(2) 1.477(4)

C(22)-H(22A) 0.9700

C(22)-H(22B) 0.9700

C(23)-N(3) 1.326(4)

C(23)-N(2) 1.335(4)

C(23)-H(23) 0.9300

C(24)-C(25) 1.353(5)

C(24)-N(3) 1.373(4)

C(24)-H(24) 0.9300

C(25)-N(2) 1.388(4)

C(25)-H(25) 0.9300

N(3)-H(3) 0.8600

O(3)-H(3A) 0.8200

Bond angles [°]

O(6)-S(2)-C(28) 108.3(4)

O(6)-S(2)-C(29) 106.7(4)

C(28)-S(2)-C(29) 97.7(4)

O(6B)-S(2B)-C(28B) 107.1(5)

O(6B)-S(2B)-C(29B) 106.9(5)

C(28B)-S(2B)-C(29B) 97.3(6)

O(6C)-S(2C)-C(28C) 90(2)

O(5)-S(1)-C(27) 108.4(5)

O(5)-S(1)-C(26) 104.9(5)

C(27)-S(1)-C(26) 101.1(5)

O(5B)-S(1B)-C(26B) 106.9(5)

O(5B)-S(1B)-C(27B) 101.9(6)

C(26B)-S(1B)-C(27B) 94.2(5)

S(1C)-C(27C)-C(26C) 52.1(14)

S(1C)-C(26C)-C(27C) 32.2(10)

C(27C)-S(1C)-O(5C) 114.2(19)

C(27C)-S(1C)-C(26C) 95.6(17)

O(5C)-S(1C)-C(26C) 115.1(15)

O(1)-C(1)-C(6) 110.6(3)

O(1)-C(1)-C(2) 125.1(3)

C(6)-C(1)-C(2) 124.3(3)

C(3)-C(2)-C(1) 115.7(3)

C(3)-C(2)-H(2) 122.1

C(1)-C(2)-H(2) 122.1

C(2)-C(3)-C(4) 121.8(3)

C(2)-C(3)-H(3) 119.1

C(4)-C(3)-H(3) 119.1

C(5)-C(4)-C(3) 121.1(3)

C(5)-C(4)-H(4) 119.5

C(3)-C(4)-H(4) 119.5

C(4)-C(5)-C(6) 118.9(3)

C(4)-C(5)-H(5) 120.6

C(6)-C(5)-H(5) 120.6

C(1)-C(6)-C(5) 118.2(3)

C(1)-C(6)-C(7) 105.6(3)

C(5)-C(6)-C(7) 136.2(3)

C(8)-C(7)-C(6) 106.7(3)

C(8)-C(7)-H(7) 126.6

C(6)-C(7)-H(7) 126.6

C(7)-C(8)-O(1) 111.0(3)

C(7)-C(8)-C(9) 136.1(3)

O(1)-C(8)-C(9) 112.7(3)

O(2)-C(9)-C(10) 119.9(3)

O(2)-C(9)-C(8) 117.2(3)

C(10)-C(9)-C(8) 122.8(3)

C(11)-C(10)-C(9) 134.0(3)

C(11)-C(10)-C(13) 109.9(2)

C(9)-C(10)-C(13) 116.1(2)

O(3)-C(11)-C(10) 133.8(3)

O(3)-C(11)-C(12) 119.4(3)

C(10)-C(11)-C(12) 106.8(2)

O(4)-C(12)-N(1) 127.1(3)

O(4)-C(12)-C(11) 125.5(3)

N(1)-C(12)-C(11) 107.4(2)

N(1)-C(13)-C(10) 102.7(2)

N(1)-C(13)-C(14) 112.2(3)

C(10)-C(13)-C(14) 113.2(3)

N(1)-C(13)-H(13) 109.5

C(10)-C(13)-H(13) 109.5

C(14)-C(13)-H(13) 109.5

C(19)-C(14)-C(15) 118.8(3)

C(19)-C(14)-C(13) 121.0(3)

C(15)-C(14)-C(13) 120.2(3)

C(14)-C(15)-C(16) 120.1(4)

C(14)-C(15)-H(15) 119.9

C(16)-C(15)-H(15) 119.9

C(17)-C(16)-C(15) 120.9(4)

C(17)-C(16)-H(16) 119.5

C(15)-C(16)-H(16) 119.5

C(16)-C(17)-C(18) 119.2(4)

C(16)-C(17)-H(17) 120.4

C(18)-C(17)-H(17) 120.4

C(17)-C(18)-C(19) 120.2(4)

C(17)-C(18)-H(18) 119.9

C(19)-C(18)-H(18) 119.9

C(14)-C(19)-C(18) 120.7(3)

C(14)-C(19)-H(19) 119.6

C(18)-C(19)-H(19) 119.6

N(1)-C(20)-C(21) 112.1(3)

N(1)-C(20)-H(20A) 109.2

C(21)-C(20)-H(20A) 109.2

N(1)-C(20)-H(20B) 109.2

C(21)-C(20)-H(20B) 109.2

H(20A)-C(20)-H(20B) 107.9

C(22)-C(21)-C(20) 114.1(3)

C(22)-C(21)-H(21A) 108.7

C(20)-C(21)-H(21A) 108.7

C(22)-C(21)-H(21B) 108.7

C(20)-C(21)-H(21B) 108.7

H(21A)-C(21)-H(21B) 107.6

N(2)-C(22)-C(21) 112.6(3)

N(2)-C(22)-H(22A) 109.1

C(21)-C(22)-H(22A) 109.1

N(2)-C(22)-H(22B) 109.1

C(21)-C(22)-H(22B) 109.1

H(22A)-C(22)-H(22B) 107.8

N(3)-C(23)-N(2) 109.0(3)

N(3)-C(23)-H(23) 125.5

N(2)-C(23)-H(23) 125.5

C(25)-C(24)-N(3) 107.3(3)

C(25)-C(24)-H(24) 126.4

N(3)-C(24)-H(24) 126.4

C(24)-C(25)-N(2) 107.0(3)

C(24)-C(25)-H(25) 126.5

N(2)-C(25)-H(25) 126.5

C(12)-N(1)-C(20) 124.5(3)

C(12)-N(1)-C(13) 113.0(2)

C(20)-N(1)-C(13) 122.5(2)


C(23)-N(2)-C(25) 108.0(3)

C(23)-N(2)-C(22) 126.2(3)

C(25)-N(2)-C(22) 125.8(3)

C(23)-N(3)-C(24) 108.8(3)

C(23)-N(3)-H(3) 125.6

C(24)-N(3)-H(3) 125.6

C(1)-O(1)-C(8) 106.1(2)

C(11)-O(3)-H(3A) 109.5

Table 4. Anisotropic displacement parameters (Å2x 103) for 11.1. The anisotropic

displacement factor exponent takes the form: -2π2[ h2a*2U11 + ... + 2 h k a* b* U12 ] U11 U22 U33 U23 U13 U12

C(1) 30(1) 19(1) 28(2) 0(1) -1(1) -2(1)

C(2) 57(2) 27(2) 28(2) -5(1) -1(2) -9(2)

C(3) 43(2) 23(2) 39(2) -4(1) -6(1) -4(1)

C(4) 30(2) 20(2) 52(2) 1(1) 4(1) -2(1)

C(5) 37(2) 25(2) 46(2) 0(1) 16(2) -3(1)

C(6) 23(1) 20(1) 35(2) -2(1) 6(1) 1(1)

C(7) 31(2) 20(1) 32(2) -2(1) 13(1) 2(1)

C(8) 26(1) 20(1) 24(1) -3(1) 3(1) 2(1)

C(9) 27(1) 20(1) 24(1) 0(1) 4(1) 1(1)

C(10) 27(1) 19(1) 22(1) 0(1) 7(1) -4(1)

C(11) 24(1) 18(1) 21(1) 0(1) 4(1) 2(1)

C(12) 26(1) 21(1) 25(1) 0(1) 6(1) -2(1)

C(13) 33(2) 20(1) 23(1) -3(1) 8(1) -5(1)

C(14) 30(2) 27(2) 28(2) -7(1) 9(1) -7(1)

C(15) 42(2) 38(2) 37(2) -2(2) 16(2) -7(2)

C(16) 46(2) 55(3) 54(2) -9(2) 29(2) -11(2)

C(17) 30(2) 44(2) 59(2) -19(2) 12(2) -6(2)

C(18) 36(2) 35(2) 51(2) -10(2) 5(2) 1(2)

C(19) 35(2) 33(2) 39(2) -2(1) 11(1) -3(1)

C(20) 36(2) 28(2) 30(2) -9(1) 14(1) -13(1)

C(21) 54(2) 26(2) 25(2) -4(1) 15(1) -16(2)

C(22) 48(2) 23(2) 29(2) 2(1) 6(1) -5(1)

C(23) 33(2) 17(1) 36(2) 5(1) 13(1) 0(1)

C(24) 45(2) 27(2) 38(2) -9(1) 19(2) -12(1)

C(25) 55(2) 29(2) 40(2) -7(1) 24(2) -18(2)

N(1) 32(1) 22(1) 25(1) -5(1) 9(1) -7(1)

N(2) 41(2) 19(1) 30(1) 0(1) 14(1) -7(1)

N(3) 39(1) 22(1) 31(1) 2(1) 17(1) 1(1)

O(1) 49(1) 22(1) 22(1) -4(1) 5(1) -10(1)

O(2) 48(1) 26(1) 24(1) -3(1) 12(1) -8(1)

O(3) 34(1) 21(1) 27(1) -4(1) 14(1) -5(1)

O(4) 34(1) 25(1) 22(1) -4(1) 8(1) -4(1)

Table 5. Hydrogen coordinates ( x 104) and isotropic displacement parameters (Å2x 103) for 11.1.

x y z U(eq)

H(2) 5992 4157 4964 47

H(3) 6950 5304 4617 44

H(4) 7479 5458 3426 41

H(5) 7068 4479 2541 42

H(7) 5934 2857 2351 32

H(13) 3550 340 3071 30

H(15) 1455 289 3604 45

H(16) -803 748 3739 59

H(17) -1995 1684 2934 52

H(18) -934 2154 1968 49

H(19) 1350 1722 1848 42

H(20A) 2087 -425 1139 36

H(20B) 1358 -342 1846 36

H(21A) 3375 -1039 2583 41

H(21B) 2386 -1533 1949 41

H(22A) 5051 -879 1767 40

H(22B) 4923 -1732 2021 40

H(23) 5165 -839 379 33

H(24) 2483 -2524 -480 42

H(25) 2570 -2468 896 47


H(3) 4117 -1526 -740 35

H(3A) 5612 1602 1256 40

Table 6. Torsion angles [°] for 11.1. C(26C)-C(27C)-S(1C)-O(5C) -120.7(17)

C(27C)-C(26C)-S(1C)-O(5C) 120(2)

O(1)-C(1)-C(2)-C(3) -177.9(3)

C(6)-C(1)-C(2)-C(3) 2.2(5)

C(1)-C(2)-C(3)-C(4) -0.8(6)

C(2)-C(3)-C(4)-C(5) -0.2(6)

C(3)-C(4)-C(5)-C(6) -0.1(5)

O(1)-C(1)-C(6)-C(5) 177.6(3)

C(2)-C(1)-C(6)-C(5) -2.6(5)

O(1)-C(1)-C(6)-C(7) -0.2(3)

C(2)-C(1)-C(6)-C(7) 179.6(3)

C(4)-C(5)-C(6)-C(1) 1.4(5)

C(4)-C(5)-C(6)-C(7) 178.3(3)

C(1)-C(6)-C(7)-C(8) 0.7(3)

C(5)-C(6)-C(7)-C(8) -176.5(4)

C(6)-C(7)-C(8)-O(1) -0.9(4)

C(6)-C(7)-C(8)-C(9) 175.0(3)

C(7)-C(8)-C(9)-O(2) -178.7(3)

O(1)-C(8)-C(9)-O(2) -2.8(4)

C(7)-C(8)-C(9)-C(10) -2.3(6)

O(1)-C(8)-C(9)-C(10) 173.5(3)

O(2)-C(9)-C(10)-C(11) -168.2(3)

C(8)-C(9)-C(10)-C(11) 15.5(5)

O(2)-C(9)-C(10)-C(13) 11.6(4)

C(8)-C(9)-C(10)-C(13) -164.7(3)

C(9)-C(10)-C(11)-O(3) -1.2(6)

C(13)-C(10)-C(11)-O(3) 179.0(3)

C(9)-C(10)-C(11)-C(12) 177.0(3)

C(13)-C(10)-C(11)-C(12) -2.9(3)

O(3)-C(11)-C(12)-O(4) 0.1(5)

C(10)-C(11)-C(12)-O(4) -178.4(3)

O(3)-C(11)-C(12)-N(1) 179.9(3)

C(10)-C(11)-C(12)-N(1) 1.4(3)

C(11)-C(10)-C(13)-N(1) 3.2(3)

C(9)-C(10)-C(13)-N(1) -176.7(3)

C(11)-C(10)-C(13)-C(14) -118.1(3)

C(9)-C(10)-C(13)-C(14) 62.1(4)

N(1)-C(13)-C(14)-C(19) -64.4(4)

C(10)-C(13)-C(14)-C(19) 51.4(4)

N(1)-C(13)-C(14)-C(15) 117.0(3)

C(10)-C(13)-C(14)-C(15) -127.3(3)

C(19)-C(14)-C(15)-C(16) -1.2(5)

C(13)-C(14)-C(15)-C(16) 177.5(3)

C(14)-C(15)-C(16)-C(17) 0.8(6)

C(15)-C(16)-C(17)-C(18) 0.5(6)

C(16)-C(17)-C(18)-C(19) -1.4(6)

C(15)-C(14)-C(19)-C(18) 0.3(5)

C(13)-C(14)-C(19)-C(18) -178.4(3)

C(17)-C(18)-C(19)-C(14) 1.0(6)

N(1)-C(20)-C(21)-C(22) -69.6(3)

C(20)-C(21)-C(22)-N(2) -67.6(4)

N(3)-C(24)-C(25)-N(2) 0.7(4)

O(4)-C(12)-N(1)-C(20) 0.4(5)

C(11)-C(12)-N(1)-C(20) -179.4(3)

O(4)-C(12)-N(1)-C(13) -179.5(3)

C(11)-C(12)-N(1)-C(13) 0.7(3)

C(21)-C(20)-N(1)-C(12) 97.3(4)

C(21)-C(20)-N(1)-C(13) -82.9(4)

C(10)-C(13)-N(1)-C(12) -2.3(3)

C(14)-C(13)-N(1)-C(12) 119.6(3)

C(10)-C(13)-N(1)-C(20) 177.8(3)

C(14)-C(13)-N(1)-C(20) -60.3(4)

N(3)-C(23)-N(2)-C(25) -0.1(4)

N(3)-C(23)-N(2)-C(22) 178.9(3)

C(24)-C(25)-N(2)-C(23) -0.4(4)

C(24)-C(25)-N(2)-C(22) -179.3(3)

C(21)-C(22)-N(2)-C(23) 124.1(4)

C(21)-C(22)-N(2)-C(25) -57.1(5)

N(2)-C(23)-N(3)-C(24) 0.5(4)

C(25)-C(24)-N(3)-C(23) -0.8(4)

C(6)-C(1)-O(1)-C(8) -0.3(3)

C(2)-C(1)-O(1)-C(8) 179.8(3)

C(7)-C(8)-O(1)-C(1) 0.8(3)

C(9)-C(8)-O(1)-C(1) -176.1(2)


Table 1. Crystal data and structure refinement for 11.2. Identification code mo_10bo_tel2_0m

Empirical formula C29 H32 Cl N3 O6 S2





Space group P21/c


b = 24.547(5) Å β= 95.182(5)°.

c = 14.313(3) Å γ = 90°.

Volume 2999.7(10) Å3

Z 4



F(000) 1296




Index ranges -11<=h<=11, -32<=k<=24, -18<=l<=19





Max. and min. transmission 0.9876 and 0.9429









C(1) 7266(2) 1394(1) 10879(1) 14(1)

C(2) 7888(2) 880(1) 11077(1) 19(1)

C(3) 8304(2) 765(1) 12020(2) 21(1)

C(4) 8080(2) 1149(1) 12728(1) 20(1)

C(5) 7445(2) 1659(1) 12517(1) 18(1)

C(6) 7037(2) 1786(1) 11566(1) 15(1)

C(7) 6365(2) 2251(1) 11065(1) 15(1)

C(8) 6230(2) 2117(1) 10140(1) 14(1)

C(9) 5494(2) 2382(1) 9278(1) 14(1)

C(10) 4892(2) 2931(1) 9311(1) 13(1)

C(11) 5256(2) 3370(1) 9907(1) 15(1)

C(12) 4364(2) 3856(1) 9487(1) 16(1)

C(13) 3728(2) 3107(1) 8501(1) 13(1)

C(14) 2197(2) 2788(1) 8456(1) 15(1)

C(15) 1552(3) 2511(1) 7661(1) 20(1)

C(16) 149(3) 2217(1) 7657(2) 24(1)

C(17) -625(3) 2197(1) 8466(2) 25(1)

C(18) -26(3) 2473(1) 9267(2) 25(1)

C(19) 1363(2) 2765(1) 9259(1) 20(1)

C(20) 2307(2) 4019(1) 8165(1) 17(1)

C(21) 2748(2) 4130(1) 7167(1) 18(1)

C(22) 4290(2) 4435(1) 7131(1) 16(1)

C(23) 4262(2) 5243(1) 8217(1) 17(1)

C(24) 3907(2) 5907(1) 7180(1) 17(1)

C(25) 3946(2) 5432(1) 6698(1) 16(1)

N(1) 3463(2) 3686(1) 8714(1) 15(1)

N(2) 4170(2) 5019(1) 7358(1) 14(1)

N(3) 4099(2) 5780(1) 8128(1) 16(1)

O(1) 6780(2) 1589(1) 9998(1) 15(1)

O(2) 5337(2) 2115(1) 8533(1) 19(1)

O(3) 6161(2) 3424(1) 10660(1) 20(1)

O(4) 4449(2) 4326(1) 9800(1) 21(1)

S(1) 2211(1) 4386(1) 4152(1) 23(1)

C(28) 1889(3) 4163(1) 1386(2) 42(1)

C(29) -754(3) 4169(1) 212(2) 37(1)

O(6) -384(2) 4862(1) 1622(1) 35(1)

S(2) -172(1) 4271(1) 1432(1) 24(1)

C(26) 2803(3) 3701(1) 4396(2) 36(1)

C(27) 143(3) 4274(1) 4035(2) 36(1)

O(5) 2554(3) 4698(1) 5048(1) 43(1)

Cl(1) 2478(1) 2526(1) 6622(1) 30(1)

Table 3. Bond lengths [Å] and angles [°] for 11.2. Bond lengths [Å]

C(1)-O(1) 1.377(2)

C(1)-C(2) 1.390(3)

C(1)-C(6) 1.402(3)

C(2)-C(3) 1.394(3)

C(2)-H(2) 0.9500

C(3)-C(4) 1.409(3)

C(3)-H(3) 0.9500

C(4)-C(5) 1.388(3)


C(4)-H(4) 0.9500

C(5)-C(6) 1.410(3)

C(5)-H(5) 0.9500

C(6)-C(7) 1.441(3)

C(7)-C(8) 1.359(3)

C(7)-H(7) 0.9500

C(8)-O(1) 1.400(2)

C(8)-C(9) 1.484(3)

C(9)-O(2) 1.249(2)

C(9)-C(10) 1.445(2)

C(10)-C(11) 1.393(3)

C(10)-C(13) 1.523(3)

C(11)-O(3) 1.277(2)

C(11)-C(12) 1.5109(16)

C(12)-O(4) 1.237(2)

C(12)-N(1) 1.357(2)

C(13)-N(1) 1.473(2)

C(13)-C(14) 1.525(3)

C(13)-H(13) 1.0000

C(14)-C(15) 1.396(3)

C(14)-C(19) 1.409(3)

C(15)-C(16) 1.402(3)

C(15)-Cl(1) 1.749(2)

C(16)-C(17) 1.386(3)

C(16)-H(16) 0.9500

C(17)-C(18) 1.390(3)

C(17)-H(17) 0.9500

C(18)-C(19) 1.390(3)

C(18)-H(18) 0.9500

C(19)-H(19) 0.9500

C(20)-N(1) 1.459(2)

C(20)-C(21) 1.535(3)

C(20)-H(20A) 0.9900

C(20)-H(20B) 0.9900

C(21)-C(22) 1.525(3)

C(21)-H(21A) 0.9900

C(21)-H(21B) 0.9900

C(22)-N(2) 1.476(2)

C(22)-H(22A) 0.9900

C(22)-H(22B) 0.9900

C(23)-N(3) 1.330(2)

C(23)-N(2) 1.341(2)

C(23)-H(23) 0.9500

C(24)-C(25) 1.357(3)

C(24)-N(3) 1.387(2)

C(24)-H(24) 0.9500

C(25)-N(2) 1.388(2)

C(25)-H(25) 0.9500

O(3)-H(3B) 0.8400

S(1)-O(5) 1.4995(17)

S(1)-C(26) 1.782(2)

S(1)-C(27) 1.787(3)

C(28)-S(2) 1.794(3)

C(28)-H(28A) 0.9800

C(28)-H(28B) 0.9800

C(28)-H(28C) 0.9800

C(29)-S(2) 1.790(2)

C(29)-H(29A) 0.9800

C(29)-H(29B) 0.9800

C(29)-H(29C) 0.9800

O(6)-S(2) 1.4912(17)

C(26)-H(26A) 0.9800

C(26)-H(26B) 0.9800

C(26)-H(26C) 0.9800

C(27)-H(27A) 0.9800

C(27)-H(27B) 0.9800

C(27)-H(27C) 0.9800

Bond angles [°]

O(1)-C(1)-C(2) 125.59(17)

O(1)-C(1)-C(6) 110.69(16)

C(2)-C(1)-C(6) 123.72(18)

C(1)-C(2)-C(3) 116.19(18)

C(1)-C(2)-H(2) 121.9

C(3)-C(2)-H(2) 121.9

C(2)-C(3)-C(4) 121.44(18)

C(2)-C(3)-H(3) 119.3

C(4)-C(3)-H(3) 119.3

C(5)-C(4)-C(3) 121.58(18)

C(5)-C(4)-H(4) 119.2

C(3)-C(4)-H(4) 119.2

C(4)-C(5)-C(6) 117.86(18)

C(4)-C(5)-H(5) 121.1

C(6)-C(5)-H(5) 121.1

C(1)-C(6)-C(5) 119.20(17)

C(1)-C(6)-C(7) 105.64(16)

C(5)-C(6)-C(7) 135.15(18)

C(8)-C(7)-C(6) 106.75(16)

C(8)-C(7)-H(7) 126.6

C(6)-C(7)-H(7) 126.6

C(7)-C(8)-O(1) 111.38(16)

C(7)-C(8)-C(9) 134.14(17)

O(1)-C(8)-C(9) 114.13(15)

O(2)-C(9)-C(10) 120.41(17)

O(2)-C(9)-C(8) 118.98(16)

C(10)-C(9)-C(8) 120.53(16)

C(11)-C(10)-C(9) 132.94(17)

C(11)-C(10)-C(13) 109.90(15)

C(9)-C(10)-C(13) 116.89(15)

O(3)-C(11)-C(10) 132.96(16)

O(3)-C(11)-C(12) 120.11(16)

C(10)-C(11)-C(12) 106.91(15)

O(4)-C(12)-N(1) 126.40(16)

O(4)-C(12)-C(11) 125.48(17)

N(1)-C(12)-C(11) 108.13(15)

N(1)-C(13)-C(10) 102.67(14)

N(1)-C(13)-C(14) 110.87(15)

C(10)-C(13)-C(14) 112.82(15)

N(1)-C(13)-H(13) 110.1

C(10)-C(13)-H(13) 110.1

C(14)-C(13)-H(13) 110.1

C(15)-C(14)-C(19) 116.89(18)

C(15)-C(14)-C(13) 124.11(18)

C(19)-C(14)-C(13) 119.00(17)

C(14)-C(15)-C(16) 122.05(19)

C(14)-C(15)-Cl(1) 120.42(16)

C(16)-C(15)-Cl(1) 117.53(16)

C(17)-C(16)-C(15) 119.3(2)

C(17)-C(16)-H(16) 120.3

C(15)-C(16)-H(16) 120.3

C(16)-C(17)-C(18) 120.2(2)

C(16)-C(17)-H(17) 119.9

C(18)-C(17)-H(17) 119.9

C(17)-C(18)-C(19) 119.8(2)

C(17)-C(18)-H(18) 120.1

C(19)-C(18)-H(18) 120.1

C(18)-C(19)-C(14) 121.71(19)

C(18)-C(19)-H(19) 119.1

C(14)-C(19)-H(19) 119.1


N(1)-C(20)-C(21) 112.87(16)

N(1)-C(20)-H(20A) 109.0

C(21)-C(20)-H(20A) 109.0

N(1)-C(20)-H(20B) 109.0

C(21)-C(20)-H(20B) 109.0

H(20A)-C(20)-H(20B) 107.8

C(22)-C(21)-C(20) 114.00(16)

C(22)-C(21)-H(21A) 108.8

C(20)-C(21)-H(21A) 108.8

C(22)-C(21)-H(21B) 108.8

C(20)-C(21)-H(21B) 108.8

H(21A)-C(21)-H(21B) 107.6

N(2)-C(22)-C(21) 113.05(16)

N(2)-C(22)-H(22A) 109.0

C(21)-C(22)-H(22A) 109.0

N(2)-C(22)-H(22B) 109.0

C(21)-C(22)-H(22B) 109.0

H(22A)-C(22)-H(22B) 107.8

N(3)-C(23)-N(2) 108.75(16)

N(3)-C(23)-H(23) 125.6

N(2)-C(23)-H(23) 125.6

C(25)-C(24)-N(3) 107.34(17)

C(25)-C(24)-H(24) 126.3

N(3)-C(24)-H(24) 126.3

C(24)-C(25)-N(2) 106.80(17)

C(24)-C(25)-H(25) 126.6

N(2)-C(25)-H(25) 126.6

C(12)-N(1)-C(20) 125.14(15)

C(12)-N(1)-C(13) 112.25(14)

C(20)-N(1)-C(13) 122.56(15)

C(23)-N(2)-C(25) 108.55(16)

C(23)-N(2)-C(22) 126.91(16)

C(25)-N(2)-C(22) 124.54(16)

C(23)-N(3)-C(24) 108.56(16)

C(1)-O(1)-C(8) 105.54(14)

C(11)-O(3)-H(3B) 109.5

O(5)-S(1)-C(26) 106.48(11)

O(5)-S(1)-C(27) 106.03(13)

C(26)-S(1)-C(27) 97.75(12)

S(2)-C(28)-H(28A) 109.5

S(2)-C(28)-H(28B) 109.5

H(28A)-C(28)-H(28B) 109.5

S(2)-C(28)-H(28C) 109.5

H(28A)-C(28)-H(28C) 109.5

H(28B)-C(28)-H(28C) 109.5

S(2)-C(29)-H(29A) 109.5

S(2)-C(29)-H(29B) 109.5

H(29A)-C(29)-H(29B) 109.5

S(2)-C(29)-H(29C) 109.5

H(29A)-C(29)-H(29C) 109.5

H(29B)-C(29)-H(29C) 109.5

O(6)-S(2)-C(29) 106.64(12)

O(6)-S(2)-C(28) 106.68(13)

C(29)-S(2)-C(28) 97.60(13)

S(1)-C(26)-H(26A) 109.5

S(1)-C(26)-H(26B) 109.5

H(26A)-C(26)-H(26B) 109.5

S(1)-C(26)-H(26C) 109.5

H(26A)-C(26)-H(26C) 109.5

H(26B)-C(26)-H(26C) 109.5

S(1)-C(27)-H(27A) 109.5

S(1)-C(27)-H(27B) 109.5

H(27A)-C(27)-H(27B) 109.5

S(1)-C(27)-H(27C) 109.5

H(27A)-C(27)-H(27C) 109.5

H(27B)-C(27)-H(27C) 109.5

Table 4. Anisotropic displacement parameters (Å2x 103) for 11.2. The anisotropic


C(1) 14(1) 14(1) 15(1) 2(1) 0(1) 0(1)

C(2) 21(1) 14(1) 21(1) 1(1) 3(1) 3(1)

C(3) 19(1) 16(1) 26(1) 7(1) -1(1) 2(1)

C(4) 18(1) 24(1) 18(1) 6(1) -4(1) -3(1)

C(5) 19(1) 19(1) 17(1) -1(1) -1(1) -3(1)

C(6) 14(1) 12(1) 18(1) 0(1) 2(1) -2(1)

C(7) 17(1) 11(1) 16(1) -1(1) 2(1) 0(1)

C(8) 15(1) 9(1) 18(1) 0(1) 2(1) 1(1)

C(9) 14(1) 10(1) 16(1) 0(1) 2(1) -1(1)

C(10) 16(1) 10(1) 13(1) 1(1) 0(1) 0(1)

C(11) 20(1) 11(1) 15(1) 1(1) 2(1) 0(1)

C(12) 22(1) 10(1) 15(1) 2(1) 3(1) -1(1)

C(13) 17(1) 9(1) 14(1) 0(1) 1(1) 0(1)

C(14) 18(1) 9(1) 19(1) 1(1) -1(1) 1(1)

C(15) 25(1) 15(1) 19(1) -1(1) 0(1) 1(1)

C(16) 25(1) 15(1) 30(1) -3(1) -5(1) 0(1)

C(17) 20(1) 17(1) 37(1) 1(1) 0(1) -1(1)

C(18) 24(1) 23(1) 28(1) 4(1) 7(1) 0(1)

C(19) 22(1) 17(1) 19(1) 2(1) 0(1) -1(1)

C(20) 18(1) 10(1) 22(1) 4(1) 0(1) 1(1)

C(21) 24(1) 11(1) 17(1) 1(1) -4(1) -1(1)

C(22) 22(1) 9(1) 19(1) 0(1) 3(1) 3(1)

C(23) 21(1) 14(1) 15(1) 1(1) -1(1) -2(1)

C(24) 20(1) 14(1) 17(1) 3(1) -1(1) -2(1)

C(25) 19(1) 14(1) 16(1) 4(1) 0(1) -2(1)

N(1) 19(1) 7(1) 17(1) 1(1) 0(1) 0(1)

N(2) 16(1) 10(1) 15(1) 2(1) 1(1) -1(1)


N(3) 20(1) 12(1) 17(1) 1(1) -1(1) -3(1)

O(1) 21(1) 9(1) 14(1) 0(1) 1(1) 3(1)

O(2) 27(1) 12(1) 16(1) -2(1) 0(1) 3(1)

O(3) 31(1) 9(1) 18(1) -4(1) -6(1) 0(1)

O(4) 37(1) 8(1) 18(1) 0(1) 0(1) 1(1)

S(1) 28(1) 19(1) 21(1) -2(1) -3(1) 1(1)

C(28) 31(1) 57(2) 38(1) -7(1) 6(1) 13(1)

C(29) 50(2) 38(1) 21(1) -6(1) 2(1) -13(1)

O(6) 35(1) 25(1) 42(1) -13(1) -3(1) 4(1)

S(2) 28(1) 23(1) 22(1) -4(1) 4(1) 1(1)

C(26) 31(1) 20(1) 55(2) -7(1) -7(1) 6(1)

C(27) 28(1) 38(1) 43(2) 3(1) 4(1) 7(1)

O(5) 82(2) 17(1) 27(1) -4(1) -20(1) -1(1)

Cl(1) 40(1) 34(1) 18(1) -9(1) 5(1) -10(1)


x y z U(eq)

H(2) 8022 621 10597 22

H(3) 8748 420 12190 25

H(4) 8370 1056 13364 24

H(5) 7291 1914 12999 22

H(7) 6074 2587 11330 18

H(13) 4211 3074 7893 16

H(16) -267 2033 7106 28

H(17) -1569 1995 8472 30

H(18) -564 2462 9819 30

H(19) 1759 2953 9810 23

H(20A) 2193 4370 8490 20

H(20B) 1280 3832 8128 20

H(21A) 2819 3778 6835 21

H(21B) 1900 4345 6826 21

H(22A) 5094 4265 7579 20

H(22B) 4644 4398 6494 20

H(23) 4418 5050 8793 20

H(24) 3774 6261 6917 21

H(25) 3839 5390 6035 20

H(3B) 6119 3746 10853 30

H(28A) 2286 4416 934 63

H(28B) 2072 3787 1191 63

H(28C) 2433 4227 2008 63

H(29A) -1873 4250 86 55

H(29B) -559 3789 44 55

H(29C) -150 4411 -162 55

H(26A) 3944 3687 4526 54

H(26B) 2484 3471 3852 54

H(26C) 2307 3568 4943 54

H(27A) -167 4092 4599 54

H(27B) -136 4043 3486 54

H(27C) -402 4624 3954 54

Table 6. Torsion angles [°] for 11.2. O(1)-C(1)-C(2)-C(3) -179.95(18)

C(6)-C(1)-C(2)-C(3) -0.5(3)

C(1)-C(2)-C(3)-C(4) 0.9(3)

C(2)-C(3)-C(4)-C(5) -0.5(3)

C(3)-C(4)-C(5)-C(6) -0.4(3)

O(1)-C(1)-C(6)-C(5) 179.15(17)

C(2)-C(1)-C(6)-C(5) -0.3(3)

O(1)-C(1)-C(6)-C(7) 0.0(2)

C(2)-C(1)-C(6)-C(7) -179.50(18)

C(4)-C(5)-C(6)-C(1) 0.8(3)

C(4)-C(5)-C(6)-C(7) 179.7(2)

C(1)-C(6)-C(7)-C(8) 0.1(2)

C(5)-C(6)-C(7)-C(8) -178.8(2)

C(6)-C(7)-C(8)-O(1) -0.2(2)

C(6)-C(7)-C(8)-C(9) 172.4(2)

C(7)-C(8)-C(9)-O(2) -168.3(2)

O(1)-C(8)-C(9)-O(2) 4.2(3)

C(7)-C(8)-C(9)-C(10) 8.5(3)

O(1)-C(8)-C(9)-C(10) -179.03(16)

O(2)-C(9)-C(10)-C(11) -158.0(2)

C(8)-C(9)-C(10)-C(11) 25.3(3)

O(2)-C(9)-C(10)-C(13) 15.3(3)

C(8)-C(9)-C(10)-C(13) -161.41(17)

C(9)-C(10)-C(11)-O(3) -5.9(4)

C(13)-C(10)-C(11)-O(3) -179.6(2)

C(9)-C(10)-C(11)-C(12) 172.3(2)


C(13)-C(10)-C(11)-C(12) -1.4(2)

O(3)-C(11)-C(12)-O(4) 1.8(3)

C(10)-C(11)-C(12)-O(4) -176.58(19)

O(3)-C(11)-C(12)-N(1) -178.19(17)

C(10)-C(11)-C(12)-N(1) 3.4(2)

C(11)-C(10)-C(13)-N(1) -0.8(2)

C(9)-C(10)-C(13)-N(1) -175.66(16)

C(11)-C(10)-C(13)-C(14) -120.23(18)

C(9)-C(10)-C(13)-C(14) 64.9(2)

N(1)-C(13)-C(14)-C(15) 119.53(19)

C(10)-C(13)-C(14)-C(15) -125.95(19)

N(1)-C(13)-C(14)-C(19) -60.6(2)

C(10)-C(13)-C(14)-C(19) 53.9(2)

C(19)-C(14)-C(15)-C(16) -0.6(3)

C(13)-C(14)-C(15)-C(16) 179.26(18)

C(19)-C(14)-C(15)-Cl(1) 178.75(15)

C(13)-C(14)-C(15)-Cl(1) -1.4(3)

C(14)-C(15)-C(16)-C(17) -0.3(3)

Cl(1)-C(15)-C(16)-C(17) -179.67(16)

C(15)-C(16)-C(17)-C(18) 0.9(3)

C(16)-C(17)-C(18)-C(19) -0.6(3)

C(17)-C(18)-C(19)-C(14) -0.3(3)

C(15)-C(14)-C(19)-C(18) 0.9(3)

C(13)-C(14)-C(19)-C(18) -178.95(18)

N(1)-C(20)-C(21)-C(22) -60.6(2)

C(20)-C(21)-C(22)-N(2) -74.8(2)

N(3)-C(24)-C(25)-N(2) -0.3(2)

O(4)-C(12)-N(1)-C(20) -6.6(3)

C(11)-C(12)-N(1)-C(20) 173.40(17)

O(4)-C(12)-N(1)-C(13) 175.88(19)

C(11)-C(12)-N(1)-C(13) -4.1(2)

C(21)-C(20)-N(1)-C(12) 114.3(2)

C(21)-C(20)-N(1)-C(13) -68.4(2)

C(10)-C(13)-N(1)-C(12) 3.1(2)

C(14)-C(13)-N(1)-C(12) 123.86(17)

C(10)-C(13)-N(1)-C(20) -174.45(16)

C(14)-C(13)-N(1)-C(20) -53.7(2)

N(3)-C(23)-N(2)-C(25) 0.2(2)

N(3)-C(23)-N(2)-C(22) 179.97(17)

C(24)-C(25)-N(2)-C(23) 0.1(2)

C(24)-C(25)-N(2)-C(22) -179.67(17)

C(21)-C(22)-N(2)-C(23) 83.3(2)

C(21)-C(22)-N(2)-C(25) -97.0(2)

N(2)-C(23)-N(3)-C(24) -0.4(2)

C(25)-C(24)-N(3)-C(23) 0.5(2)

C(2)-C(1)-O(1)-C(8) 179.35(19)

C(6)-C(1)-O(1)-C(8) -0.1(2)

C(7)-C(8)-O(1)-C(1) 0.2(2)

C(9)-C(8)-O(1)-C(1) -173.99(16)

Table 7. Hydrogen bonds for 11.2 [Å and °]. D-H...A d(D-H) d(H...A) d(D...A) <(DHA)

i. O3-H3b…N3 0.84 2.44 2.872(2) 148

ii. C2-H2…O5 0.95 2.48 3.323(3) 148

iii. C3-H3…O6 0.95 2.51 2.353(3) 148

iv. C20-H20a…O6 0.99 2.44 3.232(3) 137

v. C24-H24…O2 0.95 2.34 3.220(2) 154

vi. C24-H24…O2 0.98 2.56 3.514(3) 166

vii. C24-H24…O2 0.98 2.58 3.321(3) 132

Symmetry operators: i and vi = 1-x, 1-y, 2-z; ii and iii = 1-x, -1/2+y, 3/2-z; iv = -x, 1-y, 1-z; v = 1-x,1/2+y,3/2-z; vii =

x,y,-1+z.

Analysis of C-H...Cg (π-Ring) Interactions (H..Cg < 3.0 Ang. - Gamma < 30.0 Deg)

X-H…Cg H…Cg C-H…Cg C…Cg

i C19-H19…Cg2 2.69 99 2.989(2)

ii C22-H22a…Cg4 2.88 117 3.447(2)

iii C26-H26b…Cg5 2.81 138 3.603(3)

Symmetry operators: i = x, y, z; ii and iii = x, ½-y, ½+z.


Table 1. Crystal data and structure refinement for 11.5. Identification code mo_10bo_tel3_0ma

Empirical formula C30 H36 N3 O7 S2





Space group P21/c


b = 17.004(5) Å β= 108.601(15)°.

c = 18.512(5) Å γ = 90°.

Volume 3072.0(15) Å3

Z 4




F(000) 1300



Index ranges -14<=h<=9, -23<=k<=18, -20<=l<=24














C(1) 7218(5) 3746(3) 1205(3) 20(1)

C(2) 7046(6) 4322(3) 649(3) 29(1)

C(3) 7935(6) 4951(3) 840(3) 30(1)

C(4) 8952(5) 5006(3) 1552(3) 26(1)

C(5) 9104(5) 4427(3) 2101(3) 24(1)

C(6) 8204(5) 3782(3) 1923(3) 18(1)

C(7) 8009(5) 3080(3) 2309(3) 18(1)

C(8) 6947(5) 2686(3) 1827(3) 17(1)

C(9) 6165(4) 1958(3) 1875(3) 14(1)

C(10) 6595(5) 1443(3) 2535(3) 15(1)

C(11) 7769(5) 1380(3) 3159(3) 15(1)

C(12) 7599(5) 650(3) 3610(3) 16(1)

C(13) 5577(4) 793(3) 2562(3) 15(1)

C(14) 4270(5) 1125(3) 2659(3) 16(1)

C(15) 4293(5) 1500(3) 3344(3) 23(1)

C(16) 3091(5) 1825(3) 3393(3) 25(1)

C(17) 1866(5) 1786(3) 2803(3) 24(1)

C(18) 1850(5) 1404(3) 2133(3) 23(1)

C(19) 3063(5) 1073(3) 2065(3) 19(1)

C(20) -517(6) 1720(3) 1514(4) 39(2)

C(21) 5824(5) -373(3) 3472(3) 20(1)

C(22) 6059(5) -1108(3) 3045(3) 21(1)

C(23) 7552(5) -1343(3) 3218(3) 18(1)

C(24) 9213(5) -1312(3) 4544(3) 17(1)

C(25) 8567(5) -2354(3) 5041(3) 22(1)

C(26) 7744(5) -2297(3) 4308(3) 22(1)

C(27) 7006(6) 3141(3) 4083(4) 41(2)

C(28) 8871(6) 4288(4) 4228(4) 41(2)

C(29) 3987(5) 3809(3) 4759(3) 30(1)

C(30) 2864(6) 5015(3) 3826(3) 36(1)

N(1) 6376(4) 332(2) 3231(2) 18(1)

N(2) 8173(4) -1630(2) 4009(2) 18(1)

N(3) 9476(4) -1736(2) 5176(2) 19(1)

O(1) 6419(3) 3080(2) 1136(2) 21(1)

O(2) 5115(3) 1816(2) 1337(2) 21(1)

O(3) 8855(3) 1783(2) 3400(2) 19(1)

O(4) 8447(3) 414(2) 4198(2) 19(1)

O(5) 698(4) 1303(2) 1505(2) 31(1)

O(6) 6331(4) 4632(2) 4137(2) 39(1)

O(7) 1538(4) 3651(2) 3699(2) 34(1)

S(1) 7108(2) 4120(1) 3754(1) 29(1)

S(2) 2956(1) 3964(1) 3787(1) 25(1)


Table 3. Bond lengths [Å] and angles [°] for 11.5. Bond lengths (Ǻ)

C(1)-O(1) 1.381(5)

C(1)-C(2) 1.390(7)

C(1)-C(6) 1.393(7)

C(2)-C(3) 1.379(7)

C(2)-H(2) 0.9500

C(3)-C(4) 1.400(7)

C(3)-H(3) 0.9500

C(4)-C(5) 1.389(7)

C(4)-H(4) 0.9500

C(5)-C(6) 1.405(7)

C(5)-H(5) 0.9500

C(6)-C(7) 1.437(6)

C(7)-C(8) 1.348(6)

C(7)-H(7) 0.9500

C(8)-O(1) 1.392(5)

C(8)-C(9) 1.494(6)

C(9)-O(2) 1.238(5)

C(9)-C(10) 1.454(6)

C(10)-C(11) 1.384(6)

C(10)-C(13) 1.533(6)

C(11)-O(3) 1.265(5)

C(11)-C(12) 1.538(6)

C(12)-O(4) 1.225(5)

C(12)-N(1) 1.344(6)

C(13)-N(1) 1.475(6)

C(13)-C(14) 1.522(6)

C(13)-H(13) 1.0000

C(14)-C(19) 1.374(6)

C(14)-C(15) 1.413(7)

C(15)-C(16) 1.385(7)

C(15)-H(15) 0.9500

C(16)-C(17) 1.382(7)

C(16)-H(16) 0.9500

C(17)-C(18) 1.395(7)

C(17)-H(17) 0.9500

C(18)-O(5) 1.381(6)

C(18)-C(19) 1.411(7)

C(19)-H(19) 0.9500

C(20)-O(5) 1.443(6)

C(20)-H(20A) 0.9800

C(20)-H(20B) 0.9800

C(20)-H(20C) 0.9800

C(21)-N(1) 1.457(6)

C(21)-C(22) 1.540(7)

C(21)-H(21A) 0.9900

C(21)-H(21B) 0.9900

C(22)-C(23) 1.520(7)

C(22)-H(22A) 0.9900

C(22)-H(22B) 0.9900

C(23)-N(2) 1.481(6)

C(23)-H(23A) 0.9900

C(23)-H(23B) 0.9900

C(24)-N(2) 1.319(6)

C(24)-N(3) 1.328(6)

C(24)-H(24) 0.9500

C(25)-C(26) 1.355(7)

C(25)-N(3) 1.377(6)

C(25)-H(25) 0.9500

C(26)-N(2) 1.395(6)

C(26)-H(26) 0.9500

C(27)-S(1) 1.788(6)

C(27)-H(27A) 0.9800

C(27)-H(27B) 0.9800

C(27)-H(27C) 0.9800

C(28)-S(1) 1.769(6)

C(28)-H(28A) 0.9800

C(28)-H(28B) 0.9800

C(28)-H(28C) 0.9800

C(29)-S(2) 1.793(5)

C(29)-H(29A) 0.9800

C(29)-H(29B) 0.9800

C(29)-H(29C) 0.9800

C(30)-S(2) 1.792(5)

C(30)-H(30A) 0.9800

C(30)-H(30B) 0.9800

C(30)-H(30C) 0.9800

N(3)-H(3A) 0.8800

O(3)-H(3B) 0.8400

O(6)-S(1) 1.505(4)

O(7)-S(2) 1.513(4)

Bond angles (°)

O(1)-C(1)-C(2) 125.3(5)

O(1)-C(1)-C(6) 110.8(4)

C(2)-C(1)-C(6) 123.8(5)

C(3)-C(2)-C(1) 116.1(5)

C(3)-C(2)-H(2) 122.0

C(1)-C(2)-H(2) 122.0

C(2)-C(3)-C(4) 122.0(5)

C(2)-C(3)-H(3) 119.0

C(4)-C(3)-H(3) 119.0

C(5)-C(4)-C(3) 121.1(5)

C(5)-C(4)-H(4) 119.4

C(3)-C(4)-H(4) 119.4

C(4)-C(5)-C(6) 118.0(5)

C(4)-C(5)-H(5) 121.0

C(6)-C(5)-H(5) 121.0

C(1)-C(6)-C(5) 119.0(4)

C(1)-C(6)-C(7) 105.2(4)

C(5)-C(6)-C(7) 135.8(5)

C(8)-C(7)-C(6) 107.3(4)

C(8)-C(7)-H(7) 126.4

C(6)-C(7)-H(7) 126.4

C(7)-C(8)-O(1) 111.4(4)

C(7)-C(8)-C(9) 135.3(4)

O(1)-C(8)-C(9) 113.2(4)

O(2)-C(9)-C(10) 120.8(4)

O(2)-C(9)-C(8) 117.6(4)

C(10)-C(9)-C(8) 121.6(4)

C(11)-C(10)-C(9) 134.3(4)

C(11)-C(10)-C(13) 110.0(4)

C(9)-C(10)-C(13) 115.7(4)

O(3)-C(11)-C(10) 133.8(4)

O(3)-C(11)-C(12) 119.1(4)

C(10)-C(11)-C(12) 107.2(4)

O(4)-C(12)-N(1) 128.1(4)

O(4)-C(12)-C(11) 125.1(4)

N(1)-C(12)-C(11) 106.9(4)

N(1)-C(13)-C(14) 112.0(4)

N(1)-C(13)-C(10) 101.9(3)

C(14)-C(13)-C(10) 112.1(4)

N(1)-C(13)-H(13) 110.2


C(14)-C(13)-H(13) 110.2

C(10)-C(13)-H(13) 110.2

C(19)-C(14)-C(15) 119.9(4)

C(19)-C(14)-C(13) 119.4(4)

C(15)-C(14)-C(13) 120.7(4)

C(16)-C(15)-C(14) 118.7(5)

C(16)-C(15)-H(15) 120.7

C(14)-C(15)-H(15) 120.7

C(17)-C(16)-C(15) 122.5(5)

C(17)-C(16)-H(16) 118.7

C(15)-C(16)-H(16) 118.7

C(16)-C(17)-C(18) 118.3(5)

C(16)-C(17)-H(17) 120.8

C(18)-C(17)-H(17) 120.8

O(5)-C(18)-C(17) 124.7(5)

O(5)-C(18)-C(19) 115.0(5)

C(17)-C(18)-C(19) 120.3(5)

C(14)-C(19)-C(18) 120.3(5)

C(14)-C(19)-H(19) 119.9

C(18)-C(19)-H(19) 119.9

O(5)-C(20)-H(20A) 109.5

O(5)-C(20)-H(20B) 109.5

H(20A)-C(20)-H(20B) 109.5

O(5)-C(20)-H(20C) 109.5

H(20A)-C(20)-H(20C) 109.5

H(20B)-C(20)-H(20C) 109.5

N(1)-C(21)-C(22) 111.7(4)

N(1)-C(21)-H(21A) 109.3

C(22)-C(21)-H(21A) 109.3

N(1)-C(21)-H(21B) 109.3

C(22)-C(21)-H(21B) 109.3

H(21A)-C(21)-H(21B) 107.9

C(23)-C(22)-C(21) 114.8(4)

C(23)-C(22)-H(22A) 108.6

C(21)-C(22)-H(22A) 108.6

C(23)-C(22)-H(22B) 108.6

C(21)-C(22)-H(22B) 108.6

H(22A)-C(22)-H(22B) 107.5

N(2)-C(23)-C(22) 112.6(4)

N(2)-C(23)-H(23A) 109.1

C(22)-C(23)-H(23A) 109.1

N(2)-C(23)-H(23B) 109.1

C(22)-C(23)-H(23B) 109.1

H(23A)-C(23)-H(23B) 107.8

N(2)-C(24)-N(3) 109.1(4)

N(2)-C(24)-H(24) 125.5

N(3)-C(24)-H(24) 125.5

C(26)-C(25)-N(3) 107.4(4)

C(26)-C(25)-H(25) 126.3

N(3)-C(25)-H(25) 126.3

C(25)-C(26)-N(2) 106.2(4)

C(25)-C(26)-H(26) 126.9

N(2)-C(26)-H(26) 126.9

S(1)-C(27)-H(27A) 109.5

S(1)-C(27)-H(27B) 109.5

H(27A)-C(27)-H(27B) 109.5

S(1)-C(27)-H(27C) 109.5

H(27A)-C(27)-H(27C) 109.5

H(27B)-C(27)-H(27C) 109.5

S(1)-C(28)-H(28A) 109.5

S(1)-C(28)-H(28B) 109.5

H(28A)-C(28)-H(28B) 109.5

S(1)-C(28)-H(28C) 109.5

H(28A)-C(28)-H(28C) 109.5

H(28B)-C(28)-H(28C) 109.5

S(2)-C(29)-H(29A) 109.5

S(2)-C(29)-H(29B) 109.5

H(29A)-C(29)-H(29B) 109.5

S(2)-C(29)-H(29C) 109.5

H(29A)-C(29)-H(29C) 109.5

H(29B)-C(29)-H(29C) 109.5

S(2)-C(30)-H(30A) 109.5

S(2)-C(30)-H(30B) 109.5

H(30A)-C(30)-H(30B) 109.5

S(2)-C(30)-H(30C) 109.5

H(30A)-C(30)-H(30C) 109.5

H(30B)-C(30)-H(30C) 109.5

C(12)-N(1)-C(21) 124.3(4)

C(12)-N(1)-C(13) 113.8(4)

C(21)-N(1)-C(13) 121.8(4)

C(24)-N(2)-C(26) 108.7(4)

C(24)-N(2)-C(23) 126.7(4)

C(26)-N(2)-C(23) 124.6(4)

C(24)-N(3)-C(25) 108.6(4)

C(24)-N(3)-H(3A) 125.7

C(25)-N(3)-H(3A) 125.7

C(1)-O(1)-C(8) 105.3(4)

C(11)-O(3)-H(3B) 109.5

C(18)-O(5)-C(20) 116.4(4)

O(6)-S(1)-C(28) 106.8(3)

O(6)-S(1)-C(27) 106.2(3)

C(28)-S(1)-C(27) 98.2(3)

O(7)-S(2)-C(30) 107.0(3)

O(7)-S(2)-C(29) 107.4(2)

C(30)-S(2)-C(29) 97.4(3)

Table 4. Anisotropic displacement parameters (Å2x 103)for 11.5. The anisotropic


C(1) 21(3) 20(3) 25(3) 0(2) 18(2) 0(2)

C(2) 43(4) 28(3) 20(3) 5(2) 15(2) 1(2)

C(3) 40(4) 30(3) 30(3) 6(2) 26(3) 2(2)

C(4) 35(3) 16(3) 37(3) 0(2) 26(3) -2(2)

C(5) 25(3) 27(3) 23(3) 0(2) 13(2) 4(2)

C(6) 21(3) 18(3) 24(3) 5(2) 19(2) 7(2)

C(7) 19(3) 21(3) 17(2) 5(2) 8(2) 7(2)

C(8) 21(3) 21(3) 14(2) 3(2) 11(2) 10(2)

C(9) 13(2) 17(2) 17(2) 1(2) 9(2) 7(2)

C(10) 15(2) 16(2) 19(2) -2(2) 12(2) 2(2)

C(11) 18(3) 14(2) 18(2) -3(2) 14(2) 4(2)


C(12) 18(3) 17(2) 19(2) 0(2) 12(2) 3(2)

C(13) 16(2) 17(2) 14(2) -1(2) 6(2) 1(2)

C(15) 26(3) 24(3) 22(3) -5(2) 13(2) -3(2)

C(16) 36(3) 20(3) 29(3) -7(2) 24(2) -4(2)

C(17) 32(3) 17(3) 37(3) 0(2) 29(3) 0(2)

C(18) 23(3) 21(3) 31(3) 8(2) 17(2) -1(2)

C(19) 20(3) 21(3) 24(3) 0(2) 18(2) 1(2)

C(20) 28(3) 43(4) 50(4) -1(3) 18(3) 13(3)

C(21) 19(3) 20(3) 23(3) 3(2) 10(2) -1(2)

C(22) 27(3) 19(3) 19(3) 3(2) 9(2) -4(2)

C(23) 22(3) 20(2) 12(2) 0(2) 8(2) -3(2)

C(24) 17(3) 17(2) 19(2) 1(2) 9(2) 2(2)

C(25) 22(3) 21(3) 27(3) 4(2) 11(2) -3(2)

C(26) 23(3) 20(3) 25(3) -1(2) 11(2) -10(2)

C(27) 55(4) 29(3) 51(4) -3(3) 35(3) 1(3)

C(28) 43(4) 45(4) 38(4) 0(3) 20(3) 4(3)

C(29) 33(3) 40(3) 18(3) 3(2) 9(2) -5(2)

C(30) 55(4) 26(3) 33(3) 8(3) 24(3) -4(3)

N(1) 18(2) 16(2) 21(2) 1(2) 10(2) -3(2)

N(2) 18(2) 20(2) 16(2) -1(2) 7(2) -1(2)

N(3) 17(2) 23(2) 15(2) -4(2) 3(2) 4(2)

O(1) 28(2) 23(2) 16(2) 5(1) 11(2) 0(2)

O(2) 19(2) 26(2) 18(2) 7(1) 9(2) 2(1)

O(3) 20(2) 22(2) 14(2) 5(1) 5(1) 2(1)

O(4) 23(2) 19(2) 18(2) 2(1) 8(2) 0(1)

O(5) 22(2) 39(2) 34(2) 0(2) 13(2) 10(2)

O(6) 47(3) 31(2) 57(3) -1(2) 42(2) 8(2)

O(7) 32(2) 37(2) 34(2) 4(2) 12(2) -5(2)

S(1) 34(1) 29(1) 30(1) -4(1) 20(1) 1(1)

S(2) 32(1) 29(1) 18(1) 2(1) 14(1) 0(1)


x y z U(eq)

H(2) 6357 4284 166 35

H(3) 7855 5359 479 36

H(4) 9549 5448 1660 31

H(5) 9795 4465 2584 28

H(7) 8530 2924 2810 22

H(13) 5355 463 2091 18

H(15) 5116 1528 3762 27

H(16) 3109 2085 3851 30

H(17) 1056 2014 2851 29

H(19) 3044 814 1607 23

H(20A) -750 1571 1969 58

H(20B) -1279 1586 1057 58

H(20C) -346 2288 1522 58

H(21A) 4828 -303 3377 24

H(21B) 6266 -449 4027 24

H(22A) 5557 -1555 3174 25

H(22B) 5662 -1012 2490 25

H(23A) 7616 -1762 2859 21

H(23B) 8079 -884 3135 21

H(24) 9695 -852 4485 20

H(25) 8525 -2749 5397 27

H(26) 7019 -2642 4049 26

H(27A) 7316 3136 4642 61

H(27B) 7590 2793 3899 61

H(27C) 6055 2957 3891 61

H(28A) 9090 4839 4164 61

H(28B) 9417 3945 4012 61

H(28C) 9081 4171 4772 61

H(29A) 3514 4022 5099 45

H(29B) 4870 4076 4856 45

H(29C) 4141 3244 4854 45

H(30A) 2273 5214 3335 53

H(30B) 3785 5237 3937 53


H(30C) 2484 5167 4229 53

H(3A) 10124 -1638 5610 23

H(3B) 9375 1580 3803 28

Table 6. Torsion angles [°] for 11.5. O(1)-C(1)-C(2)-C(3) 178.4(5)

C(6)-C(1)-C(2)-C(3) 0.7(8)

C(1)-C(2)-C(3)-C(4) 0.1(8)

C(2)-C(3)-C(4)-C(5) -0.4(8)

C(3)-C(4)-C(5)-C(6) -0.1(7)

O(1)-C(1)-C(6)-C(5) -179.2(4)

C(2)-C(1)-C(6)-C(5) -1.2(7)

O(1)-C(1)-C(6)-C(7) 1.4(5)

C(2)-C(1)-C(6)-C(7) 179.3(5)

C(4)-C(5)-C(6)-C(1) 0.8(7)

C(4)-C(5)-C(6)-C(7) -179.9(5)

C(1)-C(6)-C(7)-C(8) -1.0(5)

C(5)-C(6)-C(7)-C(8) 179.7(5)

C(6)-C(7)-C(8)-O(1) 0.2(5)

C(6)-C(7)-C(8)-C(9) -175.6(5)

C(7)-C(8)-C(9)-O(2) 171.1(5)

O(1)-C(8)-C(9)-O(2) -4.6(6)

C(7)-C(8)-C(9)-C(10) -7.4(8)

O(1)-C(8)-C(9)-C(10) 176.9(4)

O(2)-C(9)-C(10)-C(11) 169.8(5)

C(8)-C(9)-C(10)-C(11) -11.8(8)

O(2)-C(9)-C(10)-C(13) -7.7(6)

C(8)-C(9)-C(10)-C(13) 170.7(4)

C(9)-C(10)-C(11)-O(3) 5.2(9)

C(13)-C(10)-C(11)-O(3) -177.2(5)

C(9)-C(10)-C(11)-C(12) -174.9(5)

C(13)-C(10)-C(11)-C(12) 2.7(5)

O(3)-C(11)-C(12)-O(4) -0.3(7)

C(10)-C(11)-C(12)-O(4) 179.8(4)

O(3)-C(11)-C(12)-N(1) -179.5(4)

C(10)-C(11)-C(12)-N(1) 0.6(5)

C(11)-C(10)-C(13)-N(1) -4.7(5)

C(9)-C(10)-C(13)-N(1) 173.4(4)

C(11)-C(10)-C(13)-C(14) 115.3(4)

C(9)-C(10)-C(13)-C(14) -66.7(5)

N(1)-C(13)-C(14)-C(19) -135.2(4)

C(10)-C(13)-C(14)-C(19) 110.9(5)

N(1)-C(13)-C(14)-C(15) 46.3(6)

C(10)-C(13)-C(14)-C(15) -67.6(6)

C(19)-C(14)-C(15)-C(16) -1.5(7)

C(13)-C(14)-C(15)-C(16) 177.0(4)

C(14)-C(15)-C(16)-C(17) 1.0(8)

C(15)-C(16)-C(17)-C(18) 0.0(7)

C(16)-C(17)-C(18)-O(5) 178.3(4)

C(16)-C(17)-C(18)-C(19) -0.5(7)

C(15)-C(14)-C(19)-C(18) 1.0(7)

C(13)-C(14)-C(19)-C(18) -177.5(4)

O(5)-C(18)-C(19)-C(14) -178.8(4)

C(17)-C(18)-C(19)-C(14) 0.1(7)

N(1)-C(21)-C(22)-C(23) 66.1(5)

C(21)-C(22)-C(23)-N(2) 68.1(5)

N(3)-C(25)-C(26)-N(2) -0.4(5)

O(4)-C(12)-N(1)-C(21) 0.8(8)

C(11)-C(12)-N(1)-C(21) 180.0(4)

O(4)-C(12)-N(1)-C(13) 177.0(4)

C(11)-C(12)-N(1)-C(13) -3.9(5)

C(22)-C(21)-N(1)-C(12) -98.3(5)

C(22)-C(21)-N(1)-C(13) 85.8(5)

C(14)-C(13)-N(1)-C(12) -114.7(4)

C(10)-C(13)-N(1)-C(12) 5.3(5)

C(14)-C(13)-N(1)-C(21) 61.5(5)

C(10)-C(13)-N(1)-C(21) -178.5(4)

N(3)-C(24)-N(2)-C(26) -0.4(5)

N(3)-C(24)-N(2)-C(23) -179.9(4)

C(25)-C(26)-N(2)-C(24) 0.5(5)

C(25)-C(26)-N(2)-C(23) 180.0(4)

C(22)-C(23)-N(2)-C(24) -117.8(5)

C(22)-C(23)-N(2)-C(26) 62.8(6)

N(2)-C(24)-N(3)-C(25) 0.1(5)

C(26)-C(25)-N(3)-C(24) 0.2(6)

C(2)-C(1)-O(1)-C(8) -179.2(5)

C(6)-C(1)-O(1)-C(8) -1.3(5)

C(7)-C(8)-O(1)-C(1) 0.6(5)

C(9)-C(8)-O(1)-C(1) 177.4(4)

C(17)-C(18)-O(5)-C(20) 9.1(7)

C(19)-C(18)-O(5)-C(20) -172.1(4)

A.4 Pyrrolidinone 13.3a

Table 1. Crystal data and structure refinement for 13.3a. Identification code mo_10bo_tel1b_0m

Empirical formula C24 H23 F N2 O4





Space group P21/c


b = 17.1711(7) Å β= 95.0980(10)°.

c = 18.0711(7) Å γ = 90°.

Volume 4216.0(3) Å3

Z 8



F(000) 1776




Index ranges -18<=h<=18, -22<=k<=22, -

23<=l<=23




Absorption correction Semi-empirical from

equivalents


Refinement method Full-matrix least-squares on

F2







for 13.3a. U(eq) is defined as one third of the trace of the orthogonalised Uij tensor. x y z U(eq)

F(2) 4446(1) 9887(1) 9257(1) 56(1)

C(1) 8753(1) 4048(1) 9029(1) 18(1)

C(2) 8639(1) 4479(1) 9670(1) 20(1)

C(3) 9259(1) 4370(1) 10312(1) 21(1)

C(4) 10004(1) 3833(1) 10297(1) 21(1)

C(5) 10143(1) 3394(1) 9681(1) 24(1)

C(6) 9504(1) 3504(1) 9044(1) 22(1)

C(7) 8061(1) 4180(1) 8336(1) 18(1)

C(8) 9093(1) 5174(1) 7679(1) 20(1)

C(9) 9107(1) 4843(1) 6896(1) 22(1)

C(10) 10039(1) 5032(1) 6528(1) 22(1)

C(11) 11106(2) 5988(1) 6009(1) 30(1)

C(12) 9326(2) 6222(1) 5909(1) 31(1)

C(13) 7362(1) 5365(1) 7912(1) 20(1)

C(14) 6567(1) 4894(1) 8247(1) 19(1)

C(15) 6986(1) 4192(1) 8480(1) 18(1)

C(16) 6570(1) 3485(1) 8745(1) 18(1)

C(17) 5637(1) 3520(1) 9109(1) 19(1)

C(18) 5085(1) 4104(1) 9344(1) 23(1)

C(19) 4286(1) 3752(1) 9688(1) 22(1)

C(20) 3483(1) 4026(1) 10043(1) 28(1)

C(21) 2864(2) 3489(1) 10321(1) 33(1)

C(22) 3022(2) 2694(1) 10247(1) 42(1)

C(23) 3817(2) 2408(1) 9904(1) 41(1)

C(24) 4433(1) 2957(1) 9634(1) 25(1)

N(1) 8202(1) 4948(1) 8008(1) 19(1)

N(2) 10175(1) 5879(1) 6372(1) 22(1)

O(1) 7240(1) 5994(1) 7602(1) 28(1)

O(2) 5701(1) 5162(1) 8228(1) 24(1)

O(3) 6984(1) 2848(1) 8694(1) 26(1)

O(4) 5264(1) 2802(1) 9286(1) 26(1)

C(25) 2580(1) 8006(1) 9104(1) 20(1)

C(26) 2904(2) 8311(1) 9793(1) 26(1)

C(27) 3536(2) 8945(1) 9852(1) 35(1)

C(28) 3833(2) 9256(1) 9210(1) 34(1)

C(29) 3556(1) 8958(1) 8517(1) 28(1)

C(30) 2920(1) 8330(1) 8469(1) 23(1)

C(31) 1855(1) 7329(1) 9045(1) 19(1)

C(32) 3069(1) 6193(1) 8965(1) 26(1)

C(33) 4052(1) 6459(1) 8711(1) 25(1)

C(34) 4055(1) 6425(1) 7873(1) 23(1)

C(35) 5046(2) 6371(1) 6816(1) 38(1)

C(36) 5535(2) 7262(1) 7835(2) 44(1)

C(37) 1632(1) 6429(1) 8069(1) 21(1)


C(38) 793(1) 7007(1) 7976(1) 19(1)

C(39) 900(1) 7506(1) 8577(1) 18(1)

C(40) 237(1) 8037(1) 8882(1) 19(1)

C(41) -730(1) 8234(1) 8488(1) 20(1)

C(42) -1123(1) 8210(1) 7775(1) 25(1)

C(43) -2066(1) 8588(1) 7752(1) 28(1)

C(44) -2816(2) 8765(1) 7198(2) 40(1)

C(45) -3639(2) 9137(1) 7403(2) 48(1)

C(46) -3735(2) 9329(1) 8138(2) 46(1)

C(47) -3004(2) 9171(1) 8703(2) 39(1)

C(48) -2175(1) 8803(1) 8479(1) 28(1)

N(3) 2237(1) 6658(1) 8661(1) 20(1)

N(4) 5074(1) 6500(1) 7631(1) 26(1)

O(5) 1726(1) 5848(1) 7686(1) 28(1)

O(6) 154(1) 6944(1) 7429(1) 23(1)

O(7) 446(1) 8344(1) 9500(1) 26(1)

O(8) -1364(1) 8598(1) 8942(1) 25(1)

F(1) 10636(1) 3740(1) 10916(1) 28(1)

Table 3. Bond lengths [Å] and angles [°] for 13.3a. Bond lengths (Ǻ)

F(2)-C(28) 1.366(2)

C(1)-C(6) 1.385(3)

C(1)-C(2) 1.395(3)

C(1)-C(7) 1.516(3)

C(2)-C(3) 1.387(3)

C(2)-H(2) 0.9500

C(3)-C(4) 1.374(3)

C(3)-H(3) 0.9500

C(4)-F(1) 1.360(2)

C(4)-C(5) 1.372(3)

C(5)-C(6) 1.393(3)

C(5)-H(5) 0.9500

C(6)-H(6) 0.9500

C(7)-N(1) 1.466(2)

C(7)-C(15) 1.512(2)

C(7)-H(7) 1.0000

C(8)-N(1) 1.453(2)

C(8)-C(9) 1.527(3)

C(8)-H(8A) 0.9900

C(8)-H(8B) 0.9900

C(9)-C(10) 1.521(2)

C(9)-H(9A) 0.9900

C(9)-H(9B) 0.9900

C(10)-N(2) 1.497(2)

C(10)-H(10A) 0.9900

C(10)-H(10B) 0.9900

C(11)-N(2) 1.492(2)

C(11)-H(11A) 0.9800

C(11)-H(11B) 0.9800

C(11)-H(11C) 0.9800

C(12)-N(2) 1.488(2)

C(12)-H(12A) 0.9800

C(12)-H(12B) 0.9800

C(12)-H(12C) 0.9800

C(13)-O(1) 1.223(2)

C(13)-N(1) 1.348(2)

C(13)-C(14) 1.521(2)

C(14)-O(2) 1.264(2)

C(14)-C(15) 1.384(2)

C(15)-C(16) 1.440(2)

C(16)-O(3) 1.238(2)

C(16)-C(17) 1.485(2)

C(17)-C(18) 1.346(3)

C(17)-O(4) 1.382(2)

C(18)-C(19) 1.434(3)

C(18)-H(18) 0.9500

C(19)-C(24) 1.384(3)

C(19)-C(20) 1.398(3)

C(20)-C(21) 1.376(3)

C(20)-H(20) 0.9500

C(21)-C(22) 1.390(3)

C(21)-H(21) 0.9500

C(22)-C(23) 1.386(3)

C(22)-H(22) 0.9500

C(23)-C(24) 1.381(3)

C(23)-H(23) 0.9500

C(24)-O(4) 1.370(2)

O(2)-H(2A) 0.8400

C(25)-C(26) 1.387(3)

C(25)-C(30) 1.391(3)

C(25)-C(31) 1.524(3)

C(26)-C(27) 1.387(3)

C(26)-H(26) 0.9500

C(27)-C(28) 1.370(3)

C(27)-H(27) 0.9500

C(28)-C(29) 1.375(3)

C(29)-C(30) 1.382(3)

C(29)-H(29) 0.9500

C(30)-H(30) 0.9500

C(31)-N(3) 1.465(2)

C(31)-C(39) 1.520(2)

C(31)-H(31) 1.0000

C(32)-N(3) 1.456(2)

C(32)-C(33) 1.526(3)

C(32)-H(32A) 0.9900

C(32)-H(32B) 0.9900

C(33)-C(34) 1.515(3)

C(33)-H(33A) 0.9900

C(33)-H(33B) 0.9900

C(34)-N(4) 1.499(2)

C(34)-H(34A) 0.9900

C(34)-H(34B) 0.9900

C(35)-N(4) 1.486(3)

C(35)-H(35A) 0.9800

C(35)-H(35B) 0.9800

C(35)-H(35C) 0.9800

C(36)-N(4) 1.483(3)

C(36)-H(36A) 0.9800

C(36)-H(36B) 0.9800

C(36)-H(36C) 0.9800


C(37)-O(5) 1.227(2)

C(37)-N(3) 1.350(2)

C(37)-C(38) 1.512(3)

C(38)-O(6) 1.263(2)

C(38)-C(39) 1.382(3)

C(39)-C(40) 1.428(2)

C(40)-O(7) 1.244(2)

C(40)-C(41) 1.482(3)

C(41)-C(42) 1.352(3)

C(41)-O(8) 1.392(2)

C(42)-C(43) 1.437(3)

C(42)-H(42) 0.9500

C(43)-C(48) 1.385(3)

C(43)-C(44) 1.400(3)

C(44)-C(45) 1.371(3)

C(44)-H(44) 0.9500

C(45)-C(46) 1.387(4)

C(45)-H(45) 0.9500

C(46)-C(47) 1.388(4)

C(46)-H(46) 0.9500

C(47)-C(48) 1.388(3)

C(47)-H(47) 0.9500

C(48)-O(8) 1.371(2)

O(6)-H(6A) 0.8400

Bond Angles (°)

C(6)-C(1)-C(2) 118.56(17)

C(6)-C(1)-C(7) 121.51(16)

C(2)-C(1)-C(7) 119.94(16)

C(3)-C(2)-C(1) 121.23(17)

C(3)-C(2)-H(2) 119.4

C(1)-C(2)-H(2) 119.4

C(4)-C(3)-C(2) 118.10(17)

C(4)-C(3)-H(3) 121.0

C(2)-C(3)-H(3) 121.0

F(1)-C(4)-C(5) 118.65(17)

F(1)-C(4)-C(3) 118.58(17)

C(5)-C(4)-C(3) 122.76(18)

C(4)-C(5)-C(6) 118.29(17)

C(4)-C(5)-H(5) 120.9

C(6)-C(5)-H(5) 120.9

C(1)-C(6)-C(5) 121.05(18)

C(1)-C(6)-H(6) 119.5

C(5)-C(6)-H(6) 119.5

N(1)-C(7)-C(15) 102.72(14)

N(1)-C(7)-C(1) 111.99(14)

C(15)-C(7)-C(1) 113.72(14)

N(1)-C(7)-H(7) 109.4

C(15)-C(7)-H(7) 109.4

C(1)-C(7)-H(7) 109.4

N(1)-C(8)-C(9) 111.08(15)

N(1)-C(8)-H(8A) 109.4

C(9)-C(8)-H(8A) 109.4

N(1)-C(8)-H(8B) 109.4

C(9)-C(8)-H(8B) 109.4

H(8A)-C(8)-H(8B) 108.0

C(10)-C(9)-C(8) 113.91(15)

C(10)-C(9)-H(9A) 108.8

C(8)-C(9)-H(9A) 108.8

C(10)-C(9)-H(9B) 108.8

C(8)-C(9)-H(9B) 108.8

H(9A)-C(9)-H(9B) 107.7

N(2)-C(10)-C(9) 114.33(15)

N(2)-C(10)-H(10A) 108.7

C(9)-C(10)-H(10A) 108.7

N(2)-C(10)-H(10B) 108.7

C(9)-C(10)-H(10B) 108.7

H(10A)-C(10)-H(10B) 107.6

N(2)-C(11)-H(11A) 109.5

N(2)-C(11)-H(11B) 109.5

H(11A)-C(11)-H(11B) 109.5

N(2)-C(11)-H(11C) 109.5

H(11A)-C(11)-H(11C) 109.5

H(11B)-C(11)-H(11C) 109.5

N(2)-C(12)-H(12A) 109.5

N(2)-C(12)-H(12B) 109.5

H(12A)-C(12)-H(12B) 109.5

N(2)-C(12)-H(12C) 109.5

H(12A)-C(12)-H(12C) 109.5

H(12B)-C(12)-H(12C) 109.5

O(1)-C(13)-N(1) 127.44(17)

O(1)-C(13)-C(14) 125.38(17)

N(1)-C(13)-C(14) 107.16(15)

O(2)-C(14)-C(15) 133.25(16)

O(2)-C(14)-C(13) 119.54(16)

C(15)-C(14)-C(13) 107.07(15)

C(14)-C(15)-C(16) 132.17(16)

C(14)-C(15)-C(7) 109.94(15)

C(16)-C(15)-C(7) 117.49(15)

O(3)-C(16)-C(15) 121.63(16)

O(3)-C(16)-C(17) 118.93(16)

C(15)-C(16)-C(17) 119.42(15)

C(18)-C(17)-O(4) 111.26(15)

C(18)-C(17)-C(16) 134.16(17)

O(4)-C(17)-C(16) 114.48(15)

C(17)-C(18)-C(19) 106.99(17)

C(17)-C(18)-H(18) 126.5

C(19)-C(18)-H(18) 126.5

C(24)-C(19)-C(20) 119.18(18)

C(24)-C(19)-C(18) 105.30(16)

C(20)-C(19)-C(18) 135.50(19)

C(21)-C(20)-C(19) 118.28(19)

C(21)-C(20)-H(20) 120.9

C(19)-C(20)-H(20) 120.9

C(20)-C(21)-C(22) 121.2(2)

C(20)-C(21)-H(21) 119.4

C(22)-C(21)-H(21) 119.4

C(23)-C(22)-C(21) 121.7(2)

C(23)-C(22)-H(22) 119.2

C(21)-C(22)-H(22) 119.2

C(24)-C(23)-C(22) 116.2(2)

C(24)-C(23)-H(23) 121.9

C(22)-C(23)-H(23) 121.9

O(4)-C(24)-C(23) 125.68(18)

O(4)-C(24)-C(19) 110.80(16)

C(23)-C(24)-C(19) 123.52(19)

C(13)-N(1)-C(8) 122.56(15)

C(13)-N(1)-C(7) 112.93(14)

C(8)-N(1)-C(7) 123.35(14)

C(12)-N(2)-C(11) 110.70(15)

C(12)-N(2)-C(10) 112.78(15)

C(11)-N(2)-C(10) 109.19(15)

C(14)-O(2)-H(2A) 109.5

C(24)-O(4)-C(17) 105.64(14)

C(26)-C(25)-C(30) 119.17(17)

C(26)-C(25)-C(31) 120.20(17)


C(30)-C(25)-C(31) 120.63(17)

C(25)-C(26)-C(27) 120.73(19)

C(25)-C(26)-H(26) 119.6

C(27)-C(26)-H(26) 119.6

C(28)-C(27)-C(26) 118.1(2)

C(28)-C(27)-H(27) 121.0

C(26)-C(27)-H(27) 121.0

F(2)-C(28)-C(27) 118.84(19)

F(2)-C(28)-C(29) 118.00(19)

C(27)-C(28)-C(29) 123.15(19)

C(28)-C(29)-C(30) 117.98(19)

C(28)-C(29)-H(29) 121.0

C(30)-C(29)-H(29) 121.0

C(29)-C(30)-C(25) 120.86(18)

C(29)-C(30)-H(30) 119.6

C(25)-C(30)-H(30) 119.6

N(3)-C(31)-C(39) 102.51(14)

N(3)-C(31)-C(25) 112.10(14)

C(39)-C(31)-C(25) 113.90(15)

N(3)-C(31)-H(31) 109.4

C(39)-C(31)-H(31) 109.4

C(25)-C(31)-H(31) 109.4

N(3)-C(32)-C(33) 113.46(16)

N(3)-C(32)-H(32A) 108.9

C(33)-C(32)-H(32A) 108.9

N(3)-C(32)-H(32B) 108.9

C(33)-C(32)-H(32B) 108.9

H(32A)-C(32)-H(32B) 107.7

C(34)-C(33)-C(32) 111.72(16)

C(34)-C(33)-H(33A) 109.3

C(32)-C(33)-H(33A) 109.3

C(34)-C(33)-H(33B) 109.3

C(32)-C(33)-H(33B) 109.3

H(33A)-C(33)-H(33B) 107.9

N(4)-C(34)-C(33) 111.94(16)

N(4)-C(34)-H(34A) 109.2

C(33)-C(34)-H(34A) 109.2

N(4)-C(34)-H(34B) 109.2

C(33)-C(34)-H(34B) 109.2

H(34A)-C(34)-H(34B) 107.9

N(4)-C(35)-H(35A) 109.5

N(4)-C(35)-H(35B) 109.5

H(35A)-C(35)-H(35B) 109.5

N(4)-C(35)-H(35C) 109.5

H(35A)-C(35)-H(35C) 109.5

H(35B)-C(35)-H(35C) 109.5

N(4)-C(36)-H(36A) 109.5

N(4)-C(36)-H(36B) 109.5

H(36A)-C(36)-H(36B) 109.5

N(4)-C(36)-H(36C) 109.5

H(36A)-C(36)-H(36C) 109.5

H(36B)-C(36)-H(36C) 109.5

O(5)-C(37)-N(3) 126.60(17)

O(5)-C(37)-C(38) 125.95(18)

N(3)-C(37)-C(38) 107.41(16)

O(6)-C(38)-C(39) 133.25(17)

O(6)-C(38)-C(37) 119.57(16)

C(39)-C(38)-C(37) 107.14(16)

C(38)-C(39)-C(40) 132.36(17)

C(38)-C(39)-C(31) 109.78(15)

C(40)-C(39)-C(31) 116.95(16)

O(7)-C(40)-C(39) 120.97(17)

O(7)-C(40)-C(41) 117.21(16)

C(39)-C(40)-C(41) 121.81(16)

C(42)-C(41)-O(8) 111.10(16)

C(42)-C(41)-C(40) 135.42(17)

O(8)-C(41)-C(40) 113.17(16)

C(41)-C(42)-C(43) 106.94(18)

C(41)-C(42)-H(42) 126.5

C(43)-C(42)-H(42) 126.5

C(48)-C(43)-C(44) 119.1(2)

C(48)-C(43)-C(42) 105.35(18)

C(44)-C(43)-C(42) 135.5(2)

C(45)-C(44)-C(43) 118.2(3)

C(45)-C(44)-H(44) 120.9

C(43)-C(44)-H(44) 120.9

C(44)-C(45)-C(46) 121.2(2)

C(44)-C(45)-H(45) 119.4

C(46)-C(45)-H(45) 119.4

C(45)-C(46)-C(47) 122.4(2)

C(45)-C(46)-H(46) 118.8

C(47)-C(46)-H(46) 118.8

C(48)-C(47)-C(46) 115.2(2)

C(48)-C(47)-H(47) 122.4

C(46)-C(47)-H(47) 122.4

O(8)-C(48)-C(43) 111.16(17)

O(8)-C(48)-C(47) 125.0(2)

C(43)-C(48)-C(47) 123.8(2)

C(37)-N(3)-C(32) 122.70(16)

C(37)-N(3)-C(31) 112.79(15)

C(32)-N(3)-C(31) 123.40(15)

C(36)-N(4)-C(35) 110.53(18)

C(36)-N(4)-C(34) 112.78(16)

C(35)-N(4)-C(34) 109.55(16)

C(38)-O(6)-H(6A) 109.5

C(48)-O(8)-C(41) 105.44(15)

Table 4. Anisotropic displacement parameters (Å2x 103)for 13.3a. The anisotropic


F(2) 68(1) 53(1) 44(1) 5(1) -7(1) -41(1)

C(1) 15(1) 18(1) 20(1) 2(1) 5(1) -2(1)

C(2) 17(1) 20(1) 23(1) 0(1) 5(1) 1(1)

C(3) 20(1) 25(1) 19(1) -2(1) 5(1) -3(1)

C(4) 18(1) 24(1) 21(1) 5(1) -1(1) -5(1)

C(5) 18(1) 23(1) 32(1) 1(1) 2(1) 4(1)

C(6) 20(1) 22(1) 24(1) -3(1) 3(1) 2(1)

C(7) 18(1) 17(1) 19(1) 0(1) 3(1) -1(1)

C(8) 17(1) 23(1) 19(1) 1(1) 4(1) -3(1)

C(9) 23(1) 21(1) 22(1) -2(1) 5(1) -3(1)


C(10) 25(1) 20(1) 22(1) -2(1) 7(1) 0(1)

C(11) 27(1) 38(1) 26(1) 7(1) 11(1) -3(1)

C(12) 30(1) 35(1) 28(1) 7(1) 3(1) 5(1)

C(13) 18(1) 20(1) 21(1) -1(1) 3(1) -2(1)

C(14) 17(1) 20(1) 19(1) 0(1) 2(1) -2(1)

C(15) 15(1) 20(1) 18(1) -1(1) 2(1) -1(1)

C(16) 18(1) 20(1) 16(1) 0(1) 1(1) -1(1)

C(17) 21(1) 18(1) 18(1) 3(1) 1(1) -5(1)

C(18) 20(1) 22(1) 26(1) 2(1) 5(1) 0(1)

C(19) 19(1) 26(1) 20(1) 2(1) 1(1) -1(1)

C(20) 22(1) 32(1) 32(1) 2(1) 6(1) 3(1)

C(21) 23(1) 44(1) 34(1) 4(1) 10(1) 1(1)

C(22) 35(1) 41(1) 54(2) 7(1) 23(1) -9(1)

C(23) 41(1) 27(1) 59(2) 3(1) 23(1) -7(1)

C(24) 21(1) 26(1) 28(1) 0(1) 8(1) -2(1)

N(1) 17(1) 20(1) 20(1) 2(1) 5(1) -1(1)

N(2) 21(1) 23(1) 23(1) 4(1) 5(1) -1(1)

O(1) 24(1) 24(1) 36(1) 10(1) 5(1) 0(1)

O(2) 19(1) 20(1) 34(1) 9(1) 4(1) 3(1)

O(3) 27(1) 19(1) 35(1) 2(1) 11(1) 2(1)

O(4) 27(1) 20(1) 35(1) 0(1) 14(1) -4(1)

C(25) 14(1) 21(1) 24(1) 4(1) 0(1) 4(1)

C(26) 27(1) 30(1) 22(1) 2(1) 2(1) -2(1)

C(27) 41(1) 38(1) 25(1) -2(1) -6(1) -13(1)

C(28) 33(1) 33(1) 34(1) 4(1) -6(1) -15(1)

C(29) 24(1) 33(1) 27(1) 10(1) -1(1) -4(1)

C(30) 21(1) 26(1) 21(1) 3(1) -2(1) 1(1)

C(31) 17(1) 19(1) 20(1) 2(1) 2(1) 2(1)

C(32) 23(1) 25(1) 27(1) 6(1) -1(1) 8(1)

C(33) 18(1) 27(1) 30(1) -1(1) -4(1) 6(1)

C(34) 17(1) 22(1) 30(1) 4(1) 1(1) 3(1)

C(35) 41(1) 40(1) 35(1) 17(1) 12(1) 10(1)

C(36) 30(1) 25(1) 76(2) 0(1) 10(1) -5(1)

C(37) 19(1) 20(1) 25(1) 4(1) 3(1) 0(1)

C(38) 16(1) 18(1) 22(1) 4(1) 2(1) -2(1)

C(39) 14(1) 19(1) 21(1) 4(1) 2(1) 1(1)

C(40) 19(1) 19(1) 19(1) 5(1) 2(1) -1(1)

C(41) 18(1) 17(1) 27(1) 3(1) 6(1) 2(1)

C(42) 23(1) 22(1) 30(1) 2(1) -2(1) 1(1)

C(43) 21(1) 19(1) 42(1) 3(1) -5(1) -2(1)

C(44) 32(1) 26(1) 58(2) 3(1) -18(1) -2(1)

C(45) 24(1) 26(1) 88(2) 8(1) -21(1) 0(1)

C(46) 16(1) 25(1) 97(2) 11(1) 3(1) 3(1)

C(47) 25(1) 29(1) 66(2) 9(1) 15(1) 5(1)

C(48) 16(1) 22(1) 45(1) 11(1) 2(1) 2(1)

N(3) 17(1) 21(1) 22(1) 1(1) -1(1) 5(1)

N(4) 24(1) 21(1) 36(1) 4(1) 6(1) 1(1)

O(5) 29(1) 22(1) 32(1) -4(1) 1(1) 4(1)

O(6) 21(1) 23(1) 24(1) -4(1) -1(1) 3(1)

O(7) 27(1) 31(1) 21(1) -2(1) 2(1) 5(1)

O(8) 19(1) 27(1) 31(1) 5(1) 8(1) 6(1)

F(1) 22(1) 35(1) 24(1) 4(1) -2(1) -2(1)

Table 5. Hydrogen coordinates ( x 104) and isotropic displacement parameters (Å2x 103) for 13.3a.

x y z U(eq)

H(2) 8127 4855 9666 24

H(3) 9172 4659 10750 25

H(5) 10663 3025 9687 29

H(6) 9583 3200 8614 27

H(7) 8171 3766 7963 21

H(8A) 9674 4981 7993 24

H(8B) 9134 5749 7660 24

H(9A) 9033 4270 6918 26

H(9B) 8534 5051 6583 26

H(10A) 10613 4846 6854 26

H(10B) 10027 4741 6054 26


H(11A) 11655 5756 6320 45

H(11B) 11229 6545 5947 45

H(11C) 11046 5734 5522 45

H(12A) 9192 5913 5456 46

H(12B) 9483 6759 5776 46

H(12C) 8744 6222 6190 46

H(18) 5201 4645 9292 27

H(20) 3369 4568 10090 34

H(21) 2318 3664 10567 40

H(22) 2574 2337 10437 51

H(23) 3932 1865 9858 49

H(2A) 5690 5615 8050 36

H(26) 2691 8082 10230 31

H(27) 3756 9158 10323 42

H(29) 3794 9176 8085 34

H(30) 2711 8117 7995 27

H(31) 1704 7169 9555 23

H(32A) 2956 5643 8816 31

H(32B) 3105 6216 9514 31

H(33A) 4186 6999 8881 31

H(33B) 4585 6122 8941 31

H(34A) 3767 5924 7691 28

H(34B) 3639 6850 7648 28

H(35A) 4606 6753 6557 57

H(35B) 4805 5844 6698 57

H(35C) 5710 6430 6657 57

H(36A) 6193 7284 7658 65

H(36B) 5588 7322 8376 65

H(36C) 5128 7682 7605 65

H(42) -832 7986 7366 30

H(44) -2755 8632 6694 48

H(45) -4154 9265 7034 57

H(46) -4321 9578 8260 55

H(47) -3068 9304 9207 47

H(6A) 305 6574 7157 34

Table 6. Torsion angles [°] for 13.3a.

C(6)-C(1)-C(2)-C(3) 0.0(3)

C(7)-C(1)-C(2)-C(3) 179.86(16)

C(1)-C(2)-C(3)-C(4) 1.1(3)

C(2)-C(3)-C(4)-F(1) 177.87(15)

C(2)-C(3)-C(4)-C(5) -1.3(3)

F(1)-C(4)-C(5)-C(6) -178.83(16)

C(3)-C(4)-C(5)-C(6) 0.3(3)

C(2)-C(1)-C(6)-C(5) -1.0(3)

C(7)-C(1)-C(6)-C(5) 179.14(17)

C(4)-C(5)-C(6)-C(1) 0.8(3)

C(6)-C(1)-C(7)-N(1) -112.07(19)

C(2)-C(1)-C(7)-N(1) 68.0(2)

C(6)-C(1)-C(7)-C(15) 132.03(18)

C(2)-C(1)-C(7)-C(15) -47.9(2)

N(1)-C(8)-C(9)-C(10) -177.84(15)

C(8)-C(9)-C(10)-N(2) -65.8(2)

O(1)-C(13)-C(14)-O(2) -1.6(3)

N(1)-C(13)-C(14)-O(2) 179.98(16)

O(1)-C(13)-C(14)-C(15) 174.62(18)

N(1)-C(13)-C(14)-C(15) -3.8(2)

O(2)-C(14)-C(15)-C(16) 4.8(4)

C(13)-C(14)-C(15)-C(16) -170.67(19)

O(2)-C(14)-C(15)-C(7) 177.2(2)

C(13)-C(14)-C(15)-C(7) 1.8(2)

N(1)-C(7)-C(15)-C(14) 0.69(19)

C(1)-C(7)-C(15)-C(14) 121.92(17)

N(1)-C(7)-C(15)-C(16) 174.38(15)

C(1)-C(7)-C(15)-C(16) -64.4(2)

C(14)-C(15)-C(16)-O(3) 155.3(2)

C(7)-C(15)-C(16)-O(3) -16.7(3)

C(14)-C(15)-C(16)-C(17) -26.5(3)

C(7)-C(15)-C(16)-C(17) 161.48(16)

O(3)-C(16)-C(17)-C(18) 169.1(2)

C(15)-C(16)-C(17)-C(18) -9.1(3)

O(3)-C(16)-C(17)-O(4) -6.8(2)

C(15)-C(16)-C(17)-O(4) 175.01(16)

O(4)-C(17)-C(18)-C(19) -1.0(2)

C(16)-C(17)-C(18)-C(19) -177.0(2)

C(17)-C(18)-C(19)-C(24) 0.4(2)

C(17)-C(18)-C(19)-C(20) 179.1(2)

C(24)-C(19)-C(20)-C(21) -0.4(3)

C(18)-C(19)-C(20)-C(21) -179.0(2)

C(19)-C(20)-C(21)-C(22) -0.6(3)

C(20)-C(21)-C(22)-C(23) 1.2(4)

C(21)-C(22)-C(23)-C(24) -0.7(4)

C(22)-C(23)-C(24)-O(4) 179.1(2)

C(22)-C(23)-C(24)-C(19) -0.3(4)

C(20)-C(19)-C(24)-O(4) -178.60(17)

C(18)-C(19)-C(24)-O(4) 0.4(2)

C(20)-C(19)-C(24)-C(23) 0.9(3)

C(18)-C(19)-C(24)-C(23) 179.8(2)

O(1)-C(13)-N(1)-C(8) -5.9(3)

C(14)-C(13)-N(1)-C(8) 172.48(15)

O(1)-C(13)-N(1)-C(7) -173.94(18)

C(14)-C(13)-N(1)-C(7) 4.4(2)

C(9)-C(8)-N(1)-C(13) -87.4(2)

C(9)-C(8)-N(1)-C(7) 79.3(2)

C(15)-C(7)-N(1)-C(13) -3.31(19)

C(1)-C(7)-N(1)-C(13) -125.71(17)

C(15)-C(7)-N(1)-C(8) -171.23(15)


C(1)-C(7)-N(1)-C(8) 66.4(2)

C(9)-C(10)-N(2)-C(12) -56.1(2)

C(9)-C(10)-N(2)-C(11) -179.64(16)

C(23)-C(24)-O(4)-C(17) 179.6(2)

C(19)-C(24)-O(4)-C(17) -0.9(2)

C(18)-C(17)-O(4)-C(24) 1.2(2)

C(16)-C(17)-O(4)-C(24) 178.04(16)

C(30)-C(25)-C(26)-C(27) 1.9(3)

C(31)-C(25)-C(26)-C(27) -177.57(19)

C(25)-C(26)-C(27)-C(28) -0.4(3)

C(26)-C(27)-C(28)-F(2) 179.2(2)

C(26)-C(27)-C(28)-C(29) -1.7(4)

F(2)-C(28)-C(29)-C(30) -178.66(19)

C(27)-C(28)-C(29)-C(30) 2.3(3)

C(28)-C(29)-C(30)-C(25) -0.7(3)

C(26)-C(25)-C(30)-C(29) -1.3(3)

C(31)-C(25)-C(30)-C(29) 178.16(17)

C(26)-C(25)-C(31)-N(3) -122.94(18)

C(30)-C(25)-C(31)-N(3) 57.6(2)

C(26)-C(25)-C(31)-C(39) 121.24(19)

C(30)-C(25)-C(31)-C(39) -58.2(2)

N(3)-C(32)-C(33)-C(34) -59.5(2)

C(32)-C(33)-C(34)-N(4) -166.50(15)

O(5)-C(37)-C(38)-O(6) -6.0(3)

N(3)-C(37)-C(38)-O(6) 175.86(15)

O(5)-C(37)-C(38)-C(39) 171.92(18)

N(3)-C(37)-C(38)-C(39) -6.3(2)

O(6)-C(38)-C(39)-C(40) 14.3(4)

C(37)-C(38)-C(39)-C(40) -163.14(18)

O(6)-C(38)-C(39)-C(31) -177.20(18)

C(37)-C(38)-C(39)-C(31) 5.34(19)

N(3)-C(31)-C(39)-C(38) -2.63(19)

C(25)-C(31)-C(39)-C(38) 118.69(17)

N(3)-C(31)-C(39)-C(40) 167.84(15)

C(25)-C(31)-C(39)-C(40) -70.8(2)

C(38)-C(39)-C(40)-O(7) 168.73(19)

C(31)-C(39)-C(40)-O(7) 0.9(2)

C(38)-C(39)-C(40)-C(41) -10.1(3)

C(31)-C(39)-C(40)-C(41) -177.97(15)

O(7)-C(40)-C(41)-C(42) 158.8(2)

C(39)-C(40)-C(41)-C(42) -22.3(3)

O(7)-C(40)-C(41)-O(8) -14.0(2)

C(39)-C(40)-C(41)-O(8) 164.92(15)

O(8)-C(41)-C(42)-C(43) 0.8(2)

C(40)-C(41)-C(42)-C(43) -172.2(2)

C(41)-C(42)-C(43)-C(48) -1.1(2)

C(41)-C(42)-C(43)-C(44) 178.6(2)

C(48)-C(43)-C(44)-C(45) -1.0(3)

C(42)-C(43)-C(44)-C(45) 179.3(2)

C(43)-C(44)-C(45)-C(46) -0.4(3)

C(44)-C(45)-C(46)-C(47) 1.1(4)

C(45)-C(46)-C(47)-C(48) -0.3(3)

C(44)-C(43)-C(48)-O(8) -178.67(17)

C(42)-C(43)-C(48)-O(8) 1.1(2)

C(44)-C(43)-C(48)-C(47) 1.9(3)

C(42)-C(43)-C(48)-C(47) -178.37(19)

C(46)-C(47)-C(48)-O(8) 179.44(18)

C(46)-C(47)-C(48)-C(43) -1.2(3)

O(5)-C(37)-N(3)-C(32) -5.1(3)

C(38)-C(37)-N(3)-C(32) 173.11(16)

O(5)-C(37)-N(3)-C(31) -173.45(18)

C(38)-C(37)-N(3)-C(31) 4.7(2)

C(33)-C(32)-N(3)-C(37) 99.3(2)

C(33)-C(32)-N(3)-C(31) -93.6(2)

C(39)-C(31)-N(3)-C(37) -1.53(19)

C(25)-C(31)-N(3)-C(37) -124.08(17)

C(39)-C(31)-N(3)-C(32) -169.81(16)

C(25)-C(31)-N(3)-C(32) 67.6(2)

C(33)-C(34)-N(4)-C(36) -63.0(2)

C(33)-C(34)-N(4)-C(35) 173.43(16)

C(43)-C(48)-O(8)-C(41) -0.6(2)

C(47)-C(48)-O(8)-C(41) 178.81(19)

C(42)-C(41)-O(8)-C(48) -0.1(2)

C(40)-C(41)-O(8)-C(48) 174.51(15)

Table 7. Hydrogen bonds for 13.3a [Å and °]. D-H...A d(D-H) d(H...A) d(D...A) <(DHA)

i. O2-H2a…N4 0.84 1.87 2.8541(19) 154

ii. O6-H6a…N2 0.84 1.85 2.646(2) 158

iii. C8-H8b…O6 0.99 2.54 3.414(2) 148

iv. C11-C11a…O5 0.98 2.47 3.082(3) 121

v. C21-H21…F1 0.95 2.44 3.341(2) 159

vi. C26-H26...O3 0.95 2.53 3.374(2) 149

vii. C35-H35a…O4 0.98 2.38 3.196(3) 137

Symmetry operators: i and v = x, y, z; ii = 1+x, y, z; iii and iv = 1+x, y, z; vi = 1-x, 1-y, 2-z; vii = 1-x, 1/2+y, 3/2-z

Analysis of Cg...Cg (π-Ring) Interactions

Cg…Cg Cg…Cg

i Cg2…Cg3 3.8331(11)

ii Cg3 …Cg7 3.9939(11

iii Cg7…Cg8 3.8301(11

Symmetry operators: i and iii = x, y, z; ii = 1-x, 1-y, 2-z

Analysis of C-H...Cg (π-Ring) Interactions (H..Cg < 3.0 Ang. - Gamma < 30.0 Deg)

X-H…Cg H…Cg C-H…Cg C…Cg

i C2-H2…Cg2 2.75 98 3.028(2)

ii C10-H10a…Cg6 2.65 125 3.311(2)

iii C29-H29…Cg2 2.89 125 3.517(2)


iv C30-H30…Cg7 2.75 98 3.027(2)

v C31-H31…Cg3 2.85 142 3.6954(19)

Symmetry operators: i and iv = x, y, z; ii and iii = 1-x, -½+y, 3/2-z; v = 1-x, 1-y, 2-z.

A.5 Pyrrolidinone 13.3b

Table 1. Crystal data and structure refinement for 13.3b. Identification code mo_12ek_tt2_0ma

Empirical formula C27 H19 F O4





Space group C 2/c


b = 5.8170(6) Å β= 99.276(4)°.

c = 35.699(4) Å γ = 90°.

Volume 4048.6(7) Å3

Z 8



F(000) 1776



Index ranges -24<=h<=26, -7<=k<=7, -47<=l<=45




Absorption correction None








for 13.3b. U(eq) is defined as one third of the trace of the orthogonalised Uij tensor. x y z U(eq)

C(11) 3212(1) 5972(4) 3610(1) 23(1)

C(12) 2636(1) 6349(3) 3276(1) 21(1)

C(13) 2094(1) 4778(4) 3202(1) 25(1)

C(14) 1560(1) 5096(4) 2899(1) 26(1)

C(15) 1585(1) 7009(4) 2673(1) 25(1)

C(16) 2113(1) 8585(4) 2730(1) 24(1)

C(17) 2639(1) 8241(4) 3034(1) 23(1)

C(20) 3614(1) 3817(4) 3530(1) 23(1)

C(21) 4290(1) 3422(4) 3788(1) 23(1)

C(22) 4684(1) 1402(4) 3699(1) 22(1)

C(23) 5283(1) 471(4) 3874(1) 26(1)

C(24) 5423(1) -1461(4) 3649(1) 24(1)

C(25) 5942(1) -3134(4) 3668(1) 30(1)

C(26) 5890(1) -4792(4) 3389(1) 30(1)

C(27) 5336(1) -4824(4) 3089(1) 29(1)

C(28) 4818(1) -3189(4) 3059(1) 27(1)

C(29) 4881(1) -1543(4) 3345(1) 22(1)

C(30) 2942(1) 5709(4) 3988(1) 26(1)

C(31) 2530(1) 7740(4) 4090(1) 26(1)

O(1) 4511(1) 4652(3) 4057(1) 31(1)

O(2) 4423(1) 197(3) 3371(1) 23(1)

O(3) 2534(1) 9635(3) 3942(1) 33(1)

F(1) 1061(1) 7357(3) 2380(1) 35(1)


O(4) 2128(1) 5327(3) 4578(1) 46(1)

C(32) 2108(1) 7344(5) 4388(1) 33(1)

C(33) 1656(1) 8766(4) 4501(1) 29(1)

C(34) 1362(1) 7666(4) 4790(1) 31(1)

C(35) 866(2) 8263(6) 5012(1) 62(1)

C(36) 719(2) 6606(8) 5275(1) 69(1)

C(37) 1034(2) 4521(7) 5306(1) 74(1)

C(38) 1507(2) 3955(6) 5097(1) 71(1)

C(39) 1666(1) 5522(4) 4836(1) 33(1)

Table 3. Bond lengths [Å] and angles [°] for 13.3b. Bond lengths (Ǻ)

C(11)-C(12) 1.525(3)

C(11)-C(20) 1.535(3)

C(11)-C(30) 1.536(3)

C(11)-H(11) 0.9800

C(12)-C(17) 1.400(3)

C(12)-C(13) 1.400(3)

C(13)-C(14) 1.396(3)

C(13)-H(13) 0.9300

C(14)-C(15) 1.380(3)

C(14)-H(14) 0.9300

C(15)-F(1) 1.365(2)

C(15)-C(16) 1.379(3)

C(16)-C(17) 1.392(3)

C(16)-H(16) 0.9300

C(17)-H(17) 0.9300

C(20)-C(21) 1.512(3)

C(20)-H(20A) 0.9700

C(20)-H(20B) 0.9700

C(21)-O(1) 1.220(3)

C(21)-C(22) 1.472(3)

C(22)-C(23) 1.360(3)

C(22)-O(2) 1.389(2)

C(23)-C(24) 1.434(3)

C(23)-H(23) 0.9300

C(24)-C(29) 1.399(3)

C(24)-C(25) 1.407(3)

C(25)-C(26) 1.379(3)

C(25)-H(25) 0.9300

C(26)-C(27) 1.404(3)

C(26)-H(26) 0.9300

C(27)-C(28) 1.390(3)

C(27)-H(27) 0.9300

C(28)-C(29) 1.389(3)

C(28)-H(28) 0.9300

C(29)-O(2) 1.371(2)

C(30)-C(31) 1.512(3)

C(30)-H(30A) 0.9700

C(30)-H(30B) 0.9700

C(31)-O(3) 1.222(3)

C(31)-C(32) 1.471(3)

O(4)-C(32) 1.354(3)

O(4)-C(39) 1.400(3)

C(32)-C(33) 1.328(3)

C(33)-C(34) 1.415(3)

C(33)-H(33) 0.9300

C(34)-C(39) 1.383(3)

C(34)-C(35) 1.400(4)

C(35)-C(36) 1.408(5)

C(35)-H(35) 0.9300

C(36)-C(37) 1.360(6)

C(36)-H(36) 0.9300

C(37)-C(38) 1.330(6)

C(37)-H(37) 0.9300

C(38)-C(39) 1.376(4)

C(38)-H(38) 0.9300

Bond angles (°)

C(12)-C(11)-C(20) 108.42(16)

C(12)-C(11)-C(30) 112.27(17)

C(20)-C(11)-C(30) 110.18(17)

C(12)-C(11)-H(11) 108.6

C(20)-C(11)-H(11) 108.6

C(30)-C(11)-H(11) 108.6

C(17)-C(12)-C(13) 118.42(18)

C(17)-C(12)-C(11) 121.23(18)

C(13)-C(12)-C(11) 120.34(18)

C(14)-C(13)-C(12) 121.27(19)

C(14)-C(13)-H(13) 119.4

C(12)-C(13)-H(13) 119.4

C(15)-C(14)-C(13) 117.77(19)

C(15)-C(14)-H(14) 121.1

C(13)-C(14)-H(14) 121.1

F(1)-C(15)-C(16) 118.47(19)

F(1)-C(15)-C(14) 118.28(19)

C(16)-C(15)-C(14) 123.26(19)

C(15)-C(16)-C(17) 118.07(19)

C(15)-C(16)-H(16) 121.0

C(17)-C(16)-H(16) 121.0

C(16)-C(17)-C(12) 121.20(19)

C(16)-C(17)-H(17) 119.4

C(12)-C(17)-H(17) 119.4

C(21)-C(20)-C(11) 116.34(17)

C(21)-C(20)-H(20A) 108.2

C(11)-C(20)-H(20A) 108.2

C(21)-C(20)-H(20B) 108.2

C(11)-C(20)-H(20B) 108.2

H(20A)-C(20)-H(20B) 107.4

O(1)-C(21)-C(22) 120.30(19)

O(1)-C(21)-C(20) 123.91(19)

C(22)-C(21)-C(20) 115.79(17)

C(23)-C(22)-O(2) 111.29(18)

C(23)-C(22)-C(21) 132.06(19)

O(2)-C(22)-C(21) 116.62(17)

C(22)-C(23)-C(24) 106.70(18)

C(22)-C(23)-H(23) 126.6

C(24)-C(23)-H(23) 126.6

C(29)-C(24)-C(25) 118.3(2)

C(29)-C(24)-C(23) 105.62(18)

C(25)-C(24)-C(23) 136.1(2)

C(26)-C(25)-C(24) 118.7(2)

C(26)-C(25)-H(25) 120.6

C(24)-C(25)-H(25) 120.6

C(25)-C(26)-C(27) 121.3(2)


C(25)-C(26)-H(26) 119.3

C(27)-C(26)-H(26) 119.3

C(28)-C(27)-C(26) 121.5(2)

C(28)-C(27)-H(27) 119.2

C(26)-C(27)-H(27) 119.2

C(29)-C(28)-C(27) 116.0(2)

C(29)-C(28)-H(28) 122.0

C(27)-C(28)-H(28) 122.0

O(2)-C(29)-C(28) 125.24(18)

O(2)-C(29)-C(24) 110.60(18)

C(28)-C(29)-C(24) 124.15(19)

C(31)-C(30)-C(11) 114.23(17)

C(31)-C(30)-H(30A) 108.7

C(11)-C(30)-H(30A) 108.7

C(31)-C(30)-H(30B) 108.7

C(11)-C(30)-H(30B) 108.7

H(30A)-C(30)-H(30B) 107.6

O(3)-C(31)-C(32) 119.8(2)

O(3)-C(31)-C(30) 124.00(19)

C(32)-C(31)-C(30) 116.2(2)

C(29)-O(2)-C(22) 105.79(15)

C(32)-O(4)-C(39) 106.86(19)

C(33)-C(32)-O(4) 110.7(2)

C(33)-C(32)-C(31) 127.7(2)

O(4)-C(32)-C(31) 121.6(2)

C(32)-C(33)-C(34) 108.4(2)

C(32)-C(33)-H(33) 125.8

C(34)-C(33)-H(33) 125.8

C(39)-C(34)-C(35) 119.3(3)

C(39)-C(34)-C(33) 105.8(2)

C(35)-C(34)-C(33) 135.0(3)

C(34)-C(35)-C(36) 116.5(3)

C(34)-C(35)-H(35) 121.8

C(36)-C(35)-H(35) 121.8

C(37)-C(36)-C(35) 121.7(3)

C(37)-C(36)-H(36) 119.2

C(35)-C(36)-H(36) 119.2

C(38)-C(37)-C(36) 121.9(3)

C(38)-C(37)-H(37) 119.1

C(36)-C(37)-H(37) 119.1

C(37)-C(38)-C(39) 118.4(3)

C(37)-C(38)-H(38) 120.8

C(39)-C(38)-H(38) 120.8

C(38)-C(39)-C(34) 122.3(3)

C(38)-C(39)-O(4) 129.4(3)

C(34)-C(39)-O(4) 108.3(2)

Table 4. Anisotropic displacement parameters (Å2x 103) for 13.3b. The anisotropic


C(11) 23(1) 21(1) 22(1) 0(1) 1(1) 1(1)

C(12) 21(1) 22(1) 21(1) -2(1) 4(1) 3(1)

C(13) 24(1) 24(1) 26(1) 3(1) 5(1) 0(1)

C(14) 21(1) 26(1) 29(1) -2(1) 4(1) -2(1)

C(15) 22(1) 31(1) 20(1) -1(1) 1(1) 6(1)

C(16) 31(1) 22(1) 21(1) 3(1) 7(1) 4(1)

C(17) 26(1) 21(1) 24(1) -2(1) 6(1) -2(1)

C(20) 23(1) 24(1) 22(1) -2(1) 2(1) 2(1)

C(21) 24(1) 24(1) 19(1) 2(1) 4(1) 0(1)

C(22) 23(1) 24(1) 19(1) 0(1) 3(1) -4(1)

C(23) 22(1) 28(1) 25(1) 1(1) 0(1) -1(1)

C(24) 20(1) 25(1) 27(1) 3(1) 2(1) -1(1)

C(25) 21(1) 31(1) 36(1) 4(1) 0(1) 4(1)

C(26) 24(1) 26(1) 42(1) 4(1) 9(1) 5(1)

C(27) 29(1) 26(1) 34(1) -4(1) 10(1) 0(1)

C(28) 24(1) 28(1) 30(1) -3(1) 3(1) -1(1)

C(29) 18(1) 23(1) 25(1) 4(1) 5(1) 2(1)

C(30) 29(1) 25(1) 23(1) 1(1) 5(1) 2(1)

C(31) 24(1) 31(1) 20(1) -2(1) -1(1) 2(1)

O(1) 30(1) 34(1) 26(1) -7(1) 0(1) 1(1)

O(2) 21(1) 24(1) 22(1) -2(1) 0(1) 3(1)

O(3) 37(1) 30(1) 31(1) 1(1) 7(1) 5(1)

F(1) 29(1) 45(1) 26(1) 2(1) -4(1) 4(1)

O(4) 50(1) 45(1) 47(1) 0(1) 17(1) 10(1)

C(32) 28(1) 49(1) 21(1) 1(1) -2(1) 0(1)

C(33) 27(1) 32(1) 27(1) 13(1) 6(1) 10(1)

C(34) 32(1) 31(1) 29(1) 2(1) 4(1) 2(1)

C(35) 50(2) 45(2) 99(3) -28(2) 32(2) -2(1)

C(36) 66(2) 103(3) 49(2) -46(2) 40(2) -48(2)

C(37) 115(3) 65(2) 46(2) 2(2) 26(2) -37(2)

C(38) 105(3) 42(2) 65(2) 24(2) 13(2) -3(2)

C(39) 38(1) 33(1) 28(1) -2(1) 3(1) 3(1)

Table 5. Hydrogen coordinates ( x 104) and isotropic displacement parameters (Å2x 103) for 13.3b.

x y z U(eq)


H(11) 3522 7297 3629 27

H(13) 2090 3498 3358 30

H(14) 1200 4053 2851 31

H(16) 2118 9842 2569 29

H(17) 3000 9287 3078 28

H(20A) 3325 2487 3547 28

H(20B) 3702 3903 3271 28

H(23) 5551 988 4097 31

H(25) 6313 -3121 3865 36

H(26) 6229 -5912 3400 36

H(27) 5316 -5966 2905 35

H(28) 4450 -3197 2860 33

H(30A) 2657 4344 3975 31

H(30B) 3328 5475 4189 31

H(33) 1550 10236 4407 34

H(35) 645 9680 4987 75

H(36) 398 6946 5431 83

H(37) 915 3460 5480 88

H(38) 1726 2535 5126 85

Table 6. Torsion angles [°] for 13.3b. C(20)-C(11)-C(12)-C(17) -112.5(2)

C(30)-C(11)-C(12)-C(17) 125.6(2)

C(20)-C(11)-C(12)-C(13) 66.6(2)

C(30)-C(11)-C(12)-C(13) -55.3(2)

C(17)-C(12)-C(13)-C(14) -1.2(3)

C(11)-C(12)-C(13)-C(14) 179.73(18)

C(12)-C(13)-C(14)-C(15) 0.3(3)

C(13)-C(14)-C(15)-F(1) -178.94(18)

C(13)-C(14)-C(15)-C(16) 0.9(3)

F(1)-C(15)-C(16)-C(17) 178.73(17)

C(14)-C(15)-C(16)-C(17) -1.2(3)

C(15)-C(16)-C(17)-C(12) 0.2(3)

C(13)-C(12)-C(17)-C(16) 1.0(3)

C(11)-C(12)-C(17)-C(16) -179.96(18)

C(12)-C(11)-C(20)-C(21) 168.03(17)

C(30)-C(11)-C(20)-C(21) -68.8(2)

C(11)-C(20)-C(21)-O(1) 2.8(3)

C(11)-C(20)-C(21)-C(22) -177.58(17)

O(1)-C(21)-C(22)-C(23) 2.9(3)

C(20)-C(21)-C(22)-C(23) -176.7(2)

O(1)-C(21)-C(22)-O(2) -175.13(18)

C(20)-C(21)-C(22)-O(2) 5.3(3)

O(2)-C(22)-C(23)-C(24) -0.2(2)

C(21)-C(22)-C(23)-C(24) -178.4(2)

C(22)-C(23)-C(24)-C(29) 0.3(2)

C(22)-C(23)-C(24)-C(25) -179.8(2)

C(29)-C(24)-C(25)-C(26) -0.8(3)

C(23)-C(24)-C(25)-C(26) 179.4(2)

C(24)-C(25)-C(26)-C(27) 0.5(3)

C(25)-C(26)-C(27)-C(28) 0.0(3)

C(26)-C(27)-C(28)-C(29) -0.2(3)

C(27)-C(28)-C(29)-O(2) -179.13(19)

C(27)-C(28)-C(29)-C(24) -0.1(3)

C(25)-C(24)-C(29)-O(2) 179.77(18)

C(23)-C(24)-C(29)-O(2) -0.4(2)

C(25)-C(24)-C(29)-C(28) 0.6(3)

C(23)-C(24)-C(29)-C(28) -179.5(2)

C(12)-C(11)-C(30)-C(31) -58.4(2)

C(20)-C(11)-C(30)-C(31) -179.34(17)

C(11)-C(30)-C(31)-O(3) -16.2(3)

C(11)-C(30)-C(31)-C(32) 164.00(18)

C(28)-C(29)-O(2)-C(22) 179.3(2)

C(24)-C(29)-O(2)-C(22) 0.2(2)

C(23)-C(22)-O(2)-C(29) 0.0(2)

C(21)-C(22)-O(2)-C(29) 178.46(17)

C(39)-O(4)-C(32)-C(33) -1.4(3)

C(39)-O(4)-C(32)-C(31) -179.6(2)

O(3)-C(31)-C(32)-C(33) 7.2(4)

C(30)-C(31)-C(32)-C(33) -173.0(2)

O(3)-C(31)-C(32)-O(4) -175.0(2)

C(30)-C(31)-C(32)-O(4) 4.8(3)

O(4)-C(32)-C(33)-C(34) 1.0(3)

C(31)-C(32)-C(33)-C(34) 179.1(2)

C(32)-C(33)-C(34)-C(39) -0.3(3)

C(32)-C(33)-C(34)-C(35) -179.4(3)

C(39)-C(34)-C(35)-C(36) 0.9(4)

C(33)-C(34)-C(35)-C(36) 179.9(3)

C(34)-C(35)-C(36)-C(37) -1.1(5)

C(35)-C(36)-C(37)-C(38) 1.4(6)

C(36)-C(37)-C(38)-C(39) -1.5(6)

C(37)-C(38)-C(39)-C(34) 1.3(5)

C(37)-C(38)-C(39)-O(4) -178.5(3)

C(35)-C(34)-C(39)-C(38) -1.0(4)

C(33)-C(34)-C(39)-C(38) 179.7(3)

C(35)-C(34)-C(39)-O(4) 178.8(2)

C(33)-C(34)-C(39)-O(4) -0.5(3)

C(32)-O(4)-C(39)-C(38) -179.1(3)

C(32)-O(4)-C(39)-C(34) 1.1(3)

A.6 Pyrrolidinone 12.2-HCl

Table 1. Crystal data and structure refinement for 12.2-HCl. Identification code mo_12ek_tt6_0ma

Empirical formula C26 H26 Cl2 N2 O5





Crystal system Orthorhombic

Space group P n a 21


b = 12.7963(14) Å β= 90°.

c = 6.2578(7) Å γ = 90°.

Volume 2442.7(5) Å3

Z 4



F(000) 1080



Index ranges -32<=h<=40, -17<=k<=17, -7<=l<=8










Absolute structure parameter -0.03(4)



for 12.2-HCl. U(eq) is defined as one third of the trace of the orthogonalised Uij tensor. x y z U(eq)

Cl(2) 2278(1) 11586(1) -6702(1) 22(1)

Cl(1) 1020(1) 8647(1) 4992(1) 23(1)

O(4) 635(1) 13619(1) 634(2) 17(1)

O(2) 1675(1) 11786(1) -3058(2) 18(1)

O(3) 827(1) 11824(1) 2632(3) 22(1)

O(5) 3512(1) 9737(1) 6125(2) 21(1)

N(2) 2741(1) 9456(1) 3549(3) 13(1)

O(1) 2064(1) 9736(1) -2907(2) 17(1)

C(31) 971(1) 11921(2) 816(3) 14(1)

N(1) 1589(1) 9456(1) -93(3) 13(1)

C(38) 398(1) 15392(2) -357(4) 22(1)

C(41) 1740(1) 8423(2) 547(3) 15(1)

C(42) 2064(1) 8469(2) 2427(3) 16(1)

C(44) 2875(1) 8674(2) 5206(3) 16(1)

C(23) 274(1) 8361(2) 2760(4) 20(1)

C(11) 1259(1) 10061(1) 1102(3) 14(1)

C(12) 1253(1) 11095(1) -82(3) 14(1)

C(43) 2493(1) 8981(2) 1725(3) 16(1)

C(26) 530(1) 9630(2) -601(3) 16(1)

C(24) 3(1) 8490(2) 1011(4) 21(1)

C(14) 1770(1) 10012(1) -1694(3) 14(1)

C(37) 405(1) 16105(2) -2022(4) 26(1)

C(35) 749(1) 14881(2) -4466(4) 21(1)

C(45) 3136(1) 9215(2) 6942(3) 19(1)

C(22) 680(1) 8858(2) 2782(3) 17(1)

C(39) 581(1) 14418(2) -802(3) 17(1)

C(47) 3140(1) 10021(2) 2742(4) 18(1)

C(36) 577(1) 15858(2) -4034(4) 25(1)

C(32) 855(1) 12845(2) -473(3) 15(1)

C(46) 3379(1) 10506(2) 4612(4) 20(1)

C(34) 750(1) 14150(2) -2799(3) 17(1)

C(21) 817(1) 9504(2) 1124(3) 15(1)

C(25) 129(1) 9123(2) -674(4) 20(1)

C(13) 1549(1) 11056(1) -1679(3) 14(1)

C(33) 923(1) 13112(2) -2547(4) 15(1)


Table 3. Bond lengths [Å] and angles [°] for 12.2-HCl. Bond lengths (Ǻ)

Cl(1)-C(22) 1.750(2)

O(4)-C(39) 1.370(2)

O(4)-C(32) 1.384(2)

O(2)-C(13) 1.328(2)

O(2)-H(2) 0.8200

O(3)-C(31) 1.225(3)

O(5)-C(45) 1.423(2)

O(5)-C(46) 1.425(3)

N(2)-C(43) 1.497(3)

N(2)-C(44) 1.499(3)

N(2)-C(47) 1.504(2)

N(2)-H(2A) 0.9100

O(1)-C(14) 1.226(2)

C(31)-C(12) 1.473(3)

C(31)-C(32) 1.474(3)

N(1)-C(14) 1.347(2)

N(1)-C(41) 1.457(2)

N(1)-C(11) 1.475(2)

C(38)-C(37) 1.386(3)

C(38)-C(39) 1.393(3)

C(38)-H(38) 0.9300

C(41)-C(42) 1.537(3)

C(41)-H(41A) 0.9700

C(41)-H(41B) 0.9700

C(42)-C(43) 1.528(3)

C(42)-H(42A) 0.9700

C(42)-H(42B) 0.9700

C(44)-C(45) 1.514(3)

C(44)-H(44A) 0.9700

C(44)-H(44B) 0.9700

C(23)-C(24) 1.381(3)

C(23)-C(22) 1.394(3)

C(23)-H(23) 0.9300

C(11)-C(12) 1.517(3)

C(11)-C(21) 1.525(3)

C(11)-H(11) 0.9800

C(12)-C(13) 1.348(3)

C(43)-H(43A) 0.9700

C(43)-H(43B) 0.9700

C(26)-C(25) 1.384(3)

C(26)-C(21) 1.399(3)

C(26)-H(26) 0.9300

C(24)-C(25) 1.385(3)

C(24)-H(24) 0.9300

C(14)-C(13) 1.497(3)

C(37)-C(36) 1.401(3)

C(37)-H(37) 0.9300

C(35)-C(36) 1.384(3)

C(35)-C(34) 1.401(3)

C(35)-H(35) 0.9300

C(45)-H(45A) 0.9700

C(45)-H(45B) 0.9700

C(22)-C(21) 1.391(3)

C(39)-C(34) 1.394(3)

C(47)-C(46) 1.511(3)

C(47)-H(47A) 0.9700

C(47)-H(47B) 0.9700

C(36)-H(36) 0.9300

C(32)-C(33) 1.358(3)

C(46)-H(46A) 0.9700

C(46)-H(46B) 0.9700

C(34)-C(33) 1.438(3)

C(25)-H(25) 0.9300

C(33)-H(33) 0.9300

Bond angles (°)

C(39)-O(4)-C(32) 105.32(16)

C(13)-O(2)-H(2) 109.5

C(45)-O(5)-C(46) 109.47(15)

C(43)-N(2)-C(44) 113.21(15)

C(43)-N(2)-C(47) 110.38(16)

C(44)-N(2)-C(47) 109.34(15)

C(43)-N(2)-H(2A) 107.9

C(44)-N(2)-H(2A) 107.9

C(47)-N(2)-H(2A) 107.9

O(3)-C(31)-C(12) 119.41(19)

O(3)-C(31)-C(32) 120.22(18)

C(12)-C(31)-C(32) 120.35(18)

C(14)-N(1)-C(41) 123.68(16)

C(14)-N(1)-C(11) 112.34(15)

C(41)-N(1)-C(11) 123.58(16)

C(37)-C(38)-C(39) 115.7(2)

C(37)-C(38)-H(38) 122.2

C(39)-C(38)-H(38) 122.2

N(1)-C(41)-C(42) 112.26(16)

N(1)-C(41)-H(41A) 109.2

C(42)-C(41)-H(41A) 109.2

N(1)-C(41)-H(41B) 109.2

C(42)-C(41)-H(41B) 109.2

H(41A)-C(41)-H(41B) 107.9

C(43)-C(42)-C(41) 110.27(17)

C(43)-C(42)-H(42A) 109.6

C(41)-C(42)-H(42A) 109.6

C(43)-C(42)-H(42B) 109.6

C(41)-C(42)-H(42B) 109.6

H(42A)-C(42)-H(42B) 108.1

N(2)-C(44)-C(45) 109.57(17)

N(2)-C(44)-H(44A) 109.8

C(45)-C(44)-H(44A) 109.8

N(2)-C(44)-H(44B) 109.8

C(45)-C(44)-H(44B) 109.8

H(44A)-C(44)-H(44B) 108.2

C(24)-C(23)-C(22) 119.1(2)

C(24)-C(23)-H(23) 120.4

C(22)-C(23)-H(23) 120.4

N(1)-C(11)-C(12) 102.59(15)

N(1)-C(11)-C(21) 111.35(15)

C(12)-C(11)-C(21) 113.67(16)

N(1)-C(11)-H(11) 109.7

C(12)-C(11)-H(11) 109.7

C(21)-C(11)-H(11) 109.7

C(13)-C(12)-C(31) 134.48(18)

C(13)-C(12)-C(11) 108.78(16)

C(31)-C(12)-C(11) 116.47(17)

N(2)-C(43)-C(42) 112.75(17)

N(2)-C(43)-H(43A) 109.0

C(42)-C(43)-H(43A) 109.0

N(2)-C(43)-H(43B) 109.0

C(42)-C(43)-H(43B) 109.0

H(43A)-C(43)-H(43B) 107.8


C(25)-C(26)-C(21) 121.56(19)

C(25)-C(26)-H(26) 119.2

C(21)-C(26)-H(26) 119.2

C(23)-C(24)-C(25) 120.41(19)

C(23)-C(24)-H(24) 119.8

C(25)-C(24)-H(24) 119.8

O(1)-C(14)-N(1) 127.39(17)

O(1)-C(14)-C(13) 126.18(18)

N(1)-C(14)-C(13) 106.42(16)

C(38)-C(37)-C(36) 122.2(2)

C(38)-C(37)-H(37) 118.9

C(36)-C(37)-H(37) 118.9

C(36)-C(35)-C(34) 117.3(2)

C(36)-C(35)-H(35) 121.4

C(34)-C(35)-H(35) 121.4

O(5)-C(45)-C(44) 112.37(17)

O(5)-C(45)-H(45A) 109.1

C(44)-C(45)-H(45A) 109.1

O(5)-C(45)-H(45B) 109.1

C(44)-C(45)-H(45B) 109.1

H(45A)-C(45)-H(45B) 107.9

C(21)-C(22)-C(23) 122.0(2)

C(21)-C(22)-Cl(1) 120.29(15)

C(23)-C(22)-Cl(1) 117.73(16)

O(4)-C(39)-C(38) 125.7(2)

O(4)-C(39)-C(34) 111.10(17)

C(38)-C(39)-C(34) 123.2(2)

N(2)-C(47)-C(46) 109.15(17)

N(2)-C(47)-H(47A) 109.8

C(46)-C(47)-H(47A) 109.8

N(2)-C(47)-H(47B) 109.8

C(46)-C(47)-H(47B) 109.9

H(47A)-C(47)-H(47B) 108.3

C(35)-C(36)-C(37) 121.5(2)

C(35)-C(36)-H(36) 119.3

C(37)-C(36)-H(36) 119.3

C(33)-C(32)-O(4) 111.83(18)

C(33)-C(32)-C(31) 133.55(19)

O(4)-C(32)-C(31) 114.62(17)

O(5)-C(46)-C(47) 111.59(17)

O(5)-C(46)-H(46A) 109.3

C(47)-C(46)-H(46A) 109.3

O(5)-C(46)-H(46B) 109.3

C(47)-C(46)-H(46B) 109.3

H(46A)-C(46)-H(46B) 108.0

C(39)-C(34)-C(35) 120.21(19)

C(39)-C(34)-C(33) 105.34(18)

C(35)-C(34)-C(33) 134.3(2)

C(22)-C(21)-C(26) 117.22(18)

C(22)-C(21)-C(11) 123.28(18)

C(26)-C(21)-C(11) 119.49(18)

C(26)-C(25)-C(24) 119.7(2)

C(26)-C(25)-H(25) 120.2

C(24)-C(25)-H(25) 120.2

O(2)-C(13)-C(12) 130.50(18)

O(2)-C(13)-C(14) 119.55(17)

C(12)-C(13)-C(14) 109.85(17)

C(32)-C(33)-C(34) 106.35(18)

C(32)-C(33)-H(33) 126.8

C(34)-C(33)-H(33) 126.8

Table 4. Anisotropic displacement parameters (Å2x 103)for 12.2-HCl. The anisotropic


Cl(2) 26(1) 18(1) 22(1) 6(1) 9(1) 7(1)

Cl(1) 23(1) 29(1) 18(1) 6(1) 1(1) -2(1)

O(4) 16(1) 14(1) 20(1) -2(1) 3(1) 4(1)

O(2) 18(1) 15(1) 20(1) 2(1) 8(1) 4(1)

O(3) 25(1) 20(1) 20(1) -1(1) 9(1) 1(1)

O(5) 15(1) 28(1) 20(1) 1(1) -1(1) 0(1)

N(2) 12(1) 15(1) 13(1) 0(1) 1(1) 2(1)

O(1) 16(1) 19(1) 17(1) 0(1) 4(1) 3(1)

C(31) 11(1) 14(1) 18(1) -3(1) 3(1) -1(1)

N(1) 12(1) 14(1) 14(1) 0(1) 2(1) 2(1)

C(38) 20(1) 20(1) 26(1) -5(1) 1(1) 5(1)

C(41) 15(1) 13(1) 18(1) 0(1) 2(1) 1(1)

C(42) 16(1) 17(1) 14(1) 1(1) 0(1) 2(1)

C(44) 16(1) 16(1) 16(1) 4(1) 0(1) 3(1)

C(23) 19(1) 14(1) 28(1) 3(1) 8(1) 0(1)

C(11) 13(1) 13(1) 15(1) 0(1) 2(1) 1(1)

C(12) 14(1) 12(1) 15(1) -1(1) 0(1) 0(1)

C(43) 15(1) 20(1) 13(1) 0(1) -2(1) 2(1)

C(26) 18(1) 14(1) 18(1) 1(1) 1(1) 0(1)

C(24) 13(1) 17(1) 33(1) -2(1) 3(1) -2(1)

C(14) 12(1) 15(1) 16(1) -2(1) -2(1) 1(1)

C(37) 27(1) 18(1) 32(1) -4(1) -5(1) 8(1)

C(35) 19(1) 23(1) 19(1) -1(1) -2(1) 2(1)

C(45) 15(1) 28(1) 15(1) 2(1) 1(1) 1(1)

C(22) 15(1) 17(1) 19(1) 1(1) 1(1) 4(1)

C(39) 12(1) 17(1) 20(1) 1(1) -2(1) -1(1)

C(47) 15(1) 19(1) 19(1) 5(1) 1(1) -1(1)

C(36) 28(1) 21(1) 27(1) 7(1) -5(1) 4(1)

C(32) 10(1) 13(1) 23(1) -4(1) 1(1) 0(1)

C(46) 18(1) 20(1) 23(1) 1(1) 0(1) -2(1)


C(34) 11(1) 18(1) 22(1) -4(1) -1(1) 2(1)

C(21) 13(1) 12(1) 18(1) -2(1) 2(1) 2(1)

C(25) 16(1) 18(1) 26(1) -2(1) -2(1) 2(1)

C(13) 14(1) 11(1) 16(1) 0(1) -2(1) 0(1)

C(33) 10(1) 15(1) 20(1) -4(1) 0(1) -1(1)

Table 5. Hydrogen coordinates ( x 104) and isotropic displacement parameters (Å2x 103) for 12.2-HCl.

x y z U(eq)

H(2) 1861 11544 -3862 26

H(2A) 2563 9934 4188 16

H(38) 280 15553 973 26

H(41A) 1882 8088 -661 18

H(41B) 1490 8001 951 18

H(42A) 1936 8868 3589 19

H(42B) 2122 7768 2942 19

H(44A) 3052 8131 4551 20

H(44B) 2617 8349 5820 20

H(23) 186 7949 3907 24

H(11) 1359 10171 2572 16

H(43A) 2676 8460 1039 19

H(43B) 2429 9520 680 19

H(26) 610 10065 -1726 20

H(24) -267 8149 965 25

H(37) 290 16770 -1797 31

H(35) 860 14715 -5809 25

H(45A) 2949 9718 7659 23

H(45B) 3228 8701 7990 23

H(47A) 3054 10561 1739 21

H(47B) 3332 9535 2008 21

H(36) 576 16363 -5104 30

H(46A) 3635 10878 4094 24

H(46B) 3188 11009 5307 24

H(25) -54 9207 -1849 24

H(33) 1055 12705 -3596 18

Table 6. Torsion angles [°] for 12.2-HCl. C(14)-N(1)-C(41)-C(42) 95.7(2)

C(11)-N(1)-C(41)-C(42) -76.6(2)

N(1)-C(41)-C(42)-C(43) -67.8(2)

C(43)-N(2)-C(44)-C(45) 178.07(16)

C(47)-N(2)-C(44)-C(45) 54.6(2)

C(14)-N(1)-C(11)-C(12) 1.7(2)

C(41)-N(1)-C(11)-C(12) 174.73(17)

C(14)-N(1)-C(11)-C(21) 123.63(17)

C(41)-N(1)-C(11)-C(21) -63.3(2)

O(3)-C(31)-C(12)-C(13) -159.1(2)

C(32)-C(31)-C(12)-C(13) 22.2(3)

O(3)-C(31)-C(12)-C(11) 14.1(3)

C(32)-C(31)-C(12)-C(11) -164.61(17)

N(1)-C(11)-C(12)-C(13) -1.1(2)

C(21)-C(11)-C(12)-C(13) -121.47(18)

N(1)-C(11)-C(12)-C(31) -175.97(16)

C(21)-C(11)-C(12)-C(31) 63.7(2)

C(44)-N(2)-C(43)-C(42) 61.6(2)

C(47)-N(2)-C(43)-C(42) -175.43(16)

C(41)-C(42)-C(43)-N(2) 155.78(16)

C(22)-C(23)-C(24)-C(25) -1.5(3)

C(41)-N(1)-C(14)-O(1) 4.1(3)

C(11)-N(1)-C(14)-O(1) 177.11(19)

C(41)-N(1)-C(14)-C(13) -174.60(17)

C(11)-N(1)-C(14)-C(13) -1.6(2)

C(39)-C(38)-C(37)-C(36) 0.9(3)

C(46)-O(5)-C(45)-C(44) 59.9(2)

N(2)-C(44)-C(45)-O(5) -57.4(2)

C(24)-C(23)-C(22)-C(21) 1.9(3)

C(24)-C(23)-C(22)-Cl(1) -178.27(16)

C(32)-O(4)-C(39)-C(38) -175.80(19)

C(32)-O(4)-C(39)-C(34) 1.9(2)

C(37)-C(38)-C(39)-O(4) 175.7(2)

C(37)-C(38)-C(39)-C(34) -1.8(3)

C(43)-N(2)-C(47)-C(46) 179.10(16)

C(44)-N(2)-C(47)-C(46) -55.7(2)

C(34)-C(35)-C(36)-C(37) -0.9(3)

C(38)-C(37)-C(36)-C(35) 0.4(4)

C(39)-O(4)-C(32)-C(33) -2.6(2)

C(39)-O(4)-C(32)-C(31) 176.80(16)

O(3)-C(31)-C(32)-C(33) -171.9(2)

C(12)-C(31)-C(32)-C(33) 6.8(3)

O(3)-C(31)-C(32)-O(4) 8.9(3)

C(12)-C(31)-C(32)-O(4) -172.46(16)

C(45)-O(5)-C(46)-C(47) -60.9(2)

N(2)-C(47)-C(46)-O(5) 59.6(2)

O(4)-C(39)-C(34)-C(35) -176.45(18)

C(38)-C(39)-C(34)-C(35) 1.3(3)

O(4)-C(39)-C(34)-C(33) -0.6(2)

C(38)-C(39)-C(34)-C(33) 177.19(18)

C(36)-C(35)-C(34)-C(39) 0.1(3)

C(36)-C(35)-C(34)-C(33) -174.4(2)

C(23)-C(22)-C(21)-C(26) -0.8(3)

Cl(1)-C(22)-C(21)-C(26) 179.43(15)

C(23)-C(22)-C(21)-C(11) 179.83(18)

Cl(1)-C(22)-C(21)-C(11) 0.0(3)


C(25)-C(26)-C(21)-C(22) -0.8(3)

C(25)-C(26)-C(21)-C(11) 178.59(18)

N(1)-C(11)-C(21)-C(22) 95.2(2)

C(12)-C(11)-C(21)-C(22) -149.51(19)

N(1)-C(11)-C(21)-C(26) -84.2(2)

C(12)-C(11)-C(21)-C(26) 31.1(3)

C(21)-C(26)-C(25)-C(24) 1.3(3)

C(23)-C(24)-C(25)-C(26) -0.1(3)

C(31)-C(12)-C(13)-O(2) -2.6(4)

C(11)-C(12)-C(13)-O(2) -176.1(2)

C(31)-C(12)-C(13)-C(14) 173.8(2)

C(11)-C(12)-C(13)-C(14) 0.3(2)

O(1)-C(14)-C(13)-O(2) -1.1(3)

N(1)-C(14)-C(13)-O(2) 177.61(16)

O(1)-C(14)-C(13)-C(12) -177.91(19)

N(1)-C(14)-C(13)-C(12) 0.8(2)

O(4)-C(32)-C(33)-C(34) 2.3(2)

C(31)-C(32)-C(33)-C(34) -177.0(2)

C(39)-C(34)-C(33)-C(32) -1.0(2)

C(35)-C(34)-C(33)-C(32) 174.0(2)


Table 1. Crystal data and structure refinement for 12.3-HCl. Identification code mo_12ek_tt4_0m

Empirical formula C26 H26 Cl F N2 O5





Space group P n


b = 20.044(15) Å β= 100.197(15)°.

c = 10.322(7) Å γ = 90°.

Volume 1172.6(15) Å3

Z 2



F(000) 524



Index ranges -7<=h<=5, -24<=k<=26, -13<=l<=13










Absolute structure parameter 0.18(9)




Cl(1) 9819(2) -701(1) 7523(1) 21(1)

C(11) 6598(8) 2181(2) 9739(4) 15(1)

C(12) 6322(7) 2609(2) 8514(4) 13(1)

C(13) 7934(7) 2410(2) 7792(4) 13(1)

C(14) 9327(8) 1842(2) 8432(4) 13(1)

C(21) 7295(8) 2587(2) 10996(4) 14(1)

C(22) 5819(8) 2622(2) 11939(4) 20(1)

C(23) 6415(8) 3008(3) 13055(4) 24(1)

C(24) 8495(8) 3359(2) 13242(4) 20(1)

C(25) 10013(9) 3341(3) 12337(4) 25(1)

C(26) 9387(8) 2954(3) 11223(4) 22(1)

C(31) 4317(7) 3077(2) 8312(4) 15(1)

C(32) 4123(7) 3623(2) 7368(4) 16(1)

C(33) 5447(8) 3853(3) 6501(4) 19(1)

C(34) 4319(8) 4445(2) 5923(4) 20(1)


C(35) 4767(9) 4914(3) 4983(4) 24(1)

C(36) 3240(9) 5441(3) 4697(5) 28(1)

C(37) 1291(9) 5518(3) 5339(4) 29(1)

C(38) 821(8) 5068(3) 6275(4) 24(1)

C(39) 2365(8) 4542(2) 6539(4) 18(1)

C(41) 9246(8) 1159(2) 10424(4) 18(1)

C(42) 8002(8) 526(2) 9847(4) 17(1)

C(43) 8871(8) -63(2) 10757(4) 15(1)

C(44) 9273(8) -1287(2) 10937(4) 15(1)

C(45) 8469(8) -1946(2) 10243(4) 18(1)

C(46) 4866(8) -1493(2) 9237(4) 19(1)

C(47) 5475(7) -813(3) 9874(4) 21(1)

N(1) 8517(6) 1727(2) 9567(3) 16(1)

N(2) 8112(6) -720(2) 10123(3) 13(1)

O(1) 10841(5) 1524(2) 7981(3) 20(1)

O(2) 8321(5) 2622(2) 6617(2) 16(1)

O(3) 2814(5) 3013(2) 9026(3) 20(1)

O(4) 2208(5) 4037(2) 7412(3) 17(1)

O(5) 5968(5) -2017(2) 10064(3) 18(1)

F(1) 9099(5) 3743(2) 14336(2) 31(1)


C(11)-N(1) 1.467(6)

C(11)-C(12) 1.513(6)

C(11)-C(21) 1.524(6)

C(11)-H(11) 0.9800

C(12)-C(13) 1.350(5)

C(12)-C(31) 1.474(6)

C(13)-O(2) 1.341(4)

C(13)-C(14) 1.479(6)

C(14)-O(1) 1.236(5)

C(14)-N(1) 1.355(5)

C(21)-C(26) 1.395(7)

C(21)-C(22) 1.403(6)

C(22)-C(23) 1.380(6)

C(22)-H(22) 0.9300

C(23)-C(24) 1.373(7)

C(23)-H(23) 0.9300

C(24)-F(1) 1.359(5)

C(24)-C(25) 1.389(6)

C(25)-C(26) 1.382(6)

C(25)-H(25) 0.9300

C(26)-H(26) 0.9300

C(31)-O(3) 1.239(5)

C(31)-C(32) 1.456(6)

C(32)-C(33) 1.356(6)

C(32)-O(4) 1.387(5)

C(33)-C(34) 1.431(7)

C(33)-H(33) 0.9300

C(34)-C(39) 1.401(6)

C(34)-C(35) 1.408(6)

C(35)-C(36) 1.372(7)

C(35)-H(35) 0.9300

C(36)-C(37) 1.409(7)

C(36)-H(36) 0.9300

C(37)-C(38) 1.382(7)

C(37)-H(37) 0.9300

C(38)-C(39) 1.376(7)

C(38)-H(38) 0.9300

C(39)-O(4) 1.368(5)

C(41)-N(1) 1.457(6)

C(41)-C(42) 1.526(6)

C(41)-H(41A) 0.9700

C(41)-H(41B) 0.9700

C(42)-C(43) 1.537(6)

C(42)-H(42A) 0.9700

C(42)-H(42B) 0.9700

C(43)-N(2) 1.501(6)

C(43)-H(43A) 0.9700

C(43)-H(43B) 0.9700

C(44)-N(2) 1.498(5)

C(44)-C(45) 1.533(6)

C(44)-H(4A) 0.9700

C(44)-H(4B) 0.9700

C(45)-O(5) 1.426(5)

C(45)-H(45A) 0.9700

C(45)-H(45B) 0.9700

C(46)-O(5) 1.430(5)

C(46)-C(47) 1.525(7)

C(46)-H(46A) 0.9700

C(46)-H(46B) 0.9700

C(47)-N(2) 1.506(5)

C(47)-H(47A) 0.9700

C(47)-H(47B) 0.9700

N(2)-H(2A) 0.9100

O(2)-H(2) 0.8200

Bond angles (°)

N(1)-C(11)-C(12) 102.7(3)

N(1)-C(11)-C(21) 110.1(4)

C(12)-C(11)-C(21) 112.5(4)

N(1)-C(11)-H(11) 110.4

C(12)-C(11)-H(11) 110.4

C(21)-C(11)-H(11) 110.4

C(13)-C(12)-C(31) 135.1(4)

C(13)-C(12)-C(11) 108.3(4)

C(31)-C(12)-C(11) 116.3(3)

O(2)-C(13)-C(12) 130.3(4)

O(2)-C(13)-C(14) 119.0(3)

C(12)-C(13)-C(14) 110.6(3)

O(1)-C(14)-N(1) 127.7(4)

O(1)-C(14)-C(13) 126.2(4)

N(1)-C(14)-C(13) 106.0(4)

C(26)-C(21)-C(22) 117.9(4)


C(26)-C(21)-C(11) 120.9(4)

C(22)-C(21)-C(11) 121.1(4)

C(23)-C(22)-C(21) 121.1(4)

C(23)-C(22)-H(22) 119.5

C(21)-C(22)-H(22) 119.5

C(24)-C(23)-C(22) 119.1(4)

C(24)-C(23)-H(23) 120.4

C(22)-C(23)-H(23) 120.4

F(1)-C(24)-C(23) 119.7(4)

F(1)-C(24)-C(25) 118.3(4)

C(23)-C(24)-C(25) 122.0(4)

C(26)-C(25)-C(24) 118.2(5)

C(26)-C(25)-H(25) 120.9

C(24)-C(25)-H(25) 120.9

C(25)-C(26)-C(21) 121.8(4)

C(25)-C(26)-H(26) 119.1

C(21)-C(26)-H(26) 119.1

O(3)-C(31)-C(32) 119.8(4)

O(3)-C(31)-C(12) 117.5(4)

C(32)-C(31)-C(12) 122.6(3)

C(33)-C(32)-O(4) 111.2(4)

C(33)-C(32)-C(31) 135.4(4)

O(4)-C(32)-C(31) 113.3(3)

C(32)-C(33)-C(34) 106.9(4)

C(32)-C(33)-H(33) 126.6

C(34)-C(33)-H(33) 126.6

C(39)-C(34)-C(35) 118.7(4)

C(39)-C(34)-C(33) 105.5(4)

C(35)-C(34)-C(33) 135.8(4)

C(36)-C(35)-C(34) 118.3(4)

C(36)-C(35)-H(35) 120.9

C(34)-C(35)-H(35) 120.9

C(35)-C(36)-C(37) 121.1(5)

C(35)-C(36)-H(36) 119.5

C(37)-C(36)-H(36) 119.5

C(38)-C(37)-C(36) 121.9(5)

C(38)-C(37)-H(37) 119.1

C(36)-C(37)-H(37) 119.1

C(39)-C(38)-C(37) 116.0(4)

C(39)-C(38)-H(38) 122.0

C(37)-C(38)-H(38) 122.0

O(4)-C(39)-C(38) 125.6(4)

O(4)-C(39)-C(34) 110.4(4)

C(38)-C(39)-C(34) 124.0(4)

N(1)-C(41)-C(42) 110.3(3)

N(1)-C(41)-H(41A) 109.6

C(42)-C(41)-H(41A) 109.6

N(1)-C(41)-H(41B) 109.6

C(42)-C(41)-H(41B) 109.6

H(41A)-C(41)-H(41B) 108.1

C(41)-C(42)-C(43) 108.7(3)

C(41)-C(42)-H(42A) 109.9

C(43)-C(42)-H(42A) 109.9

C(41)-C(42)-H(42B) 109.9

C(43)-C(42)-H(42B) 109.9

H(42A)-C(42)-H(42B) 108.3

N(2)-C(43)-C(42) 111.7(3)

N(2)-C(43)-H(43A) 109.3

C(42)-C(43)-H(43A) 109.3

N(2)-C(43)-H(43B) 109.3

C(42)-C(43)-H(43B) 109.3

H(43A)-C(43)-H(43B) 107.9

N(2)-C(44)-C(45) 108.9(3)

N(2)-C(44)-H(4A) 109.9

C(45)-C(44)-H(4A) 109.9

N(2)-C(44)-H(4B) 109.9

C(45)-C(44)-H(4B) 109.9

H(4A)-C(44)-H(4B) 108.3

O(5)-C(45)-C(44) 111.2(4)

O(5)-C(45)-H(45A) 109.4

C(44)-C(45)-H(45A) 109.4

O(5)-C(45)-H(45B) 109.4

C(44)-C(45)-H(45B) 109.4

H(45A)-C(45)-H(45B) 108.0

O(5)-C(46)-C(47) 110.8(4)

O(5)-C(46)-H(46A) 109.5

C(47)-C(46)-H(46A) 109.5

O(5)-C(46)-H(46B) 109.5

C(47)-C(46)-H(46B) 109.5

H(46A)-C(46)-H(46B) 108.1

N(2)-C(47)-C(46) 109.4(4)

N(2)-C(47)-H(47A) 109.8

C(46)-C(47)-H(47A) 109.8

N(2)-C(47)-H(47B) 109.8

C(46)-C(47)-H(47B) 109.8

H(47A)-C(47)-H(47B) 108.2

C(14)-N(1)-C(41) 123.5(4)

C(14)-N(1)-C(11) 112.4(4)

C(41)-N(1)-C(11) 123.6(3)

C(44)-N(2)-C(43) 110.8(3)

C(44)-N(2)-C(47) 109.8(3)

C(43)-N(2)-C(47) 113.0(3)

C(44)-N(2)-H(2A) 107.7

C(43)-N(2)-H(2A) 107.7

C(47)-N(2)-H(2A) 107.7

C(13)-O(2)-H(2) 109.5

C(39)-O(4)-C(32) 106.0(3)

C(45)-O(5)-C(46) 109.5(3)

Table 4. Anisotropic displacement parameters (Å2x 103) for 12.3-HCl. The anisotropic


Cl(1) 23(1) 30(1) 12(1) 1(1) 5(1) 1(1)

C(11) 17(2) 18(3) 11(2) 2(2) 2(2) -1(2)

C(12) 12(2) 16(3) 10(2) 2(2) -1(2) -1(2)

C(13) 13(2) 15(3) 9(2) -2(2) 1(2) 1(2)

C(14) 19(2) 12(3) 8(2) -2(2) 1(2) 0(2)

C(21) 22(3) 13(3) 10(2) 3(2) 7(2) 5(2)

C(22) 22(3) 21(3) 17(2) 1(2) 4(2) -2(2)

C(23) 28(3) 31(3) 14(2) 0(2) 8(2) 1(2)

C(24) 28(3) 22(3) 7(2) -5(2) -2(2) 2(2)


C(25) 19(3) 36(3) 20(2) -5(2) 5(2) -2(2)

C(26) 26(3) 30(3) 12(2) 3(2) 6(2) 3(2)

C(31) 14(2) 20(3) 10(2) -3(2) 1(2) -2(2)

C(32) 12(2) 19(3) 16(2) 0(2) 1(2) 2(2)

C(33) 16(2) 26(3) 14(2) 1(2) 2(2) 3(2)

C(34) 22(3) 20(3) 15(2) -1(2) -1(2) 3(2)

C(35) 25(3) 29(3) 17(2) 4(2) 4(2) -2(2)

C(36) 33(3) 26(3) 24(2) 13(2) 4(2) -2(2)

C(37) 33(3) 26(3) 24(2) 7(2) -3(2) 6(2)

C(38) 23(3) 26(3) 21(2) -1(2) 1(2) 8(2)

C(39) 18(3) 21(3) 15(2) -3(2) 2(2) -3(2)

C(41) 24(3) 22(3) 6(2) 0(2) 0(2) 4(2)

C(42) 19(3) 21(3) 9(2) 3(2) 0(2) -2(2)

C(43) 15(2) 20(3) 10(2) -4(2) 1(2) 1(2)

C(44) 17(2) 15(3) 13(2) 5(2) 2(2) -2(2)

C(45) 19(3) 21(3) 13(2) 4(2) 1(2) -4(2)

C(46) 22(3) 24(3) 9(2) 8(2) -1(2) -2(2)

C(47) 10(2) 31(3) 22(2) 2(2) 3(2) -2(2)

N(1) 25(2) 14(2) 8(2) 1(2) 3(2) 6(2)

N(2) 16(2) 13(2) 11(2) 3(2) 2(1) -2(2)

O(1) 27(2) 20(2) 15(2) 1(1) 7(1) 7(2)

O(2) 20(2) 20(2) 11(1) 3(1) 5(1) 6(1)

O(3) 19(2) 25(2) 16(2) -1(1) 7(1) 1(2)

O(4) 19(2) 18(2) 15(1) 2(1) 5(1) 6(1)

O(5) 16(2) 23(2) 14(1) 4(1) 3(1) -3(1)

F(1) 39(2) 38(2) 17(1) -12(1) 6(1) -8(2)


x y z U(eq)

H(11) 5146 1930 9764 18

H(22) 4416 2382 11809 24

H(23) 5421 3030 13673 28

H(25) 11413 3584 12477 30

H(26) 10387 2937 10608 27

H(33) 6825 3663 6316 22

H(35) 6065 4869 4567 28

H(36) 3495 5752 4069 33

H(37) 292 5881 5127 34

H(38) -463 5119 6701 29

H(41A) 8863 1243 11288 21

H(41B) 10941 1101 10525 21

H(42A) 8351 445 8975 20

H(42B) 6308 576 9773 20

H(43A) 10579 -51 10978 18

H(43B) 8250 -21 11568 18

H(4A) 8840 -1279 11803 18

H(4B) 10975 -1247 11042 18

H(45A) 8964 -1958 9393 21

H(45B) 9214 -2316 10763 21

H(46A) 3169 -1554 9080 23

H(46B) 5390 -1510 8395 23

H(47A) 4730 -463 9297 25

H(47B) 4891 -786 10698 25

H(2A) 8598 -733 9331 16

H(2) 9433 2415 6414 25

Table 6. Torsion angles [°] for 12.3-HCl. N(1)-C(11)-C(12)-C(13) -0.9(5)

C(21)-C(11)-C(12)-C(13) 117.5(4)

N(1)-C(11)-C(12)-C(31) 173.4(4)

C(21)-C(11)-C(12)-C(31) -68.3(5)

C(31)-C(12)-C(13)-O(2) 4.9(9)

C(11)-C(12)-C(13)-O(2) 177.6(4)

C(31)-C(12)-C(13)-C(14) -171.2(5)

C(11)-C(12)-C(13)-C(14) 1.5(5)

O(2)-C(13)-C(14)-O(1) -1.0(7)

C(12)-C(13)-C(14)-O(1) 175.6(4)

O(2)-C(13)-C(14)-N(1) -178.1(4)

C(12)-C(13)-C(14)-N(1) -1.5(5)

N(1)-C(11)-C(21)-C(26) 53.8(5)

C(12)-C(11)-C(21)-C(26) -60.2(6)

N(1)-C(11)-C(21)-C(22) -128.4(4)

C(12)-C(11)-C(21)-C(22) 117.7(5)


C(26)-C(21)-C(22)-C(23) 0.1(7)

C(11)-C(21)-C(22)-C(23) -177.8(4)

C(21)-C(22)-C(23)-C(24) -0.3(7)

C(22)-C(23)-C(24)-F(1) 179.8(4)

C(22)-C(23)-C(24)-C(25) 0.4(7)

F(1)-C(24)-C(25)-C(26) -179.7(4)

C(23)-C(24)-C(25)-C(26) -0.2(7)

C(24)-C(25)-C(26)-C(21) 0.0(7)

C(22)-C(21)-C(26)-C(25) 0.0(7)

C(11)-C(21)-C(26)-C(25) 177.9(4)

C(13)-C(12)-C(31)-O(3) 161.0(5)

C(11)-C(12)-C(31)-O(3) -11.2(6)

C(13)-C(12)-C(31)-C(32) -22.4(8)

C(11)-C(12)-C(31)-C(32) 165.3(4)

O(3)-C(31)-C(32)-C(33) 176.8(5)

C(12)-C(31)-C(32)-C(33) 0.3(8)

O(3)-C(31)-C(32)-O(4) 1.6(6)

C(12)-C(31)-C(32)-O(4) -174.8(4)

O(4)-C(32)-C(33)-C(34) -1.1(5)

C(31)-C(32)-C(33)-C(34) -176.3(5)

C(32)-C(33)-C(34)-C(39) 1.8(5)

C(32)-C(33)-C(34)-C(35) 179.7(5)

C(39)-C(34)-C(35)-C(36) -1.1(7)

C(33)-C(34)-C(35)-C(36) -178.7(5)

C(34)-C(35)-C(36)-C(37) 1.0(8)

C(35)-C(36)-C(37)-C(38) -0.3(8)

C(36)-C(37)-C(38)-C(39) -0.1(7)

C(37)-C(38)-C(39)-O(4) -179.0(4)

C(37)-C(38)-C(39)-C(34) 0.0(7)

C(35)-C(34)-C(39)-O(4) 179.7(4)

C(33)-C(34)-C(39)-O(4) -2.0(5)

C(35)-C(34)-C(39)-C(38) 0.7(7)

C(33)-C(34)-C(39)-C(38) 179.0(4)

N(1)-C(41)-C(42)-C(43) 178.4(3)

C(41)-C(42)-C(43)-N(2) -168.4(3)

N(2)-C(44)-C(45)-O(5) -59.1(4)

O(5)-C(46)-C(47)-N(2) 58.8(4)

O(1)-C(14)-N(1)-C(41) -4.0(7)

C(13)-C(14)-N(1)-C(41) 173.0(4)

O(1)-C(14)-N(1)-C(11) -176.1(4)

C(13)-C(14)-N(1)-C(11) 0.9(5)

C(42)-C(41)-N(1)-C(14) -76.8(5)

C(42)-C(41)-N(1)-C(11) 94.4(5)

C(12)-C(11)-N(1)-C(14) 0.0(5)

C(21)-C(11)-N(1)-C(14) -120.0(4)

C(12)-C(11)-N(1)-C(41) -172.2(4)

C(21)-C(11)-N(1)-C(41) 67.8(5)

C(45)-C(44)-N(2)-C(43) -179.3(3)

C(45)-C(44)-N(2)-C(47) 55.2(4)

C(42)-C(43)-N(2)-C(44) 171.5(3)

C(42)-C(43)-N(2)-C(47) -64.9(4)

C(46)-C(47)-N(2)-C(44) -55.6(4)

C(46)-C(47)-N(2)-C(43) -179.7(3)

C(38)-C(39)-O(4)-C(32) -179.6(4)

C(34)-C(39)-O(4)-C(32) 1.3(5)

C(33)-C(32)-O(4)-C(39) -0.1(5)

C(31)-C(32)-O(4)-C(39) 176.2(4)

C(44)-C(45)-O(5)-C(46) 62.2(4)

C(47)-C(46)-O(5)-C(45) -62.0(4)



Empirical formula C24 H24 Cl2 N2 O4





Space group P -1

Unit cell dimensions a = 9.8063(12) Å α= 84.013(2)°.

b = 10.3524(13) Å β= 84.700(2)°.

c = 11.6777(15) Å γ = 72.907(2)°.

Volume 1124.6(2) Å3

Z 2



F(000) 496



Index ranges -13<=h<=9, -11<=k<=13, -15<=l<=15















C(11) 7049(1) 7966(1) 7875(1) 12(1)

C(12) 7956(1) 8082(1) 6773(1) 14(1)

C(13) 8663(1) 9022(1) 6861(1) 16(1)

C(14) 8346(2) 9535(1) 8036(1) 16(1)

C(21) 5443(1) 8446(1) 7708(1) 15(1)

C(22) 4464(2) 7846(1) 8308(1) 17(1)

C(23) 3005(2) 8288(2) 8125(1) 22(1)

C(24) 2506(2) 9397(2) 7339(1) 23(1)

C(25) 3450(2) 10045(2) 6747(1) 23(1)

C(26) 4896(2) 9570(2) 6926(1) 20(1)

C(31) 8258(1) 7301(1) 5784(1) 16(1)

C(32) 7701(2) 6160(1) 5694(1) 16(1)

C(33) 7981(2) 5254(2) 4874(1) 19(1)

C(34) 7152(2) 4340(1) 5236(1) 19(1)

C(35) 6958(2) 3204(2) 4784(1) 26(1)

C(36) 6023(2) 2574(2) 5394(2) 30(1)

C(37) 5281(2) 3042(2) 6430(2) 29(1)

C(38) 5453(2) 4161(2) 6893(1) 24(1)

C(39) 6402(2) 4775(1) 6272(1) 18(1)

C(41) 6924(1) 9016(1) 9796(1) 15(1)

C(42) 7779(1) 7868(1) 10611(1) 16(1)

C(43) 9350(1) 7826(1) 10527(1) 14(1)

C(44) 9965(2) 7068(2) 12556(1) 18(1)

C(45) 11751(2) 6364(2) 10959(1) 20(1)

N(1) 7427(1) 8889(1) 8586(1) 13(1)

N(2) 10201(1) 6720(1) 11333(1) 15(1)

O(1) 8844(1) 10337(1) 8431(1) 22(1)

O(2) 9556(1) 9431(1) 6107(1) 22(1)

O(3) 9077(1) 7586(1) 4958(1) 21(1)

O(4) 6740(1) 5890(1) 6570(1) 17(1)

Cl(1) 5042(1) 6486(1) 9340(1) 23(1)

Cl(2) 10772(1) 5817(1) 8132(1) 22(1)


C(11)-N(1) 1.4722(17)

C(11)-C(12) 1.5106(18)

C(11)-C(21) 1.5312(18)

C(11)-H(11) 1.0000

C(12)-C(13) 1.3675(19)

C(12)-C(31) 1.4372(19)

C(13)-O(2) 1.3107(17)

C(13)-C(14) 1.4964(19)

C(14)-O(1) 1.2250(18)

C(14)-N(1) 1.3523(18)

C(21)-C(22) 1.390(2)

C(21)-C(26) 1.406(2)

C(22)-C(23) 1.398(2)

C(22)-Cl(1) 1.7485(14)

C(23)-C(24) 1.391(2)

C(23)-H(23) 0.9500

C(24)-C(25) 1.390(2)

C(24)-H(24) 0.9500

C(25)-C(26) 1.386(2)

C(25)-H(25) 0.9500

C(26)-H(26) 0.9500

C(31)-O(3) 1.2653(17)

C(31)-C(32) 1.456(2)

C(32)-C(33) 1.3636(19)

C(32)-O(4) 1.3890(17)

C(33)-C(34) 1.429(2)

C(33)-H(33) 0.9500

C(34)-C(39) 1.401(2)

C(34)-C(35) 1.406(2)

C(35)-C(36) 1.381(3)

C(35)-H(35) 0.9500

C(36)-C(37) 1.407(3)

C(36)-H(36) 0.9500

C(37)-C(38) 1.387(2)

C(37)-H(37) 0.9500

C(38)-C(39) 1.387(2)

C(38)-H(38) 0.9500

C(39)-O(4) 1.3757(17)

C(41)-N(1) 1.4588(17)

C(41)-C(42) 1.5321(19)

C(41)-H(41A) 0.9900

C(41)-H(41B) 0.9900

C(42)-C(43) 1.5225(19)

C(42)-H(42A) 0.9900

C(42)-H(42B) 0.9900

C(43)-N(2) 1.5008(17)

C(43)-H(43A) 0.9900

C(43)-H(43B) 0.9900

C(44)-N(2) 1.4905(17)

C(44)-H(44A) 0.9800


C(44)-H(44B) 0.9800

C(44)-H(44C) 0.9800

C(45)-N(2) 1.4897(18)

C(45)-H(45A) 0.9800

C(45)-H(45B) 0.9800

C(45)-H(45C) 0.9800

N(2)-H(1) 0.92(2)

O(2)-H(2) 0.81(3)

Bond angles (°)

N(1)-C(11)-C(12) 101.84(10)

N(1)-C(11)-C(21) 109.80(10)

C(12)-C(11)-C(21) 113.25(11)

N(1)-C(11)-H(11) 110.6

C(12)-C(11)-H(11) 110.6

C(21)-C(11)-H(11) 110.6

C(13)-C(12)-C(31) 119.84(13)

C(13)-C(12)-C(11) 109.10(11)

C(31)-C(12)-C(11) 130.71(12)

O(2)-C(13)-C(12) 129.20(13)

O(2)-C(13)-C(14) 121.00(13)

C(12)-C(13)-C(14) 109.73(12)

O(1)-C(14)-N(1) 127.25(14)

O(1)-C(14)-C(13) 127.40(13)

N(1)-C(14)-C(13) 105.32(12)

C(22)-C(21)-C(26) 117.04(13)

C(22)-C(21)-C(11) 123.62(12)

C(26)-C(21)-C(11) 119.32(12)

C(21)-C(22)-C(23) 122.52(13)

C(21)-C(22)-Cl(1) 120.06(11)

C(23)-C(22)-Cl(1) 117.41(12)

C(24)-C(23)-C(22) 118.73(14)

C(24)-C(23)-H(23) 120.6

C(22)-C(23)-H(23) 120.6

C(25)-C(24)-C(23) 120.26(14)

C(25)-C(24)-H(24) 119.9

C(23)-C(24)-H(24) 119.9

C(26)-C(25)-C(24) 119.89(14)

C(26)-C(25)-H(25) 120.1

C(24)-C(25)-H(25) 120.1

C(25)-C(26)-C(21) 121.51(14)

C(25)-C(26)-H(26) 119.2

C(21)-C(26)-H(26) 119.2

O(3)-C(31)-C(12) 119.00(13)

O(3)-C(31)-C(32) 117.70(12)

C(12)-C(31)-C(32) 123.28(13)

C(33)-C(32)-O(4) 111.74(13)

C(33)-C(32)-C(31) 130.39(14)

O(4)-C(32)-C(31) 117.86(12)

C(32)-C(33)-C(34) 106.18(13)

C(32)-C(33)-H(33) 126.9

C(34)-C(33)-H(33) 126.9

C(39)-C(34)-C(35) 119.10(15)

C(39)-C(34)-C(33) 106.27(13)

C(35)-C(34)-C(33) 134.63(15)

C(36)-C(35)-C(34) 117.62(15)

C(36)-C(35)-H(35) 121.2

C(34)-C(35)-H(35) 121.2

C(35)-C(36)-C(37) 121.79(15)

C(35)-C(36)-H(36) 119.1

C(37)-C(36)-H(36) 119.1

C(38)-C(37)-C(36) 121.74(16)

C(38)-C(37)-H(37) 119.1

C(36)-C(37)-H(37) 119.1

C(37)-C(38)-C(39) 115.60(15)

C(37)-C(38)-H(38) 122.2

C(39)-C(38)-H(38) 122.2

O(4)-C(39)-C(38) 125.60(13)

O(4)-C(39)-C(34) 110.25(13)

C(38)-C(39)-C(34) 124.15(14)

N(1)-C(41)-C(42) 113.29(11)

N(1)-C(41)-H(41A) 108.9

C(42)-C(41)-H(41A) 108.9

N(1)-C(41)-H(41B) 108.9

C(42)-C(41)-H(41B) 108.9

H(41A)-C(41)-H(41B) 107.7

C(43)-C(42)-C(41) 110.99(12)

C(43)-C(42)-H(42A) 109.4

C(41)-C(42)-H(42A) 109.4

C(43)-C(42)-H(42B) 109.4

C(41)-C(42)-H(42B) 109.4

H(42A)-C(42)-H(42B) 108.0

N(2)-C(43)-C(42) 111.77(11)

N(2)-C(43)-H(43A) 109.3

C(42)-C(43)-H(43A) 109.3

N(2)-C(43)-H(43B) 109.3

C(42)-C(43)-H(43B) 109.3

H(43A)-C(43)-H(43B) 107.9

N(2)-C(44)-H(44A) 109.5

N(2)-C(44)-H(44B) 109.5

H(44A)-C(44)-H(44B) 109.5

N(2)-C(44)-H(44C) 109.5

H(44A)-C(44)-H(44C) 109.5

H(44B)-C(44)-H(44C) 109.5

N(2)-C(45)-H(45A) 109.5

N(2)-C(45)-H(45B) 109.5

H(45A)-C(45)-H(45B) 109.5

N(2)-C(45)-H(45C) 109.5

H(45A)-C(45)-H(45C) 109.5

H(45B)-C(45)-H(45C) 109.5

C(14)-N(1)-C(41) 123.45(12)

C(14)-N(1)-C(11) 113.89(11)

C(41)-N(1)-C(11) 122.53(11)

C(45)-N(2)-C(44) 110.67(11)

C(45)-N(2)-C(43) 110.76(11)

C(44)-N(2)-C(43) 112.57(10)

C(45)-N(2)-H(1) 107.5(14)

C(44)-N(2)-H(1) 107.0(15)

C(43)-N(2)-H(1) 108.0(15)

C(13)-O(2)-H(2) 103(2)

C(39)-O(4)-C(32) 105.56(11)


displacement factor exponent takes the form: -2π2[ h2a*2U11 + ... + 2 h k a* b* U12 ]


U11 U22 U33 U23 U13 U12

C(11) 13(1) 13(1) 12(1) -3(1) -1(1) -4(1)

C(12) 13(1) 15(1) 12(1) -1(1) -1(1) -4(1)

C(13) 15(1) 18(1) 14(1) 1(1) -3(1) -5(1)

C(14) 16(1) 16(1) 17(1) 0(1) -4(1) -5(1)

C(21) 14(1) 16(1) 15(1) -7(1) -1(1) -4(1)

C(22) 18(1) 14(1) 18(1) -2(1) -1(1) -4(1)

C(23) 16(1) 23(1) 26(1) -9(1) 1(1) -5(1)

C(24) 17(1) 24(1) 28(1) -10(1) -5(1) -3(1)

C(25) 22(1) 19(1) 24(1) -3(1) -8(1) 0(1)

C(26) 18(1) 20(1) 21(1) -5(1) -3(1) -4(1)

C(31) 14(1) 17(1) 14(1) -1(1) -3(1) -2(1)

C(32) 16(1) 18(1) 13(1) -1(1) -1(1) -4(1)

C(33) 23(1) 19(1) 14(1) -4(1) -2(1) -3(1)

C(34) 24(1) 17(1) 17(1) -3(1) -5(1) -3(1)

C(35) 39(1) 19(1) 20(1) -7(1) -6(1) -6(1)

C(36) 45(1) 20(1) 28(1) -5(1) -10(1) -13(1)

C(37) 37(1) 25(1) 29(1) -2(1) -5(1) -16(1)

C(38) 29(1) 24(1) 23(1) -5(1) -1(1) -12(1)

C(39) 21(1) 15(1) 18(1) -4(1) -5(1) -5(1)

C(41) 14(1) 17(1) 14(1) -6(1) 0(1) -1(1)

C(42) 16(1) 21(1) 12(1) -2(1) -1(1) -5(1)

C(43) 15(1) 15(1) 12(1) -1(1) -2(1) -3(1)

C(44) 24(1) 20(1) 11(1) -2(1) -2(1) -6(1)

C(45) 15(1) 23(1) 20(1) -4(1) -3(1) 0(1)

N(1) 15(1) 13(1) 12(1) -3(1) -2(1) -4(1)

N(2) 16(1) 14(1) 13(1) -2(1) -3(1) -3(1)

O(1) 27(1) 22(1) 24(1) -4(1) -5(1) -14(1)

O(2) 24(1) 30(1) 17(1) 0(1) 1(1) -16(1)

O(3) 24(1) 26(1) 13(1) -3(1) 3(1) -9(1)

O(4) 20(1) 18(1) 15(1) -6(1) 1(1) -7(1)

Cl(1) 21(1) 21(1) 25(1) 5(1) 3(1) -6(1)

Cl(2) 30(1) 17(1) 20(1) -3(1) -2(1) -8(1)


x y z U(eq)

H(11) 7332 7017 8242 15

H(23) 2367 7838 8530 26

H(24) 1517 9714 7206 27

H(25) 3103 10812 6220 27

H(26) 5533 10014 6511 23

H(33) 8602 5235 4198 23

H(35) 7451 2883 4084 31

H(36) 5878 1802 5106 35

H(37) 4646 2581 6822 34

H(38) 4953 4485 7591 29

H(41A) 5907 9029 9885 18

H(41B) 6984 9893 10022 18

H(42A) 7380 8004 11414 19

H(42B) 7692 6991 10411 19

H(43A) 9749 7679 9725 17

H(43B) 9433 8710 10713 17

H(44A) 8941 7296 12786 27

H(44B) 10315 7848 12632 27

H(44C) 10483 6291 13053 27

H(45A) 11885 6138 10154 31

H(45B) 12276 5583 11450 31

H(45C) 12113 7139 11027 31

H(1) 9900(20) 5960(20) 11300(20) 38(6)

H(2) 9610(30) 8970(30) 5580(30) 64(9)




Empirical formula C25 H29 Cl N2 O6





Space group P -1


b = 10.847(5) Å β= 82.181(5)°.

c = 16.912(5) Å γ = 88.152(5)°.

Volume 1189.9(11) Å3

Z 2



F(000) 516



Index ranges -9<=h<=9, -14<=k<=14, -22<=l<=22













C(11) 8319(2) 5073(1) 8564(1) 13(1)

C(12) 8837(2) 3901(1) 8273(1) 12(1)

C(13) 7426(2) 3698(1) 7832(1) 12(1)

C(14) 5896(2) 4754(1) 7784(1) 12(1)

C(21) 5676(2) 5487(1) 6898(1) 12(1)

C(22) 3947(2) 5358(1) 6578(1) 13(1)

C(23) 3772(2) 5960(1) 5747(1) 14(1)

C(24) 5314(2) 6719(1) 5241(1) 16(1)

C(25) 7028(2) 6862(1) 5575(1) 16(1)

C(26) 7234(2) 6247(1) 6393(1) 15(1)

C(27) 1847(2) 6303(1) 4632(1) 18(1)

C(31) 5837(2) 6806(1) 8264(1) 14(1)

C(32) 3633(2) 6740(1) 8587(1) 19(1)

C(33) 2702(2) 8056(1) 8490(1) 17(1)

C(34) 2752(2) 10155(1) 8799(1) 17(1)

C(35) 2596(2) 8183(1) 9939(1) 21(1)

C(41) 7009(2) 2650(1) 7480(1) 12(1)

C(42) 8606(2) 1854(1) 7211(1) 13(1)

C(43) 10619(2) 1924(1) 7100(1) 13(1)

C(44) 11315(2) 932(1) 6719(1) 14(1)

C(45) 13182(2) 489(1) 6448(1) 17(1)

C(46) 13232(2) -529(1) 6099(1) 21(1)

C(47) 11483(2) -1113(1) 6020(1) 25(1)

C(48) 9626(2) -695(1) 6286(1) 23(1)

C(49) 9606(2) 326(1) 6631(1) 16(1)

N(1) 6660(1) 5556(1) 8245(1) 14(1)

N(2) 3347(1) 8779(1) 9053(1) 14(1)

O(1) 9262(1) 5485(1) 9012(1) 18(1)


O(2) 10450(1) 3235(1) 8466(1) 15(1)

O(3) 2016(1) 5760(1) 5493(1) 17(1)

O(4) 5299(1) 2486(1) 7369(1) 17(1)

O(5) 7940(1) 883(1) 6925(1) 15(1)

O(6) 12373(1) 3499(1) 9631(1) 19(1)

Cl(1) 7680(1) 9182(1) 9110(1) 18(1)


C(11)-O(1) 1.2332(14)

C(11)-N(1) 1.3467(16)

C(11)-C(12) 1.4999(16)

C(12)-O(2) 1.3237(15)

C(12)-C(13) 1.3508(16)

C(13)-C(41) 1.4704(16)

C(13)-C(14) 1.5162(16)

C(14)-N(1) 1.4639(14)

C(14)-C(21) 1.5183(16)

C(21)-C(22) 1.3865(17)

C(21)-C(26) 1.3998(16)

C(22)-C(23) 1.3975(16)

C(23)-O(3) 1.3667(16)

C(23)-C(24) 1.3920(17)

C(24)-C(25) 1.3937(18)

C(25)-C(26) 1.3851(17)

C(27)-O(3) 1.4332(15)

C(31)-N(1) 1.4572(15)

C(31)-C(32) 1.5212(19)

C(32)-C(33) 1.5199(18)

C(33)-N(2) 1.5015(15)

C(34)-N(2) 1.4958(16)

C(35)-N(2) 1.4865(16)

C(41)-O(4) 1.2297(16)

C(41)-C(42) 1.4673(17)

C(42)-C(43) 1.3590(19)

C(42)-O(5) 1.3848(14)

C(43)-C(44) 1.4363(16)

C(44)-C(49) 1.3981(18)

C(44)-C(45) 1.4058(18)

C(45)-C(46) 1.3852(18)

C(46)-C(47) 1.404(2)

C(47)-C(48) 1.387(2)

C(48)-C(49) 1.3847(17)

C(49)-O(5) 1.3653(15)

Bond angles (°)

O(1)-C(11)-N(1) 128.12(11)

O(1)-C(11)-C(12) 125.22(11)

N(1)-C(11)-C(12) 106.66(9)

O(2)-C(12)-C(13) 130.10(11)

O(2)-C(12)-C(11) 120.81(10)

C(13)-C(12)-C(11) 109.08(10)

C(12)-C(13)-C(41) 133.89(10)

C(12)-C(13)-C(14) 109.03(10)

C(41)-C(13)-C(14) 116.78(10)

N(1)-C(14)-C(13) 102.76(9)

N(1)-C(14)-C(21) 112.59(9)

C(13)-C(14)-C(21) 112.55(9)

C(22)-C(21)-C(26) 119.86(11)

C(22)-C(21)-C(14) 119.53(10)

C(26)-C(21)-C(14) 120.56(10)

C(21)-C(22)-C(23) 120.23(10)

O(3)-C(23)-C(24) 124.29(11)

O(3)-C(23)-C(22) 115.49(10)

C(24)-C(23)-C(22) 120.21(11)

C(23)-C(24)-C(25) 118.99(11)

C(26)-C(25)-C(24) 121.26(11)

C(25)-C(26)-C(21) 119.43(11)

N(1)-C(31)-C(32) 112.52(10)

C(33)-C(32)-C(31) 112.72(10)

N(2)-C(33)-C(32) 114.16(10)

O(4)-C(41)-C(42) 119.50(11)

O(4)-C(41)-C(13) 119.05(10)

C(42)-C(41)-C(13) 121.33(10)

C(43)-C(42)-O(5) 111.61(10)

C(43)-C(42)-C(41) 134.40(11)

O(5)-C(42)-C(41) 113.61(10)

C(42)-C(43)-C(44) 106.38(10)

C(49)-C(44)-C(45) 119.08(11)

C(49)-C(44)-C(43) 105.42(11)

C(45)-C(44)-C(43) 135.49(11)

C(46)-C(45)-C(44) 117.82(11)

C(45)-C(46)-C(47) 121.47(12)

C(48)-C(47)-C(46) 121.68(12)

C(49)-C(48)-C(47) 116.02(12)

O(5)-C(49)-C(48) 125.17(11)

O(5)-C(49)-C(44) 110.89(11)

C(48)-C(49)-C(44) 123.93(11)

C(11)-N(1)-C(31) 124.35(9)

C(11)-N(1)-C(14) 112.35(10)

C(31)-N(1)-C(14) 122.90(10)

C(35)-N(2)-C(34) 110.68(9)

C(35)-N(2)-C(33) 112.35(10)

C(34)-N(2)-C(33) 111.17(10)

C(23)-O(3)-C(27) 116.53(9)

C(49)-O(5)-C(42) 105.69(9)



C(11) 13(1) 16(1) 13(1) -6(1) -1(1) 0(1)

C(12) 12(1) 14(1) 11(1) -4(1) 0(1) 1(1)

C(13) 13(1) 12(1) 12(1) -5(1) -1(1) 1(1)

C(14) 12(1) 13(1) 13(1) -7(1) -3(1) 0(1)


C(21) 13(1) 12(1) 13(1) -7(1) -1(1) 2(1)

C(22) 12(1) 13(1) 14(1) -5(1) -1(1) 0(1)

C(23) 14(1) 14(1) 15(1) -7(1) -3(1) 2(1)

C(24) 19(1) 15(1) 13(1) -4(1) -1(1) 1(1)

C(25) 15(1) 15(1) 18(1) -5(1) 2(1) -2(1)

C(26) 12(1) 15(1) 19(1) -8(1) -2(1) 0(1)

C(27) 21(1) 21(1) 13(1) -5(1) -5(1) 3(1)

C(31) 14(1) 14(1) 18(1) -9(1) -2(1) 1(1)

C(32) 14(1) 16(1) 26(1) -10(1) 2(1) 0(1)

C(33) 14(1) 19(1) 23(1) -12(1) -4(1) 2(1)

C(34) 17(1) 13(1) 21(1) -5(1) -3(1) 1(1)

C(35) 26(1) 20(1) 16(1) -3(1) 1(1) -1(1)

C(41) 15(1) 11(1) 11(1) -3(1) -2(1) -1(1)

C(42) 17(1) 11(1) 13(1) -5(1) -4(1) 0(1)

C(43) 15(1) 12(1) 14(1) -5(1) -2(1) 0(1)

C(44) 17(1) 13(1) 13(1) -4(1) -3(1) 1(1)

C(45) 16(1) 18(1) 17(1) -5(1) -2(1) 2(1)

C(46) 21(1) 22(1) 22(1) -10(1) -2(1) 7(1)

C(47) 28(1) 22(1) 32(1) -18(1) -6(1) 7(1)

C(48) 22(1) 21(1) 32(1) -16(1) -7(1) 1(1)

C(49) 16(1) 14(1) 18(1) -7(1) -3(1) 2(1)

N(1) 13(1) 15(1) 16(1) -10(1) -4(1) 2(1)

N(2) 12(1) 14(1) 15(1) -5(1) -2(1) 0(1)

O(1) 17(1) 22(1) 20(1) -12(1) -6(1) 1(1)

O(2) 14(1) 19(1) 16(1) -9(1) -6(1) 5(1)

O(3) 14(1) 23(1) 13(1) -4(1) -5(1) -1(1)

O(4) 14(1) 16(1) 22(1) -8(1) -3(1) -1(1)

O(5) 15(1) 13(1) 21(1) -10(1) -4(1) 1(1)

Cl(1) 13(1) 19(1) 24(1) -10(1) -4(1) 0(1)


x y z U(eq)

H(22) 2900 4870 6917 15

H(24) 5202 7124 4689 19

H(25) 8054 7380 5243 20

H(26) 8397 6339 6604 18

H(27A) 562 6105 4523 27

H(27B) 2854 5951 4302 27

H(27C) 2011 7215 4494 27

H(31A) 6041 7381 7709 17

H(31B) 6541 7158 8615 17

H(32A) 2956 6272 8289 22

H(32B) 3448 6269 9168 22

H(33A) 3035 8561 7921 21

H(33B) 1271 7958 8599 21

H(34A) 3255 10524 8227 25

H(34B) 3286 10611 9135 25

H(34C) 1331 10214 8872 25

H(35A) 2992 7301 10091 32

H(35B) 1174 8231 10020 32

H(35C) 3135 8630 10279 32

H(43) 11393 2501 7243 16

H(45) 14346 867 6502 20

H(46) 14451 -833 5912 25

H(47) 11572 -1796 5784 30

H(48) 8464 -1079 6236 27

H(1) 10990(30) 3440(20) 8852(13) 43(5)

H(2) 4750(30) 8803(17) 9013(11) 29(4)

H(3A) 12400(20) 2766(18) 9937(11) 26(4)

H(3B) 11730(30) 3940(20) 9953(14) 54(6)

H(14) 4610(20) 4413(14) 8076(9) 13(3)


A.10 A co-crystallised set of salts: The hemi-HCl and hemi-oxalic acid forms of morpholine

Table 1. Crystal data and structure refinement. Identification code mo_10bo_tel9_0m

Empirical formula C9 H19 Cl N2 O5





Space group C 2/c


b = 5.685(5) Å β= 109.575(5)°.

c = 24.783(5) Å γ = 90.000(5)°.

Volume 2515(2) Å3

Z 8



F(000) 1152



Index ranges -25<=h<=25, -7<=k<=7, -33<=l<=33











Table 2. Atomic coordinates ( x 104) and equivalent isotropic displacement parameters (Å2x 103).

U(eq) is defined as one third of the trace of the orthogonalised Uij tensor. x y z U(eq)

C(1) 671(1) -947(2) 6133(1) 15(1)

C(2) 185(1) -482(2) 6501(1) 13(1)

C(3) -171(1) 3248(2) 6004(1) 12(1)

C(4) 316(1) 2631(2) 5648(1) 14(1)

C(5) -1002(1) 1521(2) 6487(1) 13(1)

C(6) -1404(1) -716(2) 6562(1) 15(1)

C(7) -2076(1) -105(2) 6741(1) 14(1)

N(1) -468(1) 1019(2) 6169(1) 11(1)

N(2) -2514(1) -2280(2) 6741(1) 14(1)

O(1) 933(1) 1181(1) 5960(1) 15(1)

C(8) -1660(1) -2155(2) 4888(1) 11(1)

C(9) -1943(1) 321(2) 4643(1) 11(1)

O(2) -1112(1) -2259(1) 5339(1) 14(1)

O(3) -2013(1) -3852(1) 4594(1) 15(1)

O(4) -2419(1) 563(1) 4175(1) 15(1)

O(5) -1608(1) 2039(1) 4987(1) 15(1)

Cl(1) -3673(1) -183(1) 7295(1) 16(1)

Table 3. Bond lengths [Å] and angles [°]. Bond lengths (Ǻ)

C(1)-O(1) 1.4260(15)

C(1)-C(2) 1.5215(15)

C(2)-N(1) 1.5018(13)

C(3)-N(1) 1.4981(16)

C(3)-C(4) 1.5177(14)

C(4)-O(1) 1.4271(13)

C(5)-N(1) 1.5030(13)

C(5)-C(6) 1.5253(17)

C(6)-C(7) 1.5224(15)

C(7)-N(2) 1.4896(16)

C(8)-O(2) 1.2474(12)

C(8)-O(3) 1.2571(14)

C(8)-C(9) 1.5550(18)

C(9)-O(4) 1.2150(13)


C(9)-O(5) 1.3113(14)

Bond angles (°)

O(1)-C(1)-C(2) 111.88(9)

N(1)-C(2)-C(1) 108.91(8)

N(1)-C(3)-C(4) 108.69(9)

O(1)-C(4)-C(3) 111.42(9)

N(1)-C(5)-C(6) 110.87(9)

C(7)-C(6)-C(5) 110.23(9)

N(2)-C(7)-C(6) 109.38(9)

C(3)-N(1)-C(2) 108.27(9)

C(3)-N(1)-C(5) 111.25(8)

C(2)-N(1)-C(5) 113.40(8)

C(1)-O(1)-C(4) 110.43(9)

O(2)-C(8)-O(3) 127.20(10)

O(2)-C(8)-C(9) 117.82(9)

O(3)-C(8)-C(9) 114.97(10)

O(4)-C(9)-O(5) 125.35(10)

O(4)-C(9)-C(8) 121.51(9)

O(5)-C(9)-C(8) 113.13(9)

Table 4. Anisotropic displacement parameters (Å2x 103). The anisotropic


C(1) 14(1) 12(1) 19(1) 2(1) 7(1) 2(1)

C(2) 12(1) 12(1) 14(1) 2(1) 3(1) 3(1)

C(3) 13(1) 9(1) 14(1) 1(1) 4(1) 0(1)

C(4) 15(1) 13(1) 16(1) 2(1) 6(1) 1(1)

C(5) 12(1) 14(1) 15(1) -1(1) 7(1) 1(1)

C(6) 14(1) 14(1) 18(1) 0(1) 8(1) 0(1)

C(7) 14(1) 14(1) 17(1) -1(1) 7(1) -1(1)

N(1) 10(1) 10(1) 11(1) 0(1) 3(1) 0(1)

N(2) 12(1) 16(1) 13(1) -1(1) 4(1) -1(1)

O(1) 13(1) 14(1) 21(1) 3(1) 7(1) 1(1)

C(8) 12(1) 10(1) 12(1) 1(1) 5(1) 1(1)

C(9) 11(1) 10(1) 13(1) 0(1) 5(1) 1(1)

O(2) 14(1) 12(1) 12(1) 0(1) 1(1) 1(1)

O(3) 16(1) 10(1) 15(1) -1(1) 1(1) -1(1)

O(4) 15(1) 13(1) 13(1) 0(1) 1(1) 2(1)

O(5) 18(1) 8(1) 15(1) 0(1) 0(1) 1(1)

Cl(1) 16(1) 17(1) 13(1) -1(1) 3(1) 0(1)

Table 5. Hydrogen coordinates ( x 104) and isotropic displacement parameters (Å2x 103). x y z U(eq)

H(1) -725(9) 200(30) 5853(7) 19(4)

H(1A) 1097 -1902 6347 18

H(1B) 384 -1826 5795 18

H(2A) 8 -1958 6604 15

H(2B) 476 319 6850 15

H(3A) 121 4099 6345 14

H(3B) -583 4244 5785 14

H(4A) 17 1812 5304 17

H(4B) 504 4065 5533 17

H(5A) -1368 2676 6277 16

H(5B) -728 2174 6860 16

H(6A) -1570 -1585 6204 18

H(6B) -1061 -1707 6851 18

H(7A) -1907 587 7120 17

H(7B) -2390 1033 6476 17

H(8A) -2908 -1926 6845 21

H(8B) -2226 -3313 6987 21

H(8C) -2672 -2902 6391 21

H(5) -1775 3303 4840 23

A.11 2,4-Dimethoxybenzaldehyde starting material

Table 1. Crystal data and structure refinement. Identification code mo_11ek_tt1_0ma

Empirical formula C9 H10 O3






Space group P 21/c


b = 3.9382(6) Å β= 113.935(4)°.

c = 14.580(3) Å γ = 90°.

Volume 790.5(2) Å3

Z 4



F(000) 352



Index ranges -18<=h<=19, -5<=k<=5, -19<=l<=19











Table 2. Atomic coordinates ( x 104) and equivalent isotropic displacement parameters (Å2x 103).

U(eq) is defined as one third of the trace of the orthogonalised Uij tensor. x y z U(eq)

C(7) 3394(1) 6059(4) 5028(1) 22(1)

C(8) 4349(1) 701(4) 3114(1) 21(1)

C(9) 1137(1) 1849(4) 382(1) 19(1)

O(1) 3985(1) 2334(2) 3768(1) 20(1)

O(2) 3171(1) 7673(3) 5617(1) 27(1)

O(3) 807(1) 3437(2) 1074(1) 19(1)

C(1) 3046(1) 3405(3) 3364(1) 16(1)

C(2) 2731(1) 5221(3) 4006(1) 16(1)

C(3) 1765(1) 6347(3) 3622(1) 17(1)

C(4) 1135(1) 5697(3) 2651(1) 17(1)

C(5) 1470(1) 3916(3) 2025(1) 15(1)

C(6) 2424(1) 2770(3) 2373(1) 16(1)

Table 3. Bond lengths [Å] and angles [°]. Bond Lengths [Å]

C(7)-O(2) 1.2197(17)

C(7)-C(2) 1.456(2)

C(8)-O(1) 1.4316(16)

C(9)-O(3) 1.4364(16)

O(1)-C(1) 1.3604(16)

O(3)-C(5) 1.3544(16)

C(1)-C(6) 1.3895(19)

C(1)-C(2) 1.4044(18)

C(2)-C(3) 1.4014(18)

C(3)-C(4) 1.3710(19)

C(4)-C(5) 1.3973(18)

C(5)-C(6) 1.3906(18)

Bond Angles [°]

O(2)-C(7)-C(2) 124.58(13)

C(1)-O(1)-C(8) 117.57(11)

C(5)-O(3)-C(9) 117.34(10)

O(1)-C(1)-C(6) 122.61(12)

O(1)-C(1)-C(2) 116.29(12)

C(6)-C(1)-C(2) 121.10(12)

C(3)-C(2)-C(1) 118.12(12)

C(3)-C(2)-C(7) 120.52(12)

C(1)-C(2)-C(7) 121.31(12)

C(4)-C(3)-C(2) 121.72(12)

C(3)-C(4)-C(5) 119.01(12)

O(3)-C(5)-C(6) 123.46(12)

O(3)-C(5)-C(4) 115.32(11)

C(6)-C(5)-C(4) 121.22(13)

C(1)-C(6)-C(5) 118.83(12)


Table 4. Anisotropic displacement parameters (Å2x 103). The anisotropic

displacement factor exponent takes the form: -2π2[ h2a*2U11 + ... + 2 h k a* b* U12 ].

U11 U22 U33 U23 U13 U12

C(7) 20(1) 24(1) 21(1) -2(1) 9(1) 1(1)

C(8) 18(1) 23(1) 25(1) -5(1) 11(1) 2(1)

C(9) 21(1) 20(1) 16(1) -1(1) 8(1) -1(1)

O(1) 15(1) 25(1) 20(1) -3(1) 7(1) 4(1)

O(2) 25(1) 36(1) 20(1) -7(1) 9(1) 1(1)

O(3) 17(1) 23(1) 15(1) -1(1) 5(1) 1(1)

C(1) 15(1) 14(1) 20(1) 2(1) 8(1) -1(1)

C(2) 18(1) 15(1) 17(1) 1(1) 9(1) -1(1)

C(3) 20(1) 16(1) 20(1) 0(1) 13(1) 0(1)

C(4) 16(1) 16(1) 21(1) 2(1) 9(1) 1(1)

C(5) 17(1) 13(1) 16(1) 2(1) 7(1) -3(1)

C(6) 17(1) 15(1) 18(1) 0(1) 10(1) -1(1)

Table 5. Hydrogen coordinates ( x 104) and isotropic displacement parameters (Å2x 103). x y z U(eq)

H(8A) 5015 68 3487 32

H(8B) 4306 2233 2585 32

H(8C) 3970 -1293 2830 32

H(9A) 604 1648 -263 28

H(9B) 1387 -369 624 28

H(9C) 1640 3205 321 28

H(7) 4055(12) 5260(40) 5224(12) 21(4)

H(3) 1547 7567 4036 20

H(4) 493 6430 2413 20

H(6) 2641 1599 1950 19

Appendix B: Biological Data

B.1 Graphical representations of the p

Figure B

showing the major species predicted at different pH ranges.

A.

B.



B.1 Schematic representation of the species predicted for compounds a.)


Neutral species

Deprotonated enol, weakly basic

Protonated

Zwitterion

Double

Neutral species


Protonated imidazole, weakly acidic

Zwitterion

Double


APPENDIX


Schematic representation of the species predicted for compounds a.)


Neutral species



Zwitterion

Double-deprotonated

Neutral species



Zwitterion

Double-deprotonated

PPENDIX B





imidazole, weakly acidic



B: BIOLOGICAL

B.1 Graphical representations of the pI and pKa



IOLOGICAL

Ka values of the pyrrolidinone compounds



IOLOGICAL DATA

values of the pyrrolidinone compounds


Page

ATA


Schematic representation of the species predicted for compounds a.) 11.1; and b.)

Page 263


; and b.) 12.1,

,


Figure B.2

showing the major species predicted at different

A.

B.

Depro

Ammonium ion

Neutral species

Double





Ammonium ion, weakly acidic

Neutral species

Double-deprotonated



Neutral species

Double-deprotonated





, weakly acidic

deprotonated

tonated enol, weakly basic

, weakly acidic

deprotonated






pH ranges.


Page


Page 264

; and b.) 14.1, ,

A.


Figure B.3


A.



Double-deprotonated

4’-O-)

Neutral species

Zwitterion (net charge, phenyl ring

Triple deprotonated


B.3 Schematic representation of the species predicted for compound a.)




deprotonated (pyrrolidinone

Neutral species

Zwitterion (net charge, phenyl ring

Triple deprotonated


Schematic representation of the species predicted for compound a.)


(pyrrolidinone ring-O- and phenyl ring

Zwitterion (net charge, phenyl ring-4’-O-)



B.

and phenyl ring-



Deprotonated enol,


Double-deprotonated

Neutral species

Triple deprotonated (

4’-O-)

Zwitterion (single net charge, phenyl ring


Zwitterion (double net charge, phenyl ring

Quadruple deprotonated





deprotonated enol (pyrrolidinone

Triple deprotonated (pyrrolidinone



Zwitterion (double net charge, phenyl ring

Quadruple deprotonated


pyrrolidinone ring-O- and phenyl ring

ring-O-, phenyl ring

Zwitterion (single net charge, phenyl ring-2’-O-)

Zwitterion (single net charge, phenyl ring-4’-O-)

Zwitterion (double net charge, phenyl ring-2’-O- and phenyl ring

Page

Schematic representation of the species predicted for compound a.) 14.3; and b.)

and phenyl ring-2’-O-

, phenyl ring-2’-O- and phenyl ring

and phenyl ring-4’-O-)

Page 265

; and b.) 14.5,

-)

and phenyl ring-

)

,

A.


Figure B.4


A.

B.



Neutral species

Double






Neutral species

Double-deprotonated



Neutral species

Double-deprotonated






deprotonated



Neutral species

deprotonated











Page


Page 266

; and b.) 16.1, ,

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