Till Olof

List of Papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals.

I Fransson, R., Botros, M., Nyberg, F., Lindeberg, G., Sandström,

A., Hallberg, M. Small Peptides Mimicking Substance P (1–7) and Encompassing a C-terminal Amide Functionality. Neuropeptides. 2008, 42, 31–37.

II Fransson, R., Botros, M., Sköld, C., Nyberg, F., Lindeberg, G., Hallberg, M., Sandström, A. Discovery of Dipeptides with High Affinity to the Specific Binding Site for Substance P 1–7. J. Med. Chem. 2010, 53, 2383-2389.

III Fransson, R., Botros, M., Sköld, C., Kratz, J. M., Svensson, R., Artursson, P., Nyberg, F., Hallberg, M., Sandström, A. Con-strained H-Phe-Phe-NH2 Analogues with High Affinity to the Substance P 1–7 Binding Site and with Improved Metabolic Stability and Cell Permeability. Manuscript.

IV Fransson, R., Nordvall, G., Botros, M., Carlsson, A., Kratz, J. M., Svensson, R., Artursson, P., Nyberg, F., Hallberg, M., Sandström, A. Discovery and Pharmacokinetic Profiling of Phenylalanine Based Carbamates as Novel Substance P 1–7 Analogues. Manuscript.

V Fransson, R., Sköld, C., Bitar, M., Larhed, M., Sandström, A. Design and Synthesis of N-Terminal Imidazole-Based H-Phe-Phe-NH2 Mimetics. Manuscript.

VI Wi ckowska, A., Fransson, R., Odell, L. R., Larhed, M. Mi-crowave-Assisted Synthesis of Weinreb and MAP Aryl Amides via Pd-Catalyzed Heck Aminocarbonylation Using Mo(CO)6 or W(CO)6. J. Org. Chem. 2011. 76, 978-981.

Reprints were made with the permission of the respective publishers.

Contents

1. Introduction ............................................................................................... 11 1.1 Neuropeptides ..................................................................................... 11 1.2 Peptides as Drug Leads ...................................................................... 12 1.3 Strategy for the Development of Peptidomimetics ............................ 13 1.4 Property-Based Design ....................................................................... 15

2. The Substance P System ........................................................................... 19 2.1 Substance P and its Bioactive Metabolites ......................................... 19 2.2 SP1–7 and its Binding Site ................................................................... 21

2.2.1 Endomorphins ............................................................................. 23

3. Aims .......................................................................................................... 25

4. SAR and Truncation Studies of SP1–7 and EM-2 ...................................... 26 4.1 Background and Strategy ................................................................... 26 4.2 Solid-Phase Peptide Synthesis ............................................................ 27 4.3 Synthesis of SP1–7 Analogs ................................................................. 29 4.4 Biological Evaluation ......................................................................... 30

4.4.1 Structure–activity relationship .................................................... 30 4.4.2 Effects of SP1–7 and its analogs ................................................... 37

4.5 Chapter Summary ............................................................................... 38

5. Design and Synthesis of Small Constrained H-Phe-Phe-NH2 Analogs .... 39 5.1 Background and Strategy ................................................................... 39 5.2 Biological Evaluation ......................................................................... 40

5.2.1 Structure–activity relationship and ADME properties ............... 40 5.3 Chapter Summary ............................................................................... 48

6. Improvement of the Pharmacokinetic Profile of Substance P1–7 Ligands . 49 6.1 Background and Strategy ................................................................... 49 6.2 Synthesis of Phenylalanine-Based Carbamates .................................. 50 6.3 Biological Evaluation ......................................................................... 51

6.3.1 Structure–activity relationship and ADME properties ............... 51 6.4 Chapter Summary ............................................................................... 55

7. Microwave-Assisted Aminocarbonylations and Direct Arylation of Imidazoles. Application to MAP Amides and SP1–7 Analogs ....................... 56

7.1 Background ........................................................................................ 58

7.1.1 Microwave irradiation in organic synthesis ................................ 58 7.1.2 Palladium-catalyzed reactions .................................................... 58 7.1.3 Design of experiments ................................................................ 61

7.2 Method Development ......................................................................... 62 7.2.1 Microwave-assisted protocol for C5 arylation of imidazole ....... 62 7.2.2 Microwave-assisted aminocarbonylation using CO-gas-free conditions ............................................................................................. 64

7.3 Application of the Developed Methods in the Synthesis of H-Phe-Phe-NH2 Mimetics ........................................................................................... 70 7.4 Chapter Summary ............................................................................... 72

8. Concluding Remarks ................................................................................. 73

Acknowledgements ....................................................................................... 75

References ..................................................................................................... 78

Abbreviations

Ac acetyl ACE the angiotensin-converting enzyme ADME absorption, distribution, metabolism and excretion Ala alanine Arg arginine Asp aspartic acid BBB blood-brain barrier Boc tert-butoxycarbonyl CDI 1,1´-carbonyldiimidazole Cha cyclohexylalanine Chg cyclohexylglycine CHO-K1 Chinese hamster ovary cell line CMD concerted metalation–deprotonation CNS central nervous system DBU 1,8-diazabicyclo[5.4.0]undec-7-ene DIEA N, N-diisopropylethylamine DMAP N, N-dimethylaminopyridine DMF N, N-dimethylformamide DMSO dimethylsulfoxide DP-IV the post-proline dipeptidyl peptidase Fmoc 9-fluorenylmethoxycarbonyl EM-1 endomorphin-1 EM-2 endomorphin-2 FHDoE focused hierarchical design of experiments GI gastrointestinal tract Gln glutamine Gly glycine HATU N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridine-1-

yl-methylene]-N-methylmethanaminium hexafluorophos-phate N-oxide

HBTU N-[(1H-benzotriazole-1-yl)-(dimethylamino)methylene]-N-methylmethanaminium hexafluorophosphate N-oxide

HPLC high-performance liquid chromatography IC50 inhibitor concentration giving 50% inhibition Ki equilibrium dissociation constant for inhibitor binding Leu leucine

Lys lysine MAP N-methyl-amino pyridyl NEP the neutral endopeptidase NK neurokinin NMM N-methylmorpholine NMR nuclear magnetic resonance Pbf 2,2,4,6,7-pentamethyldihydrobenzofuran-5-yl-sulfonyl PepT1 di/tri-peptide transporter PgP P-glycoprotein Phe phenylalanine PK pharmacokinetics PPB plasma protein binding PPCE the post-proline-cleaving enzyme Pro proline PSA polar surface area SAR structure–activity relationship SEM standard error of mean SP substance P SP1–7 substance P 1–7 SPE the substance P endopeptidase SPPS solid-phase peptide synthesis Suc succinoyl TES triethylsilane TFA trifluoroacetic acid Trp tryptophan Trt triphenylmethyl Tyr tyrosine Xantphos 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene

11

1. Introduction

1.1 Neuropeptides The classical neurotransmitters in the nervous system are amino acids and their metabolites (glutamate, aspartate, GABA and glycine), the monoamines (acetylcholine, dopamine, noradrenaline and serotonin), and “gaseous” molecules (nitric oxide and carbon monoxide). In addition to these, neuro-peptides also function as messengers. The neuropeptides often co-exist in the neurons, together with other neurotransmitters, functioning in a complemen-tary way by modulating their actions. These neurotransmitters and/or neuro-modulators constitute a large and significant group of biologically active peptides.1-3

Neuropeptides are present in all parts of the nervous system, but each has its unique distribution pattern. Thus, neuropeptides can be expressed at high levels under normal conditions and be available at any time, or they can normally be expressed at low concentrations and become up-regulated as a result of, for example, nerve injury, stress or drug abuse. It should be noted that the expression pattern of a specific neuropeptide may vary depending on the neuron in which it is expressed and the role it plays there; a specific peptide can thus exhibit different expression patterns.1 This observed modulating role of neuropeptides on the main transmitters, e.g. monoamines, is interesting from a therapeutic point of view, and targeting the functions of the neuropeptides instead of the classical neurotransmitters can be of advantage in drug development. Firstly, the “milder” effects observed for neuropeptides compared to monoamines and amino acid transmitters result in less dramatic activation or blockade effects on their receptors.1,2 Secondly, since the neuropeptides are often released from neurons under pathological conditions, antagonists may have no effect in “normal systems” but act only on unbalanced systems with increased peptide release.1,4 Thirdly, peptides in the nervous system often act on several receptors, thus offering the possibility to design selective drugs targeting a specific receptor subtype involved in the specific function.1 The targeting of neuropeptides is thus likely to lead to fewer side effects.5,6

12

The biologically active neuropeptide substance P 1–7 (SP1–7, H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-OH, Figure 1), which has been shown to attenuate naloxone-provoked withdrawal signs in morphine-dependent rats7,8 and possess antinociceptive effects,9 was the focus of the research presented in this thesis.

Figure 1. Substance P 1–7 is a bioactive neuropeptide and is the subject of this thesis.

1.2 Peptides as Drug Leads Due to their importance in many biological functions, bioactive peptides are interesting starting points in drug discovery, and can be used as valuable research tools in initial investigations of the biological mechanisms of various diseases.3 However, due to their peptide structure, they suffer from inherent drawbacks such as low bioavailability, low metabolic stability, poor absorption from the gastrointestinal tract and low permeability into the brain as a result of poor transport over the blood-brain barrier (BBB).10,11 Further-more, peptides have a large degree of conformational flexibility, and can fold into complex tertiary structures crucial for their molecular recognition and their ability to produce a biological response.

In an attempt to overcome the problems associated with peptides, low-molecular-weight and bioavailable drug-like molecules that mimic the action of peptides, i.e. peptidomimetics, are being designed and developed.12-15 Peptidomimetics are molecules with significantly reduced peptide character that mimic the bioactive conformation of peptides, and thus retain the ability to interact with the biological target and cause the same biological effect.16 These non-peptide compounds may also possess improved pharmacokinetic (PK) properties, such as better absorption, metabolic stability and bio-availability. In the work presented here, general strategies have been applied to the development of peptidomimetics by rational design, starting from a bioactive peptide.

NH

NHH2N

N

OO N

H

N

NH2

OO N

H

HN

NH2O

O

NH

H2N O

O OH

O

H2N

Substance P 1-7

13

1.3 Strategy for the Development of Peptidomimetics Rational peptide lead optimization is often a stepwise procedure, similar to the one outlined in Figure 2, employed to transform a biologically active peptide into small drug-like pseudopeptides or peptidomimetics.

Figure 2. A general strategy for development of peptidomimetics.

The process starts with investigations of the structure–activity relationships (SARs) and identification of the minimal active sequence of the peptide required for biological activity. This is normally achieved by the evaluation of binding affinities of peptide analogs to the target protein. In practice, such information is normally gathered through amino acid scans, truncations and N- and C-terminal modifications.15-18 Amino acid scans determine the importance of amino acid side chains by systematically replacing each residue within the peptide with alanine, glycine or the corresponding D-amino acid. Alanine and glycine are the smallest amino acids available, hav-ing a methyl and a hydrogen side chain, respectively, and will have small impact on the overall binding affinity. N- or C-terminal truncation removing one amino acid at a time provides information on the minimal sequence needed for biological activity. The importance of a basic N-terminal or an acidic C-terminal is determined by the introduction of capping groups. These

Bioactivepeptide

SAR-Truncation and deletion

- Amino acid scans- Terminal modifications

Bioactive conformation- Local constraints

- Global constraints- Secondary structure

replacement

Peptidomimetics

Biological testing

Computationalmodeling

14

initial investigations also lead to the identification of the pharmacophoric groups, i.e. the residues essential for biological activity. Based on the information gained from these studies, further structural modifications are undertaken to improve stability, potency and selectivity. Conformational restrictions are frequently used to explore the bioactive conformation and to enhance bioavailability by improving enzymatic stability.11,16 A constraint that leads to a reduction in the loss of conformational entropy upon interaction with the target can also increase the binding affinity.19 Global and local constraints can be achieved by cyclization,15 N-methylation,19 isosteric substitution,3,19 or by secondary structure replacement,20 as exemplified in Figure 3.

Figure 3. A) Cyclization strategies that can be performed in a peptide sequence to introduce global constraints.19 B) Positions in a peptide that can be methylated. Methylation of both the backbone atoms and the side chains can introduce local constraints.19 C) Isosteric replacement of the peptide bond can introduce local constraints. It should be noted that such replacements are not always real constraints, but alter the overall conformational behavior of the peptide backbone to varying degrees. In some cases the flexibility is increased.3,19 D) Secondary structure mimetics can induce a desired conformation when introduced into the peptide back-bone.3,21,22

15

The replacement of the scissile peptide bond by heterocyclic synthons, such as bicyclic analogs,23 or simple five-membered heterocycles, has also been reported (Figure 4).24-28 These non-peptidic scaffolds can ideally retain the bioactive conformation and, more importantly, improve the enzymatic stability, compared to the native peptides.

Figure 4. Two different peptidomimetic scaffolds based on imidazole (A)27 and 1,2,4-triazole (B).25,26 Compound C is an example of a somatostatin analog in which a tetrazole has been incorporated as a cis amide bond surrogate.29

By combining the results obtained from the rational design of peptide analogs with techniques such as nuclear magnetic resonance (NMR) spectroscopy, X-ray crystallography and computational methods, a three-dimensional pharmacophore model can be generated. This information can be used in efforts to develop peptidomimetics with the pharmacophoric groups spatially arranged with the correct orientation on a non-peptidic scaffold, thus preserving the interaction pattern.

It should be emphasized that although numerous strategies for transforming a bioactive peptide into small peptidomimetics have been reported, it is not straightforward, and often requires hard work, with no guarantee of success. The decreasing number of approved drugs during recent years has put enormous pressure on the pharmaceutical industry, resulting in a revival of interest in peptides as potential drug candidates.17 By using synthetic strategies to limit metabolism and exploring alternative routes of administration, a number of peptidic drugs have been brought to market,17 showing that it is not necessary to remove the peptide character completely, and indeed small pseudopeptides can be used as drugs.

1.4 Property-Based Design In 1988, Prentis et al. published a report showing that 39% of drugs in the drug development phase were failing due to poor PK properties and bio-availability. This posed a major economic burden on the industry since years of work on discovery and development were lost due to these failures.30 This

16

highlighted the need for earlier assessment of PK data and bioavailability already in the discovery phase and, as a consequence, ADME, i.e. investigation of the properties that affect absorption, distribution, metabolism, and excretion, has been integrated into the drug discovery process (Figure 5).31,32

Various in vitro property assays are used to assess the drug-like properties of interesting compounds. These assays measure physiochemical properties (solubility, permeability, chemical stability) and biochemical properties (metabolism, protein-binding and transport) which, in interactions with living systems, determine the pharmacokinetic profile of a specific compound.31 This change in approach in medicinal chemistry, to improve and optimize drug absorption and the pharmacokinetic properties of lead compounds early in the drug discovery process, has generated the concept of property-based design.33

Figure 5. The role of the medicinal chemists has changed during the past 20 years, from considering only affinity and specificity in lead optimization, to including the ADME properties at an earlier stage.

The activity at the target and the exposure (e.g. concentration and duration) determine the efficacy of a drug. In the body several barriers to drug exposure can be found, for example cell membranes, metabolic enzymes, efflux transporters, and binding proteins. How a compound performs at a specific barrier is connected to its drug properties (e.g. permeability, efflux transport) and due to property deficiencies (e.g. metabolic stability, solubility) this can greatly influence its PK profile.

In the gastrointestinal (GI) tract, compounds can cross the cellular membrane barrier by three major mechanisms.33 The two most common are transcellular absorption, i.e. passive transfer by diffusion across the lipid membranes, and paracellular absorption, which proceeds through aqueous pores at the tight junctions between the cells. The third mechanism is active uptake by transport proteins that usually transport nutrients across the membrane.31,33 The most important mechanism for drug absorption is passive diffusion, and about 95% of all commercial drugs are absorbed by this route.31 Metabolizing enzymes in the GI tract, such as the cytochrome P450 (CYP) enzymes, and efflux transporters, e.g. P-glycoprotein (PgP), are expressed which limit the oral absorption of compounds (Figure 6).33 As well as avoiding the efflux of PgP and metabolism by gut wall enzymes, good permeability is important to maximize the oral absorption of a

Lead identification

and optimization

Preclinical development

ADME

17

compound. Especially CYP3A4 and PgP have been shown to have a significant impact on the bioavailability of peptidic and peptidomimetic drugs.34 One means of enhancing oral bioavailability is to increase passive diffusion by changing the physiochemical properties (i.e. decreasing the hydrophilicity) of the compound.35 It should be noted that increasing the lipophilicity, in order to improve membrane permeability, can also lead to increased efflux and metabolism. Two other strategies that can be used to improve permeability are reducing hydrogen bonding and decreasing the polarity.31

Figure 6. Illustration of the barriers to drug absorption in the GI tract, that have been addressed for the compounds presented in this thesis.

In the bloodstream, enzymatic hydrolysis and plasma–protein binding (PPB) constitute barriers preventing drugs from penetrating into the tissues. The affinity of a compound to plasma proteins determines the ratio of bound to unbound (“free”) drug in solution, and only the unbound drug can enter the tissues. If a compound has a high binding affinity it can be difficult to achieve concentrations in the tissue sufficient to produce the desired phar-macological effect. High PPB also reduces the clearance of a compound and thus increases the PK half-life, since it prevents the drug from permeating into the liver and kidneys.31

A further dimension that must be considered when working with central nervous system (CNS) active agents is the uptake in the brain. Here, the im-permeable BBB constitutes an additional barrier to absorption. It has been shown that the penetration of a compound into the brain decreases with in-creasing polar surface area.36 But it is not only the physiochemical properties that influence brain uptake. Transporters, especially PgP, and metabolizing enzymes are also limiting factors. Several structure modification strategies to reduce PgP efflux have been described.31 These include reducing the number

Decomposition• enzymatic• acidic

Efflux transport

Metabolism

GI Epithelial Cell Layer

Capillary to Portal Vein

GI Lumen

= Drug molecule = Enzyme = Transporters

Drug Solid Particle

Solubility

18

of hydrogen bond donors, decreasing the hydrogen bond acceptor potential of a compound, and decreasing the overall lipophilicity of the structure.

Extensive in vitro profiling regarding the PK data of lead compounds in the early stages of development can thus provide the medicinal chemist with information on the structure–property relationship important for the further development of orally active compounds (Figure 7).

Figure 7. The development of peptidomimetics can be an iterative parallel optimiza-tion process addressing both activity and properties.

Optimization PropertyActivity

Synthesis

Redesign

In vitroSolubilityPermeabilityBBB & PgPLog P & pKa

MetabolismCYP 450 InhibitionStability

In vivoPharmacokinetics

In vivoAnimal models

In vitroCell-based assay

19

2. The Substance P System

2.1 Substance P and its Bioactive Metabolites Substance P (SP) was the first neuropeptide to be identified, and was discovered in 1931 by Von Euler and Gaddum.37 It was named a few years later based on its appearance as a powder obtained after extraction.38 In 1971, Chang et al.39 revealed the peptide sequence, H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2, and a decade later it was included in the tachykinin family.40 Together with neurokinin A (NKA) and neurokinin B (NKB), SP is the most well-known member of this family.41 Three mammalian tachykinin receptors are known today: the neurokinin (NK) receptors NK1, NK2 and NK3.42 SP is the preferred endogenous ligand for the NK1 receptor, where it acts as a neurotransmitter and a neuromodulator in both the central and peripheral nervous system.

In the brain, SP and its corresponding receptor are expressed in areas related to depression,6 anxiety43 and stress,44,45 as well as in areas involved in motivation and reward.46,47 In the spinal cord, SP is expressed in pain processing pathways.48 Although significant effort has been devoted to developing SP-related drugs for therapeutic use, the only NK1 receptor antagonist on the market today is aprepitant (MK-869), approved as an anti-emetic agent in 2003 (Figure 8). This compound was first developed as an antidepressant, and was evaluated in a clinical trial in humans. It was reported to have the same efficacy as the selective serotonin reuptake inhibi-tor (SSRI) paroxetin, but lacked some of the side effects common to SSRI drugs.6 However, the promising effect could not be reproduced in a follow-up study.

20

Figure 8. The endogenous ligand for NK1 receptor, substance P and the NK1 receptor antagonist MK-869.

Five different enzymes are mainly responsible for the degradation of SP in the human body, as illustrated in Figure 9.49-52 These are the post-proline dipeptidyl peptidase (DP-IV), the angiotensin-converting enzyme (ACE), the post-proline-cleaving enzyme (PPCE), the neutral endopeptidase (NEP), and the substance P endopeptidase (SPE).

Figure 9. A summary of the degradation paths of substance P in the human body.

Several of the degradation products of SP have been found to be bioactive, and especially the C-terminal fragments can mimic the effects of the mother peptide. For example, infusion of the C-terminal metabolite SP5–11 into the spinal cord induces nociceptive reactions53 and when injected into the dorsal periaqueductal gray matter in rats the C-terminal fragment SP6–11 was shown to produce anxiogenic effects mediated via the NK1 receptor.43 The N-terminal fragment SP1–7 is one of the major metabolites of SP and, as shown above, SPE, ACE and NEP can all generate this heptapeptide. In contrast to the C-terminal metabolites of SP, SP1–7 has been shown to oppose several effects of SP, e.g. the nociceptive effect,54 the inflammatory effect,55 and the

21

potentiating effect on opioid withdrawal symptoms.7,56 Interestingly, these effects were not found to be mediated through the NK1 receptor.

2.2 SP1–7 and its Binding Site The bioactive metabolite SP1–7 has been identified in the CNS,57,58 and the presence of the enzyme SPE has been detected in the human brain49 and in cerebrospinal fluid.50 As mentioned above, SP1–7 has been shown to modulate, and in certain cases oppose, the effects of the mother peptide.7,51,54-56 Similar counteracting behavior of metabolites has been observed for several other fragments derived from neuropeptides. For example, the selective -opioid receptor ligand dynorphin produces dyspho-ria in the reward system in the brain, but after enzymatic cleavage into its N-terminal bioactive fragment leu-enkephalin, it becomes a -opioid receptor agonist with euphoric properties.51

SP1–7 does not mediate its effects via the NK1 receptor and, although the actions of this heptapeptide are well-known, no explicit receptor has yet been identified. However, the potential existence of a specific receptor for the N-terminal partial sequences of SP in mouse spinal cord was discussed at the beginning of the 1980s,59 and in 1990 Igwe et al. characterized the specific binding site of SP1–7 in mouse brain and spinal cord.60 Binding was found to be specific, saturable and reversible, which strongly supported the existence of an N-terminal-directed SP receptor. In 2006, Nyberg and co-workers demonstrated the presence of specific binding sites for SP1–7 in rat spinal cord in accordance with previously reported results from the mouse spinal cord and brain.60,61A receptor binding assay was developed using spinal cord tissue homogenate and measurements of the binding affinity for various compounds by displacement of tritiated SP1–7 ([

3H]-SP1–7). A variety of neuroactive peptides and non-peptides were evaluated regarding their ability to competitively inhibit the binding of [3H]-SP1–7 to rat spinal cord membrane, in order to investigate possible interactions with other known neuropeptide systems. Ligands for opioid and neurokinin receptors, as well as various N-terminal SP fragments, were screened (Table 1).61

22

Table 1. Screening of various neuropeptides and ligands for the SP1–7 binding site.

Peptide/Ligand Sequence

Binding affinity, Ki (nM)

SP1–7 Arg-Pro-Lys-Pro-Gln-Gln-Phe-OH

0.8

SP1–6

Arg-Pro-Lys-Pro-Gln-Gln-OH

1831

SP1–8

Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-OH

74

SP Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2

159

D-SP1–7

Arg-D-Pro-Lys-Pro-Gln-Gln-D-Phe-OH

1.8

[Sar9, Met(O2)11]-SP

Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Sar-Leu-Met(O2)-NH2

814

R-396 Ac-Leu-Asp-Gln-Trp-Phe-Gly-NH2

> 10 000

Senktide Suc-Asp-Phe-N-Me-Phe-Gly-Leu-Met-NH2

> 10 000

DAMGO Tyr-D-Ala-Gly-N-Me-Phe-Gly-ol

13

Endomorphin-1 Tyr-Pro-Trp-Phe-NH2

1026

Endomorphin-2 Tyr-Pro-Phe-Phe-NH2

7.5

Naloxone

> 10 000

Naloxonazine

> 10 000

In comparison to SP1–7, all the ligands tested except the SP1–7 antagonist D-SP1–7, showed significantly weaker binding to this site. The N-terminal fragment SP1–8 showed twice the binding affinity of SP, although it was 100 times lower than that of SP1–7. The metabolite from enzymatic processing of NEP, the SP1–6 fragment, showed about 2000 times lower affinity for the

23

spinal cord membrane, illustrating the importance of the C-terminal phenyl-alanine. Moreover, the NK1 receptor ligand [Sar9, Met(O2)

11]-SP, the NK2 receptor ligand R396, and the NK3 receptor ligand senktide showed weak or negligible affinity to the SP1–7 binding site. To further discriminate between the identified binding sites of SP1–7 and those of other receptors, various opioid and related peptides were investigated. As can be seen in Table 1, the µ-opioid receptor agonist DAMGO showed high binding affinity, while the non-selective opioid receptor antagonist naloxone and the selective µ-opioid receptor antagonist naloxonazine were devoid of affinity. Interestingly, the high-affinity µ-opioid receptor agonists endomorphin-2 (EM-2) and endomorphin-1 (EM-1) were shown to interact differentially with the binding site of SP1–7. Thus, EM-2 had only a 10-fold lower affinity than SP1–7, whereas EM-1 had a 1400-fold lower affinity.

To rule out involvement of the µ-opioid receptor, the binding affinity of SP1–7 to the µ-opioid receptor and the ability of the heptapeptide to activate it were further investigated.61 However, no specific binding or any activation of the µ-opioid receptor was observed, which is indicative of a specific target protein for SP1–7, identical to neither the tachykinin receptors nor the µ-opioid receptor.

The pharmacological properties observed for the heptapeptide makes it very interesting from a therapeutic point of view. The finding that the tetra-peptide EM-2 retained good affinity to the SP1–7 binding site, bearing in mind its smaller size compared to the heptapeptide SP1–7, makes this a good starting point for the development of future therapeutic agents for the treatment of, for example, pain, drug addiction and inflammation.

2.2.1 Endomorphins The endomorphins were not identified until 1997, and constitute the most potent endogenous ligands for the µ-opioid receptor.62 The endomorphins differ in their structure from other endogenous opioid peptides (Figure 10). In opioid peptides the N-terminal part is usually Tyr-Gly-Gly-Phe, followed by Met or Leu, and since they are released from precursor proteins they possess a C-terminal carboxylic acid. In contrast, the endomorphins contain a C-terminal primary amide, and so far no endomorphin precursors have been identified.63,64

24

Figure 10. The structures of the µ-opioid receptor ligands EM-1 (Tyr-Pro-Trp-Phe-NH2) and EM-2 (Tyr-Pro-Phe-Phe-NH2).

The differences in binding affinity to the SP1–7 binding site observed for EM-1 and EM-2 is in accordance with other differences between these two µ-receptor agonists demonstrated in various studies. For instance, the binding characteristics and their distribution in the CNS have been reported to vary.64,65 EM-1 is widely distributed in the brain; the highest concentrations being in the thalamus, hypothalamus, cortex and striatum. EM-2 is mainly found in the spinal cord, where it has been shown to be co-localized with SP.66,67 Different binding affinities of EM-1 and EM-2 to the different subtypes of µ-receptors have also been reported.68-70 The relationship between SP and SP1–7, as well as that between SP1–7 and EM-2, is very interesting from a pharmacological point of view, and in order to study this connection in complex animal models, low-molecular-weight research tools, i.e. SP1–7 peptide mimetics, are highly desirable. Neither SP1–7 nor EM-2 has been addressed in any SP1–7 medicinal chemistry program. Consequently, they were chosen as starting points in a rational drug design program targeting the SP1–7 system, which was initiated by the research presented in this thesis.

25

3. Aims

The start of the present research was also the initiation of the first drug dis-covery project related to SP1–7. The long term goal was to develop small drug-like molecules for further development and as research tools for com-prehensive studies of the physiological functions related to SP1–7 and in par-ticular its role in chronic pain and opioid withdrawal response.

The overall objectives of the present study were:

To establish structure–activity relationship of SP1–7 and EM-2 regard-

ing their binding to the SP1–7 binding site. To design and develop stable, bioavailable, and potent SP1–7 peptido-

mimetics. To develop synthetic methods that allows for efficient preparation of

the designed SP1–7 analogs. To investigate the structure–property relationship for the new SP1–7

analogs.

During the course of the above investigations the following specific aims were formed:

To develop a synthetic method for the introduction of an imidazole

scaffold into a peptide sequence. To develop a fast microwave protocol for direct arylation of imid-

azoles. To develop a carbonylative method for synthesis of MAP aryl amides.

26

4. SAR and Truncation Studies of SP1–7 and EM-2

4.1 Background and Strategy As mentioned in the introduction, peptides are not suitable as drugs due to their inherent instability. Stepwise structural modifications and biological evaluation can provide crucial information for the transformation of bio-active peptides into small drug-like compounds.

Besides the interesting mechanistic aspects of the SP1–7 and EM-2 correlation, the study by Botros et al.61 also led to the identification of EM-2 as a lead compound in the development of low-molecular-weight ligands to the SP1–7 binding site. Although the affinity of EM-2 was 10 times lower than SP1–7, the smaller size of EM-2 motivated the use of this tetrapeptide for further development.

A SAR study of the two peptide leads, SP1–7 (Paper I) and EM-2 (Paper II), was planned, with the intention of identifying the pharmacophoric groups. This design strategy included Ala scans, truncations and C- and N-terminal modifications of the two target peptides (Figure 11). Thus, a series of peptide analogs, in which each amino acid residue of the two target pep-tides was replaced sequentially with an alanine, was synthesized. An Ala scan was chosen since the incorporation of a glycine residue is more likely to affect the peptide conformation. In the truncation studies, one amino acid at a time was removed from the N-terminal. Both C-terminal carboxylic acids and carboxamides were included.

Furthermore, two sets of small peptides were designed, one to investigate the preferred configuration by introduction of D-amino acids, and the other to explore the effect of small variations in side chain size and polarity on affinity. The latter set was obtained using focused hierarchical design of experiments (FHDoE) strategy.71 This statistical design method is used to select a diverse set of analogs based on an active peptide. Each peptide in the set is designed to retain a high resemblance to the active peptide by substituting one or more of the original amino acids in the active peptide with residues of similar size and hydrophobic/hydrophilic properties. The aim of using focused design was to increase the probability of obtaining new active analogs.

27

Figure 11. Illustration of the modifications of A) SP1–7 and B) EM-2, used in the SAR study.

4.2 Solid-Phase Peptide Synthesis In 1963, R.B. Merrifield introduced solid-phase peptide synthesis (SPPS), which simplified peptide chemistry considerably.72 Before this, peptide synthesis had been carried out in solution, which involved additional coupling and deprotection steps, in combination with time-consuming intermediate isolation and purification steps.3 The SPPS technique normally involves attachment of the C-terminal amino acid via its carboxy group by a covalent bond to a non-soluble support, i.e. a resin, as illustrated in Figure 12. The desired sequence is then obtained by stepwise attachment of amino acids from the C-terminal to the N-terminal. The advantage of SPPS is that soluble reagents can be used in large excess and can be easily removed together with by-products by simple washing and filtration after each syn-thetic step, since the peptide remains anchored to the resin throughout the process. Hence, no tedious and time-consuming isolation and purification of the intermediates are needed. After completion, the desired peptide is cleaved from the solid support and isolated. A temporary protecting group that can be selectively removed after each step is used for the N -amino function, usually the 9-fluorenylmethoxycarbonyl group (Fmoc).73

28

The Fmoc/t-butyl scheme is a two-dimensional orthogonal scheme, where the Fmoc group can be removed under mild basic conditions, while side-chain deprotection and peptide–resin cleavage require treatment with a strong acid such as trifluoroacetic acid (TFA).3 One further advantage of using the Fmoc protecting group is that it allows the retention of stereoche-mistry at the amino acid -carbon by suppressing the formation of oxazolone from the activated amino acid, which can undergo racemization due to de-protonation at the -carbon. Hence, aminolysis of the racemized oxazolone intermediate results in peptide epimers that can be avoided using urethane-protected NH groups (R-O-CO-NH), e.g. the Fmoc group.3

Today, several functionalized resins are available for SPPS providing a wide variety of ways of obtaining the desired peptide sequences and C-terminal functional groups. In the work presented in this thesis three different functionalized resins were used, depicted in Figure 13, in order to obtain different C-terminal functional groups, i.e. carboxylic acid (A), primary amide (B) and secondary amide (C).

Figure 12. The strategy used in solid-phase peptide synthesis.

LinkerX

LinkerX

O

NH

R1

Fmoc

LinkerX

O

H2N

R1

LinkerX

O

NH

R1O

R2

HN

Fmoc

LinkerX

O

NH

R1O

Rn

HN

O

H2N

Rn+1

n

X

O

NH

R1O

Rn

HN

O

H2N

Rn+1

n

1. Anchoring of the first Fmoc-protected amino acid

2. Removal of the N-protection group

3. Coupling of the next Fmoc-protected amino acid

4. Repeated couplings (steps 2 and 3)

5. Removal of side-chain protection andfinal cleavage of the peptide from the resin

29

Figure 13. Functionalized resins used in the synthesis of the peptides presented in this thesis.

4.3 Synthesis of SP1–7 Analogs The two lead peptides, SP1–7 (1) and EM-2 (2), and the Ala-substituted pep-tides 3–13, the N- and C-terminally modified analogs 14–16 and 28–29, the truncated analogs 17–27, the (D) and (L) variants 30–32, and the FHDoE-derived peptides 33–39 (Tables 2 and 3) were prepared using standard Fmoc SPPS (Scheme 1). The starting polymers were H-Phe-2-Cl-trityl resin, Rink amide MBHA resin, or H-Ala-2-Cl-trityl resin. Coupling was carried out in N,N-dimethylformamide (DMF), using N-[(1H-benzotriazole-1-yl)-(dimethylamino)methylene]-N-methylmethanaminium hexafluorophosphate N-oxide (HBTU) as coupling reagent and N-methylmorpholine (NMM) or N,N-diisopropylethylamine (DIEA) as base. The Fmoc group was removed by treatment with 20% piperidine in DMF before coupling with the next amino acid. Acetylation of the peptides 14 and 29 was performed by allow-ing the resin to react with acetic anhydride solution directly after the last Fmoc deprotection. The final peptides were cleaved from the resin by the addition of triethylsilane (TES) and 95% aqueous TFA, which also removed the protecting groups Pbf, Boc and Trt from the Arg, Lys and Gln side chains, respectively. The crude peptides were precipitated in diethyl ether and purified using reversed-phase high-performance liquid chromatography (RP-HPLC).

30

Scheme 1.

X = functional group of the resin, Fmoc-AA-OH = Fmoc protected amino acid

4.4 Biological Evaluation 4.4.1 Structure–activity relationship The binding affinities of the compounds in Tables 2 and 3 were assessed in a radioligand binding assay, using spinal cord membrane from male Sprague-Dawley rats and the analog [3H]-SP1–7 as tracer.61 The competition experi-ments were performed at six different concentrations, run in triplicate, and each assay was repeated at least three times on different days. The binding affinities are reported as equilibrium dissociation constants (Ki values).

The outcome of the biochemically evaluated SP1–7 analogs 1–39 (Tables 2 and 3) gave a broad range of values, extending from inactive analogs (Ki > 10 000 nM) to analogs more potent (Ki value of 0.3 nM) than SP1–7 (Ki = 1.6 nM), and was thus very informative.

1. 20% Pip/DMF2. Fmoc-AA-OH, HBTU, DIEA or

NMM, DMF3. 20% Pip/DMF

95% TFA, TES

linkerXlinkerX

FmocHN

R1

FmocHN

R1OH

O

HBTU, DIEA or NMM

linkerXNH

R1HN

O

RnH2N

O

Rn+1

nrepeated steps

n = 0-5 1-13, 15-28 and 30-39

1.

2. 95% TFA, TES

O

O O

n = 0 and 5 14 and 29

1.

O

O

31

Table 2. Ki values of SP1–7 and EM-2 analogs for inhibition of [3H]-SP1–7 binding to rat spinal cord membrane.

Compound Sequence Ki ± SEM (nM) 1 (SP1–7) H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-OH

1.6 ± 0.1

2 (EM-2) H-Tyr-Pro-Phe-Phe-NH2 8.7 ± 0.1

Alanine-substituted SP1–7 3 H-Ala-Pro-Lys-Pro-Gln-Gln-Phe-OH

12.3 ± 0.4

4 H-Arg-Ala-Lys-Pro-Gln-Gln-Phe-OH

1.7 ± 0.1

5 H-Arg-Pro-Ala-Pro-Gln-Gln-Phe-OH

2.8 ± 0.1

6 H-Arg-Pro-Lys-Ala-Gln-Gln-Phe-OH

2.8 ± 0.1

7 H-Arg-Pro-Lys-Pro-Ala-Gln-Phe-OH

78.6 ± 5.1

8 H-Arg-Pro-Lys-Pro-Gln-Ala-Phe-OH

365 ± 3

9 H-Arg-Pro-Lys-Pro-Gln-Gln-Ala-OH

>10 000

Alanine-substituted EM-2 10 H-Ala-Pro-Phe-Phe-NH2

11.5 ± 0.1

11 H-Tyr-Ala-Phe-Phe-NH2

10.2 ± 0.3

12 H-Tyr-Pro-Ala-Phe-NH2

9.4 ± 0.1

13 H-Tyr-Pro-Phe-Ala-NH2

1460 ± 15

Terminally Modified SP1–7 and EM-2 14 Ac-Arg-Pro-Lys-Pro-Gln-Gln-Phe-OH

7.1 ± 0.0

15 H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-NH2

0.3 ± 0.0

16 H-Tyr-Pro-Phe-Phe-OH 30.2 ± 1.7 Continued on next page

32

Compound Sequence Ki ± SEM (nM) Truncated SP1–7 Peptides

17 H-Pro-Lys-Pro-Gln-Gln-Phe-OH

29.6 ± 0.8

18 H-Pro-Lys-Pro-Gln-Gln-Phe-NH2

2.8 ± 0.25

19 H-Lys-Pro-Gln-Gln-Phe-OH

30.9 ± 0.4

20 H-Lys-Pro-Gln-Gln-Phe-NH2

4.4 ± 0.1

21 H-Pro-Gln-Gln-Phe-OH

26.2 ± 0.7

22 H-Pro-Gln-Gln-Phe-NH2

4.5 ± 0.3

23 H-Gln-Gln-Phe-OH

20.4 ± 0.8

24 H-Gln-Gln-Phe-NH2

1.9 ± 0.05

Truncated EM-2 Peptides 25 H-Pro-Phe-Phe-NH2

10.9 ± 0.7

26 H-Phe-Phe-NH2 1.5 ± 0.1

27 H-Phe-NH2 5028 ± 31

The results were remarkably consistent for SP1–7 and EM-2 peptides (Table 2). Substitution with alanine in the N-terminal part of SP1–7 was well tole-rated without affecting the binding affinity significantly (cf. 1 with 4, 5 and 6), although replacement of the basic amino acid arginine rendered a 10-fold lower affinity (cf. 1 and 3). Likewise, the three N-terminal amino acid resi-dues in EM-2 (tyrosine, proline and the internal phenylalanine) could be substituted with an alanine and still retain binding affinity (cf. 2 with 10, 11 and 12). However, removal of the two primary amide functions of the side chains of the glutamine in the C-terminal of SP1–7 resulted in a considerable decrease in affinity (7 and 8). The C-terminal phenylalanine was absolutely crucial for strong affinity in both SP1–7 and EM-2. Replacement of the C-terminal phenylalanine in SP1–7 gave analog 9, which was devoid of affinity. Making the same substitution in EM-2 resulted in compound 13 with a Ki value of 1460 nM. The finding that the C-terminal phenylalanine plays such an important role in binding affinity is in line with the peptide scan discussed above, where the C-terminal fragment SP1–6 possessed very weak binding affinity to the SP1–7 binding site.60,61 Moreover, the potency of SP1–7 showed a five-fold increase upon amidation of the terminal carboxyl group (15). Similarly, the binding affinity of EM-2 was reduced by a factor of four upon removal of the amide function (16). Since SP1–7 is a proteolytic product of SP resulting in a C-terminal carboxylic acid it was surprising that the ami-dated analog 15 led to improved binding affinity.

33

When considering that the endomorphins are the only endogenous opioids that comprise an amide function, it is enticing to propose that some of the in vivo effects observed for SP1–7 might be attributed to amidated SP1–7. It is not known whether SP1–7 can be enzymatically amidated, but since the endo-morphins contain an amidated phenylalanine it is not unlikely.

As deduced from the two Ala scans, the N-terminal parts of SP1–7 and EM-2 are not engaged in molecular recognition or binding to the target pro-tein, a fact that prompted the synthesis of the truncated analogs 17–24 and 25–27. For SP1–7, removal of the N-terminal arginine rendered the hexapep-tide 17 with a 20-fold reduction in affinity compared to SP1–7 and a little more than 2-times lower affinity than the alanine derivative 3. The affinity could, however, be recovered by amidation of 17, resulting in peptide 18, which was 10 times more potent. Further truncation down to the tripeptide level was possible without loss of affinity, and C-terminal amidation of all the truncated SP1–7 analogs improved the affinity 5–10 fold. Hence, the tri-peptide H-Gln-Gln-Phe-NH2 (24) exhibited a Ki of 1.9 nM.

For EM-2, simultaneous removal of the two N-terminal amino acids tyro-sine and proline improved the binding affinity six times, resulting in the notable discovery of the dipeptide H-Phe-Phe-NH2 (26) with a Ki value simi-lar to that of SP1–7 itself. It should be emphasized that the Tyr-Pro sequence is the critical fragment for binding to the µ-receptor, whereas the two C-terminal phenylalanines are not, facts that highlight the double nature of EM-2.74-76 In the Ala scan of EM-2, substitution of the internal phenylalanine was accepted (12), but truncation down to a single phenylalanine resulted in 27 with no binding affinity (Ki of 5028 nM).

The binding features of the new dipeptide lead compound H-Phe-Phe-NH2 were further explored via the synthesis of peptides 28–39 (Table 3). As observed for SP1–7 and EM-2, the C-terminal function should be a primary amide. The corresponding carboxylic acid was devoid of activity (cf. 26 with 28). All four stereoisomers of H-Phe-Phe-NH2 (26, 30, 31 and 32) were syn-thesized and evaluated. The natural L-Phe-L-Phe isomer (26) was found to be preferred, followed by the D,D compound (31), although with a 40-fold low-er affinity. As mentioned above, incorporation of D-amino acids into neuro-peptides can change their biological function from an agonist to an antagon-ist, e.g. D-SP1–7, which makes it interesting to evaluate the analogs 30, 31 and 32 in animal studies concerning their functional activities.

Additionally, a few closely related analogs to H-Phe-Phe-NH2, generated from the focused design, encompassing amino acids with similar physio-chemical properties, were evaluated (33–39).

34

Table 3. Ki values of Phe-Phe analogs for inhibition of [3H]-SP1–7 binding to rat spinal cord membrane.

Compound Sequencea Ki ± SEM (nM)

26 1.5 ± 0.1

Terminally Modified Phe-Phe Peptides

28 > 10 000

29 18.5 ± 1.7

Phe-Phe Analogs

30 540 ± 20

31 64 ± 2

32 175 ± 13

Continued on next page

H2N (S)

HN (S)

NH2

O

O

H2N (S)

HN (S)

OH

O

O

H2N (S)

HN (R)

NH2

O

O

H2N (R)

HN (R)

NH2

O

O

H2N (R)

HN (S)

NH2

O

O

35

Compound Sequencea Ki ± SEM (nM) FHDoE generated Phe-Phe Analogs

33 > 10 000

34 10.2 ± 1.0

35 > 10 000

36 251 ± 4

37b 2247 ± 115

38b 182 ± 7

39

> 10 000

a S configuration = L configuration and R configuration = D configuration. b Both dia-stereomers were obtained due to racemization during the SPPS.

H2N

HN

NH2

O

O

36

The FHDoE-generated substitution pattern is illustrated in Figure 14, and is based on a combination of two design layers. The first design layer deter-mines whether a substitution should be made (depicted in Figure 14 as co-lored), and the second layer determines how the substitution should be made (depicted in Figure 14 as +/-), i.e. in which direction to go regarding proper-ties related to size and hydrophilicity when substituting the phenylalanine residue with another amino acid.

Figure 14. The design matrix used in the FHDoE to generate the dipeptides 33–39. White shading indicates that substitution was performed, and the symbols (+/-) indi-cate whether properties related to size and hydrophilicity were increased or de-creased, with respect to Phe (which can be seen as a center point).

Unfortunately, the peptides showed no, or considerably less, affinity than the native H-Phe-Phe-NH2, which indicates that the binding site is sensitive to side chain modifications. Changing the C-terminal phenylalanine by reduc-ing the carbon side chain by one carbon, giving phenylglycine (33), resulted in no affinity. Furthermore, substitution with the bioisosteric thiophene, as seen in peptide 36, reduced the affinity 170 fold. The N-terminal phenylala-nine seemed less sensitive to modifications since replacing phenylalanine with leucine did not affect the affinity significantly (cf. 26 with 34). Shorten-ing of the N-terminal phenylalanine side chain by one carbon resulted in 120-fold or 1500-fold lower affinity, depending on the configuration of the

Cmpd.

Position 1 Position 2

Sequence

SizeHydro-philicity

SizeHydro-philicity

26- + - +

H-Phe-Phe-NH2

33+ + - -

H-Phe-Phg-NH2

34- + + -

H-Leu-Phe-NH2

35+ + + +

H-Tyr(OMe)-Phe(2-Me)-NH2

26- - - -

H-Phe-Phe-NH2

36+ - - +

H-Phe-Thi-NH2

38- - + +

H-Phg-Phe-NH2

39+ - + -

H-Cha-Phe(3-F)-NH2

37

phenylglycine (cf. 26 with 37 and 38). However, the decrease was not as great as that seen for the same modification in the C-terminal. A modifica-tion of both the side chains concurrently was devastating for the affinity (cf. 26 with 35 and 39). Taken together, these observations indicate the presence of a discrete binding pocket in the SP1–7 binding site matching the H-Phe-Phe-NH2 compound very well.

Due to the smaller size of H-Phe-Phe-NH2 in comparison to the heptapep-tide SP1–7 lower selectivity can be expected. Moreover, H-Phe-Phe-NH2 resembles ligands for the NK3 receptor.77,78 Hence, the possible binding affinity of H-Phe-Phe-NH2 (26) to the human neurokinin receptors NK1 and NK3 was studied. Binding was evaluated in agonist radioligand binding assays relying on the displacement of [Sar9, Met(O2)

11]-SP from NK-1 recep-tors, and [MePhe7]-NKB from NK-3 receptors.79,80 The dipeptide was tested at a concentration of 10 µM, but showed no affinity for any of the receptors.

4.4.2 Effects of SP1–7 and its analogs As mentioned in the introduction, SP1–7 has been shown to influence opioid withdrawal symptoms and possess antinociceptive properties. Notably, the synthesized compounds 15 and 26 have been evaluated in different in vivo models by our collaborators. The amidated C-terminal analog SP1–7-NH2 (15) was demonstrated to attenuate the expression of naloxone-precipitated withdrawal in morphine-dependent rats when administered intracerebroven-tricularly.8,56 In agreement with the binding affinities obtained in the SAR study, the C-terminal amide analog is more efficient in reducing opioid withdrawal symptoms than SP1–7.

SP1–7-NH2 (15) and also H-Phe-Phe-NH2 (26) have been further tested re-garding their potential antinociceptive effect in both non-diabetic and diabet-ic mice after intrathecal administration (Figure 15).9,81-84 The use of diabetic mice for evaluation is due to their reduced pain threshold compared to non-diabetic mice, a reduction thought to arise from hyperalgesia, which makes them a good model for studying neuropathic pain. Interestingly, morphine was unable to induce any antinociceptive effect in the diabetic mice, whereas SP1–7 showed a dose-dependent antinociceptive effect in both diabetic and non-diabetic mice.9 The effect was higher in diabetic mice, which suggests that the compound is more effective on neuropathic pain and that SP1–7 ame-liorates signs of hyperalgesia (Figure 15). In agreement with the results ob-tained from the opioid withdrawal test, SP1–7-NH2 proved to be more effi-cient in reducing pain than the native heptapeptide. It was also demonstrated that the dipeptide H-Phe-Phe-NH2 (26), which possessed the same binding affinity as SP1–7 (1), showed greater antinociceptive potency in diabetic mice than SP1–7.

81,82,84

38

Figure 15. The antinociceptive effect of SP1–7 (1), SP1–7-NH2 (15) and H-Phe-Phe-NH2 (26) in non-diabetic (left) and diabetic (right) mice. The antinociceptive effect was evaluated by the AUC calculated from the time-response curve of tail-flick latency. Each column represents the mean with S.E.M. (n=6).

4.5 Chapter Summary The optimization process described above, starting with the heptapeptide SP1–7 and the tetrapeptide EM-2, resulted in the remarkable discovery of the dipeptide H-Phe-Phe-NH2 (26), which was equipotent with endogenous SP1–7 and had a higher binding affinity than EM-2. The C-terminal phenylalanine amide seems to be crucial for good binding affinity. Moreover, modification of the phenylalanine side chain must be carried out carefully in order to re-tain good binding affinity. It is gratifying that the two most potent peptides discovered here, SP1–7-NH2 (15) and H-Phe-Phe-NH2 (26), have also been shown to possess interesting pharmacological properties, i.e. attenuation of naloxone-provoked withdrawal symptoms in morphine-dependent rats and antinociceptive effects. Considering the fact that no satisfactory treatment of neuropathic pain is available today, these findings are very promising and these small peptides may perhaps serve as lead compounds in the develop-ment of future agents for the treatment of neuropathic pain.

Non-diabetic mice Diabetic mice

SP1-7–NH2

H-Phe-Phe-NH2

SP1-7

39

5. Design and Synthesis of Small Constrained H-Phe-Phe-NH2 Analogs

5.1 Background and Strategy The potent dipeptide lead H-Phe-Phe-NH2 (26), discussed above, was chosen for further optimization studies with the overall aim of developing metaboli-cally stable and selective SP1–7 analogs to be used as research tools in com-plex animal models (Paper III). As mentioned in Section 1.3, the introduc-tion of local constraints can enhance stability, selectivity and bioavailability. The intestinal permeability is an important factor in the development of oral-ly bioavailable drugs. In the intestine, the di/tri-peptide transporter PepT1 enables the absorption of small peptides from the digestion of dietary pro-teins. This transport system has also been shown to transport a variety of peptidomimetic drugs, such as -lactam antibiotics and ACE inhibitors and might be exploited in order to increase the absorption of our small com-pounds.85-87 A known problem with peptides is their susceptibility to efflux. For peptides targeting functions in the CNS, uptake in the brain, i.e. crossing the BBB, is a crucial factor. As a defense mechanism preventing harmful substances from entering the brain, the BBB is equipped with efflux trans-porters.88 PgP is one of the most important, and can actively transport sub-stances out of the brain.89 Such transporters can be an obstacle to entering the CNS.

A series of H-Phe-Phe-NH2 analogs incorporating different types of con-straints were designed, synthesized and evaluated regarding their binding affinity, stability, uptake and permeability (Figure 16). N-methyl and -methyl amino acids were incorporated, substituting one residue at a time. Furthermore, -methylation of the phenylalanine side chain was used to re-duce the conformational flexibility, which can be of advantage upon binding. This approach has been successful in other projects in obtaining neuropep-tide analogs resistant to metabolism while still retaining their biological ac-tivity.11 Both N- and C-terminal rigidifications were accomplished by the introduction of a 3-phenylproline derivative.3 Finally, some modifications of the C-terminal phenylalanine were made in order to supplement the FHDoE-generated library. The peptides were synthesized by SPPS as described in Section 4.3.

40

The synthesized peptides were further explored by pharmacophore modeling analysis in order to find a plausible bioactive conformation.90,91

Figure 16. Overview of the modification strategy presented in Paper III.

5.2 Biological Evaluation 5.2.1 Structure–activity relationship and ADME properties The binding affinities of the dipeptides 40–53 were evaluated as described in Section 4.4.1. The dipeptide lead H-Phe-Phe-NH2 was retested with the new peptides for a more accurate comparison of the Ki values. The metabolic stability was evaluated by incubating the peptides with pooled human liver microsomes. In vitro half-life (t1/2) and in vitro intrinsic clearance (Clint) were calculated using previously reported models.92,93 The Ki values and metabol-ic stabilities (t1/2 and Clint) of the methylated analogs 40–44 are presented in Table 4, while the results of the rigidified and C-terminal-phenylalanine-modified analogs 45–53 are presented in Table 5.

As mentioned above, a pharmacophore search was conducted in order to further investigate the SAR and, if possible, arrive at a bioactive binding conformation. The compounds with Ki values of 10 nM or less and known stereochemistry were defined as active (i.e. 26, 42, 51 and 53), while com-pounds with the lowest binding affinity and known stereochemistry were defined as inactive (40, 41 and 44). A pharmacophore model for the com-pounds was found (Figure 17). Six important interaction features common to all high-affinity ligands were identified: two hydrogen bond acceptors, two hydrogen donors, and two aromatic rings.

Notably, the stereochemistry of compounds 45–50 (not included in the set generating the active binding conformation) was estimated based on the model generated and knowledge obtained from the previous investigation of the influence of the stereochemistry of H-Phe-Phe-NH2 on optimal binding (see Table 3).

41

Table 4. Binding affinity (Ki values) and metabolic stability (Clint and t1/2) of the methylated H-Phe-Phe-NH2 analogs

Compound Structure Binding affinity Ki ± SEM (nM)

Clearancec

Clintd (µl/min/mg)

Half-lifec

t1/2e (min)

26 8.4 ± 0.4a

(1.5 ± 0.1)b 121 ± 39 12 ± 4

40 189 ± 3 2.7 ± 1.5 597 ± 40

41 70 ± 3 64 ± 11 22 ± 4

42 9.4 ± 0.1 92 ± 0 15 ± 1

43 26 ± 1 38 ± 15 40 ± 16

44 136 ± 2 175 ± 5 7.9 ± 0.2

a Ki value determined on the same occasion as for 40–53. b Previously reported and deter-mined Ki value (Paper II). c The metabolic stability data are expressed as mean ± SD. d Clint = in vitro intrinsic clearance. e t1/2 = in vitro half-life.

42

Figure 17. The pharmacophore generated with the high-affinity lead compound H-Phe-Phe-NH2 (26) aligned.

In the methylated series, only compound 42 with internal N-methylation, retained binding affinity comparable to that of H-Phe-Phe-NH2 (26); unfor-tunately no significant improvement in the metabolic stability was achieved. Methylation of the N-terminal resulted in 22 times lower affinity, but was found to have a pronounced impact on the stability, which increased the half-life 50 times (cf. 26 and 40). As can be observed when inspecting the phar-macophore model (Figure 18), the methyl group on the N-terminus is posi-tioned in the direction of the hydrogen bond donor vector from the amine to the target protein, which could explain the decrease in affinity.

Figure 18. Compound 40 aligned in the pharmacophore model. The N-terminal methyl group interferes with the hydrogen bond donor vector from the amine to the target, necessary for optimal binding, which results in a 22-fold decrease in binding affinity.

43

C-terminal methylation (44) was also accompanied by a roughly 20-fold decrease in binding affinity, but without improvement of the stability. The reason for the reduced binding affinity of 44 might be that the secondary amide loses an important hydrogen bond donor feature (Figure 19).

Figure 19. Inspection of compound 44 aligned in the pharmacophore model shows how the C-terminal methyl group on the amide is positioned in the same direction as the hydrogen bond vector in the pharmacophore, which results in the loss of an im-portant hydrogen bond interaction between the ligand and the target protein.

Incorporation of an -methyl amino acid (41 and 43) also reduced the bind-ing affinity, but not as much as seen for the N-methyl amino acids in the terminal parts of H-Phe-Phe-NH2. A 2- to 3-fold increase in the half-life was thus observed for the -carbon methylated analogs.

Table 5. Binding affinity (Ki values) and metabolic stability (Clint and t1/2) of the rigidified and C-terminal-modified H-Phe-Phe-NH2 analogs

Compound Structure Binding affinity Ki ± SEM (nM)

Clearancec

Clintd (µl/min/mg)

Half-lifec

t1/2e (min)

45a 34 ± 3 28 ± 3 50 ± 6

46a 51 ± 2 16 ± 9 103 ± 6

47a 2.4 ± 0.6b 16 ± 3 88 ± 15

Continued on next page

44

Compound Structure Binding affinity Ki ± SEM (nM)

Clearancec

Clintd (µl/min/mg)

Half-lifec

t1/2e (min)

48a 93 ± 0 7.6 ± 1.9 187 ± 5

49a 18 ± 1 39 ± 4 36 ± 3

50a 68 ± 1 16 ± 7 98 ± 4

51 6.2 ± 0.2 44 ± 6 32 ± 5

52 12 ± 0 99 ± 5 14 ± 1

53 3.3 ± 0.2 107 ± 12 13 ± 1

a The stereochemistry of each diastereomer pair was estimated from the pharmacophore mod-el. b IC50 value. Analog 47 was tested at a 2:1 ratio with analog 46. It was tested once, at six different concentrations, in triplicate. c The metabolic stability data are expressed as mean ± SD. d Clint = in vitro intrinsic clearance. e t1/2 = in vitro half-life.

When the cis 3-phenylproline derivative was incorporated into the N-terminal part of H-Phe-Phe-NH2 replacing phenylalanine (45 and 46), the binding affinity decreased 4 and 6 times, respectively. Both these diastereo-mers showed low fitness scores to the pharmacophore model, which indi-cates that these compounds have problems adopting the optimal binding conformation. Replacement of the C-terminal phenylalanine by the cis 3-phenylproline moiety with the estimated (S,S,S) configuration (47) resulted in a more potent ligand than H-Phe-Phe-NH2, which was well positioned in the active binding site (Figure 20). This rigidification also increased the half-life 7 times (cf. 26 and 47).

H2N (S)

HN

O

(S)

NH2O

F

45

Figure 20. Comparison of the alignment of compounds, 47 (above) with a IC50 value of 2.4 nM and 48 (below) with the Ki value 93 nM, to the pharmacophore model. This clearly illustrates the difference in binding affinity towards the SP1–7 binding site. Compound 47 with the proposed (S,S,S) configuration fits the pharma-cophore nicely and picks up all the important binding interactions to the target pro-tein, while compound 48 with the proposed (S,R,R) configuration does not.

-Methylation of the C-terminal phenylalanine gave compounds 49 and 50, with lower binding affinity than H-Phe-Phe-NH2. When comparing the me-tabolic stability in all the diastereomeric pairs (45 vs. 46, 47 vs. 48, and 49 vs. 50), the natural (S,S,S) configuration was more easily metabolized. In-corporation of D-amino acids (amino acids with S configuration) is a know strategy to improve metabolic stabilization.10,11

As observed for the peptides generated by the FHDoE strategy discussed above, the C-terminal part seemed to be sensitive to modifications. Shorten-ing of the side chain by one carbon resulted in a compound 33 with no affini-ty, and bioisosteric replacement of the phenyl ring with a thiophene (36) was not well tolerated. Furthermore, simultaneous modification of both the phe-nylalanine residues was devastating for affinity (35 and 39). Therefore, to further explore the SAR around the C-terminal phenylalanine, compounds 51–53 were prepared (Table 5). Contrary to the shortening of the C-terminal phenylalanine side chain, elongation by one carbon in the same position resulted in a compound (51) with similar binding affinity to H-Phe-Phe-NH2. When the same modification of the C-terminal phenylalanine was made as in compounds 35 and 39 (Table 3), but with retention of the native phenylala-nine in the N-terminal part, compounds (52 and 53) with high binding

46

affinity, Ki = 12 nM and Ki = 3.3 nM, respectively, were obtained. This illu-strates the unpredictability of changing both the terminal parts concurrently.

The physiochemical properties of all the synthesized compounds were further explored by evaluation of their intestinal epithelial permeability. This was determined from transport rates across a Caco-2 cell monolayer, and is expressed as the apparent permeability coefficient (Papp).

94 A good relation-ship between the permeability across the Caco-2 monolayer and the extent of absorption in vivo has been reported.95 Each compound was investigated in the apical to basolateral (a–b) and basolateral to apical (b–a) direction. One of several efflux transporters present in the Caco-2 cells is PgP. Measure-ment of the efflux (the ba/ab ratio) can thus indicate whether or not the com-pounds are substrates for PgP.

Since uptake transporters can enhance absorption, the possibility of the peptides being actively transported was studied in Chinese hamster ovary (CHO) cells stably transfected with the PepT1 transporter (CHO)-PepT1, using CHO-K1 cells as control. The results are expressed as pmol/mg of protein/min. For peptides being actively transported the PeptT1/K1 ratio should be greater than one. Uptake and permeability are reported in Tables 6 and 7.

Table 6. Active uptake and permeability data for the methylated H-Phe-Phe-NH2 analogs.

Compd.

Uptakea

(pmol/mg protein/min)Caco-2 permeabilitya

Pappb (10-6 cm/s)

CHO-PepT1 CHO-K1

Ratio PepT1/

K1 a–bc b–ad Ratio

ab/ba Ratio ba/ab

26 0.3 ± 0.0 0.3 ± 0.0 1.0 0.02 ± 0.00 0.2 ± 0.0 0.1 11

40 2.2 ± 0.7 2.4 ± 0.5 0.9 14 ± 1 124 ± 21 0.1 9

41 1.1 ± 0.2 1.5 ± 0.2 0.7 18 ± 0 87 ± 0 0.2 5

42 19 ± 0 23 ± 3 0.8 9.0 ± 2.2 124 ± 19 0.1 14

43 33 ± 2 32 ± 9 1.0 20 ± 2 224 ± 5 0.1 11

44 0.3 ± 0.1 0.4 ± 0.1 0.7 0.3 ± 0.1 0.9 ± 0.2 0.3 3 a The results are expressed as mean ± SD. b Papp = apparent permeability coefficient. c a–b = apical to basolateral. d b–a = basolateral to apical.

A Papp value below 0.2 x 10-6 cm/s indicates low permeability, a Papp value ranging from 0.2 x 10-6 cm/s to 1.6 x 10-6 cm/s indicates moderate permea-bility and a Papp value above 1.6 x 10-6 cm/s indicates high permeability.96 The permeability in the a–b direction increased substantially for all the me-thylated analogs (ranging from 0.3 x 10-6 cm/s to 20 x 10-6 cm/s) compared to

47

H-Phe-Phe-NH2 (26, 0.02 x 10-6 cm/s). All the compounds, except 44, were classified as having high permeability with the highest permeability ob-served for the -methylated analogs 41 and 43, 18 x 10-6 cm/s and 20 x 10-6

cm/s, respectively. However, the methylated compounds were not actively transported, as can be seen from the PepT1/K1 ratio. Furthermore, the pep-tides also displayed efflux (the ba/ab ratio ranging from 3 to 14), where compounds 42 and 43 showed the highest tendency towards efflux and were equal to 26.

Compared to the methylated analogs, the rigidified and the C-terminal-phenylalanine-modified analogs possessed much lower permeability (rang-ing from 0.02 x 10-6 cm/s to 4.4 x 10-6 cm/s). Satisfyingly, the high affinity analog 47 turned out to have high permeability (3.6 x 10-6 cm/s). In this se-ries the efflux was much higher; 8–95 times, in the b–a direction than in the a–b direction. The stereochemistry of the compounds also seemed to influ-ence the predisposition for efflux. Thus, the compounds 45, 47 and 49 all showed lower efflux ratios than their diastereomeric counterparts 46, 48 and 50. The replacement of the phenylalanine in the N- or C-terminal by the 3-phenylproline moiety seemed advantageous in improving the active uptake of these compounds, since a slight increase in the PepT1/K1 ratio was ob-served (cf. 26 vs. 45, 46, 47 and 48).

Table 7. Active uptake and permeability data for the rigidified and C-terminal-modified H-Phe-Phe-NH2 analogs.

Compd.

Uptakea

(pmol/mg protein/min)Caco-2 permeabilitya

Pappb (10-6 cm/s)

CHO-PepT1 CHO-K1

Ratio PepT1/

K1 a–bc b–ad Ratio

ab/ba Ratio ba/ab

45 31 ± 4 22 ± 2 1.4 0.5 ± 0.0 26 ± 1 0.0 53

46 15 ± 2 11 ± 1 1.4 0.8 ± 0.1 76 ± 1 0.0 95

47 13 ± 2 11 ± 1 1.2 3.6 ± 0.4 75 ± 2 0.1 21

48 11 ± 1 8.7 ± 0.8 1.3 0.6 ± 0.0 51 ± 2 0.0 86

49 0.6 ± 0.1 0.7 ± 0.0 0.9 0.7 ± 0.0 5.4 ± 0.4 0.1 8

50 25 ± 4 22 ± 3 1.1 4.4 ± 0.1 171 ± 6 0.0 39

51 0.6 ± 0.0 0.9 ± 0.2 0.7 0.1 ± 0.0 0.9 ± 0.3 0.1 9

52 0.8 ± 0.1 1.3 ± 0.3 0.6 0.02 ± 0.00 0.3 ± 0.0 0.1 15

53 0.2 ± 0.0 0.3 ± 0.0 0.7 0.1 ± 0.0 0.8 ± 0.1 0.1 8 a The results are expressed as mean ± SD. b Papp = apparent permeability coefficient. c a–b = apical to basolateral. d b–a = basolateral to apical.

48

5.3 Chapter Summary One approach used in drug development is the introduction of local con-straints to enhance the potency and improve the pharmacokinetic properties. The dipeptide lead compound H-Phe-Phe-NH2, which was found to be as potent as the endogenous SP1–7 (1), showed poor metabolic stability and permeability, and suffered from high efflux rates. The introduction of local constraints by incorporation of the cis 3-phenylproline into the C-terminal of H-Phe-Phe-NH2, provided a compound with retained binding affinity and substantially improved pharmacokinetic properties. Thus, an increase in permeability and improvement in metabolic stability were achieved.

Figure 21. Comparison of compound 26 and the constrained analog 47.

The constraint in compound 47 has locked the dipeptide in a conformation that seems to be well accepted in the target protein. This conformation is also in agreement with the proposed pharmacophore model. Although highly active compounds with improved pharmacokinetic properties were obtained, they still suffered from efflux, a problem that requires further attention.

49

6. Improvement of the Pharmacokinetic Profile of Substance P1–7 Ligands

6.1 Background and Strategy As discussed above, two small peptides (26 and 47) with high binding affini-ty to the SP1–7 binding site were identified. From the initial ADME investiga-tion it was seen that the compounds were associated with high efflux, which is a problem for drugs targeting pathological conditions in the CNS. Efforts were then made to improve the pharmacokinetic properties by reducing the efflux and increasing the lipophilicity (Paper IV). Aiming at compounds with good cell permeability and with the potential to cross the BBB, a new type of structure (illustrated in Figure 22) was identified as possible SP1–7 ligands. It was thought that replacement of the N-terminal basic amine func-tion with this phenylalanine-based carbamate could result in improved ADME properties.

Figure 22. Binding affinity of SP1–7, 26 and 47. Structure A was investigated as a new type of compound for SP1–7 binding affinity.

H2N

O

OHN

NH2NH

NHH2N

N

OO N

H

N

NH2

OO N

H

HN

NH2O

O

NH

H2N O

O OH

O

H2N

K i = 1.5 nM

K i= 1.6 nM

SP1-7

26

O

O

OHN

NH2

H2NN

OO NH2

IC50 = 2.4 nM

47

A

50

Structure A was synthesized and its affinity to the SP1–7 binding site was investigated. The pharmacokinetic properties of the new structure were eva-luated and compared to the lead compounds 26 and 47. In order to obtain a detailed ADME profile of the three compounds, several important parame-ters were investigated such as lipophilicity, PPB, permeability including efflux, metabolic stability and their influence on the CYP enzymes. Subse-quently, a set of compounds based on the prototype structure, in which the C-terminal primary amide was replaced by different C-terminal functional groups (Figure 23), was synthesized and evaluated regarding their binding affinity, stability, uptake and permeability. Bioisosteric substitution is fre-quently used in drug discovery and can, for example, improve potency, se-lectivity or the ADME profile.97,98

Figure 23. Illustration of the investigated C-terminal groups.

6.2 Synthesis of Phenylalanine-Based Carbamates The target compounds were synthesized as outlined in Scheme 2. Compound 54, with a primary amide, was prepared through the reaction of C-terminally activated benzyloxycarbonyl-protected phenylalanine (Z-Phe-OSu) with ammonia. The acyl cyanamide, 55, was obtained by activation of Z-Phe-OH by N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridine-1-yl-methylene]-N-methylmethanaminium hexafluorophosphate N-oxide (HATU) followed by coupling with the cyanamide. The reaction was run for 3 days at 40 °C. Preactivation of Z-Phe-OH with 1,1´-carbonyldiimidazole (CDI) at 60 °C prior the coupling with methylsulfonamide, generated the acyl sulfonamide, 56, at a yield of 50%. Activation of Z-Phe-OH with isobutyl chloroformate at low temperature and coupling with hydroxylamine hydrochloride gave the hydroxamic acid 57 at a reasonable yield. The diacyl hydrazine (58) was prepared under standard peptide coupling conditions between Z-Phe-OH and benzhydrazide, using HATU and DIEA as couplings reagents. The 1,3,4-oxadiazole, 59, was synthesized from the diacyl hydrazine compound, 58, which was dehydrated using Burgess reagent99 in order to render the hetero-cyclic compound 59. All the target compounds were purified by RP-HPLC.

51

Scheme 2.

Reagents: (a)(i) Z-Phe-OSu, NH3, THF, 0 °C to r.t. or (ii) Z-Phe-OH, cyanamide, HATU, DIEA, DMF, 3 days at 40 °C, or (iii) Z-Phe-OH, methylsulfonamide, CDI, DBU, THF, 3 h or (iv) Z-Phe-OH, hydroxylamine hydrochloride, NMM, isobutylchloroformate, Et3N, THF, DMF, 2 h or (v) Z-Phe-OH, benzhydrazide, HATU, DIEA, DCM, r.t. (b) 58, Burgess reagent, THF, 75 °C.

6.3 Biological Evaluation 6.3.1 Structure–activity relationship and ADME properties The synthesized compounds were biologically evaluated. The binding affini-ties determined in the SP1–7 binding assay are given in Tables 8 and 9. The pharmacokinetic properties of compounds 26, 47 and 54 were evaluated in an extensive in vitro profile program100-102 and are reported in Table 8. The metabolic stability (Clint and t1/2) and the apparent permeability of com-pounds 55–58 were determined as described above and are given in Tables 9 and 10.

Despite substitution of the basic amine with the lipophilic benzyloxycar-bonyl moiety, compound 54 was still able to interact properly with the target protein and retain good binding affinity (Ki = 5.2 nM). The reduced peptide character of compounds 47 and 54 compared to 26, together with their in-creased lipophilicity, should promote permeability across the BBB. As dis-cussed above, compound 47 showed higher permeability than 26 but gener-ally much lower than the methylated analogs. Compound 47 also suffered from efflux. As can be seen in Table 8, substitution of the N-terminal pheny-lalanine with the carbamate function resulted in a large increase in the per-meability (30 x 10-6 cm/s) but, most importantly, the efflux was considerably reduced (efflux ratio 1). The metabolic stability and the PPB were evaluated using both human and rat tissues since species differences can occur. How-ever, no difference was observed for these compounds. Comparison of the metabolic stability and PPB, revealed that both compounds 47 and 54 pos-sessed lower clearance and PPB than 26 (Table 8). The constrained analog 47 was found to be very stable and showed only 40% plasma–protein

52

binding. All three compounds showed weak or moderate inhibition of the CYP enzymes.

Table 8. The in vitro pharmacokinetic profiles of compounds 26, 47 and 54.

Pysicochemical properties ClogP 1.02 1.67 2.15 Polar surface area (PSA)

107 94 88

Soluble dried DMSO (µM)

> 400 257 97

Plasma–protein bindingc, d

Human PPB Fu % 10 µM

NV 60 19

Rat PPB Fu % 10 µM

NV 67 18a

Permeability and efflux Papp (10-6 cm/s) NVb 2.8 31 Efflux ratio NVb 4.2 1 Metabolisme, f, h

Clint (Rat Mic) 102.8 8.3 21.2 Clint (Human Mic) 97.3 13.9 22.6 Clint (Rat Hep) NV 7.1 39.4 Cytochrome P450 inhibitioni, j

CYP2D6 MS or F Weak Weak Weak CYP3A4 MS or F Weak Moderate Weak CYP1A2 MS or F Weak Weak Weak CYP2C9 MS or F Weak Weak Weak NV = No value. a Using protease inhibitors. b Below level of quantification. c PPB = Plasma protein binding. d Fu = Fraction unbound. e Mic = Microsomes. f Clint = clearance intrinsic. h Hep = Hepatocytes. i Weak : IC50 > 20 µM. j Moderate: IC50 2–20 µM.

53

Encouraged by the high binding affinity and the reduced efflux of compound 54, a set of analogs incorporating different C-terminal groups was prepared. These analogs were evaluated regarding SP1–7 binding affinity, metabolic stability, uptake and permeability including efflux (Tables 9 and 10).

Table 9. Binding affinity (IC50 values) and metabolic stability (Clint and t1/2) for the compounds comprising different C-terminal groups.

Compound Structure Binding affinity

IC50

Clearancea

Clintb

(µl/min/mg)

Half-lifea

t1/2c (min)

26 9.8d 121 ± 39 12 ± 4

54 6.1 21e ND

55 17 11 ± 4 136 ± 46

56 61 17 ± 4 84 ± 19

57 4.2 23 ± 1 60 ± 3

58 6.5 65 ± 13 22 ± 4

59 9.0 287 ± 4 4.8 ± 0.1

a The metabolic stability data are expressed as mean ± SD. b Clint = in vitro intrinsic clearance. c t1/2 = in vitro half-life. d IC50 value determined at the same occasion as for 54–59. Corre-sponds to the Ki value 8.4 nM (Table 4). e Value obtained from Table 8 and measured at a different laboratory. Comparisons with the other values should thus be made with caution.

H2N

HN

NH2

O

O

54

The prepared carbamate-based phenylalanine analogs with different C-terminal groups showed good to moderate binding affinities (IC50 ranging from 4 to 61 nM). Replacing the primary amide in compound 54 with an acyl cyanamide (55) or an acyl sulfonamide (56) resulted in 3 and 10 times lower affinity, respectively. As previously discussed, a C-terminal carboxyl-ic acid is not well tolerated. Considering that the pKa of these compounds is in the same range as that of carboxylic acid, this verifies that acidity is not favorable. Both compounds were found to be more stable regarding clear-ance and half-life than compound 26 (H-Phe-Phe-NH2), and comparable to the prototype compound (cf. 54 and 56, Table 9) or even better (cf. 54 and 55, Table 9). Compound 55 also showed very good permeability (52 x 10-6 cm/s) and low efflux (Table 10). In compound 57, where the primary amide was replaced by a hydroxamic acid, retaining the possibility for two hydro-gen bond interactions, the binding affinity was slightly increased compared to compound 54 (Table 9). Interestingly, this compound was also found to have the highest permeability (60 x 10-6 cm/s) and an efflux ratio below one (Table 10). The hydrazine-based analog, 58, and the 1,3,4-oxadiazole, 59, showed reasonably preserved binding affinity (Table 9). The diacyl hydra-zine, 58, offers both H-bond-donating and H-bond-accepting atoms that can interact with the target protein, and the 1.3,4-oxadiazole moiety in 59 can provide new interactions within the binding site. Both 58 and 59 were classi-fied as having high permeability but unfortunately the incorporation of the 1,3,4-oxadiazole moiety in 9 turned out to be unfavorable for the metabolic stability (Table 9 and 10).

Table 10. Uptake and permeability data for the compounds comprising different C-terminal groups.

Compd.

Uptakea

(pmol/mg protein/min)Caco-2 permeabilitya

Pappb (10-6 cm/s)

CHO-PepT1 CHO-K1

Ratio PepT1/

K1a–bc b–ad Ratio

ab/ba Ratio ba/ab

26 0.3 ± 0.0 0.3 ± 0.0 1.0 0.02 ± 0.00 0.2 ± 0.0 0.1 11

55 4.1 ± 0.9 4.0 ± 1.5 1.0 52 ± 2 62 ± 1 1 1.2

56 90 ± 1 77 ± 4 1.2 6.2 ± 0.4 3.3 ± 0.1 1.9 0.5

57 5.2 ± 0.2 4.4 ± 0.9 1.2 60 ± 2 45 ± 0 1 0.7

58 127 ± 7 156 ± 10 0.8 11 ± 0 14 ± 1 0.8 1.3

59 1082 ± 53 1128 ± 74 1.0 13.3 ± 0 7.4 ± 0.6 1.8 0.6 a The results are expressed as mean ± SD. b Papp = apparent permeability coefficient. c a–b = apical to basolateral. d b–a = basolateral to apical.

55

6.4 Chapter Summary The replacement of the basic N-terminal amine in H-Phe-Phe-NH2 with a benzyl carbamate rendered active SP1–7 ligands (IC50 6–61 nM) with im-proved permeability (6–60 x 10-6 cm/s) and substantially reduced efflux (ef-flux ratio 0.5–1). Substitution of the C-terminal primary amide with hydrox-amic acid or hydrazine-based groups was well accepted in combination with the more lipophilic N-terminal carbamate group (Figure 24). These new types of compounds seem to bind to the target protein in a different way since they do not match the pharmacophore model previously generated, but still show good binding affinity. The question thus arises as to whether these compounds would work analogously to the endogenous SP1–7, or would act in an antagonistic way. In vivo studies of this new class of SP1–7 analogs to establish their functional mode are yet to be performed.

Figure 24. The analogs 54, 57 and 58 possessed the highest binding affinity and all were classified as having high permeability. The efflux ratios were 8 to 15 times lower than for H-Phe-Phe-NH2 (26).

56

7. Microwave-Assisted Aminocarbonylations and Direct Arylation of Imidazoles. Application to MAP Amides and SP1–7 Analogs

As mentioned in the introduction, heterocycles can be used as a non-peptidic scaffold in peptidomimetics. The use of decorated 1,3-azoles (thiazole, im-idazole and oxazoles) as dipeptide mimetics was reported by Gordon and coworkers.103 They envisioned that the dipeptide shown in Figure 25 could be replaced by an 1,3-azole moiety because of the similarity in three-dimensional structure.

Figure 25. Gordon and colleagues proposed a cyclization including the carbonyl carbon of tryptophan and the N, -carbon and -carbon of phenylalanine would generate a new conformationally constrained dipeptide mimetic.

A set of substituted azole-based moieties was synthesized and the dipeptide mimetics were inserted into the hexapeptide H-Pro-Trp-Phe-Trp-Leu-Phe-NH2, which is a known SP antagonist. The new analogs were biologically evaluated and were shown to be up to ten times more potent than the native hexapeptide.103

The findings that the basic N-terminal amine in compound 26 could be replaced by the phenylalanine-based carbamate group in compound 54 with retained binding affinity, but with significantly reduced efflux, initiated the investigation of the incorporation of an imidazole moiety into the N-terminal backbone of the dipeptide H-Phe-Phe-NH2, as illustrated in Figure 26. The outcome of a more rigidified 1,3-azole moiety regarding stability, efflux and binding affinity was explored. A fast and simple route to obtain multi-decorated imidazoles for introduction into the N-terminus of a peptide was desired (Paper V). We envisioned that palladium-catalyzed direct arylation could be used to obtain C-5 arylated imidazoles (Figure 27, step one).

57

Figure 26. Binding affinity and pharmacokinetic properties of compounds 26 and 54, together with an illustration of the potential mimetic incorporating a imidazole moiety.

In addition, we wanted to develop a convenient method for the insertion of the heterocyclic scaffold into the N-terminal of a peptide backbone. A palla-dium(0)-catalyzed aminocarbonylation approach utilizing microwave irrad-iation and a solid carbon monoxide source was identified as a suitable strate-gy (Figure 27, step two).

Figure 27. The proposed synthetic route to obtain heterocyclic-based dipeptide analogs.

In parallel with the medicinal chemistry project, the use of a solid carbon monoxide source in microwave-assisted aminocarbonylation was also inves-tigated for the preparation of a set of acylating agents, as depicted in Scheme 3 (Paper VI).

Scheme 3.

N

HN

NH2

O

OO

O

HN

NH2

O

O

HN

NH2

OHN

N

K i = 1.5 nM

Clint = 97 µl/min/mg

Papp = 0.02 x 10-6 cm/s

Efflux ratio = 11

K i = 5.2 nM

Clint = 23 µl/min/mg

Papp = 31 x 10-6 cm/s

Efflux ratio = 1

26 54

H

H

O

HN

NH2

ON

NH

58

7.1 Background 7.1.1 Microwave irradiation in organic synthesis In 1986 a new heating technique, microwave heating, was implemented in organic chemistry as a complement to conventional heating, i.e. oil baths and hot plates.104,105 Initially, reactions were performed in domestic microwave ovens, but dedicated microwave reactors designed for organic synthesis have since been developed.106 These advanced reactors feature magnetic stirrers, explosion-proof cavities, and direct measurement of temperature and pres-sure. The advantages of microwave heating compared to classical heating are rate enhancement, higher yields and fewer by-products, which have led to a tremendous increase in the number of studies using microwave-assisted or-ganic synthesis.107 Normally, microwave-assisted reactions are conducted in sealed vessels that allow a solvent to be heated far above its boiling point, which can be favorable for the reaction, i.e. reducing the reaction time and increasing the yield.

The heating process in microwave irradiation acts by two mechanisms: ionic conduction and dipolar polarization. The applied oscillating electric field forces ions and dipoles to align to the field, causing molecular friction and increased collision rate, i.e. kinetic energy that will be lost as heat. The ability of a solvent to absorb microwave energy and convert it into heat is determined by the so-called loss tangent value (tan ). Polar ethanol, which has a high value of tan , is a good absorber, while hexane and toluene are very poor absorbers. However, by using polar reaction components or add-ing small amounts of additives (e.g. ionic salts) low-absorbing solvents can also be used in microwave-assisted reactions.106,107 During the past decade the beneficial effects of microwave irradiation have led to its increasing ap-plication in lead optimization and in process chemistry in order to speed up the reactions and to improve the yields and purity profiles.108

7.1.2 Palladium-catalyzed reactions 7.1.2.1 Direct arylation of aromatic heterocycles Palladium-catalyzed coupling reactions have had an enormous impact in organic chemistry for the preparation of carbon–carbon and carbon–heteroatom bonds, as evidenced by last year’s Nobel Prize in Chemistry (2010). An effective and straightforward method of making aryl–heteroaryl bonds is the palladium-catalyzed direct arylation of heteroaryl C–H bonds with aryl halides.109-111 Since it was first reported at the beginning of the 1980s, direct arylation has emerged as a valuable and efficient method of obtaining aryl functionalized heterocyclic compounds.110-113 Today, numer-ous five-and six-membered heterocycles have been reported to undergo di-rect arylation with a variety of aryl halides.109,114

59

The general mechanism of palladium-catalyzed direct arylation involves a Pd0 catalytic cycle, as outlined in Figure 28.110,114

Figure 28. The general catalytic cycle for palladium-catalyzed direct arylation of heteroaromatics.

The first step is the oxidative addition of the electrophile, i.e. aryl halide, to the Pd0 species, which generates a PdII complex. Normally, the order of reac-tivity of the aryl halides is I > Br > Cl. Several mechanisms have been pro-posed for the next step, two of which have gained most support. The first is electrophilic aromatic substitution (SEAr) with electron-rich substrates, and the second is concerted metalation–deprotonation (CMD) with electron-deficient heterocycles.109,110,115 However, Fagnou and coworkers recently demonstrated that when the conditions developed for CMD-type direct aryla-tion, i.e. the addition of pivalic acid, were applied to electron-rich hetero-aromatics, the reaction rates increased and the reaction outcomes improved.111 Thus, the pivalic acid is thought to enhance the CMD step, as illustrated in Figure 29.

60

Figure 29. The addition of pivalic acid is thought to assist in the concerted metala-tion–deprotonation step.111

However, it is most likely that both SEAr and CMD can occur, depending on the nature (i.e. the electron density) of the heterocycle and the reaction con-ditions applied. The last step in the catalytic cycle is reductive elimination, which liberates the product and regenerates the Pd0 species.

7.1.2.2 The Heck carbonylation reaction In the early 1970s, Heck and coworkers discovered that vinyl and aryl ha-lides could form acyl-palladium intermediates in the presence of carbon mo-noxide, and that these could further react with different nucleophilic alcohols or amines to provide esters116 and amides.117 Since then, a number of differ-ent methods have been reported for the carbonylation of different aryl and vinyl halides and triflates.118 One advantage with the carbonylation reaction is the facile access to different kinds of functional groups, i.e. different car-boxylic acid derivatives, simply by varying the nucleophile, as illustrated in Figure 30.

Figure 30. A general scheme for obtaining carboxylic acid and derivatives by car-bonylation of aryl-X compounds.

A general catalytic mechanism for palladium-catalyzed carbonylation is depicted in Figure 31. It consists of three main steps: oxidative addition (I), carbon monoxide insertion (II) and nucleophilic attack (III).119 As described above, the PdII complex is generated in situ via the oxidative addition of the aryl halide. In the next step carbon monoxide is inserted into the aryl-palladium bond. Electron-withdrawing substituents in the 4-position of the aryl group have been reported to slightly decrease the carbon monoxide in-sertion rate in comparison to electron-rich substituents.119 In the third and final step the acyl-palladium complex is attacked by the nucleophile, which releases the product and regenerates the Pd0 species. When working with sterically hindered or poor nucleophiles (e.g. aniline) acyl-transfer regents such as N,N-dimethylaminopyridine (DMAP) or imidazole can be used to facilitate the carbonylation reaction.120

61

Figure 31. The catalytic mechanism for carbonylation of aryl halides.

7.1.3 Design of experiments In method development, for example an organic synthesis protocol, several parameters often need to be investigated in order to determine their influence on the reaction outcome. Moreover, the interaction between these factors must also be studied. One way of systematically developing a method is to use an experimental design.121,122 In order to obtain as much information as possible, it is important to choose an experimental design that meets the requirements defined by the chemist.122 In the work presented here, a factori-al design was used as it allows the evaluation of both experimental factors and interaction effects on the response. In a full factorial design, all the main effects, as well as the interaction effects, are studied, and as the number of experimental factors increases so does the number of interaction effects. The number of practical experiments required to investigate all the effects of k variables (2k) thus increases dramatically. Normally, it is not necessary to investigate all the interaction effects. Instead, a fractional factorial design (2k-p) can be used, were p is the size of the fraction. The size of the fraction determines how many effects that can be studied, and the number of practic-al experiments needed (Figure 32).121,122 In this work, a 24-1 fractional fac-torial design was used, which is half of the factorial design with 4 variables, which means 8 experiments are required instead of 16.

62

Figure 32. An illustration of the transformation of a full factorial design with 3 factors (23) into a fractional factorial design (23-1), where only a fraction (half) of all possible experiments have been carried out. The grey dot inside the cube depicts replicated center-point experiments that are carried out to estimate the experimental error.

7.2 Method Development 7.2.1 Microwave-assisted protocol for C5 arylation of imidazole Efforts were made to find a method for substitution of the imidazole by dif-ferent types of aryl halides; the overall aim being to develop substituted im-idazole moieties for incorporation into the N-terminal backbone of a peptide sequence. A literature search revealed an appealing method that offered a convenient way to selectively perform direct C5 arylation of N-protected imidazoles.123-125 Substituted imidazoles are commonly synthesized by cycli-zation reactions resulting directly in a 1.5, 1.2 or 4.5 substitution pattern.125 The advantage of the method chosen here compared to cyclization reactions was the possibility of using the cheap and easily available starting material, 1-benzyl-1H-imidazole, and decorating it in a single step, using a number of different aryl bromides. The protocol described employed conventional heat-ing and the reported reaction times ranged from 27 to 88 hours, delivering 5-aryl-1H-imidazoles at yields of 43–73%.123 It was decided to investigate whether the reaction could be performed with microwave irradiation in order to reduce the reaction time and, if possible, improve the yields.126,127 In the initial investigations of the microwave-assisted protocol the reaction condi-tions reported by Bellina et al.123 were used, and after considerable expri-mentations, product 60 was obtained at an isolated yield of 35%. The reac-tion was performed in sealed vessels in the presence of 5% catalyst and 30% pivalic acid, with microwave heating at 140 C for 75 minutes. Pivalic acid was added based on the report that the acid could accelerate direct arylation of heterocycles and lead to higher yields.111

Encouraged by the initial results, a 24-1 fractional factorial design was set up in order to screen various reaction conditions such as temperature, time, Pd catalyst loading, and amount of aryl bromide, in a systematic way, as described in Section 7.1.3. The reaction outcome was based on product yield, amount of diarylated by-product and nonconsumed starting material. The reaction outcome was determined by 1H-NMR ratios.

63

Table 11. The setup of the experimental design used to screen the reaction condi-tions for microwave-assisted direct arylation of N-protected imidazole.

Exp. no. Time (min)

Temp. (ºC) Pd (%)

Ar-X (equiv.)

Product 60 (%)a

Starting material

(%)a

By-product (%)a

1 45 110 2 1 20 80 0

2 105 110 2 5 33 67 0 3 45 140 2 5 75 16 9 4 105 140 2 1 66 29 5 5 45 110 10 5 22 78 0 6 105 110 10 1 26 74 0 7 45 140 10 1 20 80 0 8 105 140 10 5 68 0 32 9b 75 125 6 3 71 21 8

10b 75 125 6 3 71 22 7 Constant in all experiments: 0.3 equiv. PivOH, 3 equiv. K2CO3, DMF (2.5 mL). a Values determined from the NMR ratio. b Experiments 9 and 10 were run under the same conditions to assess the experimental error.

Experiment 1 (Table 11), in which all the investigated reaction conditions were minimized, provided a low yield of 60 (20%). Running the reaction at a low temperature (110 ºC) resulted in overall poor yields (Table 11, experi-ments 1–2 and 5–6). Full conversion of the starting material was achieved when all the reaction parameters were maximized (Table 11, experiment 8). However, this also resulted in increased formation of the by-product. Expe-riments 3 and 9, resulted in high yields and acceptable amounts of by-product. The conditions used in experiment 3 provided a reaction time of 45 minutes and a catalyst loading of 2%, and were chosen for the synthesis of C5-substituted imidazoles, without further optimization. Gratifyingly, both electron-deficient and electron-rich aryl bromides, as well as sterically hin-dered aryl bromides, provided isolated yields of C5-substituted imidazoles in the same range as reported with conventional heating (Table 12). Microwave irradiation reduced the reaction times from 27–88 hours to 45 minutes with-out affecting the reaction yields.

64

Table 12. Microwave-assisted palladium-catalyzed direct arylation of imidazoles.

Entry Aryl bromide Product Yield (%)

1

60a 64

2

60b 57

3 60c 56

4

60d 32

5

60e 60

Reaction conditions: 1 mmol 1-bensyl-1H-imidazole, 5 equiv. aryl bromide, 0.02 equiv. Pd(OAc)2, 0.04 equiv. P(2-furyl)3, 0.3 equiv. PivOH, 3 equiv. K2CO3, DMF (4 mL), 140 ºC, 45 min, MW.

7.2.2 Microwave-assisted aminocarbonylation using CO-gas-free conditions 7.2.2.1 Carbon monoxide sources One disadvantage of the early carbonylation methods was the use of toxic and flammable gaseous carbon monoxide. These procedures required careful handling and storage, and made carbonylation reactions rather inconvenient. In an endeavor to overcome the problems of working with gaseous carbon monoxide, alternative carbon monoxide sources delivering the gas in situ have been developed. Formate, formamide, aldehydes and metal carbonyls can all generate carbon monoxide in situ, and these methods benefit from low cost and preparative ease.128 A solid source of carbon monoxide, molyb-denum hexacarbonyl (Mo(CO)6) was used in this work for aminocarbonyla-tion reactions. Mo(CO)6 has been successfully used in various carbonylation reactions, at our department120,129-131 and others.128,132,133

F3C

Br

Br

65

7.2.2.2 Synthesis of MAP aryl amides (Paper VI) The N-methyl-amino pyridyl (MAP) amides were discovered to be acylating agents in 1978 by Meyer and Comins.134,135 Depending on the organometallic reagent applied, the MAP amides can be useful in the synthesis of different types of aldehydes, ketones, alcohols, amides and esters.135-137 As discussed above, aryl halides can be converted into amides by palladium-catalyzed carbonylation reactions with primary and secondary amines. We wanted to explore if this strategy could be applied in the synthesis of MAP aryl amides, using a CO-gas-free approach. In initial attempts to synthesize MAP amides from aryl halide precursors, the conditions used by our group for the prepa-ration of Weinreb amides from aryl iodides and O,N-dimethylhydroxylamine were examined.138 Hence, the reaction between 4-bromo-1,1´-biphenyl and 2-methylaminopyridine was studied using Pd(OAc)2 as the precatalyst, Xantphos as the ligand, K3PO4 as the base, Mo(CO)6 as the CO source, DMAP as an acyl-transfer reagent and microwave irradiation as the heating source. In the reaction the amine was used in excess relative to the aryl ha-lide. However, this resulted in only a moderate yield and incomplete conver-sion of the starting materials. Although attempts were made to optimize this procedure (by varying the time, temperature, solvent, catalyst and ligand) full conversion of the starting material was not achieved. We speculated that this was caused by the poor nucleophilicity of 2-methylaminopyridine, and the problem was addressed by adding the nucleophile at a large excess. To our surprise, this resulted in complete inhibition of the reaction, thus indicat-ing that the 2-methylaminopyridine coordinates to palladium and forms an inactive Pd–pyridine complex that decreases the availability of active Pd species in the reaction. By inverting the stoichiometric ratio, using an excess of the aryl halide, with respect to the amine, the MAP aryl amides could be obtained at satisfactory yields (41–92%). Gratifyingly, good yields could be obtained with both electronically and sterically diverse aryl bromides (Table 13).

66

Table 13. Microwave-assisted carbonylation of aryl bromides with 2-methylaminopyridine.

Entry Aryl bromide Product Isolated

yield (%)

1

61a 61

2

61b 93

3 61c 92

4

61d 80

5

61e 41

6

61f 79

7

61g 59

8

61h 92

9

61i 91

10

61j 85

Reaction conditions: 0.25 mmol 2-methylaminopyridine, 5.0 equiv. aryl bromide, 0.05 equiv. Pd(OAc)2, 0.1 equiv. Xantphos, 2 equiv. Mo(CO)6, 2 equiv. DMAP, 3 equiv. K3PO4, 1,4-dioxane (2 mL), 120 C, 30 min, MW.

The next step was to investigate aryl iodides instead of aryl bromides as coupling partners in the synthesis of MAP aryl amides. Since aryl iodides are more prone to undergo oxidative addition than aryl bromides, this could

N N

O

O

Ph

67

compensate for the catalyst poisoning effects of the amine, and therefore the aryl iodide was defined as the limiting reagent. After initial screening, MAP aryl amides could be obtained from electron-rich, electron-poor and sterical-ly demanding aryl iodides at moderate to good yields (40–69%, Table 14). However, the reaction required higher palladium catalyst loading than is normal for aryl iodide substrates. This can be attributed to trapping of the catalyst in the Pd–pyridine complex. In summary, MAP aryl amides could be obtained at good yields from a variety of different aryl halides, applying a palladium-catalyzed aminocarbonylation protocol using Mo(CO)6 as the source of the CO gas.

Table 14. Microwave-assisted carbonylation of aryl iodides with 2-methylaminopyridine.

Entry Aryl iodide Product

Isolated yield (%)

1

62a 59

2 62b 66a

69

3

62c 63

4

62d 40

5

62e 50

6

62f 57

7

62g 62

Reaction conditions: 0.25 mmol aryl iodide, 3 equiv. 2-methylaminopyridine, 0.1 equiv. Pd(OAc)2, 0.2 equiv. Xantphos, 0.5 equiv. Mo(CO)6, 3 equiv. K3PO4, 1,4-dioxane (2.5 mL), 100 C, 20 min, MW. a 1 equiv. Mo(CO)6.

N N

O

O

Ph

68

7.2.2.3 Aminocarbonylation using an amino acid as nucleophile The traditional method of forming an amide bond is to couple a carboxylic acid to an amine using coupling reagents, such as HATU or CDI, to generate in situ activation of the carboxylic acid. Converting the carboxylic acid into the more reactive acid chloride analog is also a well-known approach.

In our aspiration to produce SP1–7 mimetics incorporating an imidazole in the peptide backbone (Figure 27), we wanted to investigate whether an im-idazole halide could be coupled to an amino acid by aminocarbonylation with the formation of an amide bond. Wannberg et al. previously reported on aminocarbonylation of phenyl iodide with C-terminally protected or non-protected glycine and with free leucine.131 The protocol used Mo(CO)6 as the solid source of CO gas. The yields were moderate (46–60%) and no exam-ples with heteroaryl halides were reported. There is little in the literature on the use of 1,3-azole halides in carbonylation reactions. Letavic and Ly re-cently published a report on microwave-assisted palladium-catalyzed amino-carbonylation of hetero-aromatic bromides using Mo(CO)6.

139 However, they gave only two examples of 1,3-azole bromides. Thus, we wanted to investi-gate whether amino acids with a C-terminal primary amide could be coupled to an N-protected imidazole halide. In our initial investigation we combined the reaction conditions reported by Wannberg et al.131 and Letavic and Ly.139 Consequently, the reaction between N,N-dimethyl-4-iodo-1H-imidazole-1-sulfonamide and phenylalanine amide was studied using Pd(OAc)2 as preca-talyst, tri(tert-butylphosphonium)tetrafluoroborate ([(t-Bu)3PH]BF4) as li-gand, DBU as base, Mo(CO)6 as CO source and dioxane as solvent. The reaction was performed in sealed vessels using microwave heating for 5 minutes at different temperatures. When the reaction was run at 100 C for 5 minutes (Table 15, entry 3) compound 63 could be obtained at a yield of 42%. This corresponds well with the yields (50–60%) reported in the litera-ture for carbonylation with 1,3-azole halides.139,140

Having demonstrated that aminocarbonylation works with non-substituted imidazoles, we wanted to determine whether the procedure could be applied to more sterically congested imidazoles. In previous work, the sterically hindered 2,6-dimethylated aryl chloride could be coupled with the poor nuc-leophile aniline by aminocarbonylation giving an acceptable yield (65%).130 However, to the best of the author’s knowledge, sterically hindered het-eroaryl substrates have not been explored in carbonylation reactions, a fact that promoted attempts using the aryl-substituted imidazole 64. Screening of reaction conditions (time, temperature, Pd catalyst, ligand and acylating transfer agent) was performed, showing that product indeed could be formed, although at low yields (0–15%, Table 16).

69

Table 15. Screening of temperature for aminocarbonylation of N-protected imida-zole.

Entry Temperature ( C) Isolated yield (%)

1 60 n.d.a

2 80 25 3 100 42 4 120 40

5 140 34 Reaction conditions: 0.2 mmol N,N-dimethyl-4-iodo-1H-imidazole-1-sulfonamide, 2.5 equiv. phenylalanine amide hydrochloride, 0.1 equiv. Pd(OAc)2, 0.2 equiv. [(t-Bu)3PH]BF4, 1 equiv. Mo(CO)6, 6 equiv. DBU, 1,4-dioxane (1 mL), 5 min, MW. a Not determined because LC-MS showed very low conversion.

Table 16. Screening of reaction conditions for aminocarbonylation of sterically hindered imidazoles.

Entry [Pd] Ligand Ar-X Additive (2 equiv.)

Temp. ( C)

Time (min)

Isolated yield (%)

1 Herrmanns [(t-Bu)3PH]BF4 Br -- 170 30 11

2 Herrmanns [(t-Bu)3PH]BF4 Br -- 150 30 15 3 Herrmanns [(t-Bu)3PH]BF4 Br Imidazole 150 60 13 4 Herrmanns [(t-Bu)3PH]BF4 Br Imidazole 150 90 10 5 Pd(OAc)2 [(t-Bu)3PH]BF4 Br DMAP 150 30 15 6a Pd(OAc)2 Xantphos Br -- 140 45 0

7 Pd(OAc)2 [(t-Bu)3PH]BF4 I Imidazole 80 90 14 Reaction conditions: 1 equiv. aryl halide, 2 equiv. phenylalanine amide, 0.1 equiv. [Pd], 0.2 equiv. ligand, 1 equiv. Mo(CO)6, 6 equiv. DBU, MW. a 6 equiv. K3PO4 was used as base instead of DBU.

70

Due to the slower oxidative addition to the Pd(0)–complex of aryl bromides, with respect to aryl iodides, the temperature and the reaction time were in-creased. Herrmann’s catalyst141 was evaluated as palladium precatalyst in the aminocarbonylation step since it is known to be more thermostable than Pd(OAc)2 at temperatures above 150 C (Table 16, entries 1–4). However, this did not influence the reaction outcome. As mentioned above, the addi-tion of an acyl transfer agent can facilitate the aminocarbonylation of poor or sterically hindered nucleophiles, but no such effect was observed in the cur-rent transformation (Table 16, entries 3–5 and 7). The reason may be that the steric congestion caused by the ortho aryl group makes CO insertion the rate-limiting step.119 Switching ligands from [(t-Bu)3PH]BF4 to the one used in the protocol to obtain the MAP aryl amides, Xantphos, resulted in no product formation. Finally, the iodide analog was tested to ascertain whether this would be a better substrate, but the outcome was the same as for the bromo analog (Table 16, entry 7).

7.3 Application of the Developed Methods in the Synthesis of H-Phe-Phe-NH2 Mimetics The methods developed, i.e. the novel microwave-assisted direct arylation and an accessible aminocarbonylation with an amino acid using Mo(CO)6 as CO source, were applied in the synthesis of numerous H-Phe-Phe-NH2 ana-logs (Scheme 4). 1-Benzyl-1H-imidazole could be decorated with different aryl groups using the direct arylation protocol (Table 12) to yield compounds 66–70. In the next step, a bromine was introduced in the C4 position using a very small molar excess of N-bromosuccinimide (NBS) in MeCN at room temperature.123 The reaction is more likely to proceed via an electrophilic aromatic substitution rather than the radical mechanism usually involved when using NBS. The compounds 71–76 were obtained at very good yields, except for the more sterically congested 72. An attempt was made to intro-duce an iodine substituent in 70 using N-iodosuccinimide (NIS) in dichloro-methane (DCM), but this reaction was sluggish and required a reaction time of 4 days and a temperature of 40 C. The product (76) was only obtained at a 31% yield, which is much lower than for its bromo analog 75 (78%). After the successful introduction of a halide substituent in the C4 position of the imidazoles, subsequent aminocarbonylation with the phenylalanine amide was attempted in order to prepare the dipeptide mimetics 77–82. The reac-tion conditions used to obtain entry 5 in Table 16 were used, rendering the compounds at yields of 5–16% after RP-HPLC purification. Several attempts were made to improve the reaction outcome, but due to the sluggish CO insertion and difficult purification, these were not successful. However, the final products were produced at amounts sufficient for biological evaluation.

71

Scheme 4.

Benzyl deprotection of 81 furnished the more H-Phe-Phe-NH2-like com-pound 82. This was achieved by refluxing 81 with Pd(OH)2/C and HCOONH4 for 24 h, yielding 82 at 20% yield.123 Once again, the steric hin-drance of the ortho aryl substituent presumably influenced the reaction out-come negatively.

Scheme 5.

The synthesized analogs 77–82 will soon be biologically evaluated. Howev-er, compound 81 has been subjected to preliminary evaluation regarding its binding to the SP1–7 binding site, and the results were very promising (IC50 value = 26 nM). Despite its extra steric bulk from the benzyl protecting group, this analog shows only about 3 times lower binding affinity than compounds 26 and 54 (Table 17). Consequently, it appears that the imida-zole moiety is accepted in the binding site, and that the N-terminal phenyl

N

N

Ar

Ar-X, Pd(OAc)2, P(2-furyl)3

K2CO3, DMF, MW, 140 oCN

N

66, Ar=4-MeC6H4 (64%)67, Ar=2-MeC6H4 (57%)68, Ar=4-CF3C6H4 (56%)69, Ar=4-MeOC6H4 (32%)70, Ar=C6H5 (60%)

N

N

Ar

Br or I

N

NAr

HN

O

NH2

O

NBS, MeCN, rt

or

NIS, DCM, 40 oC

71, Ar=4-MeC6H4 (79%)72, Ar=2-MeC6H4 (33%)73, Ar=4-CF3C6H4 (81%)74, Ar=4-MeOC6H4 (98%)75, Ar=C6H5 (78% with Br)76, Ar=C6H5 (31% with I)

77, Ar=4-MeC6H4 (16%)78, Ar=2-MeC6H4 (9%)79, Ar=4-CF3C6H4 (7%)80, Ar=4-MeOC6H4 (5%)81, Ar=C6H5 (15% from 75)81, Ar=C6H5 (14% from 76)

Pd(OAc)2, [(t-Bu)3PH]BF4

Mo(CO)6, DBU, Dioxane,

150 oC, 30 min, MW

H2NNH2

O

72

group can be placed in the right position for important interactions with the target protein. It also appears that the 1,3-azole moiety may be a good non-peptidic scaffold for the design of future SP1–7 analogs.

Table 17. Binding affinity (IC50 values) of compounds 26, 54 and 81.

Compound Structure IC50 (nM)

26 9.8a

54 6.1b

81 26

a The IC50 value from the retesting of H-Phe-Phe-NH2, as described above. Corresponds to the Ki value = 8.4 nM. b The IC50 value corresponds to the Ki value = 5.2 nM.

7.4 Chapter Summary Two new methods were developed with the intention of synthesizing H-Phe-Phe-NH2 mimetics: the first, a fast microwave-assisted protocol for direct arylation of N-protected imidazole, and the second, an aminocarbonylation reaction utilizing a solid source of CO for incorporation of an imidazole moiety into the N-terminal of a peptide sequence. These methods generated six new H-Phe-Phe-NH2 mimetics, which will be biological evaluated re-garding their potential as SP1–7 analogs. Preliminary results seem promising. The use of a solid source of CO in aminocarbonylation reactions was further investigated for the synthesis of the acylating agent MAP amide. Various aryl bromides and aryl iodides could be coupled with 2-methylamino-pyridine to obtain numerous MAP amides at good yields.

73

8. Concluding Remarks

This thesis describes the first medicinal chemistry studies on the neuropep-tide SP1–7. The design and synthesis of SP1–7 analogs have led to extensive structure–activity and structure–property relationships. The optimization process is outlined below.

The initial SARs revealed that the C-terminal amino acids, and especially the C-terminal phenylalanine in both EM-2 and SP1–7, are crucial for binding affinity. Moreover, the C-terminal functional group should preferably be a primary amide.

74

Small SP1–7 analogs in the low-nanomolar range, incorporating a C-terminal primary amide, were obtained and resulted in the remarka-bly discovery of the new lead compound H-Phe-Phe-NH2 (26). The importance of the natural (S,S) configuration in these small analogs for high binding affinity was indicated by the decreased potency of analogs with different stereochemistry.

Rigidification in the N- or the C-terminal part of the dipeptide lead 26 rendered analogs with retained potency and improved cell per-meability and metabolic stability (e.g. 47).

Replacement of the basic N-terminal amine generated a new class of SP1–7 analogs with good binding affinity (e.g. 54). The advantages of the carbamate group compared to the phenylalanine regarding drug-like properties have been demonstrated in a detailed in vitro pharma-cokinetic study. These new analogs were shown to have substantial-ly increased cell permeability and significantly reduced efflux.

A convenient and robust carbonylative method for the preparation of MAP aryl amide acylating agents has been demonstrated. The proto-col provides MAP amides at good yields from both aryl iodides and aryl bromides.

A fast and expedient process for direct arylation of imidazoles, using a microwave-assisted protocol, has been developed. Furthermore, this method has been used together with a carbonylative method for coupling imidazole scaffolds with amino acids to generate novel H-Phe-Phe-NH2 mimetics.

The imidazole-based H-Phe-Phe-NH2 mimetic (81) was found to be a promising scaffold in the development of SP1–7 analogs.

In a future perspective the findings presented in this thesis, together with the fact that some of the generated SP1–7 analogs have been shown to reproduce the biological effects of SP1–7 in animal models, might result in future thera-peutic agents in the treatment of neuropathic pain and opiate abuse, and to overcome the problem with accompanying addiction.

75

Acknowledgements

The work presented in this thesis was definitively not a “one-girl job”, and I would like to express my sincere gratitude to the following people for making my PhD studies a lot easier and much more enjoyable:

Dr. Anja Sandström, my excellent supervisor. Thank you for accepting me as a PhD student. No matter what the question was, you always had a good answer, and you always had the time to discuss my problems. I have had many ideas and ongoing parallel projects during the years, and although I know you were sometimes a little worried about me, your trust, encouragement and wise guidance have given me valuable scientific training. You are a true role model!

Prof. Anders Hallberg, my co-supervisor, for being such an inspiration and for shar-ing your deep knowledge in medicinal chemistry with me. I am especially grateful for your support towards the end of this work, and the fact that your “med-chem eyes” never missed a thing. It was also a real pleasure to work with you on the Uni-versity Board and in the Academic Senate.

Dr. Christian Sköld, my co-supervisor, for taking on this project with such enthu-siasm, and for sharing your knowledge and skills in computational chemistry with me. Your support from “next door” during the writing of this thesis has been invalu-able.

Prof. Anders Karlén, my co-supervisor, for sharing your vast knowledge in medicin-al chemistry with me, and for good advices during our peptidomimetic group meet-ings. You are really a positive character.

My advisors and co-authors: Dr. Gunnar Lindeberg, for introducing me to peptide chemistry, for being such a good teacher, and for your skilful help in the lab, for always taking the time to answer and discuss my questions. You have given me tremendous support over the years. Dr. Mathias Hallberg, for always being so opti-mistic, for valuable discussions, and for taking your time to answer all my biology related questions.

I am sincerely grateful for all the hard work of my co-authors: Dr. Milad Botros for providing the binding data; Anna Carlsson, for providing me with all the in vivo

76

data, and for nice discussions, it was really nice to have a team mate; Prof. Fred Nyberg, for fruitful collaboration and for sharing you knowledge in the SP1–7 field; Jadel M Kratz, Richard Svensson and Prof. Per Arthursson, for performing the sta-bility and permeability tests on the compounds; Dr. Gunnar Nordvall for providing me with all the PK data.

In the palladium projects I would like to thank Prof. Mats Larhed for introducing me to the carbonylation reaction field, for all your good advices and for always taking time for questions and discussions. Dr. Anna Wi ckowska for being such a good chemist and collaborator and Dr. Luke Odell for all your input in the MAP project.

Rebecka Strand, Pontus Raak and Madeleine Bitar for skilful assistance during your diploma and summer work.

All staff at the department for your hard work and for maintaining the good working atmosphere. Gunilla Eriksson, for making this one of the best working places at the university, for all your support over the years and for just being you. Dr. Eva Åker-blom, for accepting me as a master student and for introducing me to medicinal chemistry. Sorin Sorbu, for being a great sysadmin. Dr. Uno Svensson for sharing your tremendous knowledge. Eva Strömberg for all the practical arrangement re-garding the upcoming defense.

Helen Sheppard, for excellent linguistic revision of my thesis. All linguistic mis-takes are certainly a result of own last minute changes. Gunnar, Johan, Luke and Anna L for constructive criticism and comments on my thesis. Per for technical assistance, what would I do without you?

Petra and Marco, for a great year in the PhD student´s Board. Petra you are a true source of joy and a good friend! Coco, Carl-Gustaf and Iréne, for all the good times in the Academic Senate.

Värmlands nation and the Swedish Pharmaceutical Society, for financially support-ing my participation in scientific meetings.

To all my past and present colleagues and friends for making this such an enjoyable working place. Thank you for all good times and fun memories. I would particularly like to express my gratitude to:

Hanna for being such a good friend and for the happy times in Montreal and NY, my credit card is still crying. Riina for making these years so memorable and fun, and for dragging me to the gym early in the morning. I really miss you here in Sweden! Per for taking such a good care of your little sister, especially in Copenhagen. Anna W and Krzysztof for all the crazy times and for putting me in the polish Trabant! See you in Canada soon. Anna-Karin for your consideration during these last

77

months. You are a true support of “nödraketer”! Kristina, Riina, and Anna L for a fabulous appearance at Vienna City Hall and for an unforgettable (!) night in the Bermuda triangle. Johan, Jonas and Jonas, for introducing us city girls to the wilder-ness of California. It was one of the best trips ever!

Rosa Klubben, i.e. Gunnar, Riina and Anna L, it should be pink Thursday with pink bubbles everyday! Maratongänget, i.e. Anneli, Anna L, Anna W, Krzysztof, one of the best and worse ideas ever! Running from Knivsta, seriously…

Anna L, my colleague and one of my best friends. What would these years been like without “balladlistan”, “sportpåsarna”, NY, and all the gossip?? Thank you for all your support, especially during these last months. You are invaluable!

Det finns även personer utanför detta jobb som jag vill tacka, alla mina kära vänner och släktingar som gör livet underbart. Speciellt,

Mamma och pappa för att ni alltid har trott på mig och stöttat mig i alla mina beslut. Det är tack vare er jag är där jag är idag. Jag älskar er, hur mycket som helst!

Benjamin för att du är den bästa lillebror man kan tänka sig. Tack för alla söndags-middagar med vin och fotboll som har lyst upp min tillvaro under dessa tunga må-nader och tack Karolin för att du förgyllde dem.

Anita, kan man ha en bättre svärmor? Tack för din ständiga omtanke, dina middagar har gett mig många behövliga pauser.

Maria och Marianne för att ni är världens finaste vänner! Tack för alla roliga stunder i Milano, Prag, Uppsala och Stockholm. Tur att jag har mina egna guider, hur skulle jag annars känna igen slottet! Vad blir det härnäst?

Lina, min vän! Tack för att du har funnits där i alla år, du betyder så otroligt mycket för mig…

Noel, the one and only! Dina fredagar, lördagar och söndagar (!) fyller mig med energi. Tack för allt ditt stöd under åren och speciellt nu i slutet. Vi gjorde det! Pöss

Olof, tack för att du alltid finns där och alltid ställer upp! Jag vet att jag inte har varit den lättaste personen att leva med de senaste månader men nu börjar äventyret i Montreal. Eftersom du själv aldrig kommer skriva en avhandling i Inters färger, så gör jag det….älskar dig!

Rebecca 24/3-2011

78

References

(1) Hökfelt, T.; Broberger, C.; Xu, Z. Q. D.; Sergeyev, V.; Ubink, R.; Diez, M. Neuropeptides - an overview. Neuropharmacology 2000, 39, 1337-1356.

(2) Hökfelt, T.; Bartfai, T.; Bloom, F. Neuropeptides: opportunities for drug discovery. Lancet neurology 2003, 2, 463-472.

(3) Sewald, N.; Jakubke. H.-D. Peptides: Chemistry and Biology. Wiley-VCH Verlag GmbH: Weinheim: 2002.

(4) Hökfelt, T. Neuropeptides in Perspective - the Last 10 Years. Neuron 1991, 7, 867-879.

(5) Madaan, V.; Wilson, D. R. Neuropeptides: relevance intreatment of depression and anxiety disorders. Drug News & Perspectives 2009, 22, 319-324.

(6) Kramer, M. S.; Cutler, N.; Feighner, J.; Shrivastava, R.; Carman, J.; Sramek, J. J.; Reines, S. A.; Liu, G.; Snavely, D.; Wyatt-Knowles, E.; Hale, J. J.; Mills, S. G.; MacCoss, M.; Swain, C. J.; Harrison, T.; Hill, R. G.; Hefti, F.; Scolnick, E. M.; Cascieri, M. A.; Chicchi, G. G.; Sadowski, S.; Williams, A. R.; Hewson, L.; Smith, D.; Carlson, E. J.; Hargreaves, R. J.; Rupniak, N. M. J. Distinct mechanism for antidepressant activity by blockade of central substance P receptors. Science 1998, 281, 1640-1645.

(7) Kreeger, J. S.; Larson, A. A. Substance P-(1-7), a substance P metabolite, inhibits withdrawal jumping in morphine-dependent mice. Eur. J. Pharmacol. 1993, 238, 111-115.

(8) Zhou, Q.; Carlsson, A.; Botros, M.; Fransson, R.; Sandström, A.; Gordh, T.; Hallberg, M.; Nyberg, F. The C-terminal amidated analogue of the Substance P (SP) fragment SP1-7 attenuates the expression of naloxone-precipitated withdrawal in morphine dependent rats. Peptides 2009, 30, 2418-2422.

(9) Carlsson, A.; Ohsawa, M.; Hallberg, M.; Nyberg, F.; Kamei, J. Substance P1-7 induces antihyperalgesia in diabetic mice through a mechanism involving the naloxone-sensitive sigma receptors. Eur. J. Pharmacol. 2010, 626, 250-255.

(10) Humphrey, M. J.; Ringrose, P. S. Peptides and Related Drugs - a Review of Their Absorption, Metabolism, and Excretion. Drug Metab. Rev. 1986, 17, 283-310.

(11) Veber, D. F.; Freidinger, R. M. The Design of Metabolically-Stable Peptide Analogs. Trends Neurosci. 1985, 8, 392-396.

(12) Olson, G. L.; Bolin, D. R.; Bonner, M. P.; Bos, M.; Cook, C. M.; Fry, D. C.; Graves, B. J.; Hatada, M.; Hill, D. E.; et al. Concepts and

79

progress in the development of peptide mimetics. J. Med. Chem. 1993, 36, 3039-49.

(13) Vagner, J.; Qu, H. C.; Hruby, V. J. Peptidomimetics, a synthetic tool of drug discovery. Curr. Opin. Chem. Biol. 2008, 12, 292-296.

(14) Ripka, A. S.; Rich, D. H. Peptidomimetic design. Curr. Opin. Chem. Biol. 1998, 2, 441-452.

(15) Hruby, V. J. Designing peptide receptor agonists and antagonists. Nat. Rev. Drug Discov. 2002, 1, 847-858.

(16) Grauer, A.; Konig, B. Peptidomimetics - A Versatile Route to Biologically Active Compounds. Eur. J. Org. Chem. 2009, 5099-5111.

(17) Vlieghe, P.; Lisowski, V.; Martinez, J.; Khrestchatisky, M. Synthetic therapeutic peptides: science and market. Drug Discov. Today 2010, 15, 40-56.

(18) Wiley, R. A.; Rich, D. H. Peptidomimetics Derived from Natural-Products. Med. Res. Rev. 1993, 13, 327-384.

(19) Rizo, J.; Gierasch, L. M. Constrained peptides: models of bioactive peptides and protein substructures. Annu. Rev. Biochem. 1992, 61, 387-418.

(20) Kim, H. O.; Kahn, M. A merger of rational drug design and combinatorial chemistry: Development and application of peptide secondary structure mimetics. Comb. Chem. High Throughput Screening 2000, 3, 167-183.

(21) Rosenström, U.; Sköld, C.; Lindeberg, G.; Botros, M.; Nyberg, F.; Karlen, A.; Hallberg, A. Design, synthesis, and incorporation of a beta-turn mimetic in angiotensin II forming novel pseudopeptides with affinity for AT(1) and AT(2) receptors. J.Med.Chem 2006, 49, 6133-6137.

(22) Schmidt, B.; Kuhn, C. Racemic, yet diastereomerically pure azido acids as both gamma-turn and inverse gamma-turn mimetics for solid-phase peptide synthesis. Synlett 1998, 1240-1242.

(23) Hanessian, S.; McNaughton-Smith, G.; Lombart, H.-G.; Lubell, W. D. Design and synthesis of conformationally constrained amino acids as versatile scaffolds and peptide mimetics. Tetrahedron 1997, 53, 12789-12854.

(24) Yu, K. L.; Johnson, R. L. Synthesis and chemical properties of tetrazole peptide analogs. J. Org. Chem. 1987, 52, 2051-9.

(25) Hitotsuyanagi, Y.; Motegi, S.; Fukaya, H.; Takeya, K. A cis Amide Bond Surrogate Incorporating 1,2,4-Triazole. J. Org. Chem. 2002, 67, 3266-3271.

(26) Hitotsuyanagi, Y.; Motegi, S.; Hasuda, T.; Takeya, K. Semisynthesis of an Analogue of Antitumor Bicyclic Hexapeptide RA-VII by Fixing the Ala-2/Tyr-3 Bond to Cis by Incorporating a Triazole cis-Amide Bond Surrogate. Org. Lett. 2004, 6, 1111-1114.

(27) Petit, S.; Fruit, C.; Bischoff, L. New Family of Peptidomimetics Based on the Imidazole Motif. Org. Lett. 2010, 12, 4928-4931.

80

(28) Abell, A. D. Heterocyclic-based peptidomimetics. Lett. Pept. Sci. 2001, 8, 267-272.

(29) Beusen, D. D.; Zabrocki, J.; Slomczynska, U.; Head, R. D.; Kao, J. L. F.; Marshall, G. R. Conformational Mimicry - Synthesis and Solution Conformation of a Cyclic Somatostatin Hexapeptide Containing a Tetrazole Cis Amide Bond Surrogate. Biopolymers 1995, 36, 181-200.

(30) Prentis, R. A.; Lis, Y.; Walker, S. R. Pharmaceutical Innovation by the 7 Uk-Owned Pharmaceutical Companies (1964-1985). Br. J. Clin. Pharmacol. 1988, 25, 387-396.

(31) Kerns, E. H.; Di, L. Drug-like Properties: Concepts, Structure Design and Methods. Elsevier Inc.: London, UK, 2008.

(32) Borchardt, R. T.; Kerns, E. H.; Lipinski, C. A.; Thakker, D. R.; Wang, B.; Editors. Pharmaceutical Profiling in Drug Discovery for Lead Selection. Arlington, VA: AAPS Press: 2004; p 451-466.

(33) van de Waterbeemd, H.; Smith, D. A.; Beaumont, K.; Walker, D. K. Property-based design: optimization of drug absorption and pharmacokinetics. J. Med. Chem. 2001, 44, 1313-1333.

(34) Wacher, V. J.; Silverman, J. A.; Zhang, Y. C.; Benet, L. Z. Role of P-glycoprotein and cytochrome P450 3A in limiting oral absorption of peptides and peptidomimetics. J. Pharm. Sci. 1998, 87, 1322-1330.

(35) Wang, B.; Nimkar, K.; Wang, W.; Zhang, H.; Shan, D.; Gudmundsson, O.; Gangwar, S.; Siahaan, T.; Borchardt, R. T. Synthesis and evaluation of the physicochemical properties of esterase-sensitive cyclic prodrugs of opioid peptides using coumarinic acid and phenylpropionic acid linkers. J. Pept. Res. 1999, 53, 370-382.

(36) Van de Waterbeemd, H.; Camenisch, G.; Folkers, G.; Chretien, J. R.; Raevsky, O. A. Estimation of blood-brain barrier crossing of drugs using molecular size and shape, and H-bonding descriptors. J. Drug Targeting 1998, 6, 151-165.

(37) Von Euler, U. S.; Gaddum, J. H. An unidentified depressor substance in certain tissue extracts. J. Physiol. 1931, 72, 74-87.

(38) Gaddum, J. H.; Schild, H. Depressor substances in extracts of intestine. J. Physiol. 1935, 83, 1-14.

(39) Chang, M. M.; Leeman, S. E.; Niall, H. D. Amino-Acid Sequence of Substance P. Nature-New Biol 1971, 232, 86-87.

(40) Erspamer, V. The tachykinin peptide family. Trends Neurosci. 1981, 4, 267-9.

(41) Harrison, S.; Geppetti, P. Substance P. Int. J. Biochem. Cell Biol. 2001, 33, 555-576.

(42) Otsuka, M.; Yoshioka, K. Neurotransmitter functions of mammalian tachykinins. Physiol. Rev. 1993, 73, 229-308.

(43) De Araujo, J. E.; Huston, J. P.; Brandao, M. L. Opposite effects of substance P fragments C (anxiogenic) and N (anxiolytic) injected

81

into dorsal periaqueductal gray. Eur. J. Pharmacol. 2001, 432, 43-51.

(44) Culman, J.; Unger, T. Central Tachykinins - Mediators of Defense Reaction and Stress Reactions. Can. J. Physiol. Pharm. 1995, 73, 885-891.

(45) Ebner, K.; Muigg, P.; Singewald, G.; Singewald, N. Substance P in stress and anxiety: NK-1 receptor antagonism interacts with key brain areas of the stress circuitry. Ann. N. Y. Acad. Sci. 2008, 1144, 61-73.

(46) Huston, J. P.; Hasenoehrl, R. U.; Boix, F.; Gerhardt, P.; Schwarting, R. K. W. Sequence-specific effects of neurokinin substance P on memory, reinforcement, and brain dopamine activity. Psychopharmacology 1993, 112, 147-62.

(47) Hasenohrl, R. U.; Gerhardt, P.; Huston, J. P. Naloxone blocks conditioned place preference induced by substance P and [pGlu6]-SP(6-11). Regul. Pept. 1991, 35, 177-87.

(48) Zubrzycka, M.; Janecka, A. Substance P: transmitter of nociception (Minireview). Endocr. Regul. 2000, 34, 195-201.

(49) Lee, C.-M.; Sandberg, B. E. B.; Hanley, M. R.; Iversen, L. L. Purification and characterization of a membrane-bound substance P-degrading enzyme from human brain. Eur. J. Biochem. 1981, 114, 315-327.

(50) Nyberg, F.; Le Greves, P.; Sundqvist, C.; Terenius, L. Characterization of substance P(1-7) and (1-8) generating enzyme in human cerebrospinal fluid. Biochem. Biophys. Res. Commun. 1984, 125, 244-50.

(51) Hallberg, M.; Nyberg, F. Neuropeptide conversion to bioactive fragments-an important pathway in neuromodulation. Curr. Protein Peptide Sci. 2003, 4, 31-44.

(52) Persson, S.; Le Greves, P.; Thoernwall, M.; Eriksson, U.; Silberring, J.; Nyberg, F. Neuropeptide converting and processing enzymes in the spinal cord and cerebrospinal fluid. Prog. Brain Res. 1995, 104, 111-30.

(53) Skilling, S. R.; Smullin, D. H.; Larson, A. A. Differential-Effects of C-Terminal and N-Terminal Substance-P Metabolites on the Release of Amino-Acid Neurotransmitters from the Spinal-Cord - Potential Role in Nociception. J. Neurosci. 1990, 10, 1309-1318.

(54) Sakurada, C.; Watanabe, C.; Sakurada, T. Occurrence of substance P(1-7) in the metabolism of substance P and its antinociceptive activity at the mouse spinal cord level. Methods Find. Exp. Clin. Pharmacol. 2004, 26, 171-176.

(55) Wiktelius, D.; Khalil, Z.; Nyberg, F. Modulation of peripheral inflammation by the substance P N-terminal metabolite substance P1-7. Peptides 2006, 27, 1490-1497.

(56) Zhou, Q.; Frandberg, P.-A.; Kindlundh, A. M. S.; Le Greves, P.; Nyberg, F. Substance P(1-7) affects the expression of dopamine D2

82

receptor mRNA in male rat brain during morphine withdrawal. Peptides 2003, 24, 147-153.

(57) Sakurada, T.; Le Greves, P.; Stewart, J.; Terenius, L. Measurement of substance P metabolites in rat CNS. J. Neurochem. 1985, 44, 718-722.

(58) Rimon, R.; Legreves, P.; Nyberg, F.; Heikkila, L.; Salmela, L.; Terenius, L. Elevation of Substance P-Like Peptides in the CSF of Psychiatric-Patients. Biol. Psychiat. 1984, 19, 509-516.

(59) Piercey, M. F.; Dobry, P. J. K.; Einspahr, F. J.; Schroeder, L. A.; Masiques, N. Use of Substance-P Fragments to Differentiate Substance-P Receptors of Different Tissues. Regul. Pept. 1982, 3, 337-349.

(60) Igwe, O. J.; Kim, D. C.; Seybold, V. S.; Larson, A. A. Specific binding of substance P aminoterminal heptapeptide [SP(1-7)] to mouse brain and spinal cord membranes. J. Neurosci. 1990, 10, 3653-3663.

(61) Botros, M.; Hallberg, M.; Johansson, T.; Zhou, Q.; Lindeberg, G.; Frändberg, P.-A.; Toemboely, C.; Toth, G.; Le Greves, P.; Nyberg, F. Endomorphin-1 and endomorphin-2 differentially interact with specific binding sites for substance P (SP) aminoterminal SP1-7 in the rat spinal cord. Peptides 2006, 27, 753-759.

(62) Zadina, J. E.; Hackler, L.; Ge, L.-J.; Kastin, A. J. A potent and selective endogenous agonist for the micro -opiate receptor. Nature 1997, 386, 499-502.

(63) Goldberg, I. E.; Rossi, G. C.; Letchworth, S. R.; Mathis, J. P.; Ryan-Moro, J.; Leventhal, L.; Su, W.; Emmel, D.; Bolan, E. A.; Pasternak, G. W. Pharmacological characterization of endomorphin-1 and endomorphin-2 in mouse brain. J. Pharmacol. Exp. Ther. 1998, 286, 1007-1013.

(64) Zadina, J. E. Isolation and distribution of endomorphins in the central nervous system. Jap. J. Pharmacol. 2002, 89, 203-208.

(65) Horvath, G. Endomorphin-1 and endomorphin-2: pharmacology of the selective endogenous mu-opioid receptor agonists. Pharmacol. Ther. 2000, 88, 437-63.

(66) Martin-Schild, S.; Zadina, J. E.; Gerall, A. A.; Vigh, S.; Kastin, A. J. Localization of endomorphin-2-like immunoreactivity in the rat medulla and spinal cord. Peptides 1997, 18, 1641-1649.

(67) Sanderson Nydahl, K.; Skinner, K.; Julius, D.; Basbaum Allan, I. Co-localization of endomorphin-2 and substance P in primary afferent nociceptors and effects of injury: a light and electron microscopic study in the rat. Eur. J. Neurosci. 2004, 19, 1789-1799.

(68) Pasternak, G. W.; Wood, P. J. Multiple mu opiate receptors. Life Sci. 1986, 38, 1889-98.

(69) Sakurada, S.; Hayashi, T.; Yuhki, M.; Orito, T.; Zadina, J. E.; Kastin, A. J.; Fujimura, T.; Murayama, K.; Sakurada, C.; Sakurada, T.; Narita, M.; Suzuki, T.; Tan-no, K.; Tseng, L. F. Differential antinociceptive effects induced by intrathecally administered

83

endomorphin-1 and endomorphin-2 in the mouse. Eur. J. Pharmacol. 2001, 427, 203-210.

(70) Tseng, L. F.; Narita, M.; Suganuma, C.; Mizoguchi, H.; Ohsawa, M.; Nagase, H.; Kampine, J. P. Differential antinociceptive effects of endomorphin-1 and endomorphin-2 in the mouse. J. Pharmacol. Exp. Ther. 2000, 292, 576-583.

(71) Muthas, D.; Lek, P. M.; Nurbo, J.; Karlen, A.; Lundstedt, T. Focused hierarchical design of peptide libraries-follow the lead. J. Chemom. 2007, 21, 486-495.

(72) Merrifield, R. B. Solid Phase Peptide Synthesis .1. Synthesis of a Tetrapeptide. J. Am. Chem. Soc. 1963, 85, 2149-&.

(73) Atherton, E.; Fox, H.; Harkiss, D.; Logan, C. J.; Sheppard, R. C.; Williams, B. J. A mild procedure for solid phase peptide synthesis: use of fluorenylmethoxycarbonylamino-acids. J. Chem. Soc. 1978, 537-9.

(74) Okada, Y.; Fujita, Y.; Motoyama, T.; Tsuda, Y.; Yokoi, T.; Li, T.; Sasaki, Y.; Ambo, A.; Jinsmaa, Y.; Bryant Sharon, D.; Lazarus Lawrence, H. Structural studies of [2',6'-dimethyl-L-tyrosine1]endomorphin-2 analogues: enhanced activity and cis orientation of the Dmt-Pro amide bond. Biorg. Med. Chem. 2003, 11, 1983-1994.

(75) Kruszynski, R.; Fichna, J.; do-Rego, J.-C.; Janecki, T.; Kosson, P.; Pakulska, W.; Costentin, J.; Janecka, A. Synthesis and biological activity of N-methylated analogs of endomorphin-2. Biorg. Med. Chem. 2005, 13, 6713-6717.

(76) Fichna, J.; Janecka, A.; Costentin, J.; Do Rego, J.-C. The endomorphin system and its evolving neurophysiological role. Pharmacol. Rev. 2007, 59, 88-123.

(77) Boden, P.; Eden, J. M.; Hodgson, J.; Horwell, D. C.; Howson, W.; Hughes, J.; McKnight, A. T.; Meecham, K.; Pritchard, M. C.; et al. The rational development of small molecule tachykinin NK3 receptor selective antagonists - the utilization of a dipeptide chemical library in drug design. Bioorg. Med. Chem. Lett. 1994, 4, 1679-1684.

(78) Boden, P.; Eden, J. M.; Hodgson, J.; Horwell, D. C.; Pritchard, M. C.; Raphy, J.; Suman-Chauhan, N. The development of a novel series of non-peptide tachykinin NK3 receptor selective antagonists. Bioorg. Med. Chem. Lett. 1995, 5, 1773-1778.

(79) Anthes, J. C.; Chapman, R. W.; Richard, C.; Eckel, S.; Corboz, M.; Hey, J. A.; Fernandez, X.; Greenfeder, S.; McLeod, R.; Sehring, S.; Rizzo, C.; Crawley, Y.; Shih, N. Y.; Piwinski, J.; Reichard, G.; Ting, P.; Carruthers, N.; Cuss, F. M.; Billah, M.; Kreutner, W.; Egan, R. W. SCH 206272: a potent, orally active tachykinin NK1, NK2, and NK3 receptor antagonist. Eur. J. Pharmacol. 2002, 450, 191-202.

(80) Heuillet, E.; Menager, J.; Fardin, V.; Flamand, O.; Bock, M.; Garret, C.; Crespo, A.; Fallourd, A. M.; Doble, A. Characterization of a

84

Human Nk1 Tachykinin Receptor in the Astrocytoma Cell-Line U-373 Mg. J. Neurochem. 1993, 60, 868-876.

(81) Nyberg, F.; Hallberg, A.; Hallberg, M.; Sandström, A.; Fransson, R. Therapeutic methods and compositions employing peptide compounds. 2009-IB530202010004535, 20090710, 2010.

(82) Ohsawa, M.; Carlsson, A.; Koizumi, T.; Asato, M.; Fransson, R.; Sandström, A.; Hallberg, M.; Nyberg, F.; Kamei, J. Changes of Opioidergic Systems in Diabetic Painful Neuropathy. In INRC 2010, Malmö, Sweden, 2010.

(83) Ohsawa, M.; Carlsson, A.; Asato, M.; Koizumi, T.; Nakanishi, Y.; Fransson, R.; Sandström, A.; Hallberg, M.; Nyberg, F.; Kamei, J. The effect of substance P1-7 amide on nociceptive threshold in diabetic mice. Peptides 2011, 32, 93-98.

(84) Fransson, R.; Carlsson, A.; Sköld, C.; Nyberg, F.; Hallberg, M.; Sandström, A. Design and Synthesis of Small Substance P (1-7) Mimetics. In INRC 2010, Malmö, Sweden, 2010.

(85) Brandsch, M. Transport of drugs by proton-coupled peptide transporters: pearls and pitfalls. Expert Opin. Drug Met. 2009, 5, 887-905.

(86) Brodin, B.; Nielsen, C. U.; Steffansen, B.; Frokjaer, S. Transport of peptidomimetic drugs by the intestinal di/tri-peptide transporter, PepT1. Pharmacol. Toxicol. 2002, 90, 285-296.

(87) Rubio-Aliaga, I.; Daniel, H. Mammalian peptide transporters as targets for drug delivery. Trends Pharmacol. Sci. 2002, 23, 434-440.

(88) Witt, K. A.; Gillespie, T. J.; Huber, J. D.; Egleton, R. D.; Davis, T. P. Peptide drug modifications to enhance bioavailability and blood-brain barrier permeability. Peptides 2001, 22, 2329-2343.

(89) Giacomini, K. M.; Huang, S. M.; Tweedie, D. J.; Benet, L. Z.; Brouwer, K. L. R.; Chu, X. Y.; Dahlin, A.; Evers, R.; Fischer, V.; Hillgren, K. M.; Hoffmaster, K. A.; Ishikawa, T.; Keppler, D.; Kim, R. B.; Lee, C. A.; Niemi, M.; Polli, J. W.; Sugiyama, Y.; Swaan, P. W.; Ware, J. A.; Wright, S. H.; Yee, S. W.; Zamek-Gliszczynski, M. J.; Zhang, L.; Transporter, I. Membrane transporters in drug development. Nat. Rev. Drug Discov. 2010, 9, 215-236.

(90) Phase, 3.1; Schrödinger, LLC: New York, NY, 2009. (91) Maestro, 9.0; Schrödinger, LLC: New York, NY, 2009. (92) Houston, J. B. Utility of in-Vitro Drug-Metabolism Data in

Predicting in-Vivo Metabolic-Clearance. Biochem. Pharmacol. 1994, 47, 1469-1479.

(93) Obach, R. S. Prediction of human clearance of twenty-nine drugs from hepatic microsomal intrinsic clearance data: An examination of in vitro half-life approach and nonspecific binding to microsomes. Drug Metab. Disposition 1999, 27, 1350-1359.

(94) Hubatsch, I.; Ragnarsson, E. G. E.; Artursson, P. Determination of drug permeability and prediction of drug absorption in Caco-2 monolayers. Nature Protocols 2007, 2, 2111-2119.

85

(95) Stewart, B. H.; Chan, O. H.; Lu, R. H.; Reyner, E. L.; Schmid, H. L.; Hamilton, H. W.; Steinbaugh, B. A.; Taylor, M. D. Comparison of Intestinal Permeabilities Determined in Multiple in-Vitro and in-Situ Models - Relationship to Absorption in Humans. Pharm. Res. 1995, 12, 693-699.

(96) Bergström, C. A. S.; Strafford, M.; Lazorova, L.; Avdeef, A.; Luthman, K.; Artursson, P. Absorption classification of oral drugs based on molecular surface properties. J. Med. Chem. 2003, 46, 558-570.

(97) Chen, X.; Wang, W. The use of bioisosteric groups in lead optimization. Annu. Rep. Med. Chem. 2003, 38, 333-346.

(98) Thornber, C. W. Isosterism and Molecular Modification in Drug Design. Chem. Soc. Rev. 1979, 8, 563-580.

(99) Brain, C. T.; Paul, J. M.; Loong, Y.; Oakley, P. J. Novel procedure for the synthesis of 1,3,4-oxadiazoles from 1,2-diacylhydrazines using polymer-supported Burgess reagent under microwave conditions. Tetrahedron Lett. 1999, 40, 3275-3278.

(100) Alelyunas, Y. W.; Liu, R. F.; Pelosi-Kilby, L.; Shen, C. Application of a Dried-DMSO rapid throughput 24-h equilibrium solubility in advancing discovery candidates. Eur. J. Pharm. Sci. 2009, 37, 172-182.

(101) Sohlenius-Sternbeck, A. K. Determination of the hepatocellularity number for human, dog, rabbit, rat and mouse livers from protein concentration measurements. Toxicol. In Vitro 2006, 20, 1582-1586.

(102) Sohlenius-Sternbeck, A. K.; Afzelius, L.; Prusis, P.; Neelissen, J.; Hoogstraate, J.; Johansson, J.; Floby, E.; Bengtsson, A.; Gissberg, O.; Sternbeck, J.; Petersson, C. Evaluation of the human prediction of clearance from hepatocyte and microsome intrinsic clearance for 52 drug compounds. Xenobiotica 2010, 40, 637-649.

(103) Gordon, T.; Hansen, P.; Morgan, B.; Singh, J.; Baizman, E.; Ward, S. Peptide Azoles - a New Class of Biologically-Active Dipeptide Mimetics. Bioorg. Med. Chem. Lett. 1993, 3, 915-920.

(104) Gedye, R.; Smith, F.; Westaway, K.; Ali, H.; Baldisera, L.; Laberge, L.; Rousell, J. The use of microwave ovens for rapid organic synthesis. Tetrahedron Lett. 1986, 27, 279-82.

(105) Giguere, R. J.; Bray, T. L.; Duncan, S. M.; Majetich, G. Application of commercial microwave ovens to organic synthesis. Tetrahedron Lett. 1986, 27, 4945-8.

(106) Kappe, C. O. Synthetic methods. Controlled microwave heating in modern organic synthesis. Angew. Chem., Int. Ed. 2004, 43, 6250-6284.

(107) Lidstrom, P.; Tierney, J.; Wathey, B.; Westman, J. Microwave assisted organic synthesis - a review. Tetrahedron 2001, 57, 9225-9283.

(108) Wathey, B.; Tierney, J.; Lidstrom, P.; Westman, J. The impact of microwave-assisted organic chemistry on drug discovery. Drug Discov. Today 2002, 7, 373-380.

86

(109) Satoh, T.; Miura, M. Catalytic direct arylation of heteroaromatic compounds. Chem. Lett. 2007, 36, 200-205.

(110) Seregin, I. V.; Gevorgyan, V. Direct transition metal-catalyzed functionalization of heteroaromatic compounds. Chem. Soc. Rev. 2007, 36, 1173-1193.

(111) Liegault, B.; Lapointe, D.; Caron, L.; Vlassova, A.; Fagnou, K. Establishment of Broadly Applicable Reaction Conditions for the Palladium-Catalyzed Direct Arylation of Heteroatom-Containing Aromatic Compounds. J. Org. Chem. 2009, 74, 1826-1834.

(112) Ames, D. E.; Opalko, A. Palladium-Catalyzed Cyclization of 2-Substituted Halogenoarenes by Dehydrohalogenation. Tetrahedron 1984, 40, 1919-1925.

(113) Nakamura, N.; Tajima, Y.; Sakai, K. Direct Phenylation of Isoxazoles Using Palladium Catalysts Synthesis of 4-Phenylmuscimol. Heterocycles 1982, 17, 235-245.

(114) Alberico, D.; Scott, M. E.; Lautens, M. Aryl-aryl bond formation by transition-metal-catalyzed direct arylation. Chem. Rev. 2007, 107, 174-238.

(115) Gorelsky, S. I.; Lapointe, D.; Fagnou, K. Analysis of the concerted metalation-deprotonation mechanism in palladium-catalyzed direct arylation across a broad range of aromatic substrates. J. Am. Chem. Soc. 2008, 130, 10848-10849.

(116) Schoenbe.A; Bartolet.I; Heck, R. F. Palladium-Catalyzed Carboalkoxylation of Aryl, Benzyl, and Vinylic Halides. J. Org. Chem. 1974, 39, 3318-3326.

(117) Schoenbe.A; Heck, R. F. Palladium-Catalyzed Amidation of Aryl, Heterocyclic, and Vinylic Halides. J. Org. Chem. 1974, 39, 3327-3331.

(118) Brennfuehrer, A.; Neumann, H.; Beller, M. Palladium-Catalyzed Carbonylation Reactions of Aryl Halides and Related Compounds. Angew. Chem. Int. Ed. 2009, 48, 4114-4133.

(119) Barnard, C. F. J. Palladium-Catalyzed Carbonylation-A Reaction Come of Age. Organometallics 2008, 27, 5402-5422.

(120) Odell, L. R.; Savmarker, J.; Larhed, M. Microwave-promoted aminocarbonylation of aryl triflates using Mo(CO)(6) as a solid CO source. Tetrahedron Lett. 2008, 49, 6115-6118.

(121) Lundstedt, T.; Seifert, E.; Abramo, L.; Thelin, B.; Nystrom, A.; Pettersen, J.; Bergman, R. Experimental design and optimization. Chemometrics Intellig. Lab. Syst. 1998, 42, 3-40.

(122) Eriksson, L.; Johansson, E.; Kettaneh-Wold, N.; Wikström, C.; Wold, S. Design of experiments. Principle and applications. 3 ed.; MKS Umetrics AB: Umeå, 2008.

(123) Bellina, F.; Cauteruccio, S.; Di Fiore, A.; Marchetti, C.; Rossi, R. Highly selective synthesis of 4(5)-aryl-, 2,4(5)-diaryl-, and 4,5-diaryl-1H-imidazoles via Pd-catalyzed direct C-5 arylation of 1-benzyl-1H-imidazole. Tetrahedron 2008, 64, 6060-6072.

87

(124) Bellina, F.; Cauteruccio, S.; Di Fiore, A.; Rossi, R. Regioselective synthesis of 4,5-diaryl-1-methyl-1H-imidazoles including highly cytotoxic derivatives by Pd-catalyzed direct C-5 arylation of 1-methyl-1H-imidazole with aryl bromides. Eur. J. Org. Chem. 2008, 5436-5445.

(125) Bellina, F.; Cauteruccio, S.; Rossi, R. Synthesis and biological activity of vicinal diaryl-substituted 1H-imidazoles. Tetrahedron 2007, 63, 4571-4624.

(126) Larhed, M.; Hallberg, A. Microwave-assisted high-speed chemistry: a new technique in drug discovery. Drug Discov. Today 2001, 6, 406-416.

(127) Larhed, M.; Moberg, C.; Hallberg, A. Microwave-accelerated homogeneous catalysis in organic chemistry. Acc. Chem. Res. 2002, 35, 717-727.

(128) Morimoto, T.; Kakiuchi, K. Evolution of carbonylation catalysis: no need for carbon monoxide. Angew. Chem., Int. Ed. 2004, 43, 5580-5588.

(129) Georgsson, J.; Hallberg, A.; Larhed, M. Rapid palladium-catalyzed synthesis of esters from aryl halides utilizing Mo(CO)(6) as a solid carbon monoxide source. J. Comb. Chem. 2003, 5, 350-352.

(130) Lagerlund, O.; Larhed, M. Microwave-promoted aminocarbonylations of aryl chlorides using Mo(CO)(6) as a solid carbon monoxide source. J. Comb. Chem. 2006, 8, 4-6.

(131) Wannberg, J.; Larhed, M. Increasing rates and scope of reactions: Sluggish amines in microwave-heated aminocarbonylation reactions under air. J. Org. Chem. 2003, 68, 5750-5753.

(132) Liptrot, D.; Alcaraz, L.; Roberts, B. New Synthesis of Aryl and Heteroaryl N-Acylureas via Microwave-Assisted Palladium-Catalysed Carbonylation. Adv. Synth. Catal. 2010, 352, 2183-2188.

(133) Liptrot, D.; Alcaraz, L.; Roberts, B. Microwave-assisted palladium-catalysed carbonylations of aryl and heteroaryl halides with sulfamide nucleophiles utilising a solid CO source. Tetrahedron Lett. 2010, 51, 5341-5343.

(134) Comins, D.; Meyers, A. I. Facile and Efficient Formylation of Grignard-Reagents. Synthesis 1978, 403-404.

(135) Meyers, A. I.; Comins, D. L. N-Methylamino Pyridyl Amides (MAPA)-Efficient Acylating Agent for Various Nucleophiles and Sequential Addition to Unsymmetrical Tertiary-Alcohols. Tetrahedron Lett. 1978, 5179-5182.

(136) Meyers, A. I.; Babiak, K. A.; Campbell, A. J.; Comins, D. L.; Fleming, M. P.; Henning, R.; Heuschmann, M.; Hudspeth, J. P.; Kane, J. M.; Reider, P. J.; Roland, D. M.; Shimizu, K.; Tomioka, K.; Walkup, R. D. Total Synthesis of (-)-Maysine. J. Am. Chem. Soc. 1983, 105, 5015-5024.

(137) Comins, D. L.; Dernell, W. A One Pot Synthesis of Unsymmetrical Secondary Alcohols from 2 Grignard-Reagents. Tetrahedron Lett. 1981, 22, 1085-1088.

88

(138) Wieckowska, A.; Fransson, R.; Odell, L. R.; Larhed, M. Microwave-Assisted Synthesis of Weinreb and MAP Aryl Amides via Pd-Catalyzed Heck Aminocarbonylation Using Mo(CO)(6) or W(CO)(6). J. Org. Chem. 2011, 76, 978-981.

(139) Letavic, M. A.; Ly, K. S. Microwave assisted, palladium catalyzed aminocarbonylations of heteroaromatic bromides using solid Mo(CO)6 as the carbon monoxide source. Tetrahedron Lett. 2007, 48, 2339-2343.

(140) Roberts, B.; Liptrot, D.; Alcaraz, L. Novel Aryl and Heteroaryl Acyl Sulfamide Synthesis via Microwave-Assisted Palladium-Catalyzed Carbonylation. Org. Lett. 2010, 12, 1264-1267.

(141) Beller, M.; Fischer, H.; Herrmann, W. A.; Oefele, K.; Brossmer, C. Coordination chemistry and mechanism of metal-catalyzed C-C coupling reactions. Part 6. Palladacycles as efficient catalysts for aryl coupling reactions. Angew. Chem. Int. Ed. 1995, 34, 1848-9.