SYNTHESIS, CHARACTERIZATION AND BIOLOGICAL STUDIES OF NEW FERROCENE BASED GUANIDINES  

 

  

 

A DISSERTATION SUBMITTED TO THE DEPARTMENT OF CHEMISTRY, GOMAL UNIVERSITY, DERA ISMAIL KHAN, IN PARTIAL FULFILLMENT

OF THE REQUIREMENTS FOR THE DEGREE OF  

 

Doctor of Philosophy

In

Chemistry

By

Rukhsana Gul Department of Chemistry

Gomal University, Dera Ismail Khan 2013

SYNTHESIS, CHARACTERIZATION AND BIOLOGICAL STUDIES OF NEW FERROCENE BASED GUANIDINES

 

 

 

 

 

 

 

By

Rukhsana Gul  

Department of Chemistry Gomal University, Dera Ismail Khan

2013

In the Name of ALLAH,

The most Gracious, The most Merciful

  

Contents Page Acknowledgement I

List of Figures III

List of Schemes VIII

List of Tables X

List of Abbreviation XI

Abstract XII

Chapter-1 Introduction 1-35 1 Introduction to guanidines 1

1.1 Methods for the synthesis of substituted guanidines 2

1.1.1 Guanylation methods for the synthesis of guanidines 2

1.1.1.1 Synthesis of guanidines from thiourea 2

1.1.1.2 Synthesis of guanidines from isothiourea 4

1.1.1.3 Synthesis of guanidines from carbodiimdes 5

1.1.1.4 Synthesis of guanidines from triazoles 7

1.1.1.5 Synthesis of cyclic guanidines 7

1.1.1.6 Synthesis of acyl guanidines 8

1.1.1.7 Synthesis of guanidines from amino amides 8

1.1.2 Guanidinylation methods for the synthesis of guanidines 9

1.1.2.1 Through carboxamidine reagents 9

1.1.2.2 Through alkyl halide 9

1.1.2.3 Through alcohols 10

1.1.2.4 Through esters, to form cyclic guanidines 10

1.2 Applications of guanidines 11

1.2.1 Biological applications 11

1.2.2 Non biological applications of guanidines 16

1.2.3 Supramolecular chemistry of guanidines 17

  

1.3 Introduction to ferrocene derivatives 19

1.3.1 Biological applications of ferrocene derivatives 19

1.3.2 Non biological applications 21

1.4 Drug-DNA interaction study 23

1.4.1 Techniques to study non-covalent interaction 26

1.4.1.1 DNA binding study by UV-Visible spectroscopy 26

1.4.1.2 DNA binding study by cyclic voltammetery 27

Aims of study 29

References 30

Chapter-2 Experimental and Charracterization 36-76 2.1 Chemicals 36

2.2 Instrumentation 36

2.3 Synthesis of ferrocenyl guanidines 37

2.3.1 Synthesis of nitrophenyl ferrocene (a & b) 37 2.3.2 Synthesis of ferrocenylanilines (c & d) 38 2.3.3 Synthesis of N, N disubstituted phenylthioureas. 40

2.3.3.1 Phenylthioureas derived from benzoic acid (e1-18) 40 2.3.3.2 Phenylthioureas derived from chlorobenzoic acid (f1-6) 41 2.3.4 Synthesis of ferrocenyl guanidines from benzoylthioureas 42

2.3.4.1 para-Ferrocenylguanidines (g1-18) 43 2.3.4.2 meta-Ferrocenylguanidines (h1-18) 54 2.3.5 Synthesis of ferrocenyl guanidines from chlorobenzoyl thioureas. 64

2.3.5.1 para-Ferrocenyl guanidines (i1-6) 64 2.3.5.2 meta-Ferrocenyl guanidines (j1-6) 68 2.3.6 Synthesis of tetra-substituted ferroceylguanidine 71

2.3.6.1 Synthesis of tetra substituted N-isopropyl-N-(4-ferrocenylphenyl)-

N'-(2,6-diethylphenyl)-N''-benzoylguanidine (k-1) 71

2.4 DNA binding study 72

2.5 DPPH free radical scavenging activity 73

2.6 Antibacterial assay 74

2.7 Antifungal assay 74

  

References 76

Chapter-3 Results and Discussion 77-1043.1 Syntheses 77

3.2 Elemental analysis 79

3.3 UV-Visible Spectroscopy 79

3.4 Cyclic Voltammetery 80

3.5 FT-IR Spectroscopy 82

3.6 Multi-nuclear (1H, 13C) NMR Spectroscopy 82

3.7 Single crystal X-ray diffraction analysis and supramolecular

chemistry

83

3.7.1 N-(4-ferrocenylphenyl)-N’-(3-triflourometylphenyl)-N”-

benzoylguanidine (g-2) 84

3.7.2 N-(4-ferrocenylphenyl)-N’-(4-methylphenyl)-N”-benzoylguanidine

(g-5) 88

3.7.3 N-(4-ferrocenylphenyl-N’-(2-chlorophenyl)-N”-benzoylguanidine

(g-10) 91

3.7.4 N-(4-ferrocenylphenyl-N’-(2,6-dichlorophenyl)-N”-

benzoylguanidine (g-14) 94

3.7.5 N-(3-ferrocenylphenyl-N’-(2,3-dichlorophenyl)-N”-

benzoylguanidine (h-11) 96

3.7.6 N-(4-ferrocenylphenyl-N'-(2,3-dichlorophenyl)-N"-(3-

chlorobenzoyl) guanidine (i-3) 98

3.7.7 N-isopropyl-N-(4-ferrocenylphenyl)-N'-(2,6-diethylphenyl)-N''-

benzoylguanidine (k-1) 101

References 104

Chapter-4 Biological Screening 105-127

4 Biological screening 105

4.1 DNA Binding studies 105

4.1.1 DNA binding study by UV-Visible spectrophotometer 105

4.1.2 DNA binding study by Cyclic Voltammetry 111

4.2 Antioxidant activity 119

  

4.3 In vitro antibacterial activity 121

4.4 In vitro antifungal activity 123

Conclusions 126

Future plans 126

References 127

List of publications 128

I  

Acknowledgement

First of All I bow down my head to the Omniscient, Omnipotent and Omnipresent AL-

MIGHTY ALLAH Who provided me the opportunity of exploring texture of His

natural beauties at the molecular level, and all respects for the Holy Prophet Hazrat

Muhammad (Peace be upon him) for enlightening our conscious with essence of faith

in ALLAH, covering all His kindness and mercy upon him.

I wish to express vehement sense of thankfulness to my supervisor, Prof. Dr. Azim

Khan, Chairman, Department of Chemistry, Gomal University, Dera Ismail Khan, for

his enthusiastic interest and support.

I owe a deep depth of heartiest regards to my affectionate research co-supervisor Prof.

Dr.Amin Badshah, Chairman Department of Chemistry, Quaid-i-Azam University,

Islamabad, whose excellent supervision, valuable suggestions, constant encouragement

and precious attentions throughout the course of these investigations resulted in the

successful completion of this research work. I am also very thankful for his valuable

support during my stay at the Department of Chemistry, Quaid-i-Azam University,

Islamabad.

I would also like to extend a wholehearted thanks to Prof. Dr. Saqib Ali, Prof. Dr.

Javed Hassan Zaidi, Prof. Dr. Siddiq, Dr. Afzal Shah and Dr. Zia-ur-Rahman for

their guidance and Prof. Dr. Nawaz Tahir for single crystal analyses.

I would like to express my deepest gratitude to Dr.Asghari Bano and Rabia Naz for

carrying out microbial activities.

My special thanks are extended to my lab fellows for providing me valuable assistance,

moral supports and many smiling moments during my stay at the Department of

Chemistry, Quai-i-Azam University Islamabad.

I am thankful to all the technical and non-technical staff of the Department of Chemistry Quaid-i-Azam University as well as Gomal University, especially Mr Shareef Chohan,

II  

Mr Shamas Perviz, Mr Naqeeb ullah and Mr Ramzan Babar who helped me in the

completion of my research work and thesis.

I highly appreciate Higher Education Commission (HEC) of Pakistan for financial

support.

I would like to say special thanks to Mrs Mumtaz Amin and my cousin Mrs Abida

Kalsoom for their kind and loving support during my stay in Islamabad.

I would like to express my heartiest gratitude and regards to my Mother,, my sister

Miss Saima Hayat, my Mother in Law, my brother in law Muhammad Arsalan, my

uncle Mr Javed Iqbal and my sweet daughters Manal Gul, Bushra Nawal and my

whole family. Their prayers love and support over the years has enabled me to complete

my task. Special thanks to my husband Dr.Asif Junaid for his love and caring attitude.

Without his co-operation it was impossible for me to achieve my target.

My cordial thanks to all those who helped me and pray for me.

RUKHSANA GUL

III  

List of figures

Figure Title Page 1.1 Resonance structures of conjugate acid of guanidine 1

1.2 pKa of some of the common guanidine 1

1.3 Drugs for the treatment of ulcer 11

1.4 Drugs for the treatment of influenza 11

1.5 Drugs to controll the sugar level in body 11

1.6 Amiloride, an antihypertensive drug 12

1.7 Drugs used to control blood pressure 12

1.8 Tree diagram of the cancer causes 13

1.9 Anticancer Netamines C 13

1.10 Some other organic compounds having anticancer activity 14

1.11 (a) Variolin, (b) Meriolin 14

1.12 Chlorohexadine, an effective antibiotic 15

1.13 Pyrrolidinebis-cyclic guanidine 15

1.14 Different damage take place in the body as a result of free radicals 15

1.15 Mercaptoethylguanidine, A free radical scavenger. 16

1.16 Guanidine derivatives, promising candidates for artificial sweeteners 17

1.17 Guanidine salts used as ionic liquids or as catalyst 17

1.18 Non-covalent interactions responsible for the formation of a

supramolecule

18

1.19 Guanidine based dendrimers 18

1.20 Ferrocene derivatives used as antibiotics 19

1.21 Ferrocene analog of tamoxifin (a) used as anticancer agents 20

1.22 Water soluble anticancer ferrocenyl derivatives 20

1.23 Anticancer agents in the form of salts 20

1.24 Antimalarial ferrocene derivative 21

1.25 Ferrocerone 21

1.26 Ferrocenyl derivatives as catalysts 21

1.27 Ferrocenyl derivative as corrosion inhibitors 22

IV  

1.28 Ferrocenyl derivative as biosensors 22

1.29 Biosencers 22

1.30 Glucose biosencers 23

1.31 Structure of DNA molecule 24

1.32 Different types of interactions by which a drug affects the DNA 25

1.33 Netropsine 25

1.34 Different analytical schemes used to study the DNA Binding potency of

a compound

26

2.1 Different (R1) groups used in the synthesis of benzoylthiourea 41 2.2 Different (R1) groups used in the synthesis of chlorobenzoyl

phenylthiourea

42

3.1 Comparison of the UV-Visible spectra of different ferrocenyl guanidines 80

3.2 Cyclic voltammogram of ferrocenyl guanidine (g-1) 81 3.3 a) Voltamograms of 2 mM and b) 4 mM solutions of different ferrocenyl

guanidines in DMSO

82

3.4 ORTEP diagram of compound (g-2) with atomic numbering scheme. b) The L shaped geometry of the compound

84

3.5 Different non-covalent interactions found in compound (g-2) 86 3.6 Rectangular shape macrocycle = Two Cp-π---H—C6H4-CF3 = 2.872 Å,

composition = C22 H2N4Fe2 = 30-membered ring, Dimension, cavity

dimension = 13.041 x 9.324 Å

86

3.7 Another non-ferrocene containing macrocycle mediated by two C6H4-

F2CF---H-C6H3 2.577 Å to form C18H2N4F2 = 26-membered

86

3.8 Supramolecular sheet having macrocycles 87

3.9 Cylindrical structure originated from the placement of macrocycles on

one another

87

3.10 Supramolecular structure of compound (g-2) having cylindrical cavities 88 3.11 a) ORTEP diagram of (g-5) with atomic numbering scheme. b) 89 The T- shaped geometry of the compound

3.12 Different non-covalent interactions found in compound (g-5) 89 3.13 Chain mediated two H(2A)---O(1) = 2.428 Å and two π--- π = 3.296 Å. 91

V  

These interactions as 0.1 Å are less than the van der Waal’s radii

3.14 ORTEP diagram of (g-10) with atomic numbering scheme 91 3.15 a) Showing connection of reference molecule (light green) with five

other molecule. b) Sixteen membered ring mediated by intermolecular hydrogen bonds

93

3.16 Supramolecular structure of compound (g-10) 93 3.17 Space filled diagram of compound (g-10) showing cavities along b-axis 94 3.18 a) ORTEP diagram of (g-14) with atomic numbering, b) The crystal

structure with intramolecular hydrogen bonding represented by dotted

lines

94

3.19 Supramolecular structure of the compound (g-14) 96 3.20 Supramolecular structure of the compound showing cylindrical cavities

(g-14) 96

3.21 (a) ORTEP diagram of (h-11) with atomic numbering scheme, (b) The crystal packing with hydrogen bonding represented by dotted lines

97

3.22 Supramolecular structure of compound (h-11) mediated by non-covalent interactions

97

3.23 (a) ORTEP diagram of compound (i-3) with atomic numbering, (b) The crystal packing with hydrogen bonding represented by dotted lines

99

3.24 Supramolecular structure of the compound (i-3) 99 3.25 Space filled diagram of the compound (i-3) showing cavities 100

3.26 ORTEP diagram of compound (k-1) with atomic numbering 101 3.27 Packing diagram of the compound (k-1) with secondary interactions 102 4.1 Comparison between the UV-visible spectrum of the ferrocenyl

guanidine and phenyl guanidine without ferrocene

106

4.2 UV-Vis spectrum of the compound (g-2) in the absence (A) and presence (B 30; C 40; D 50; E 60; F 70 μM) of DNA

107

4.3 UV-Vis spectrum of the compound (g-4) in the absence (A) and presence (B 30; C 40; D 50; E 60 μM) of DNA

107

4.4 UV-Vis spectrum of the compound (g-5) in the absence (A) and presence (B 30; C 40; D 50; E 60; F 70; G 80; H 90 μM) of DNA

108

VI  

4.5 UV-Vis spectrum of the compound (g-8) in the absence (A) and presence (B 30; C 40; D 50 μM) of DNA

108

4.6 UV-Vis spectrum of the compound (g-17) in the absence (A) and presence (B 30; C 40; D 50; E 60; F 70; G 80; H 90 μM) of DNA

109

4.7 UV-Vis spectrum of the compound (i-1) in the absence (A) and presence (B 30; C 40; D 50; E 60; F 70; G 80; H 90 μM) of DNA

109

4.8 UV-Vis spectrum of the compound (j-5) in the absence (A) and presence (B 30; C 40; D 50; E 60; F 70; G 80; H 90 μM) of DNA

110

4.9 UV-Vis spectrum of the compound phenylguanidine without ferrocene in

the absence (A) and presence (B 30; C 40; D 50; E 60; F 70; G 80; H 90

μM) of DNA

110

4.10 Voltammogram of the compound (g-2) in the absence (A) and presence (B 30; C 60; D 90; E 120; F 150; G 180 μM) of DNA

113

4.11 Voltammogram of the compound (g-17) in the absence (A) and presence (B 30; C 60; D 90; E 120 μM) of DNA

113

4.12 Voltammogram of the compound (g-17) in the absence (A) and presence (B 30; C 60; D 90; E 120 μM) of DNA

114

4.13 Voltammogram of the compound (g-17) in the absence (A) and presence (B 30; C 60; D 90; E 120 μM) of DNA

114

4.14 Voltammogram of the compound (g-5) in the absence (A) and presence (B 30; C 60; D 90; E 120; F 150; G 180 μM) of DNA

115

4.15 Voltammogram of the compound (g-8) in the absence (A) and presence (B 30; C 60; D 90; E 120; F 150; G 180 μM) of DNA

115

4.16 Voltammogram of the compound (h-13) in the absence (A) and presence (B 30; C 60; D 90; E 120; F 150; G 180 μM) of DNA

116

4.17 Voltammogram of the compound (k-1) in the absence (A) and presence (B 30; C 60; D 90; E 120; F 150; G 180 μM) of DNA

116

4.18 Voltammogram of simple phenyl guanidine without ferrocene in the

absence (A) and presence (B 30; C 60; D 90; E 120; F 150; G 180 μM)

of DNA

117

4.19 UV-Visible spectrum of the DPPH in the absence (A) and presence (B 120

VII  

3.12; C 6.25; D 12.5; E 25; F 50; G 100 μM) of ferrocenyl guanidine 4.20 UV-Visible spectrum of the DPPH in the absence (A) and presence (B

3.12; C 6.25; D 12.5; E 25; F 50; G 100 μM) of ferrocenyl guanidine 121

4.21 Antifungal assay chart of selected synthesized ferrocenyl guanidines with a comparison to Terbinafine (PC, standard)

124

VIII  

List of Schemes

Schemes Title Page 1.1 Guanylation through Mercuric chloride 2

1.2 Use of carbodimide for the synthesis of guanidine 2

1.3 Water soluble carbodiimides used in guanidine synthesis 3

1.4 Guanidine synthesis by a DIC reagent 3

1.5 Conversion of resin based thiourea to guanidines 3

1.6 Use of EDCl reagent to synthesize guanidine 4

1.7 Mukayama reagent for the synthesis of guanidine 4

1.8 Conversion of isothiourea to guanidine 4

1.9 Use of diazonium resin in guanidine synthesis 5

1.10 Mercuric chloride as a coupling reagent for the conversion of

isothiourea to guanidine

5

1.11 Conversion of isothiourea to guanidine in microwave 5

1.12 Conversion of carbodiimides to guanidines 6

1.13 Synthesis of chiral guanidines 6

1.14 Use of supported carbodiimide in guanidine synthesis 6

1.15 Conversion of benzotriazole to guanidine 7

1.16 Cyclic guanidines 7

1.17 Synthesis of guanidine through free radical mechanism 8

1.18 Synthesis of acyl guanidines (Lipophylic guanidines) 8

1.19 Amino amide conversion to guanidines 9

1.20 Use of carboxamidine as guanidinylating agent 9

1.21 Using alkyl halide for guanidinylation 10

1.22 Mitsunobu protocol for the use of alcohol to synthesis new

guanidines

10

1.23 Synthesis of cyclic guanidines by guanidinylation methods 10

2.1 Synthesis of nitrophenylferrocenes (a & b), nitro group is substituted at para position in a and meta in b, from ferrocene *PTC = Phase Transfer Catalyst i.e. cetyltrimethylammonium bromide (CTAB)

37

IX  

2.2 Synthesis of para (c) and meta (d) ferrocenyl aniline. 39 2.3 Synthesis of N, N disubstituted phenylthioureas (e) starting from 40

benzoic acid.

2.4 Synthesis of N, N disubstituted phenylthioureas (f) starting from chloro benzoic acid.

41

2.5 Synthesis of ferrocenyl guanidines (g & h), ferrocene moity is substituted at para position in (g) and meta in (h), from the NH of guanidine functionality.

42

2.6 Synthesis of ferrocenyl guanidines (i & j), ferrocene moity is substituted at para position in (i) and meta in (j) from the NH of guanidine functionality.

64

2.7 Synthesis of tetra substituted ferrocenyl guanidine from

trisubstituted ferrocenyl guanidine.

72

3.1 Overall synthetic scheme for the synthesis of ferrocenyl guanidines. 77

3.2 Mechanism for the synthesis of ferrocenyl guanidines. 78

3.3 Synthetic scheme for the conversion of trisubstituted ferrocenyl

guanidine to tetra-substituted ferrocenyl guanidines.

79

3.4 Ferrocenyl guanidine skeleton. 81

4.1 Increase in conjugation due to the presence of ferrocene at para

position.

111

4.2 (a) Diphenylpicrylhydrazyl (b) Diphenylpicrylhydrazine. 120

4.3 (a) Hydrophilic character of ferrocenyl guanidine. (b) Decrease in

hydrophilicity and increase in lipophilicity due to the delocalization

of the lone pair of electrons of nitrogen atoms by electron

withdrawing group (R) and ferrocene.

123

X  

List of Tables

Tables Title Page3.1 Crystal data and structure refinement parameters for (g-2) 85 3.2 Crystal data and structure refinement parameters for (g-5) 90 3.3 Crystal data and structure refinement parameters for compound (g-10) 92 3.4 Crystal data and structure refinement parameters for (g-14). 95 3.5 Crystal data and structure refinement parameters for compound (h-11) 98 3.6 Crystal data and structure refinement parameters for compound (i-3) 100 3.7 Crystal data and structure refinement parameters for compound (k-1) 103 4.1 Binding constant values for selected compounds 118

4.2 Antibacterial activity of selected ferrocenyl phenylguanidine 122

4.3 Antifungal activity of selected ferrocenyl phenylguanidine 125

XI  

List of abbreviations

TBAP Tetrabutylammoniumperchlorate

DNA Deoxyribonucleic acid

DPPH 1,1-diphenyl-2-picrylhydrazyl

DMSO Dimethyl sulfoxide

ATCC American type culture collection

TLC Thin layer chromatography

DMF N, N-dimethyl formamide

HDTAB Hexadecyltrimetyl ammonium bromide

PTC

UV-VIS

CV

FTIR

NMR

TMS

Cp

DEAD

Phase transfer catalyst

Ultra Violet- Visible Spectroscopy

Cyclic Voltammetery

Fourier Transform Infrared Spectroscopy

Nuclear Magnetic Resonance Spectroscopy

Tetrametyl silane

Cyclopentadienyl

Diethyl azodicarboxylate

XII  

Abstract

Four series of trisubstituted ferrocenyl guanidines g(1-18), h (1-18), i (1-6) & j (1-6) of general formula [RC6H5CONC(HN'C6H4C5H4FeC5H5)(HN''C6H5R1)] where R=3-Cl and R1= H, 3-

CF3, 4-CF3, 4-NO2, 4-CH3, 2-CH3, 2,6-C2H5, 2-OCH3, 3-OCH3, 2-Cl, 2,3- (Cl)2, 2,4- (Cl)2,

2,5- (Cl)2, 2,6- (Cl)2, 3,4- (Cl)2, 3,5- (Cl)2, 2,4,5- (Cl)3, 2,4- (Br)2 have been synthesized and

characterized by using elemental analysis, FT-IR, multinuclear (1H and 13C) NMR

spectroscopy, UV-Visible spectrophotometery and cyclic voltammeter. Single crystal XRD

was used for structural elucidation of some of the synthesized ferrocenyl guanidines. Based

on the single crystal X-ray analysis most of the synthesized ferrocenyl guanidine have been

stabilized by intermolecular as well as intramolecular hydrogen bonding and possesses

interesting supramolecular chemistry having cylindrical cavities and empty spaces. In

addition, a tetra substituted ferrocenyl guanidine (N-isopropyl-N-(4-ferrocenylphenyl)-N'-(2,

6-diethylphenyl)-N''-benzoyl guanidine) has also been synthesized and fully characterized.

The preliminary investigation of the anticancer potency of the synthesized ferrocenyl

guanidines has been carried out by determining their ability to bind with DNA and by the free

radical scavenging activity. The DNA interaction studies performed by cyclic voltammetry

and UV-Visible spectroscopy are in close agreement with the binding constants K (0.79 - 5.4)

×105 M-1 (CV) and (0.72 - 5.1) ×105 M-1 (UV-Visible). The results reveal that the ferrocenyl

guanidines have strong binding ability with DNA as compared to the guanidines having no

ferrocene. The presence of ferrocene is concluded to enhance the DNA binding activity of

guanidines. This may be due to the fact that in the presence of ferrocene the delocalization of

lone pair of nitrogen extended to Cp ring of ferrocene due to which the nitrogen become more

polarized, stable and favorable for electrostatically bind with negatively charged DNA. The

binding constants results show that the compounds having ferrocene at para position have

slightly larger binding constants values as compared to the meta-ferrocenyl guanidines. This

may be due to more delocalization of electron when the Cp ring of ferrocene is at para

position. The compounds having electron withdrawing groups on the phenyl ring also have

higher binding constant values as compared to those compounds having electron donating

XIII  

groups. This may also be due to the delocalization of lone pair of nitrogen on phenyl ring and

making the nitrogen more polar.

The free radical scavenging potentials of the selected synthesizes compounds was

determined on a UV-Visible spectrophotometer by using DPPH as a free radical. The activity

of ferrocene incorporated guanidines was found to be higher than guanidines without

ferrocene. The compounds which have electron withdrawing groups showed an increase in the

free radical scavenging potency. This might be due to the stabilization of resulting guanidine

free radical in the presence of electronegative groups.

Antimicrobial activities of the selective synthesized compounds were tested against

five representatives, gram-positive (Staphylococcus aureus, Pseudomonas aerugnosa and

Bacillus subtilis) and gram-negative (Klebsiella pneumonia and Escherchia coli) bacterial

strains by disc diffusion method. Three fungal strains, fusarium moniliforme, aspergillus

fumigates, aspergillus flavus were tested by using well diffusion method. The results revealed

that the compounds having ferrocene and electron withdrawing groups showed moderate to

good antibacterial activity as compared to the standard drug penicillin used. Significant

antifungal activity was observed against aspergillus flavus and good against fusarium

moniliforme and aspergillus fumigatus. The antifungal activity of these compounds was found

comparable with the standard drug used (Terbinafin). Other compounds having electron

donating groups were found to have a moderate or less activity against the tested bacteria and

fungi. Exact mechanism of the structure-activity relationship was not yet developed but this

might be due to a decrease in basicity, in turn an increase in the lipophilicity of the

compounds in the presence of ferrocene and electron withdrawing substituents. Lipophilic

compounds have more penetrating power across the cell membrane.

1  

Chapter 1 Introduction

1 Introduction of Guanidines Guanidines are the compounds containing CN3 unit. The first guanidine was synthesized by

Adolph Streeker in 1861 by the oxidation of guanine [1]. Guanidines contain three nitrogen

atoms and show the strongest bronsted basicity (Pka around 12.5) among the amine

derivatives. The resonance stability of its conjugate acid [C (NH2)3]+ which is due to the

delocalization of π-electron across the almost symmetric CN3 unit is the basic reason for the

high basicity of guanidines (Figure 1.1) [2].

Figure 1.1: Resonance structures of conjugate acid of guanidine

The basic nature of guanidines can be changed by the substitution on the nitrogens of

guanidine. The introduction of electron donating groups on one of the guanidine nitrogen

atoms increases its basicity where as electron withdrawing groups decreases its basicity. Due

to high basicity, the substituted guanidines are included in super bases and are extensively

used in organic synthesis. Some of the common guanidines with their pKa values [3] are

given in figure (1.2).

Figure 1.2: pKa of some of the common guanidines.

2  

1.1 Methods for the synthesis of substituted guanidines. Synthesis of substituted guanidines can be divided into two major categories by Batey and his

Co-workers [4].

1.1.1 Guanylation methods for the synthesis of guanidines. In guanylation reaction nucleophilic amine is treated with electrophilic amidine or

carbodiimide species to produce a new guanidine.

1.1.1.1 Synthesis of guanidines from thiourea. Thiourea moiety can be converted into guanidine functionality by the use of different

coupling reagents.

1 By the use of Mercuric chloride The conversion of thiourea into guanidines by treating it with primary and secondary amines

using mercuric chloride is one of the well known methods for the synthesis of guanidines. The

process has been found to be effective with the thioureas containing at least one conjugated

substituent on nitrogen atom such as the guanidines having N-aryl substituents [5] as shown

in scheme (1.1).

Scheme1.1: Guanylation through Mercuric chloride.

2 By the use of polymer supported carbodiimide.

Carbodiimide can also be used as an activating reagent for thiourea with polymer supported

trisamine as scavenger (Scheme 1.2) [6].

Scheme 1.2: Use of the carbodimide for the synthesis of guanidine.

3  

3 By the use of 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride reagent. N-(tert-butoxycarbonyl) thiourea on combination with amine hydrochloride in the presence of

water soluble carbodiimide, 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride

with stirring at room temperature provide N-(tert-butoxycarbonyl guanidine, and at the last,

the product is treated with an acid to give guanidine as shown in scheme (1.3) [7].

Scheme 1.3: Water soluble carbodiimides used in the guanidine synthesis

4 By the use of N, N-diisopropylcarbodiimide (DIC) reagent. Fmoc 4-(aminomethyl) benzoic acid is allowed to react with Boc-protected thioureas in

dichloroethane and dimethyl formamide in the presence of DIC reagent to give the

corresponding guanidines (Scheme 1.4) [8].

Scheme 1.4: Guanidine synthesis by a DIC reagent.

Guanidines can be obtained by the use of Rink amide resin to generate a resin based

thiourea and then by the use of DIC as a coupling reagent (Scheme 1.5) [9].

Scheme 1.5: Conversion of resin based thiourea to guanidines. 5 By the use of (1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride) EDCl. The synthesis of ethyl carbamate protected guanidine using a coupling reagent EDCl has been

reported by Anslyn et al. (Scheme 1.6) [10].

4  

Scheme 1.6: Use of EDCl reagent for the synthesis of guanidine.

6 By the use of 2-chloro-1-methylpyridinium iodide (Mukayama’s reagent).

Mukayama’s reagent, an acid activating reagent used for the synthesis of guanidines from

thiourea was extensively used by Lipton [11] and by Burgess for the synthesis of guanidines

(Scheme 1.7) [12].

Scheme 1.7: Mukayama reagent for the synthesis of guanidine.

1.1.1.2 Synthesis of guanidines from isothiourea. Flygare and his co-workers reported the synthesis of guanidines from methyl isothiourea

piperidine (Scheme 1.8) [13].

Scheme 1.8: Conversion of isothiourea to guanidine.

A wide range of substituted guanidine can be achieved by the attachment of primary

amines to T2* diazonium resin which further on treatment with ammonia, primary and

secondary amines gives guanidines (Scheme 1.9) [14].

5  

Scheme 1.9: Use of diazonium resin for the synthesis of guanidine.

Guanidines can be synthesized by the guanylation of primary and secondary amines

with S-alkyl isothiourea in the presence of Mercuric chloride and triethylamine (Scheme 1.10) [15].

Scheme 1.10: Mercuric chloride as a coupling reagent for the conversion of isothiourea to guanidine.

A direct reaction of isothiourea with amines and anilines using only triethylamine in

different solvents like THF, DMF, MeOH and EtOH lead to the formation of guanidines [16].

N,N'-diarylcyanoguanidines have been synthesized from corresponding isothiourea under

microwave conditions (Scheme 1.11) [17].

Scheme 1.11: Conversion of isothiourea to guanidine under microwave conditions.

1.1.1.3 Synthesis of guanidines from carbodiimdes. Carbadiimide on treating with primary and secondary aryl amines give the corresponding

guanidines.

6  

Imido vanadium complexes are used as catalysts in this reaction (Scheme 1.12) [18].

Scheme 1.12: Conversion of carbodiimides to guanidines.

Carbodiimides are very important reagents for the synthesis of guanidines [19]. The

chiral amines are treated with n-butyllithium to form lithium amides and then with

carbodiimide to give lithiated product which are hydrolysed to form guanidines (Scheme

1.13).

Scheme 1.13: Synthesis of chiral guanidines.

D.H. Drewry proposed solid phase synthesis of trisubstitutedguanidines via aza-wittig

coupling of a solid supported alkyl iminophosphorane with an aryl or alkyl isothiocayante to

generate the corresponding solid supported carbodiimide which is then treated with primary

or secondary amine to obtain the desired tri-substituted guanidines (Scheme 1.14) [20].

Scheme 1.14: Use of supported carbodiimide in guanidine synthesis.

7  

1.1.1.4 Synthesis of guanidines from triazoles. In this reaction benzotriazole is treated with cyanogens bromide and as a result di-

(benzotriazol-yl) methamine is obtained, which is further treated with amines, displacement of

benzotriazolemoiety takes place and guanidine is obtained as a final product (Scheme 1.15) [21].

Scheme 1.15: Conversion of benzotriazole to guanidine.

1.1.1.5 Synthesis of cyclic guanidines. 1 By the use of Isatoic anhydride R.Giridhar and his Co-workers synthesized five, six and seven membered cyclic guanidines

by the use of isatoic anhydride. Isatoic anhydride was treated with primary amine to obtain 2-

aminobenzamide, which on reaction with cyanogen bromide gives the desired product

(Scheme 1.16) [22].

Scheme 1.16: Cyclic guanidines.

2 By the use of Luotonin A through a free radical mechanism. Luotonin A is converted to guanidine via the formation of aminyl radical (B), iminyl radical

(C) and tricyclic radical (D) (Scheme 1.17) [23].

8  

Scheme 1.17: Synthesis of guanidine through free radical mechanism.

1.1.1.6 Synthesis of acyl guanidines. Acyl guanidines can be synthesized by different synthetic approaches (Scheme 1.18) [24] which includes

(i). A reaction of isocyanide dichloride with amines.

(ii). A reaction of alkoxy-hexametylmetane triamines with amides.

(iii). A reaction of N-chloroamides with tetramethyl alkoxymethane diamines.

Scheme 1.18: Synthesis of acyl guanidines.

1.1.1.7 Synthesis of guanidines from amino amides. Rigid cyclic α-amino amides (b), derived from L-proline or L-pipecolic acid (a), are

promising candidates for the formation of guanidines. The practical synthesis of guanidine has

9  

been achieved by the addition of lithium amino amide (c) to the carbodiimide (Scheme 1.19) [25].

Scheme 1.19: Conversion of Amino amide to guanidines.

1.1.2 Guanidinylation methods for the synthesis of guanidines. Guanidinylation is referred to the reaction in which an electrophile is treated with a

nucleophilic guanidine to produce a new substituted guanidine.

1.1.2.1 Through carboxamidine reagents One step conversion of primary and secondary amines into their corresponding guanidines by

using 4-benzyl-3,5-dimethyl-1-pyrazole-1-caroxamidine and 3,5 dimethyl-n-nitro-1-pyrazole-

1-carboxamidine as guanidinylating agents was described by B.Drake [25], F.Y.Yong [26]

and W. Solodinko (Scheme 1.20) [27].

Scheme 1.20: Use of carboxamidine as guanidinylating agent.

1.1.2.2 Through alkyl halide The alkylation of carbamate protected guanidines with alkyl bromide in the presence of phase

transfer catalyst Tetrabutylammonium iodide (Bu4NI) using a mixture of KOH,

dichloromethane and water was described by Batey and his Co-workers. They also studied the

effect of different alkyl halides and found that alkyl bromides are the better reagents as

compaired to the other alkyl halides (Scheme 1.21) [4].

10  

Scheme 1.21: Using alkyl halide for guanidinylation.

1.1.2.3 Through alcohols. An efficient method for the conversion of guanidines into new protected alkylated guanidines

by the treatment with a variety of a primary and secondary alcohol was reported by Goodman

and his Co-workers (Scheme 1.22) [28].

Scheme 1.22: Synthesis of new guanidines from alcohol.

1.1.2.4 Through esters, to form cyclic guanidines. Cyclic guanidines can be synthesized by the guanidylation of simple guanidine with ester (Scheme 1.23).

Scheme 1.23: Synthesis of cyclic guanidines by guanidinylation method.

11  

1.2 Applications of guanidines. 1.2.1 Biological applications 1 Antihistamines drugs Famotidine (a), cimetidine (b) and dispacamide (c) are well known medicines used for the

treatment of ulcer (Figure1.3) [29-31].

Figure 1.3: Drugs for the treatment of ulcer.

2 Influenza inhibitor Zanamivir is a drug containing guanidine moiety and is effective against all types of flue

(Figure 1.4) [32].

Figure 1.4: Drugs for the treatment of influenza

3 Antidiabetic drugs Metformin (a), Phenformin (b), N-(2-methyl, 5-chloro-1H-indol-3-yl)-guanidine (c) [33] and 3-guanidinopropionicacid (d) are used for the treatment of diabetes (Figure 1.5).

Figure 1.5: Drugs controlling sugar level in the body.

12  

4 Anti-inflamatory activities Compounds (1-(3,4-dimethyl-2-chlorobenzylidineamino)-guanidine), 1,3-Bis-

(phenylethylidineamino) guanidine hydrochloride and Mercaptoethyl guanidine are known to

possess anti-anflimatory activity [34-36].

5 Antihypertensive agents Amiloride (3, 5-diamino-6-chloro-N-(diaminomethylene) pyrazine is a potassium sparing

diuretic approved for use in 1967 by the name of ‘‘MK870’’ is used in hypertension and heart

failure (Figure 1.6) [37]. Doxazosin is a drug used in high blood pressure. It is also called alpha-adrenergic blockers. It relaxes veins and arteries so that the blood can more easily pass

through them and also relaxes the muscles in the prostate and bladder neck, making the urine

discharge easier [38].

Figure 1.6: Amiloride, an antihypertensive drug.

Guanadrel (a), Guanabenz (b), guanoxan (c) can be used as antihypertensive drugs (Figure

1.7) [39, 40].

Figure 1.7: Drugs used to control blood pressure.

6 Anticancer activities Cancer is the uncontrolled cell growth which harms the body by forming lumps or masses of

tissues called tumors, except leukemia. Cancer becomes more dangerous when its cells

manage to move throughout the body (invasion) and when they form new blood vessels in

order to feed themselves (angiogenesis). There are different causes of cancer as shown in

figure (1.8).

13  

Figure 1.8: Cancer causes tree

Cisplatin is an important anticancer drug but it is having many therapeutic drawbacks

as well, owing to its toxicity. A lot of research work is being done in this field and new

compounds are being synthesized having more potent and less toxic nature. Many naturally

occurring as well as synthesized guanidines are known to possess anticancer activity.

Netamines C has been found to have promising activity against human tumor cell line (Figure

1.9) [41].

Figure 1.9: Anticancer Netamines C

(2-(2, 5-dimethoxyphenylthio)-6-methoxybenzylideneamino) guanidine (a), Spergualin (b)

and a series of 1-[2-alkylthio-5-(azol-2 or 5-yl)-4-chlorobenzenesulfonyl]-3-

hydroxyguanidines (c) are considered to possess anticancer activity (Figure 1.10) [42-44].

14  

Figure 1.10: Compounds having anticancer activity

Compound 1-amino-2-(4-chloro-2-mercaptobenzene-sulfonyl) guanidine derivatives

and some novel guanidine based inhibitors of inosin monophosphate dehydrogenase has been

synthesized by Edwin J. Iwanowicz [45-47]. R. Scott and his Co-workers have studied the

biological activities of some variolin and meriolin.Varioline (a) has been found to have

modest activities against leukemia cell line. Meriolin (b) displays considerabaly enhanced

cyclin-dependent kinase inhibition and cytotoxicity (Figure 1.11) [48].

Figure 1.11: (a) Variolin, (b) Meriolin

7 Antibiotics Chlorhexidine is an antiseptic and effective on both gram-positive and Gram negative bacteria

(Figure 1.12). It is often used as an active ingredient in mouthwash in order to reduce dental plaque and oral bacteria [49].Streptomycin is a bactericidal antibiotic [50] and Akacid plus is

a new member of disinfectants having less toxicity belonging to biocides which are different

from antibiotics in their mode of action [51]. Polyhexanide (polyhexamethylenebiguanide,

PHMB) is a polymer used as a disinfectant antiseptic and in dermatological preparations [52].

15  

Figure 1.12: Chlorohexadine an effective antibiotic.

Pyrrolidinebis-cyclic guanidines have antimicrobial activities against drug-resistant Gram-

positive pathogens (Figure 1.13).

Figure 1.13: Pyrrolidinebis-cyclic guanidine.

8 As antioxidant Antioxidants are the molecules which inhibit the oxidation of other molecules. Oxidation

produces free radicals which are checked by different enzymes in the body called antioxidant

enzymes or by antioxidant nutrients like vitamin C, vitamin E, beta carotene and

bioflavonides. If generation of the free radical increases then troubles arise in the body in the

form of diseases like bronchitis, arthritis, heart diseases, peptic ulcer, wrinkles and aging and

if free radical affects the DNA then it becomes mutated and produces cancer, leukemia,

diabetes, liver and kidney problems (Figure 1.14).

Figure 1.14: Different damages take place in the body as a result of free radicals.

16  

Antioxidants should contain hydroxyl or NH groups and should also contain a lot of

delocalization in order to stabilize the resulting free radical. Guanidines are the compounds

having NH proton to scavenge the free radical formed. Y. Naito proposed T-593 ((+/-)-(E)-1-

[2-hydroxy-2-(4-hydroxyphenyl) ethyl]-3'-[2-[[[5-(methylamino) methyl-2-furyl] methyl]

thio] ethyl]-2"-(methylsulfonyl) guanidine, a new histamine H2-receptor antagonist as a free

radical scavenger [53]. Aminoguanidine and a high concentration (50.1 mM) of

methylguanidine have free radical scavenging activities against O2, HOCl, hydroxyl radical

and peroxynitrite [54]. Mercaptoethylguanidine (Figure 1.15) is a combined inhibitor of nitric oxide synthesis and peroxynitrite free radicals [55].

Figure 1.15: Mercaptoethylguanidine, a free radical scavenger.

1.2.2 Non-biological applications of guanidines.

1 Supper bases in organic synthesis Substituted guanidines are the super bases due to their strong basicity and are used in organic

synthesis as catalysts. One of the important guanidine which is extensively used in base

catalyzed reactions is the 1,1,3,3-tetramethylguanidine (TMG) [56]. Pentaalkylguanidines

(known as Barton’s bases) are used as sterically hindered organic bases in organic synthesis

reported by Barton et al. [57]. 1,5,7-triazabicycle [4.4.0] dec-5-ene (TBD) and its N-methyl

analogue (MTBG) are the important bicyclic guanidine based organic bases introduced by

Schwesinger [58].

2 As artificial sweeteners Some guanidines like lugduname (a), sucrononate (b) and carrelame (c) are very sweet in taste

even 160,000-225,000 times sweeter than sucrose can be used as artificial sweeteners but their

use in food items is under investigation (Figure 1.16).

17  

Figure 1.16: Guanidine derivatives promising candidates for artificial sweeteners.

3 Use of guanidine salt Guanidine salts nominated as guanidine-type ionic liquids show unique physico-chemical

characteristics and are used in hydrogenation, hydroformylation, aldol reaction and palladium

catalyzed Heck reaction (Figure 1.17) [59].

Figure 1.17: Guanidine salts used as ionic liquids or as catalyst.

1.2.3 Supramolecular chemistry of guanidines. A supermolecule is a large and complex entity formed from other molecules [60]. The

molecules which comprise the supermolecular property interact with each other via non-

covalent interactions such as hydrogen bonding, hydrophobic interactions, coordination or

even spatial interactions to form new entities with novel properties and functions. Non-

covalent interactions play an important role in organizing structural units in natural as well as

in artificial systems [61]. Different types of supramolecular interactions are responsible for the formation of supramolecular structures and their strength are shown in the figure (1.18).

18  

Figure 1.18: Non-covalent interactions responsible for the formation of a supramolecule.

Guanidines are of special interest due to their structural features and hydrogen

bonding array provided by the molecules which suggest that they are good building blocks for

the formation of supramoleular entities mediated by three pairs of directional hydrogen

bonding interactions. However, their significance in the generation of multi-dimensional

networks has been recently appreciated [62-64]. Said et.al. have reported supramolecular

structure of bis (N, N′, N″-triisopropylguanidinium) phthalate [65]. Guanidine moiety is

lipophylic in nature and thus can be used for the formation of dendrimers. C. V. Bonduelle

and his co-worker describe some guanidine based dendrimers (Figure 1.18) [65].

Figure 1.19: Guanidine based dendrimers.

19  

1.3 Introduction of ferrocene derivatives Ferrocene an orange compound was first discovered in 1951[66]. Due to the favorable

electronic properties of ferrocene, stability in solution state and its easy derivatization have

made ferrocenyl compounds very popular molecules for biological and non biological uses. Ferrocene is more widely used in drug design as compared to other metallocenes [67-69].

1.3.1 Biological applications of ferrocene derivatives 1 As antibiotic E. I. Edward’s research group synthesized a series of ferrocene based antibiotics in 1970’s,

including ferrocenylpenicillin (a), ferrocenyl-cephalosporin (b) which are effective against all

types of gram positive and gram negative bacteria (Figure 1.20) [70].

Figure 1.20: Ferrocene derivatives used as antibiotics.

2 As anticancer Ferrocifen (b) and ferrocephane (c), the ferrocene analog of tamoxifin (a) are the molecules

have been shown to be active against both hormones dependent and hormones independent

breast cancer cells (Figure 1.21) [71-73].

20  

Figure 1.21: Ferrocene analog of tamoxifin (a) used as anticancer agent.

Ferrocene containing compounds have been reported to have antitumor activity due to

metabolic formation of ferrocenium ions. Water soluble ferrocenyl derivatives can also be

used as anti-cancer agents (Figure 1.22) [74].

Figure 1.22: Water soluble anticancer ferrocenyl derivatives.

Ferrocenium tetra fluoroborate salt (a) and ferrocenium iodate (b) also posseses

anticancer activity (Figure 1.23) [75, 76].

Figure 1.23: Anticancer agents in the form of salts.

3 As antimalarial (7-chloro-4-[(2-N, N-dimethyl-aminomethyl) ferrocenylmethylamino] quinoline) commonly

known as ferroquine can be used as antiplasmodial agent (Figure 1.24) [77].

21  

Figure 1.24: Antimalarial ferrocene derivative.

4 As antianeamic Ferrocerone, a sodium salt of o-carboxybenzoyl ferrocene can be used to treat various forms

of iron deficiency (Figure 1.25) [78].

Figure 1.25: Ferrocerone.

1.3.2 Non biological applications 1 As catalysts Chesney et al. synthesized two ligands, derived from L-(S-methyl)-cysteine (a) and L

methionine (b) which were found to be effective in palladium-catalysed substitution reactions

of allylic acetate with diethyl malonate (Figure 1.26) [79].

Figure 1.26: Ferrocenyl derivatives as catalyst.

2 As corrosion inhibitor Diformylferrocene (a), diacetyl ferrocene (b) and 2-benzimidazolylthioacetyl ferrocene (c)

show inhibitory effect against mild steel corrosion (Figure 1.27) [80].

22  

Figure 1.27: Ferrocenyl derivative as corrosion inhibitors.

3 As biosensors Beer and his Co-workers designed ferrocene containing amide molecules (a & b) having the

ability to bind with and electrochemically sense anionic substrates (Figure 1.28) [81].

Figure 1.28: Ferrocenyl derivative as biosensors.

N-meta ferrocenyl benzoyl dipeptide ester (a) and ferrocene carboxylic acid-crown ether (b) [82] can be used as a material in biomolecular sensor devices (Figure 1.29).

Figure 1.29: Biosencers

2-ferrocenyl-4-nitrophenol (a) and N-(4-hydroxy benzylidene)-4-ferrocenyl aniline (b)

were used as glucose and alcohol biosensor described by J. Razumiene and his Co-workers

(Figure 1.30) [83].

23  

Figure 1.30: Glucose and alcohol biosencers

By considering the biological applications of guanidine derivatives as well as of the

ferrocene derivatives, it was noted that both these groups have promising anticancer and

antimicrobial activities. It was thus decided to incorporate ferrocene in guanidine moiety, in

order to increase its stability, lipophylicity, electrochemical properties and to decrease its

toxicity.

Before moving towards expensive cell line studies, it is better to know about the

potency of the compounds acting as an anti cancer as well as antimicrobial agents by using

simple and cheap assays. Highly rash free radicals and reactive oxygen species (ROS) found

in biological systems are liable for the oxidation of nucleic acids, proteins, lipids and DNA

and can also initiate degenerative disease like cancer. In the light of the above it was decided

to do antioxidant screening of the targeted compounds. As different cancerous cell growth can

be controlled by controlling DNA [84]. Hence keeping in mind the importance of DNA

binding with different compounds it was decided to screen the target compounds for their

DNA binding potency.

1.4 Drug-DNA Interaction Study. DNA is a molecule of great biological significance (Figure 1.31), being carrier of the genetic information it is a major target for drug interaction mainly because of the ability to interfere

with transcription (gene expression and protein synthesis) and DNA replication, a major step

in cell growth and division.

24  

Figure 1.31: Structure of DNA molecule.

The binding interaction of the small molecules with DNA is amongst the most

important aspects of biological studies in drug discovery and pharmaceutical development

processes. Moreover, this study is helpful to understand the structural properties of DNA, the

mutation of genes, the origin of some diseases, and the action mechanism of some antitumor

and antivirus drugs and to design a new and more efficient DNA targeted drugs dealing with

genetic diseases [85]. Further, when complex formation occurs between DNA and a drug,

there is a change in the thermodynamic stability and functional properties of DNA [86, 87].

A drug interacts with DNA in three different ways.

Through control of transcription factors and polymerases.

Through RNA binding to DNA double helix to form nucleic acid triple helical

structure exposing DNA single strand regions.

Through a small molecule that binds to DNA double helical structures directly.

The third one is the most important for anti-cancer agents having two kinds of interactions.

1 Covalent interaction In covalent interactions, the small molecules make a covalent or coordinate covalent bond with different components of DNA. For example cis-platin used as

an anticancer drug bindes covalently and makes an intra and inter strand cross-link through

the replacement of chlorides with the nitrogen's on the DNA bases. Covalent interactions are

irreversible having complete inhibition of DNA and as a result cells death occurs.

2 Non-covalent interactions In non- covalent interaction, there is no permanent affiliation of the molecules with DNA and hence cause temporary conformational changes in DNA. This

25  

kind of interaction is reversible and is preferred over a covalent interaction because covalent

binding has many toxic side effects.

The non-covalent interactions are further classified into three categories (Figure 1.32) [88, 89].

(i) Intercalation In this type of interaction, the planer groups in a molecule penetrate between the base pairs of DNA double strand and introduce strong disturbance in DNA double helical

structure. For examples, Nogalamycin and ethidium bromide are the two important

intercalating drugs.

Figure 1.32: Different types of interactions by which a drug affect the DNA.

(ii) Groove binding It is the attachment of small drug molecules with DNA by promoting Vander Waals interactions and hydrogen bonds to the bases. Groove binders are usually

crescent shaped. Most of the groove binders bind at the AT (adenine and thymine) base pair

of the DNA i.e., Netropsin (Figure 1.33), Distamycin etc.

Figure 1.33: Netropsin

26  

(iii) Electrostatic interaction. It is the ionic type of interaction between the negative charge of DNA phosphates and a positively charged molecule which cannot intercalate into the DNA

helical structure due to structural make up.

1.4.1 Techniques used to study non-covalent interaction Variety of techniques can be used to evidence drug-DNA interactions as shown in the scheme

(1.34) [90].

Figure 1.34: Analytical techniques used to study the DNA binding potency of a compound.

Keeping in view the available facilities it has been tried to explore the DNA binding behavior

of the synthesized compounds using UV-visible spectrophotometry and cyclic voltammetry.

1.4.1.1 DNA binding study by UV-Visible Spectrophotometry UV-visible spectrophotometric method is highly sensitive, efficient, fast, and low cost

technique for studying the interaction between small molecules and DNA. The evidence for

the interaction mechanism, nature of the complex formed, binding constant, binding site size

can be provided by UV-visible spectrophotometric signal related to DNA interactions. The

27  

interaction can be detected by measuring the changes in the absorption properties of the

interacting compound or the DNA molecules. The UV-Vis absorption spectrum of DNA

exhibits a broad band at 200-350 nm in the UV region with maxima at 260 nm. This

maximum is a consequence of the chromophoric groups in purine and pyrimidine moieties

responsible for the electronic transitions. The probability of these transitions is high and thus

the molar absorptivity (ε) is of the order of 6600 M-1cm-1 as discussed in our paper [91]. So

the compounds absorbing in other than this region can easily be studied for DNA interaction

by UV-visible spectrophotometeric titration by keeping concentration of the compound

constant and varying the DNA concentration. When a compound interacts with DNA, its

electronic structure changes and thus changes in the electronic spectrum take place. There are

different kinds of changes e.g a shift in the wavelength up-to 70 nm towards red or blue and

also a Hyperchromic or hypochromic effect. In the case of poor associations, only

hyperchromic or hypochromic effect is observed without noteworthy changes of shifts in the

spectra. Increase or a decrease in the absorbance along with red shift in UV-visible spectra is

a typical characteristic of an intercalating mode. Where as the change in the absorbance along

with blue shift in UV-visible spectra is a typical characteristic of an ionic or electrostatic

mode of interaction [92].

The quantitative association of the compound with DNA can be achieved on the basis

of changes observed on increasing the amount of DNA. The equilibrium constants (binding

constant) is calculated by fitting data in the Benesi-Hildebrand equation (Equation 1.1) [93].

Equation 1.1

Where Ao and A are the absorbance of the free compound and of the compound–DNA

complex, εG and εH-G is the molar extinction coefficients of free compound and of the compound–DNA complex respectively.

1.4.1.2 Drug-DNA binding study using Cyclic Voltammetery Cyclic voltammetry is one of the most versatile techniques used for the study of drug-DNA

interaction [94]. In Cyclic voltammetry, the peak potential and peak current of the compound

changes in the presence of DNA if the compound interacts with nucleic acid. The variation in

28  

peak potential and peak current can be used for the determination of binding parameters.

Positive peak potential shift indicates intercalation of the drug with DNA. Negative peak

potential shift is observed for electrostatic mode of interaction and groove binding mode is

witnessed by the decrease in peak current accompanied with no shift in peak position.

Based upon the change in peak current, the binding constant value is calculated according to

the following equation (1.2) [95].

Equation 1.2

Where K is binding constant, I and Io are the peak currents with and without DNA and A is

proportionality constant. The plot of 1/DNA versus 1/(1-I/Io) yields binding constant (K).

29  

Aims of the study Guanidines and ferrocene derivatives are well known for their biological applications

specifically in cancer treatment. The incorporation of ferrocene is always appreciated to make

the biologically active synthetic compounds stable, less toxic and more lipophylic. Keeping

the significances in view, our aim was;

To synthesize some novel ferrocenyl guanidines in good yield.

To fully characterize these compounds by using UV-visible spectroscopy elemental

analysis, NMR, FT-IR measurement and single crystal X-ray diffraction analysis.

To investigate preliminary anticancer potency of the synthesized compounds for their

antioxidant activity and DNA binding.

To find anti-mictobial activity of the synthesized compounds.

To find structure-activity relationship among the designed compounds and their

biological activities.

30  

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Chapter 2 Experimental and Characterization

2.1 Chemicals All chemicals, reagents and organic solvents were purchased from Sigma-Aldrich, Fluka and

E. Merck. Benzoic acid, 3-chlorobenzoic acid, potassium thiocyanate, thionyl chloride,

mercuric chloride, 2-chloroaniline, 3-chloroaniline, 4-chloroaniline, 2,3-dichloroaniline, 2,4-

dichloroaniline, 2,5-dichloroaniline, 2,6-dichloroaniline, 3,4-dichloroaniline, 3,5-

dichloroaniline, 2,3,5-trichloroaniline, 2,4,6-trichloroaniline, 4-nitroaniline, 3-

triflouromethylaniline, 4-triflouromethylaniline, 2-methylaniline, 4-methylaniline, 2,6-

diethylaniline, 2-methoxyaniline, 3-methoxyaniline, and 2,4-dibromoaniline were used as

received without further purification. Organic solvents acetone, dichloromethane, chloroform,

alcohols, petroleum ether and n-hexane were distilled, purified & dried according to reported

methods and were saturated with nitrogen, stored over molecular sieves 4 Å and degassed

before use.

2.2 Instrumentation Melting points were determined using melting point apparatus; model Bio Cote SMP10- UK.

NMR spectra were recorded on Bruker AV-300 MHz spectrometer using deuterated solvents. 1H NMR (300 MHz): internal standard solvent CDCl3 (7.28 ppm from TMS; 13C NMR (75.47

MHz): internal standard solvent CDCl3 (77.0 ppm from TMS). The splitting of proton

resonance in the 1H NMR spectra are defined as s = singlet, d = doublet, t = triplet, q = quartet

and m = multiplet etc; coupling constants are reported in Hz.

Infrared absorption spectra were recorded in the range of 4000-400 cm-1 as KBr discs

on Bio-Rad Excalibur FT-IR Model FTS 3000 MX and Bruker-Tensor 37 spectrophotometer.

The NICOLET 6700, Thermo Scientific FT-IR spectrophotometer was used to record the

spectra in the range of 200-400 cm-1 using ATR. CHNS analyses were performed on Fisons

EA1108 CHNS analyzer and LECO-183 CHNS analyzer. The crystallographic data of all the

crystalized guanidines were collected on a Bruker kappa APEXII CCD diffractometer.

DNA binding potency of the compounds was measured by cyclic voltammetry and UV-Vis

spectroscopy.

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Cyclic voltammetric measurements were performed using Biolog sp 300 Potentiostate.

Tetrabutylammoniumperchlorate (TBAP) having 99% purity supplied by Fluka and was used

as an electrolyte. UV-Visible spectra of the selected synthesized guanidines were recorded on

UV-1800 Shimadzu spectrophotometer.

2.3 Synthesis of Ferrocenyl Guanidines Ferrocenyl guanidines were synthesized in four steps.

2.3.1 Synthesis of nitrophenyl ferrocene. Two nitrophenyl ferrocenes (a & b) were synthesized in the laboratory by the coupling of ferrocene with diazonium salt of nitroanilines in ether/water mixture using PTC as a phase

transfer catalyst [1] as given in the scheme (2.1).

Scheme 2.1: Synthesis of nitrophenylferrocenes (a &b), nitro group is substituted at para position in a and at meta position in b with respect to ferrocene.

*PTC (Phase Transfer Catalyst) i.e. Cetyltrimethylammonium bromide (CTAB).

2.3.1.1 para- Nitrophenyl ferrocene (a).

(a)

Compound (a) was synthesized by applying the reported method. 4-Nitroaniline (14.1 g, 100 mmol), 35 ml of water and 35 ml of concentrated hydrochloric acid were mixed together and

were cooled to 0-5 °C using salt-ice mixture in a water bath. A solution of sodium nitrite (7.2

g, 100 mmol) in 50 ml of water was added drop wise to the above mixture with constant

stirring. After complete addition, the resulting solution was stirred for 20 minutes and kept

below 5 °C to obtain diazonium salt solution. Ferrocene (9.6 g, 50 mmol) and PTC (0.5 g,

1.38 mmol) were added to 155 ml diethyl ether and cooled to 0-5 °C. The above prepared

diazonium salt solution was added drop wise to ferrocene solution with constant stirring and

38  

keeping the temperature below 5 °C. The reaction mixture was stirred overnight at room

temperature and was made concentrated on a rotary evaporator. The residue was washed with

water and the crude solid was then steam distilled for the removal of unreacted ferrocene. The

product was recrystallized from n-hexane to give 4-nitrophenylferrocene (a) as violet pellets (14.3 g, 73.7 %), FTIR (KBr, υ cm-1): 2992.4, 1592.2, 1507.8, 1343.5, 1286.3, 1105.9, 1081.6, 847.2, 756.1, 696.8, 505.6, 494.2, 472.8; 1H NMR (400 MHz, CDCl3, ppm) δ 4.31 (s, 5H, C5H5), 4.68 (t, J = 1.8 Hz, 2H, C5H4), 4.99 (t, J = 1.8 Hz, 2H, C5H4), 7.46 (d, J = 8.8

Hz, 2H, Ar-H), 8.16 (d, J = 8.8 Hz, 2H, Ar-H); 13CNMR (101 MHz, CDCl3, ppm) δ 67.1 (2C), 68.4 (2C), 69.5 (5C), 81.7 (C), 123.8 (2C), 125.9 (2C), 148.2, 145.5; Anal. Calcd. For

C16H13FeNO2 C, 62.57; H, 4.27; N, 4.56 found C, 62.53; H, 4.25; N, 4.59 %;

2.3.1.2 meta-Nitrophenylferrocene (b).

(b) Yield 15.4 g (75 %), FTIR (KBr, υ cm-1): 3083.5, 2935.1, 1527.7, 1342.9, 1105.3, 742.1, 723.6, 675.9, 498.4, 462.5; 1H NMR (400 MHz, CDCl3, ppm) δ 4.23 (s, 5H, C5H5), 4.51 (t, J = 1.8 Hz, 2H, C5H4), 4.78 (t, J = 1.8 Hz, 2H, C5H4), 7.44 (t, J = 7.9 Hz, 1H, Ar-H), 7.76 (d, J

= 7.9 Hz, 1H, Ar-H), 8.03 (d, J = 8.2 Hz, 1H, Ar-H), 8.28 (s, 1H, Ar-H); 13CNMR (101 MHz, CDCl3, ppm) δ 66.8 (2C), 68.4 (2C), 69.8 (5C), 81.1, 120.4, 120.5, 129.2, 131.6, 139.6, 145.1;

Anal. Calcd. For C16H13FeNO2 C, 62.57; H, 4.27; N, 4.56; found C, 62.55; H, 4.25; N, 4.58

%;

2.3.2 Synthesis of ferrocenylanilines (c & d). Nitrophenylferrocenes (a & b) already synthesized were reduced to their respective anilines (c & d) by dissolving nitrophenyl ferrocene in ethanol, a small amount of palladium charcoal was added with constant stirring. Hydrazine 70% was added and the mixture was refluxed.

The progress of the reaction was monitored on TLC. After complete reduction a yellow color

mixture was obtained which was filtered and was evaporated under reduced pressure. The

residue was re-dissolved in dichloromethane (20 ml) and was washed with water (4 x 30 ml)

39  

and dried the organic phase over anhydrous magnesium sulphate. The solvent was evaporated

to obtain solid ferrocenyl aniline as given in scheme (2.2) [2].

Scheme 2.2: Synthesis of para (c) and meta (d) ferrocenyl anilines.

2.3.2.1 para-Ferrocenylaniline (c).

(c)

Yield 1.68 g (97 %). m.p; 156-157 ºC. FT-IR (KBr, υ cm-1): 3435.2, 3372.6, 3354.3, 1616.5, 1528.9, 1455.7, 1103.9, 999.1, 814.4.747.8, 628.2, 565.7, 520.1, 494.6, 472.3, 447.1.1H NMR (400 MHz, CDCl3, ppm) δ 3.66 (bs, 2H), 4.08 (s, 5H, C5H5), 4.28 (t, J = 1.8 Hz, 2H, C5H4),

4.59 (t, J = 1.8 Hz, 2H, C5H4), 6.67 (d, J = 8.2 Hz, 2H, Ar-H), 7.33 (d, J = 8.2 Hz, 2H, Ar-H); 13CNMR (101 MHz, CDCl3, ppm) δ 65.6 (2C), 68.2 (2C), 69.3 (5C), 84.6, 115.2 (2C), 127.2 (2C),130.1, 144.5; Anal. Calcd. For C16H15FeN; C, 69.34; H, 5.46; N, 5.05 found C, 69.29; H,

5.41; N, 5.09 %;

2.5.2.2 meta-Ferrocenylaniline (d)

(d)

Yield 1.6 g (94 %), FT-IR (KBr, υ cm-1): 3398.6, 3311.8, 3252.2, 2925.1, 1595.2, 1527.1, 1433.8, 1116.2, 987.6, 812.4, 544.2. 495.1, 473.4, 442.2. 1H NMR (400 MHz, CDCl3, ppm) δ 3.67 (bs, 2H), 4.15 (s, 5H, C5H5), 4.29 (t, J = 1.9 Hz, 2H, C5H4), 4.71 (t, J = 1.9 Hz, 2H,

C5H4), 6.53 (d, J = 7.9 Hz, 1H, Ar-H), 6.84 (s, 1H, Ar-H),6.91 (d, J = 7.9 Hz, 1H, Ar-H),7.09

(t, J = 7.3 Hz, 1H, Ar-H); 13CNMR (101 MHz, CDCl3, ppm) δ 66.5 (2C), 67.4 (2C), 69.7

40  

(5C), 82.5, 120.1, 120.4, 123.4, 129.8, 131.6, 142.2; Anal. Calcd. For C16H15FeN; C, 69.34;

H, 5.46; N, 5.05 found C, 69.32; H, 5.44; N, 5.07 %;

2.3.3 Synthesis of N, N disubstituted phenylthioureas. A number of substituted phenylthioureas were synthesized in the laboratory from benzoic acid

and chlorobenzoic acids by the reported method [3].

2.3.3.1 Phenylthioureas derived from benzoic acid and different amines (e1-18). Benzoyl chloride was obtained by the treatment of benzoic acid with thionyl chloride and was

added to the suspension of potassium thicayanate in acetone. The reaction mixture was heated

for 15 minutes and then stirred at room temperature for 1-2 hours and obtained benzoyl

isothiocyanate. Amines having different (R1) groups given in figure (2.1) were then added to the reaction mixture with continous stirring and got the desired thioureas. Progress of the

reaction was monitored by TLC at regular time intervals till completion of the reaction. The

reaction mixture was poured into an ice cooled water to remove the impurities. The solid

product obtained was then filtered and washed with deionized water. Thiourea so obtained

was dried in air and recrystallized in acetone to obtain the fine fibers which were used as such

in further reactions (Scheme 2.3).

Scheme 2.3: Synthesis of N, N disubstituted phenylthioureas (e).

41  

Figure 2.1: Different (R1) groups used in the synthesis of benzoylthiourea.

2.3.3.2 Phenylthioureas derived from chlorobenzoic acid and different amines (f1-6). Phenyl thioureas (f) were synthesized by applying the same method qouted earlier except that 3-chlorobenzoyl chloride was used instead of benzoyl chloride (Scheme 2.4).

Scheme 2.4: Synthesis of N, N disubstituted phenylthioureas (f).

42  

Figure 2.2: Different (R1) groups used in the synthesis of chlorobenzoyl phenylthiourea.

2.3.4 Synthesis of ferrocenyl guanidines from benzoylthioureas The benzoylphenylthiourea (e & f) were mixed with the ferrocenyl aniline (c & d) in DMF in equimolar ratio with two equivalents of triethylamine. The temperature was maintained below

5 ˚C using an ice bath and one equivalent of mercuric chloride was added to the reaction

mixture with vigorous stirring. The ice bath was removed after 30 minutes while the stirring

continued overnight. The progress of the reaction was monitored by TLC till the completion

of reaction. Chloroform (20 ml) was added to the reaction mixture and the suspension was

filtered through a sintered glass funnel to remove the mercuric sulphide residue. The solvents

from filterate were evaporated under reduced pressure and residue was redissolved in

dichloromethane (20 ml) and was washed with water (4 x 30 ml) and dried the organic phase

over anhydrous magnesium sulphate. The solvent was evaporated and the residue was purified

by column chromatography for obtaining ferrocenyl guanidines (g & h) as given in scheme (2.5) [4].

Scheme 2.5: Synthesis of ferrocenyl guanidines (g & h), ferrocene moity is substituted at

para position in (g) and at meta position in (h) with respect to the NH of guanidine functionality.

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2.3.4.1 para-Ferrocenylguanidines (g 1-18). Eighteen ferrocenyl guanidines of the type (g) were synthesized. Charracterization data for each of the compounds along with structural formula is given below.

(g-1)

N-(4-ferrocenylphenyl-N’-phenyl-N”-benzoylguanidine (g-1). Yield: 2.59 g, 72%; m. P. 151-152 °C; FT-IR (KBr, cm-1): 3340.3, 3278.7, 2995.4, 1673.7, 1583.1, 1517.5, 1465.4, 1449.6, 1394.1, 1331.5, 1280.5, 1243.9, 1179.7, 1102.1, 1078.3,

1033.1, 877.3, 860.5, 800.1, 730.2, 660.4, 599.5, 579.2, 480.7, 448.3, 420.1; 1H NMR (300 MHz, CDCl3, 25 °C): δ 4.11 (s, 5H, C5H5), 4.27 (t, 2H, J = 1.9 Hz, C5H4), 4.79 (t, 2H, J = 1.9

Hz, C5H4), 7.24 (d, 2H, J = 8.2 Hz. Ar-H), 7.33 (d, 2H, J = 8.1 Hz, Ar-H), 7.46-7.91 (m, 10H,

Ar-H), 10.38 (s, H, N-H), 11.39(s, H, N-H); 13C NMR (75.47 MHz, CDCl3, 25 °C): δ 66.1 (2C), 67.01 (2C), 67.5 (5C), 81.6, 121.4 (2C), 122.0, 122.3, 124.6, 129.1, 129.2 (2C), 129.8

(2C), 130.4 (2C), 130.6, 134.2, 134.5, 135.5, 140.1, 140.6, 156.8 (CN3), 176.3 (C=O; Anal.

Calcd. For C30 H25 N3 Fe O (499.13): C,72.15; H, 5.05; N, 8.41; Found: C,72.09; H, 5.01; N,

8.45 %.

(g-2)

N-(4-ferrocenylphenyl)-N’-(3-triflourometylphenyl)-N”-benzoylguanidine (g-2). Yield: 79%; m. P. 159-159.5 °C; FT-IR (KBr, cm-1): 3342.1, 3283.6, 3030.6, 1675.5, 1599.8, 1536.6, 1486.6, 1421.4, 1385.8, 1248.3, 1178.9, 1060.6, 1015.5, 988.6, 906.3, 803.9, 725.5,

690.2, 570.3, 558.6, 525.1, 475.5, 446.9, 412.1; 1H NMR (300 MHz, CDCl3): δ 4.14 (s, 5H, C5H5), 4.33 (t, 2H, J = 1.9Hz C5H4 ), 4.82 (t, J = 1.9Hz 2H, C5H4), 7.28 (d, 2H, J = 8.1Hz, Ar-

H), 7.35 (d, 2H, J = 8.1Hz, Ar-H), 7.50 (s, H, Ar-H), 7.55-8.21 (m, 8H, Ar-H), 10.60 (s, H,

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N-H), 11.62 (s, H, N-H); 13C NMR (75.47 MHz, CDCl3): δ 66.9 (2C), 67.7 (2C), 68.4 (5C), 84.6, 120.1, 121.9 (2C), 122.2, 124.2 (q, J = 3.5 Hz, CF3), 124.7, 129.1 (2C), 129.7 (2C),

130.6 (2C), 132.1, 134.7, 135.5, 136.4, 139.9, 140.2, 143.6, 160.8 (CN3), 179.2 (C=O).Anal.

Calcd. For C31H24N3F3FeO (567.38): C, 65.62; H, 4.26; N, 7.41; Found: C, 65.59; H, 4.25; N,

7.42%.

(g-3)

N-(4-ferrocenylphenyl)-N’-(4-triflourometylphenyl)-N”-benzoylguanidine (g-3). Yield: 78%; m. p. 158-159 °C; FT-IR (KBr, cm-1): 3345.3, 3286.1, 3033.6, 2916.1, 1677.4, 1597.7, 1562.9, 1532.1, 1486.8, 1422.1, 1340.9, 1248.6, 1231.3, 1178.8, 1062.8, 1015.7,

980.1, 785.3, 738.6, 688.4, 585.1, 569.7, 537.9, 475.4, 435.2, 407.1; 1H NMR (300 MHz, CDCl3): δ 4.21 (s, 5H, C5H5), 4.33 (t, 2H, J = 1.9Hz C5H4), 4.81 (t, 2H, J = 1.9Hz C5H4),

7.29(d, 2H, J = 8.1Hz, Ar-H), 7.35 (d, 2H, J = 8.1Hz, Ar-H), 7.57 (d, 2H, J = 8.2 Hz, Ar-H),

7.61 -8.01 (m, 5H, Ar-H), 8.22 (d, 2H, J = 8.2 Hz, Ar-H), 10.56 (s, H, N-H), 11.61 (s, H, N-

H); 13C NMR (75.47 MHz, CDCl3): δ 67.1 (2C), 67.8 (2C), 68.5 (5C), 84.7, 121.6 (2C), 122.3 (2C), 124.2 (q, J = 3.5 Hz, CF3), 125.0, 128.6 (2C), 129.2 (2C), 129.8 (2C), 130.6 (2C),

134.7, 136.0, 139.9, 141.6, 145.3, 160.7 (CN3), 179.4 (C=O). Anal.Calcd. For

C31H24N3F3FeO (567.38): C, 65.62; H, 4.26; N, 7.41; Found: C, 65.60; H, 4.25; N, 7.42%.

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(g-4)

N-(4-ferrocenylphenyl)-N’-(4-nitrophenyl)-N”-benzoylguanidine (g-4). Yield: 74%; m. p. 156-156.5 °C; FT-IR (KBr, cm-1): 3356.4, 3088.1, 1678.4, 1581.7, 1510.2, 1470.2, 1346.8, 1250.9, 1178.6, 1105.2, 1068.7, 1029.1, 1002.7, 979.5, 906.3, 835.9, 802.9,

795.0, 690.2, 555.3, 532.8, 498.4, 484.6, 455.9, 435.8, 410.6; 1H NMR (300 MHz, CDCl3): δ 4.21 (s, 5H, C5H5), 4.37 (t, 2H, J = 1.8Hz, C5H4), 4.89 (t, 2H, J = 1.8Hz C5H4), 7.30 (d, 2H, J

= 8.1Hz, Ar-H), 7.38 (d, 2H, J = 8.1Hz, Ar-H), 7.55 (d, 2H, J = 8.2 Hz, Ar-H), 7.62-8.12 (m,

7H, Ar-H), 10.53 (s, H, N-H), 11.55 (s, H, N-H); 13C NMR (75.47 MHz, CDCl3): δ 66.9 (5C), 67.8 (2C), 68.48 (2C), 84.6, 121.8 (2C), 124.1 (2C), 127.5 (2C), 129.3 (2C), 129.7 (2C),

130.8 (2C), 134.7, 135.9, 139.1, 139.9, 141.8, 146.6, 160.1 (CN3), 179.3 (C=O). Anal.Calcd.

For C30H24N4FeO3 (544.3): C, 66.19; H, 4.44; N, 10.29; Found: C, 66.18; H, 4.44; N, 10.30%;

(g-5)

N-(4-ferrocenylphenyl)-N’-(4-methylphenyl)-N”-benzoylguanidine (g-5). Yield: 74%; m. p. 158-159 °C; FT-IR (KBr, cm-1): 3342.9, 3255.3, 3085.6, 1671.5 , 1586.2, 1568.8, 1465.8, 1419.7, 1342.6, 1275.4, 1265.2, 1167.7, 1104.6, 1079.5, 1057.9, 990.1, 910.2,

807.3, 735.5, 679.9, 608.4, 591.6, 566.2, 490. 4, 451.6, 411.1; 1H NMR (300 MHz, CDCl3): δ 2.42 (s, 3H, Me-H), 4.11 (s, 5H, C5H5), 4.28 (t, 2H, J = 1.9Hz C5H4), 4.81 (t, 2H, J = 1.9Hz

C5H4), 7.23 (d, 2H, J = 8.2 Hz, Ar-H), 7.30 (d, 2H, J = 8.2 Hz, Ar-H), 7.38 (d, 2H, J = 8.1Hz,

Ar-H), 7.48 (d, 2H, J = 8.1Hz, Ar-h), 7.52-7.92 (m, 5H, Ar-H), 10.49 (s, H, N-H), 11.31 (s,

H, N-H); 13C NMR (75.47 MHz, CDCl3): δ 16.0 (CH3), 66.0 (2C), 67.01 (2C), 67.75 (5C),

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82.3, 121.1 (2C), 122.5 (2C), 128.5, 129.2 (2C), 129.6 (2C), 129.8 (2C),130.2 (2C), 134.5,

136.5, 139.3, 140.2, 140.8, 156.5 (CN3), 176.1 (C=O). Anal.Calcd. For C31H27N3FeO (513.4):

C, 72.52; H, 5.30; N, 8.18; Found: C, 72.51; H, 5.29; N, 8.19%.

(g-6)

N-(4-ferrocenylphenyl)-N’-(2-methylphenyl)-N”-benzoylguanidine (g-6). Yield: 78%; m. p. 155-156 °C; FT-IR (KBr, cm-1): 3341.2, 3254.6, 3094.7, 3001.1, 2920.8,

1670.6, 1605.4, 1595.2, 1528.8, 1465.7, 1419.1, 1385.5, 1342.6, 1265.4, 1172.7, 1144.3,

1079.8, 1057.5,1039.1, 1001.3, 982.7, 893.1, 807.8, 788.6, 679.5, 608.3, 566.4, 520.6, 490.9,

460.1; 1H NMR (300 MHz, CDCl3): δ 2.42 (s, 3H, Me-H), 4.12 (s, 5H, C5H5), 4.29 (t, 2H, J = 1.9Hz C5H4), 4.80 (t, 2H, J = 1.9Hz C5H4), 7.25 (d, 2H, J = 8.1 Hz, Ar-H), 7.31 (d, 2H, J =

8.1 Hz, Ar-H), 7.33-7.92 (m, 9H, Ar-H), 10.45 (s, H, N-H), 11.36(s, H, N-H); 13C NMR (75.47 MHz, CDCl3): δ 16.1, 66.2 (2C), 67.01 (2C), 67.5 (5C), 82.1, 121.6 (2