synthesis of indole and tryptamine derivatives and their

i

Synthesis of Indole and Tryptamine Derivatives and Their

Bioactivities

A Dissertation Submitted to the Faculty of Science for the

Partial Fulfillment for the Requirement of the Degree of Doctor of Philosophy

by

KANWAL

H. E. J. Research Institute of Chemistry

International Center for Chemical and Biological Sciences

University of Karachi, Karachi-75270

Pakistan

ii

Dedicated

To

My Lovable Parents (My inspiration)

Mr. Haji Ahmed

&

Mrs. Rabia Bano

My Niece (Ms. Aiza Noor)

My Siblings

and

My Teachers

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THESIS CERTIFICATE

This to certify that the dissertation entitled, “Synthesis of Indole and Tryptamine Derivatives and

Their Bioactivities,” has been submitted to the Board of Advance Studies and Research (BASR),

University of Karachi, by Ms. Kanwal D/o Mr. Haji Ahmed for the award of Doctor of Philosophy

(Ph.D.) degree in Chemistry. This research has been conducted under my supervision, and I certify

the originality of the work. The work presented in this thesis meets the criteria of the University

of Karachi, for the award of Ph. D. degree. Thesis and work presented here have not been

previously submitted to any institution for any degree.

Prof. Dr. Khalid M. Khan, Sitara-i-Imtiaz, Tamgha-i-Imtiaz

H. E. J. Research Institute of Chemistry

International Center for Chemical and Biological Sciences

University of Karachi

Karachi-75270

Pakistan

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List of Contents

PERSONAL INTRODUCTION ............................................................................................... xiii

ACKNOWLEDGEMENTS ...................................................................................................... xiv

SUMMARY ................................................................................................................................. xv

Chapter 1 ....................................................................................................................................... 1

Synthesis of Tryptamine Derivatives and Their Bioactivities................................................... 1

1 General Introduction ............................................................................................................. 2

1.1 Introduction ...................................................................................................................... 2

1.2 Biological Importance of Tryptamine .............................................................................. 4

1.2.1 Anti-ulcerogenic Activity ......................................................................................... 4

1.2.2 Antihepatitis activity ................................................................................................. 5

1.2.3 CDK4/ cyclin D1 inhibitors ........................................................................................ 5

1.2.4 As Hallucinogens ...................................................................................................... 5

1.2.5 As 5-HT6 receptor ligands ......................................................................................... 6

1.2.6 Antioxidant activity .................................................................................................. 7

1.2.7 As 5-HT1D receptor agonists ..................................................................................... 7

1.2.8 Antileishmanial activity ............................................................................................ 7

1.2.9 β-Adrenergic receptors agonists ............................................................................... 8

1.2.10 H5-HT2A Antagonists ................................................................................................ 8

1.3 Synthetic strategies for tryptamine ................................................................................... 9

1.3.1 By reacting chloroalkylalkyne with substituted aryl hydrazines ............................ 12

1.3.2 Fischer synthesis of tryptamines ............................................................................. 13

1.3.3 Grandberg Reaction for the Synthesis of Tryptamine ............................................ 14

PART-A ....................................................................................................................................... 15

2 Synthesis of Schiff bases of Tryptamine ............................................................................ 15

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2.1 Results and Discussion ................................................................................................... 15

2.1.1 Chemistry ................................................................................................................ 15

2.2 Characteristic Spectral Feature of Representative Compound 60 .................................. 16

2.2.1 1H NMR Spectroscopy ............................................................................................ 16

2.2.2 Mass Spectrometry.................................................................................................. 18

2.3 Biological Screening ...................................................................................................... 22

2.3.1 Antileishmanial Activity ......................................................................................... 22

2.3.2 Antiglycation activity.............................................................................................. 25

2.3.3 Di-peptidyl peptidase (DPP-IV) activity ................................................................ 30

2.3.1 NTPDase (Nucleotide triphosphate diphospho hydrolase) inhibitory activity ....... 32

2.4 Conclusion ...................................................................................................................... 35

2.5 Physical Data for the Synthesized Compounds.............................................................. 36

2-((2-(1H-Indol-3-yl)ethylimino)methyl)-4,6-dichlorophenol (60) ...................................... 36

2-((2-(1H-Indol-3-yl)ethylimino)methyl)phenol (61) ........................................................... 36

4-((2-(1H-Indol-3-yl)ethylimino)methyl)-2-bromo-6-methoxyphenol (62) ......................... 37

2-((2-(1H-Indol-3-yl)ethylimino)methyl)-4-chlorophenol (63) ............................................ 37

2-((2-(1H-Indol-3-yl)ethylimino)methyl)-6-bromo-4-chlorophenol (64) ............................. 37

2-((2-(1H-Indol-3-yl)ethylimino)methyl)-4-fluorophenol (65) ............................................. 37

N-(2-Bromo-4,5-dimethoxybenzylidene)-2-(1H-indol-3-yl)ethanamine (66) ...................... 38

4-((2-(1H-Indol-3-yl)ethylimino)methyl)-2-iodo-6-methoxyphenol (67) ............................. 38

4-((2-(1H-Indol-3-yl)ethylimino)methyl)-2,6-dimethoxyphenol (68) .................................. 38

4-((2-(1H-Indol-3-yl)ethylimino)methyl)benzene-1,3-diol (69) ........................................... 38

2-((2-(1H-Indol-3-yl)ethylimino)methyl)-4,6-di-tert-butylphenol (70) ................................ 39

2-((2-(1H-Indol-3-yl)ethylimino)methyl)-3-bromo-6-methoxyphenol (71) ......................... 39

N-(2-Fluoro-4-methoxybenzylidene)-2-(1H-indol-3-yl)ethanamine (72) ............................. 39

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2-(1H-Indol-3-yl)-N-(4-(methylthio)benzylidene)ethanamine (73) ...................................... 39

N-(Furan-2-ylmethylene)-2-(1H-indol-3-yl)ethanamine (74) ............................................... 40

N-(3,4-Dimethoxybenzylidene)-2-(1H-indol-3-yl)ethanamine (75) ..................................... 40

4-((2-(1H-Indol-3-yl)ethylimino)methyl)-2-bromophenol (76) ............................................ 40

3-((2-(1H-Indol-3-yl)ethylimino)methyl)benzene-1,2-diol (77) ........................................... 40

1-((2-(1H-Indol-3-yl)ethylimino)methyl)naphthalen-2-ol (78) ............................................. 41

5-((2-(1H-Indol-3-yl)ethylimino)methyl)-2-methoxyphenol (79) ........................................ 41

2-(1H-Indol-3-yl)-N-(naphthalen-1-ylmethylene)ethanamine (80) ....................................... 41

N-(Anthracen-9-ylmethylene)-2-(1H-indol-3-yl)ethanamine (81) ........................................ 41

4-((2-(1H-indol-3-yl)ethylimino)methyl)-2-chlorophenol (82) ............................................ 42

2-((2-(1H-Indol-3-yl)ethylimino)methyl)-4-nitrophenol (83) ............................................... 42

2-((2-(1H-Indol-3-yl)ethylimino)methyl)-4-bromophenol (84) ............................................ 42

2-((2-(1H-Indol-3-yl)ethylimino)methyl)-5-methoxyphenol (85) ........................................ 43

Part-B ........................................................................................................................................... 44

3 Urea and Thiourea Derivatives of Tryptamine ................................................................ 44


3.1.1 Chemistry ................................................................................................................ 44

3.2 Characteristic Spectral Features of Representative Compound 100 .............................. 45

3.2.1 1H NMR Spectroscopy ............................................................................................ 45



3.3.1 Urease Inhibitory Activity ...................................................................................... 48

3.3.2 Carbonic anhydrase inhibitory Activity .................................................................. 52

3.3.3 Bacterial multidrug resistance (MDR) activity ....................................................... 55


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3.3.5 Antiepileptic Activity.............................................................................................. 60

3.4 Conclusion ...................................................................................................................... 61


1-(2-(1H-Indol-3-yl)ethyl)-3-(naphthalen-1-yl)urea (87) ...................................................... 62

1-(2-(1H-Indol-3-yl)ethyl)-3-phenylthiourea (88) ................................................................ 62

1-(2-(1H-Indol-3-yl)ethyl)-3-(4-nitrophenyl)urea (89) ......................................................... 62

1-(2-(1H-Indol-3-yl)ethyl)-3-(3-chlorophenyl)urea (90) ...................................................... 63

1-(2-(1H-Indol-3-yl)ethyl)-3-(2-(trifluoromethyl)phenyl)urea (91) ...................................... 63

1-(2-(1H-Indol-3-yl)ethyl)-3-(3-(trifluoromethyl)phenyl)urea (92) ...................................... 63

1-(2-(1H-Indol-3-yl)ethyl)-3-(4-nitrophenyl)thiourea (93) ................................................... 63

1-(2-(1H-Indol-3-yl)ethyl)-3-(3-fluorophenyl)thiourea (94) ................................................. 64

1-(2-(1H-Indol-3-yl)ethyl)-3-(5-chloro-2-methylphenyl)thiourea (95) ................................ 64

1-(2-(1H-Indol-3-yl)ethyl)-3-(2-bromophenyl)thiourea (96) ................................................ 64

1-(2-(1H-Indol-3-yl)ethyl)-3-(2-fluorophenyl)thiourea (97) ................................................. 65

1-(2-(1H-Indol-3-yl)ethyl)-3-(2,6-dimethylphenyl)thiourea (98) ......................................... 65

1-(2-(1H-Indol-3-yl)ethyl)-3-(3-bromophenyl)thiourea (99) ................................................ 65

1-(2-(1H-Indol-3-yl)ethyl)-3-(o-tolyl)thiourea (100) ............................................................ 65

1-(2-(1H-Indol-3-yl)ethyl)-3-(2,5-dichlorophenyl)thiourea (101) ........................................ 66

1-(2-(1H-Indol-3-yl)ethyl)-3-(4-chlorophenyl)thiourea (102) .............................................. 66

1-(2-(1H-Indol-3-yl)ethyl)-3-(2,4-dimethoxyphenyl)thiourea (103) .................................... 66

1-(2-(1H-Indol-3-yl)ethyl)-3-(2-methoxyphenyl)thiourea (104) .......................................... 67

1-(2-(1H-Indol-3-yl)ethyl)-3-(2-chlorophenyl)thiourea (105) .............................................. 67

1-(2-(1H-Indol-3-yl)ethyl)-3-(2,4-difluorophenyl)thiourea (106) ......................................... 67


1-(2-(1H-Indol-3-yl)ethyl)-3-(3,5-dimethylphenyl)thiourea (108) ....................................... 68

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1-(2-(1H-Indol-3-yl)ethyl)-3-(2,3-dichlorophenyl)thiourea (109) ........................................ 68


1-(2-(1H-Indol-3-yl)ethyl)-3-(4-bromophenyl)thiourea (111) .............................................. 68

References ................................................................................................................................. 69

Chapter 2 ..................................................................................................................................... 75

Synthesis of Indole Derivatives and Their Structure-Activity Relationship Studies ............ 75

4 Introduction .................................................................................................................... 76

4.1 General introduction to indoles ...................................................................................... 76

4.1.1 Reactivity of Indole................................................................................................. 78

4.2 Biological activities of Indole ........................................................................................ 79

4.3 Synthetic strategies of indole ......................................................................................... 83

4.3.1 Sigmatropic rearrangements ................................................................................... 83

4.3.2 Nucleophilic cyclization ......................................................................................... 85

4.3.3 Electrophilic cyclization ......................................................................................... 86

4.3.4 Nitrene Cyclization ................................................................................................. 86

4.3.5 Oxidative cyclization .............................................................................................. 86

PART-A ....................................................................................................................................... 87

5 Synthesis of Indole-3-acetamides ....................................................................................... 87


5.1.1 Chemistry ................................................................................................................ 87

5.1.2 Plausible mechanism for the synthesis of indole-3-acetamides .............................. 88

5.2 Spectral Characterization of Representative Analogue 55 ............................................. 88

5.2.1 1H NMR Spectroscopic characterization ................................................................ 88



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5.3.1 Urease Inhibitory Activity ...................................................................................... 93


5.3.3 Anticancer Activity ................................................................................................. 95

5.3.4 Bacterial Multi Drug Resistance ............................................................................. 96

5.3.5 Antiglycation activity.............................................................................................. 98

5.4 Conclusion ...................................................................................................................... 98


2-(1H-Indol-3-yl)-N-phenylacetamide (55) ........................................................................... 99

N-(4-Bromophenyl)-2-(1H-indol-3-yl) acetamide (56) ......................................................... 99

N-(2,5-Dimethoxyphenyl)-2-(1H-indol-3-yl) acetamide (57) ............................................... 99

N-(4-Ethylphenyl)-2-(1H-indol-3-yl) acetamide (58) ......................................................... 100

2-(1H-Indol-3-yl)-N-(3-methoxy-4-methylphenyl) acetamide (59) .................................... 100

N-(4-Fluorophenyl)-2-(1H-indol-3-yl) acetamide (60) ....................................................... 100

N-(3-Fluorophenyl)-2-(1H-indol-3-yl) acetamide (61) ....................................................... 100

N-(3-Bromophenyl)-2-(1H-indol-3-yl) acetamide (62) ....................................................... 101

N-(4-Butylphenyl)-2-(1H-indol-3-yl) acetamide (63) ......................................................... 101

2-(1H-Indol-3-yl)-N-(3-(methylthio) phenyl) acetamide (64) ............................................. 101

2-(1H-Indol-3-yl)-N-(4-(methylthio) phenyl) acetamide (65) ............................................. 101

N-(5-Chloro-2-methylphenyl)-2-(1H-indol-3-yl) acetamide (66) ....................................... 102

2-(1H-Indol-3-yl)-N-(4-iodophenyl) acetamide (67) ........................................................... 102

N-(2,4-Difluorophenyl)-2-(1H-indol-3-yl) acetamide (68) ................................................. 102

N-(4-Chlorophenyl)-2-(1H-indol-3-yl) acetamide (69) ....................................................... 102

2-(1H-Indol-3-yl)-N-(4-(octyloxy) phenyl) acetamide (70) ................................................ 103

N-(4-Butoxyphenyl)-2-(1H-indol-3-yl) acetamide (71) ...................................................... 103

2-(1H-Indol-3-yl)-N-(4-methoxyphenyl) acetamide (72) .................................................... 103

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2-(1H-Indol-3-yl)-N-(3-iodophenyl) acetamide (73) ........................................................... 103

N-(3,4-Dimethylphenyl)-2-(1H-indol-3-yl) acetamide (74) ................................................ 104

N-(3,4-Difluorophenyl)-2-(1H-indol-3-yl) acetamide (75) ................................................. 104



N-(5-Chloro-2,4-dimethoxyphenyl)-2-(1H-indol-3-yl)acetamide (78) ............................... 105

2-(1H-Indol-3-yl)-N-(3-nitrophenyl) acetamide (79) .......................................................... 105

2-(1H-Indol-3-yl)-N-(2-methoxyphenyl) acetamide (80) .................................................... 105

2-(1H-Indol-3-yl)-N-(p-tolyl) acetamide (81) ..................................................................... 105

Part-B ......................................................................................................................................... 107

6 Synthesis of Indole acrylonitriles ..................................................................................... 107

6.1 Results and Discussion ................................................................................................. 107

6.1.1 Chemistry .............................................................................................................. 107

6.2 Representative structure elucidation by 1H NMR and mass spectroscopy of compound

113................................................................................................................................... 109

6.2.1 1H NMR Spectroscopy .......................................................................................... 109

6.2.2 Mass Spectrometry................................................................................................ 111

6.3 Biological Screening .................................................................................................... 115

6.3.1 Antiepileptic Activity............................................................................................ 115

6.3.2 Anticancer Activity (HeLa cancer cell lines) ........................................................ 118

6.3.3 Antiinflammatory activity ..................................................................................... 121

6.4 Conclusion .................................................................................................................... 124

6.5 Physical Data for the Synthesized Compounds............................................................ 124

3-(2,3-Dimethoxyphenyl)-2-(1H-indole-3-carbonyl)acrylonitrile (83) ............................... 124

3-(2,4-Dichlorophenyl)-2-(1H-indole-3-carbonyl)acrylonitrile (84) .................................. 124

viii

3-(1H-Indol-3-yl)-2-(1H-indole-3-carbonyl)acrylonitrile (85) ........................................... 125

3-(4-Ethoxy-3-methoxyphenyl)-2-(1H-indole-3-carbonyl)acrylonitrile (86) ..................... 125


3-(2-Bromo-4,5-dimethoxyphenyl)-2-(1H-indole-3-carbonyl)acrylonitrile (88) ................ 125


3-(4-Hydroxy-3-iodo-5-methoxyphenyl)-2-(1H-indole-3-carbonyl)acrylonitrile (90) ....... 126

3-(5-Hydroxy-2-nitrophenyl)-2-(1H-indole-3-carbonyl)acrylonitrile (91) ......................... 126

2-(1H-Indole-3-carbonyl)-3-(3,4,5-trimethoxyphenyl)acrylonitrile (92) ............................ 126

3-(2-Chloro-5-nitrophenyl)-2-(1H-indole-3-carbonyl)acrylonitrile (93) ............................ 126


3-(3-Bromo-4, 5-dimethoxyphenyl)-2-(1H-indole-3-carbonyl)acrylonitrile (95) ............... 127

3-(5-Bromo-2-methoxyphenyl)-2-(1H-indole-3-carbonyl) acrylonitrile (96) ..................... 127

2-(1H-Indole-3-carbonyl)-3-phenylacrylonitrile (97) ......................................................... 128

3-(3-Bromo-4,5-dimethoxyphenyl)-2-(1,2-dimethyl-1H-indole-3-carbonyl)acrylonitrile (98)

............................................................................................................................................. 128

2-(1H-Indole-3-carbonyl)-3-(2,3,4-trimethoxyphenyl)acrylonitrile (99) ............................ 128

2-(1H-Indole-3-carbonyl)-3-(3-methoxyphenyl) acrylonitrile (100) .................................. 128

3-(2-Chloro-3,4-dimethoxyphenyl)-2-(1H-indole-3-carbonyl)acrylonitrile (101) .............. 129

3-(2,3-Dimethoxyphenyl)-2-(1,2-dimethyl-1H-indole-3-carbonyl)acrylonitrile (102) ....... 129

3-(2,4-Dimethoxyphenyl)-2-(1,2-dimethyl-1H-indole-3-carbonyl)acrylonitrile (103) ....... 129

2-(1,2-Dimethyl-1H-indole-3-carbonyl)-3-(2-fluoro-4-methoxyphenyl)acrylonitrile (104)

............................................................................................................................................. 129

3-(4-Bromo-2-fluorophenyl)-2-(1,2-dimethyl-1H-indole-3-carbonyl)acrylonitrile(105) ... 130

3-(4-Bromo-2-fluorophenyl)-2-(1H-indole-3-carbonyl)acrylonitrile (106) ........................ 130

3-(5-Bromo-2-methoxyphenyl)-2-(1,2-dimethyl-1H-indole-3-carbonyl)acrylonitrile (107)

............................................................................................................................................. 130

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3-(3-Bromo-4-methoxyphenyl)-2-(1,2-dimethyl-1H-indole-3-carbonyl)acrylonitrile (108)

............................................................................................................................................. 131

2-(1,2-Dimethyl-1H-indole-3-carbonyl)-3-(3,4,5-trimethoxyphenyl)acrylonitrile (109) ... 131

3-(2-Bromo-5-fluorophenyl)-2-(1H-indole-3-carbonyl) acrylonitrile (110) ....................... 131

3-(Anthracen-9-yl)-2-(1H-indole-3-carbonyl) acrylonitrile (111) ...................................... 131

3-(3-Bromo-4-methoxyphenyl)-2-(1H-indole-3-carbonyl) acrylonitrile (112) ................... 132

2-(1,2-Dimethyl-1H-indole-3-carbonyl)-3-phenylacrylonitrile (113) ................................. 132

3-(4-Fluoro-3-methoxyphenyl)-2-(1H-indole-3-carbonyl) acrylonitrile (114) ................... 132

2-(1,2-Dimethyl-1H-indole-3-carbonyl)-3-(naphthalen-2-yl) acrylonitrile (115) ............... 132

3-(4-Bromophenyl)-2-(1H-indole-3-carbonyl) acrylonitrile (116) ..................................... 132

3-(2-Bromo-4,5-dimethoxyphenyl)-2-(1,2-dimethyl-1H-indole-3-carbonyl)acrylonitrile

(117)..................................................................................................................................... 133

3-(2-Chloro-3,4-dimethoxyphenyl)-2-(1,2-dimethyl-1H-indole-3-carbonyl)acrylonitrile

(118)..................................................................................................................................... 133

References ............................................................................................................................... 134

Chapter-3 ................................................................................................................................... 139

Synthesis and biological activities of Bis(indolyl) methanes ................................................. 139

7 General Introduction ......................................................................................................... 140

7.1 Introduction .................................................................................................................. 140

7.2 Biological activities of Bisindolyl methanes ................................................................ 141

7.3 Synthesis of Bisindolyl methanes and its derivatives................................................... 143

7.4 Results and Discussion ................................................................................................. 148

7.4.1 Chemistry .............................................................................................................. 148

7.5 Spectral Characterization of Representative Compound 44 ........................................ 149

7.5.1 1H NMR Spectroscopy .......................................................................................... 149

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7.5.2 Mass Spectrometry................................................................................................ 150

7.6 Biological Screening .................................................................................................... 157

7.6.1 Anticancer Activity ............................................................................................... 157

7.6.2 Antiglycation Activity .......................................................................................... 158

7.6.3 Antiinflammatory Activity.................................................................................... 159

7.7 Conclusion .................................................................................................................... 161

7.8 Physical Data for the Synthesized Compounds............................................................ 161

3,3′-(Phenylmethylene)bis(1H-indole) (38) ........................................................................ 161

3,3′-(Naphthalen-2-ylmethylene)bis(1H-indole) (39) ......................................................... 162

3,3′-((2,3-Dimethoxyphenyl)methylene)bis(1H-indole) (40) .............................................. 162

3,3′-((2-Methoxyphenyl)methylene)bis(1H-indole) (41) .................................................... 162

4-(Di(1H-Indol-3-yl)methyl)-2-methoxyphenol (42) .......................................................... 162

3,3′-((2-Bromo-5-fluorophenyl)methylene)bis(1H-indole) (43) ......................................... 163


2,4-Dichloro-6-(di(1H-indol-3-yl)methyl)phenol (45) ........................................................ 163

3,3′-((2-Nitrophenyl)methylene)bis(1H-indole) (46) .......................................................... 164

4-(Di(1H-indol-3-yl)methyl)-2-iodo-6-methoxyphenol (47) .............................................. 164

3,3′-((5-Bromo-2-methoxyphenyl)methylene)bis(1H-indole) (48) ..................................... 164

3,3′-((3,4,5-Trimethoxyphenyl)methylene)bis(1H-indole) (49) .......................................... 164


3,3′-((2,3,4-Trimethoxyphenyl)methylene)bis(1H-indole) (51) .......................................... 165

3,3′-((4-Nitrophenyl)methylene)bis(1H-indole) (52) .......................................................... 165


3,3′-((4-Bromophenyl)methylene)bis(1H-indole) (54) ....................................................... 166

3,3′-((3-Methylthiophen-2-yl)methylene)bis(1H-indole) (55) ............................................ 166

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3,3′-(Pyren-1-ylmethylene)bis(1H-indole) (56) .................................................................. 166

3,3′-((2-Fluoro-4-methoxyphenyl)methylene)bis(1H-indole) (57) ..................................... 166

4-(Di(1H-indol-3-yl)methyl)-2-methoxyphenyl acetate (58) .............................................. 167


3,3′-((4-Bromo-2-fluorophenyl)methylene)bis(1H-indole) (60) ......................................... 167

2-Bromo-4-(di(1H-indol-3-yl)methyl)phenol (61) .............................................................. 167

2-Bromo-4-(di(1H-indol-3-yl)methyl)-6-methoxyphenol (62) ........................................... 168


2-(Di(1H-indol-3-yl)methyl)-5-fluorophenol (64) .............................................................. 168

3,3′-((2-Chlorophenyl)methylene)bis(1H-indole) (65) ....................................................... 168

3,3′-(Furan-2-ylmethylene)bis(1H-indole) (66) .................................................................. 169

3,3′-((1H-Indol-2-yl)methylene)bis(1H-indole) (67) .......................................................... 169

3,3′-(Naphthalen-1-ylmethylene)bis(1H-indole) (68) ......................................................... 169

3,3′-(o-Tolylmethylene)bis(1H-indole) (69) ....................................................................... 169

3,3′-((4-Bromo-3,5-dimethoxyphenyl)methylene)bis(1H-indole) (70) ............................... 170

3,3′-((4-Nitrophenyl)methylene)bis(1H-indole-5-carbonitrile) (71) ................................... 170

3,3′-((2,4-Dimethoxyphenyl)methylene)bis(1,2-dimethyl-1H-indole) (72) ........................ 170


3,3′-((4-Bromo-3,5-dimethoxyphenyl)methylene)bis(1,2-dimethyl-1H-indole) (74) ......... 171

3,3′-((2-Bromo-4,5-dimethoxyphenyl)methylene)bis(1,2-dimethyl-1H-indole) (75) ......... 171

3,3′-(Naphthalen-2-ylmethylene)bis(1,2-dimethyl-1H-indole) (76) ................................... 171

3,3′-((3,4,5-Trimethoxyphenyl)methylene)bis(1,2-dimethyl-1H-indole) (77) .................... 171

4-(Bis(1,2-dimethyl-1H-indol-3-yl)methyl)-2-methoxyphenol (78) ................................... 172

3,3′-((4-Bromophenyl)methylene)bis(1,2-dimethyl-1H-indole) (79) .................................. 172

3,3′-((5-bromo-2-fluorophenyl)methylene)bis(1,2-dimethyl-1H-indole) (80) .................... 172

xii


3,3′-((4-Nitrophenyl)methylene)bis(1,2-dimethyl-1H-indole) (82) .................................... 173

2-(Bis(1,2-dimethyl-1H-indol-3-yl)methyl)-3-bromo-6-methoxyphenol (83) .................... 173

3,3′-(Phenylmethylene)bis(1,2-dimethyl-1H-indole) (84) .................................................. 173


4-(Bis(1,2-dimethyl-1H-indol-3-yl)methyl)-2-iodo-6-methoxyphenol (86) ....................... 174

3,3′-((4-(Benzyloxy)phenyl)methylene)bis(1,2-dimethyl-1H-indole) (87) ......................... 174

4-(Bis(1,2-dimethyl-1H-indol-3-yl)methyl)-2-bromo-6-methoxyphenol (88) .................... 174

3,3′-((2-Fluoro-4-methoxyphenyl)methylene)bis(1,2-dimethyl-1H-indole) (89) ............... 174

3,3′-((4-Methoxyphenyl)methylene)bis(1,2-dimethyl-1H-indole) (90) .............................. 175

3,3′-((5-Methylfuran-2-yl)methylene)bis(1,2-dimethyl-1H-indole) (91) ............................ 175

3,3′-(Thiophen-3-ylmethylene)bis(1,2-dimethyl-1H-indole) (92) ...................................... 175

References ............................................................................................................................... 176

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PERSONAL INTRODUCTION

My name is Kanwal and I was born in Lyari, one of the largest town in Karachi. I had a

dream of becoming doctor and it was my father’s dream also and I usually used to say that I

will become a doctor either by doing MBBS or Ph.D. I had done my matriculation from

Bright Public School which was situated on walking distance from my home and during my

schooling my principal Sir Ghulam Nabi Qureshi (Late) motivated and supported me for

higher studies. My intermediate was from Govt. College for Women Shahra-e-Liaquat (Pre-

medical) and got B grade which was not enough for admission in medical college. At that

time I was little hurt but then I admitted to the Govt. Sindh D. J Science College for

graduation. I took admission in Chemistry department of Karachi University, at that time my

father had taken a promise from me to continue my studies until M.Phil. During my BS

program, I often used the H.E.J. library for the study purpose and at that time, I dreamed to

be the part of this great institution. I got privileged to be the part of Prof. Dr. Khalid M.

Khan’s group. He has played the vital role in polishing my research skills. He is kind,

generous and like a father to me, he helped and guided me in every failure of mine. I was

lucky to learn from the great teachers during my whole period of Ph.D.

My stay at this institute will always be memorable for me. My dream wouldn’t be

accomplished without the kind support of Sir Ghulam Nabi Qureshi (Late) and my friend

cum sister Zeenat.

Kanwal

Karachi, 2017

xiv

ACKNOWLEDGEMENTS

First and foremost, countless thanks to Almighty ALLAH for blessings, much more than I

deserve and for awarding me everything “The best” beyond my thinking. I also want to pay

gratitude to ALLAH for sending me in the ummat of Hazrat Muhammad Sallallaho-

Alaihi-Wasallum. Countless Salam and Durood Pak to beloved Holy Prophet Hazrat

Muhammad Sallallaho-Alaihi-Wasallum who enlightened my conscience with the

essence of faith in ALLAH (SWT).

I want to extend my gratitude to Prof. Salimuzzaman Siddiqui, FRS, S.I., H.I. founder of H. E.

J. Research Institute of Chemistry and Prof. Atta-ur-Rahman, FRS, N.I., H.I., S.I., T.I. for their

determination in establishing an excellent and world class institution. I am also grateful to

Prof. Dr. M. Iqbal Choudhary, H.I., S.I., T.I. Director, International Center for Chemical and

Biological Sciences, University of Karachi, for running this institute with excellence, and

for the support and collaboration regarding biological studies.

My heartiest gratitude and respect to the person who is my spiritual father and teacher, Prof.

Dr. Khalid M. Khan, S.I., T.I. for his valued suggestions, proficient research supervision and

active co-operations to accomplish this thesis without which this work would just have

remained a mere dream. He taught me everything that a researcher should know. I would

like to prolong my thanks to all faculty members, especially Prof. Dr. Viqar-uddin Ahmed,

Prof. Dr Bina S. Siddiqui, Prof. Dr. Abdul Malik, and Prof Dr. Shaiq Ali.

I would also like to thank all my senior and junior lab fellows for being associated with me,

especially, Dr. Imran Fakhri, and Dr. Anila Karim. I also wants to thank my close and dearest

friends Mrs. Zeenat, Mrs. Bilquees Bano, Mrs. Bibi Fatima, Ms Asma Mukhtar, Mrs Sadia

Razi Khan, Ms Deenish Dabeer, Ms Uzma Salar, Ms Arshia, Ms Huma Bano, Ms Mahwish

Siddiqui, Ms Qurat-ul-ain Qadeer, and all my batch fellows for their moral support. I am

also grateful to all those colleagues who collaborated with me regarding biological

screening.

I would like to pay special thanks to Mr. Shamim Khan and Mr. Javaid Versiani for their

cooperation during the course of my research work. I am thankful to all the technical and

non-technical staff of the H. E. J. Research Institute of Chemistry.

Kanwal

xv

SUMMARY

This dissertation describes the syntheses of indole and tryptamine derivatives via various

synthetic strategies and screening of their biological activities. All synthetic compounds

were characterized by different spectroscopic techniques such as 1H-, and 13C-NMR, EI-

MS/FAB-MS and EI-HRMS.

This dissertation has been divided into three chapters and each chapter has its own

numbering of compounds, figures, tables, schemes, and references.

Chapter-1 comprises of the syntheses of Schiff bases of tryptamine 60-85 (Part A), urea

and thiourea analogues of tryptamine 87-99 (Part B) and evaluation of their biological

activities. Compound 78 (IC50 = 206.90 ± 0.88 μM) showed a potent activity against

antiglycation activity, while, other thirteen compounds showed good to moderate activity.

Chapter-2 describes the broad literature survey regarding the general introduction of indole,

its biological importance and various synthetic schemes. It includes synthetic strategies of

indole-3-acetamides 55-81 (Part A) and Knoevengal condensates of indole 83-118 (Part B)

and evaluation of their biological activities. Eight compounds 84, 85, 88, 94, 106, 111, 115,

and 118 were found to be weakly active against antiepileptic activity. Compound 83 (IC50 =

4.03 ± 0.05 μM) and 91 (IC50 = 4.89 ± 0.05 μM) were found to be the most active members

of the series.

Chapter-3 deals with the syntheses of bis(indolyl)methanes derivatives 38-92. The

synthesized analogues were subjected for the random screening and were found to be active

against anticancer and anti-inflammatory activities. Five analogues 64 (IC50 = 2.7 ± 0.1 μM),

47 (IC50 = 5.4 ± 0.8 μM), 52 (IC50 = 6.7 ± 0.6 μM), 78 (IC50 = 7.1 ± 1.3 μM), and 42 (IC50 =

7.2 ± 1.2 μM) were found to have potent activity as compared to standard ibuprofen (IC50 =

11.2 ± 1.9 μM), while compounds 45 (IC50 = 17.1 ± 0.2 μM) and 41 (IC50 = 26.6 ± 2.4 μM)

possess weak antiinflammatory activity. However, seventeen compounds showed a weak

anticancer activity.

1

Chapter 1

Synthesis of Tryptamine Derivatives and Their

Bioactivities

2

Summary

This chapter describes the synthesis of Schiff bases, urea, and thiourea derivatives of

tryptamine. The synthetic compounds were evaluated for various biological activities

including urease inhibition, antiepileptic, DPPIV inhibition, antiglycation, bacterial MDR

assay, and antileisminicidal activity.

1 General Introduction

Heterocyclic compounds are cyclic compounds which comprise of one or more non-carbon

atoms and these atoms are referred to as heteroatoms, commonly these heteroatom may be

sulphur, oxygen, nitrogen, phosphorus, etc. Heterocyclic chemistry constitutes a number of

novel compounds with various biological activities, principally because the resulting

compounds have unique power to mimic the peptides social system and to bind reversibly

to proteins (Franzen, 2000). In medicinal chemistry, these diverse biologically active

compounds are the key to synthesize a library based on one principal framework and to

screen those libraries against different receptors to find out active compounds which may

act as lead molecules in drug discovery. The fusion of these heterocyclic moieties can give

rise to unlimited combinations of novel polycyclic frameworks which leads to various

physical, chemical and biological aspects. Thus, the development of efficient methodologies

which leads to the formation of polycyclic moieties from biologically active heterocyclic

compounds are always of great interest to both organic and medicinal chemists (Kaushik et

al., 2013).

1.1 Introduction

Alkaloids were first isolated in the beginning of 19th century and have drawn the

considerable attention of chemists due to their structural diversity, their biological, and

pharmaceutical properties. Narcotine was probably the first isolated alkaloid followed by the

isolation of morphine from opium, strychnine, piperine, caffeine, and so on. Similarly,

coniine was the first alkaloid that was synthesized and structurally elucidated. They

commonly exist in crystalline form either in free state or as their salts, their solubility may

3

also vary depending on their structural features. Alkaloids are mainly classified into two

major groups non-heterocyclic also known as protoalkaloids or biological amines and

heterocyclic alkaloids which is again sub-divided into fourteen groups depending on the ring

structure they contain. The core structure of alkaloids composed of carbon, hydrogen,

nitrogen, and oxygen. In broad sense, the alkaloids may have a nitrogen atom either primary,

secondary, tertiary, or quaternary as shown in mescaline, ephedrine, atropine, and

tubocurarine, respectively.

A huge number of indole alkaloids are derived from an amino acid tryptophan and its

decarboxylated product tryptamine and are significantly important comprising of two

nitrogen atoms one is enclosed in the indole ring and other at ethylamine chain present at the

ß-position of indole ring. When tryptamine undergoes condensation with an aldehyde may

give rise to the formation of ß-carboline or indolinine depending on the attack at α or ß

position, respectively, these compounds exhibit psychomimetic properties (Allen and

Holmstedt, 1980). Strychnine and reserpine are some of the pharmaceutically important

alkaloids which are derived from tryptamine.

4

Tryptamine is an aromatic heterocyclic compound and was first isolated from shoots and

flowers of Acacia species (E. White, 1946), it is a bicyclic alkaloid moiety containing indole

ring system. The N-methyl substituted and N,N-dimethyl derivatives of tryptamine are found

naturally in many plants and are biologically active, it subsists in orange to red crystalline

powdery form. Tryptamine participates in many metabolic pathways (Friedrich, Brase, and

O Connor, 2009), it is a metabolite of an amino acid tryptophan and a precursor for

biosynthesis of many alkaloids comprising of indole nucleus (Wakahara, Fujiwara, and

Tomita, 1973). Tryptophan upon decarboxylation yields tryptamine which possess

sympathomimetic activity and found in trace amounts in central nervous system (Zucchi,

Chiellini, Scanlan, and Grandy, 2006). It is basic having pK value 10.2, due to presence of

an amino group (Onal, Tekkeli, and Onal, 2013). Tryptamine and its analogous compounds

constitute a family of naturally occurring as well as marketed drugs which possess important

pharmacological significance (Rene and Fauber, 2014). Tryptamine analogues are

commonly known as 5HT agonists and are found to be active against migraine (Brandes et

al., 2007) and obesity (Salikov, Belyy, Khusnutdinova, Vakhitova, and Tomilov, 2015).

1.2 Biological Importance of Tryptamine

1.2.1 Anti-ulcerogenic Activity

Schiff base 1 derived from 5-chlorosalicylaldehyde and tryptamine and their copper and

nickel complexes had shown remarkable activity as compared to the standard cimetidine

against ulcers that were induced in rats (Mustafa, Hapipah, Abdulla, and Ward, 2009).

5

1.2.2 Antihepatitis activity

N,N-disubstituted 2 and N-oxide 3 derivatives of tryptamine found to be active against

hepatitis B virus having potent antiviral activity and low cytotoxicity (Qu et al., 2011).

(IC50 = 0.4 µM) (CC50 = 40.6 µM) (IC50 = <1 µM) (CC50 = >25 µM)

1.2.3 CDK4/ cyclin D1 inhibitors

Compound 4, 5, and 6 possess CDK4/ cyclin D1 inhibitory activity with IC50 values 9.2, 11.2,

and 9 μM, respectively (Jenkins et al., 2008).

1.2.4 As Hallucinogens

A wide variety of tryptamines are found to exhibit hallucinogen effect, they act as 5HT2a

agonists and are capable of changing, mood, sensory perceptions, and thoughts in humans.

6

1.2.5 As 5-HT6 receptor ligands

The recent research on 5-HT6 receptors have remarkable and fascinating results showing

their role in treating various diseases associated with central nervous system like

Schizophrenia, Alzheimer′s, obesity, anxiety, appetite control, and epilepsy etc. It is most

recently discovered receptor of serotonin receptor family, expressed mainly in the striatum,

olfactory tubercle, hippocampus, and nucleus accumbens Table-1.

Table-1: Representing 5HT6 receptor binding data for compounds 7a-7t

Compound R R1 Salt % Inhibition at 100 nM

concentration

7a 5-F H HCl 11.46

7b 5-Cl H HCl 40.49

7c 5-Cl 4′-Cl HCl 28.87

7d 5-Br 4′-Cl HCl 43.75

7e H 4′-F - 10.23

7f H H HCl 20.16

7g 5-F 4′-Cl HCl 14.85

7h 5,7-Di-F H - 20.04

7i 4-Cl, 7-CH3 H HCl 54.55

7j 5-Cl 4′-OCH3 - 43.84

7k 5-OCH3 2′-Br - 18.1

7l H 4′-Cl HCl -

7m 5-Br H HCl -

7n H 4′-OCH3 - -

7

7o 5-F 4′-OCH - -

7p 5-OCH3 4′-Cl - -

7q 4-Cl,7-CH3 4′-Cl - -

7r 5,7-Di-F 4′-Cl - -

7s H 4′-CH3 - -

7t 5-OCH3 H - -

1.2.6 Antioxidant activity

The tryptamine derivatives synthesized via the treatment of diphenyl chlorophosphine

resulting in the formation of an intermediate which upon further reaction with various aryl

substituted azide derivatives affords the compound 8. These compounds were evaluated for

antioxidant activity and few of the analogues were found to possess good antioxidant

activity.

1.2.7 As 5-HT1D receptor agonists

A series of 5-(2-or 3-thienyl) tryptamine derivatives 9 possess potent and selective activity

against 5-HT1D receptor agonists and are therefore potentially active against migraine (Meng

et al., 2000).

1.2.8 Antileishmanial activity

Other derivatives of tryptamine 10 and 11 are found to be active against leishmaniasis and

demonstrated up to 100% inhibition against promastigotes and about 98% inhibition against

8

amastigotes at a concentration of 10 μg/ml (Gupta, Talwar, Palne, Gupta, and Chauhan,

2007).

1.2.9 β-Adrenergic receptors agonists

Tryptamine derivative 12 found to be agonist for human β-adrenergic receptors (ARs). 7-

Methanesulfonyloxy tryptamine was found to have high potency and high selectivity against

β3 ARs (Mizuno et al., 2004).

1.2.10 H5-HT2A Antagonists

Some 2-aryltryptamines 13 are synthesized and recognized as high affinity H5-HT2A

antagonists being meaningful in the treatment of schizophrenia (Stevenson et al., 2000).

9

1.3 Synthetic strategies for tryptamine

Tryptamines can be prepared by following different ways:

Palladium catalyzed reaction of various o-iodoaniline 14 with aldehyde 15 in the presence

of DABCO (1,4-diazabicycl[2,2,2]octane) as a base and DMF as a solvent at 85 ºC gives the

derivatives of tryptamine 16. The palladium catalyst used in this reaction was Pd(OAc)2 in

a catalytic amount of (0.5mol %) (Hu, Qin, Cui, and Jia, 2009) Scheme-1.

Scheme-1: Palladium catalyzed synthesis of tryptamine

Cyanoguanidine derivatives of tryptamines can be prepared in several steps by using

tryptamine derivatives 17 as starting material which were reacted with isothiocyanates 18

using CH2Cl2 as a solvent and Et3N as a base affording thiourea derivatives 19 which were

then methylated by using CH3I in acetone produced thioether derivatives 20. These

compounds were then treated with cyanamide in boiling butanol in the presence of catalytic

amount of a strong base DABCO (1,4-diazabicyclo[2,2,2]octane) as a result desired

cyanoguanidine derivatives 21 were obtained (Novak, 2001) Scheme-2.

10

Scheme-2: Synthesis of cyanoguanidines via tryptamine

C-1 substituted terahydro-β-carbolines were prepared from tryptamine using domino Heck-

Aza-Micheal reaction. The nitrogen of tryptamine 22 was first protected by a tosyl group

using tosyl chloride and triethyl amine in CH2Cl2 that generated sulfonamide 23. The

bromination of this sulfonamide at C-2 position of indole ring afforded 2-bromoindole 24.

It then undergoes Pd-catalyzed domino Heck-Aza-Micheal reaction and the desired product

25 was obtained (Priebbenow, Henderson, Pfeffer, and Stewart, 2010) Scheme-3.

Scheme-3: Synthesis of β-carbolines via Pd catalyzed domino Heck-Aza-Micheal reaction.

Tryptamine 26 and N-ω-methyltryptamine 27 when reacted with o/m/p-benzoyl chlorides 28

yields key intermediates 29. Intermediates 29a and 29d were subjected to Suzuki-Miyaura

coupling with a range of 4-substituted boronic acid that affords para-biphenyl derivatives

30a-k. Ortho-biphenyl derivatives 31a-e were synthesized by reacting 29c with the

11

particular phenylboronic acid. Meta-biphenyl derivative 32 was synthesized from the key

intermediate 29b (Jenkins et al., 2008) Scheme-4.

Scheme-4: Synthesis of tryptamine analogues via Suzuki-Miyaura coupling.

Analogs of naturally occurring indole alkaloid 43 can be synthesized from tryptamine in

several steps involving Knoevenagel or Wittig-Horner reactions (Boumendjel, Nuzillard,

and Massiot, 1999) Scheme-5.

12

Scheme-5: Synthesis of indole alkaloid via tryptamine

1.3.1 By reacting chloroalkylalkyne with substituted aryl hydrazines

One pot synthesis of substituted tryptamines was carried out via domino reaction sequence

starting from titanium catalyzed amination of chloroalkynes 44 resulting in the formation of

aryl hydrazones 46 which upon [3+3] sigmatropic rearrangement followed by nucleophilic

substitution of chloride by ammonia affords tryptamines 47 (Khedkar, Tillack, Michalik,

and Beller, 2004) Scheme-6.

13

Scheme-6: Synthesis of tryptamine via [3,3] sigmatropic rearrangement.

Tryptamine was converted to imine through condensation with ketone which undergoes

cyclization in the presence of iodine to afford 1,1-disubstituted tetrahydro ß-carbolines 49.

This reaction is also referred to as Pictet-Spengler cyclization Scheme-7.

Scheme-7: Synthesis of tetrahydro ß-carbolines via pictet Spengler cyclization.

1.3.2 Fischer synthesis of tryptamines

Tryptamines were synthesized by Fischer method for the first time by reacting acetals of

aminobutanals 51 with arylhydrazines 50 in the presence of zinc chloride at 180 °C Scheme-

8.

Scheme 8: Fischer synthesis of tryptamine and its derivatives

14

1.3.3 Grandberg Reaction for the Synthesis of Tryptamine

The reaction of 5-chloro-2-pentanone 54 with phenylhydrazine in the presence of aqueous

ethanol affords 2-methyl tryptamine 55 in good yields in high purity (Slade et al., 2007)

Scheme-9.

Scheme-9: Grandberg synthesis of tryptamines

Melatonin (N-acyl-5-methoxytryptamine) 57 was synthesized through one pot rodonium

catalyzed hydroformylation of N-allylacetamide 56 in the presence of protic solvent

Scheme-10.

Scheme-10: Rodonium catalyzed synthesis of Melatonin

15

PART-A

2 Synthesis of Schiff bases of Tryptamine

2.1 Results and Discussion

2.1.1 Chemistry

Schiff bases of tryptamine were synthesized by reacting tryptamine with different substituted

benzaldehydes in methanol. The change in the color of reaction mixture indicated the

progression of reaction and the completion of reaction was monitored via TLC. The solvent

was then evaporated under vacuum resulting in the formation of a crude product which had

been purified through crystallization from methanol. The synthetic analogues (60-85) were

characterized by different spectroscopic techniques such as EI-MS, HREI-MS, 1H-, and 13C-

NMR Scheme-11. Compounds 60, 62, 64-85 are newly synthesized compounds, while,

compounds 61 and 63 are already reported.

Scheme-11: Synthesis of Schiff Bases of Tryptamine 60-85

Mechanism

The mechanism starts with the protonation of aldehyde with acidic proton thus making

carbonyl carbon more electrophilic. The amine group of tryptamine then attack on carbonyl

carbon followed by dehydration resulting in the formation of iminic bond Figure-1.

16

Figure-1: Mechanism of formation of Schiff base

2.2 Characteristic Spectral Feature of Representative

Compound 60

2.2.1 1H NMR Spectroscopy

The 1H NMR was recorded on a 300 MHz instrument in DMSO. A sharp singlet for OH

appeared at δ 14.60 the most downfield signal of the spectrum. Another sharp singlet for NH

of indole appeared at δ 10.10. There are nine aromatic protons present, imine proton

appeared at δ 8.45 while H-4 of indole ring appeared as doublet at δ 7.58 showing coupling

with H-5 having coupling constant J4,5 = 7.8 Hz, H-4′ appeared at δ 7.52 as doublet showed

meta coupling with H-6′ having coupling constant J4′,6′ = 2.7 Hz, however, H-7 and H-2

appeared at δ 7.31 as multiplet. H-6′ appeared as doublet at δ 7.16 showing meta coupling

with H-4′ having coupling constant J6′,4′ = 1.8 Hz, while, H-5 appeared at δ 7.06 as triplet

showing coupling with H-4 and H-6 having coupling constant J5,4/5,6 = 7.8 Hz. H-6 of indole

moiety appeared at δ 6.96 as triplet showing coupling with H-5 and H-7 having coupling

constant J6,7/6,5 = 7.8 Hz. As structure contains four methylene protons, thus a triplet for two

protons adjacent to nitrogen appears at δ 3.91 having coupling constant J = 6.6 Hz while, the

signal of other two protons appeared at δ 3.09 as triplet having coupling constant J = 6.9 Hz

Figure-2.

17

Figure-2: Representative 1H NMR signals of compound 60

13C-NMR broad-band decoupled spectrum (DMSO-d6) indicated total 17 carbon signals

containing two methylene, eight methine, and seven quaternary carbons. Methine of iminic

group was the most downfield signal appeared at C 165.2, while, the C-2′ quaternary carbon

attached to an electronegative oxygen atom appeared at C 163.9 and quaternary C-9

resonated at C 136.2. The methine C-4′ appeared at C 132.8, however, C-2 adjacent to

electronegative nitrogen atom appeared as downfield signal and resonated at C 130.2. All

remaining aromatic carbons appeared in the usual aromatic range of C 110.4-134.5. The

methylene adjacent to iminic nitrogen appeared at C 53.9 while another signal of methylene

was most up field and appeared at C 25.7 Figure-3.

Figure-3: Representative 13C NMR signal of compound 60

The 13C/1H NMR chemical shifts assigned on the basis of HSQC and HMBC correlations.

The iminic double bond was found to have (E) stereochemistry after observing the NOESY

interaction of iminic hydrogen with H-2 and with H-6′ of the benzene ring.

18

2.2.2 Mass Spectrometry

The EI-MS spectra of compound 60 showed the [M+] at m/z 332 for molecular formula

C17H14Cl2N2O. The significant fragment appears at m/z 202 by the loss of 3-methyl indole.

The fragment which showed formation of 3-methyl indole appeared at m/z 130, another

fragment appeared at m/z 144 confirms the formation of 3-ethyl indole, while, the formation

of 2, 4-dichloro-6-(λ2-methyl) phenol appears at m/z 175. The key fragments are presented

in Figure-4.

Figure-4: Representative fragmentation pattern of compound 60

19

Table-2: Synthesized analogues of Schiff bases 60-85

Compound Structure Compound Structure

60

73

61

74

62

75

63

76

64

77

20

65

78

66

79

67

80

68

81

69

82

21

70

83

71

84

72

85

22

2.3 Biological Screening

2.3.1 Antileishmanial Activity

Leishmaniasis is a vector-borne protozoan disease spread by sand flies, it is currently

amongst the six endemic diseases considered as high priorities worldwide (Veland et al.,

2012). Three distinct types of leishmaniasis are visceral, cutaneous, and mucocutaneous. It

is the second common cause of death and third most common cause of morbidity after

malaria. This protozoan infection has clinical symptoms ranging from asymptomatic

infection to fatal visceral leishmaniasis also known as kala-azar (Savoia, 2015). Among them

the most common type is cutaneous leishmaniasis exhibiting a wide range of symptoms

ranging from small cutaneous lumps to gross mucosal tissue destruction (Reithinger et al.,

2007) and the most severe type is visceral leishmaniasis in which vital organs are being

targeted by the parasite. The major symptoms includes prolonged fever, splenomegaly,

hypergammaglobulinemia, and pancytopenia. It is a fatal disease, if being untreated

(Boelaert et al., 2000). The female phlebotomine sand flies which serve as the vector for the

protozoan transfer the disease when it bites the host that are commonly mammals. The

disease is then spread via macrophages in the liver, spleen, and bone marrow (Kamhawi,

2006). Leishmania parasites are found to be dimorphic organisms, having two

morphological forms in their life cycle, amastigotes in the mononuclear phagocytic system

of the mammalian host, and promastigotes in the digestive organs of the vector (Burchmore

and Barrett, 2001). The common drugs used for the treatment of leishmaniasis are

glucantime, pentostam, pentamidine, and amphotericin B (Croft, Barrett, and Urbina, 2005).

In recent years the leishmania/HIV co-infection has been noticed, so it is one of the

opportunistic infections that attack HIV-infected individuals. Visceral form is common

among co-infections. The mortality rate for visceral leishmaniasis are as high as 100%, if

left untreated, and is spreading in several areas of the world due to increase numbers of AIDS

victims (Mahendra). Leishmania and HIV co-infections have found to be endemic in 35

countries (Cruz, Nieto, Moreno, and Canavate, 2006).

23

Structural-Activity Relationship

Compound 60-85 were categorized in four different categories including, nitro, halogen,

and alkoxy and hydroxyl substituted derivatives. The effect of variation in ring size was

also observed to develop a better structure-activity relationship.

a) Nitro substituted derivatives

All the synthetic compound were screened for their antileishmanial activity. Compound 83

(IC50 = 54.6 ± 2.6 μM) showed some activity several folds less than standards, this compound

possess hydroxyl group at its ortho position and an electron withdrawing nitro group at meta

position so the activity may be due to the presence of these substituents.

b) Variation in the ring size

The second active compound of the series was compound 78 (IC50 = 54.6 ± 2.7 μM)

possessing naphthalene ring and hydroxyl substituents, thus, the activity may be due to the

presence of electron rich ring. While, variation in ring size in other compounds 81 and 74

possessing anthracene and thiophene rings, respectively, showed no activity.

24

c) Alkoxy and hydroxy substituents

Among all alkoxy substituents only two compounds showed weak activity. Compound 68

(IC50 = 70.3 ± 2.6 μM) which possess two methoxy substituents at meta positions and an

hydroxy group at para position was found to be more active as compared to compound 79

(IC50 = 88.2 ± 2.9 μM) having one methoxy substituent and an hydroxy group at meta

position. This shows that the presence of two methoxy substituents increases the activity on

the other hand it can also be observed that the shifting of hydroxyl position may have some

impact on the activity. However, compound 77 (IC50 = 86.4 ± 2.9 μM) having two hydroxyl

groups showed similar activity like 79.

d) Halogen substituted analogues

Among twelve analogues only three were found to be active. Compound 76 (IC50 = 60.3 ±

2.4 μM) was found to be most active analogues among these possessing para substituted

hydroxyl and meta substituted bromine while compound 84 (IC50 = 74.6 ± 3 μM) possessing

meta substituted hydroxyl and bromine it showed the change in position of substituents also

affect the activity. Compound 67 (IC50 = 72.4 ± 2.8 μM) having iodo group at meta position

and para hydroxyl group but, the decreased activity may be due to the presence of one

methoxy substituent.

25

Table-3: Antileishmanial activity of Schiff bases of tryptamine 60-85.

Compound IC50 ± SEM (μM) Compound IC50 ± SEM (μM)

60 - 73 -

61 - 74 -

62 - 75 -

63 - 76 60.3 ± 2.4

64 - 77 86.4 ± 2.9

65 - 78 54.6 ± 2.7

66 - 79 88.2 ± 2.9

67 72.4 ± 2.8 80 -

68 70.3 ± 2.6 81 -

69 - 82 -

70 - 83 54.6 ± 2.6

71 - 84 74.6 ± 2.3

72 - 85 -

Amphotericin B(std) 0.29 ± 0.05 Pentamidine (std) 5.09 ± 0.09 SEMa is the standard error of the mean, (-) Not active, Amphotericin B(std) and Pentamidine(std) are standard inhibitor for

anti-leishmanial activity.

2.3.2 Antiglycation activity

The non-enzymatic reaction between a protein and reducing sugar is referred to as glycation

(Nagai, Mori, Yamamoto, Kaji, and Yonei, 2010). The glycation and glycoxidation of

proteins results in the development of oxidative stress causing the damage of cell membranes

which results in the aging of skin. Thus, glycation stress promotes aging inside and outside

of the body. (Hori et al., 2012). A series of complex reactions results in the formation of

advanced glycation end products (AGEs) including formation of Schiff bases and Amadori

products. The sugar and proteins undergoes irreversible, non-enzymatic reaction called

Maillard reaction. The carbonyl group of reducing sugar reacts with amino terminal of

26

protein which results in the formation of Schiff base. The Schiff base is then modified to

ketoamine through Amadori rearrangement, the ketoamine act as an intermediate in the

formation of AGEs. The accumulation of AGEs in tissues may cause long term complication

of diabetes mellitus. It is also involved in three major diabetic complications nephropathy,

neuropathy, and retinopathy (Radoi, Lixandru, Mohora, and Virgolici, 2012). A prolonged

hyperglycemic condition favours in the enhanced production of AGEs which alters the

structures of blood vessel by inducing cross link process in the structure of life span protein

collagen (Flier, Underhill, Brownlee, Cerami, and Vlassara, 1988).

Receptor for advanced glycation end products (RAGE) are the specific receptors for the

binding of AGEs. AGEs can modify the intracellular signals and responses by activating the

cells signals that generate inflammatory cytokines and are thus playing a pathogenetic role,

it can also modify the action of hormones and free radicals. AGEs along with intracellular

activities have ability to modify extracellular matrix (Nagai et al., 2010) (Brownlee, 1995)

Figure-5.

Figure-5: Pathway for the formation of AGEs (Advanced glycation end products).

AGEs are formed endogenously but smoking, processed food, cooking (microwave) are

some of the exogenous sources of it. A controlled diet along with some physical exercises

can minimize the formation and consumptions of these AGEs and thus minimize the chances

of various complications caused by their formation.

In vitro BSA-MG Antiglycation Assay

The 10 mg/mL concentration of BSA and 14 mM of methylglyoxal were prepared in 0.1 M

phosphate buffer having pH 7.4, 3 mM sodium azide (NaN3) was used as an antimicrobial

agent. The assay was performed in triplicate and all the test compounds (1 mM) were

dissolved in DMSO. Reaction mixtures were prepared by adding 50 μL methylglyoxal, 50

27

μL BSA, 20 μL test compound, and 80 μL phosphate buffer of pH 7.4. Reaction mixture

were incubated at 37 ºC for 9 days under sterile conditions. The fluorescence was measured

at 420 and 330 nm for emission and excitation by using Microtitre plate reader (Spectra Max

M2, Molecular Devices, CA, USA) (Choudhary, Adhikari, Rasheed, Marasini, Hussain,

Kaleem, and Atta-ur-Rahman, 2011; Wu and Yen, 2005). The given formula was used for

the calculation of percent inhibition:

% Inhibition= (1- Fluorescence of test sample/Fluorescence of the control) x 100

The test compounds were evaluated at various concentration of IC50 values (1000-50 μM)

by using EZ-FIT Enzyme Kinetics Program (Perrella Scientific Inc., Amherst, USA). The

standard drug used was rutin with IC50 of 294 ± 1.5 μM in protein model system (BSA-MG

glycation model).

Structure-Activity Relationship

All synthetic compounds 60-85 were screened for their antiglycation activity, few

compounds showed activity in potent range while, other were weakly or moderately active.

These compounds were categorized in three different categorizes in order to develop a better

structure-activity relationship. The most active compound was 78 (IC50 = 206.9 ± 0.88 μM)

having potent activity possessing an ortho hydroxyl substituted naphthyl ring which may be

reason for the activity of this compound.

a) Dihydroxy derivatives

The second most active compound 69 (IC50 = 244.5 ± 1.4 μM) possessing the ortho and para

hydroxyl substituents, thus, the activity may be due to the presence of these substituents.

However, compound 77 (IC50 = 407.4 ± 1.5 μM) also possessing two hydroxyl group but

28

due to the change in position of hydroxyl from ortho to meta has decreased the activity of

compound.

29

b) Halogen substituents

The third most active compound of the class is compound 82 (IC50 = 274.3 ± 1.5 μM) with

hydroxyl group at para and chloro at meta positions may be the reason for activity.

Compound 82 when compared with 76 (IC50 = 340.7 ± 1.6 μM) showed a decreases activity

when chloro is replaced with bromo substituents. However, compound 84 (IC50 = 662.3 ±

2.6 μM) showed several folds less activity when the position of hydroxyl is changed to ortho.

c) Methoxy substituents

Compounds 71 (IC50 = 328.7 ± 1.2 μM) and 62 (IC50

= 407.4 ± 1.5 μM) having bromo,

hydroxyl and methoxy substituents the shift of hydroxyl from para to ortho and bromo from

meta to ortho results a decreased activity. While, compound 67 (IC50 = 392.3 ± 2.9 μM)

having an iodo substituent showed comparable activity to 71 but enhanced activity than

compound 62. Compound 68 (IC50 = 823.0 ± 3.5 μM) possessing two methoxy substituent

showed increased activity when compared with compound 79 (IC50 = 850.6 ± 6.4 μM).

30

Table-4: Antiglycation activity of synthetic analogues 60-85


60 327.7 ± 2.5 73 -

61 - 74 -

62 444.7 ± 2.9 75 -

63 - 76 340.8 ± 1.6

64 310.6 ± 0.6 77 407.4 ± 1.5

65 - 78 206.9 ± 0.88

66 - 79 850.6 ± 6.4

67 392.3 ± 2.9 80 -

68 823.0 ± 3.5 81 -

69 244.5 ± 1.4 82 274.3 ± 1.5

70 - 83 294.5 ± 1.5

71 328.7 ± 1.2 84 662.4 ± 2.6

72 - 85 -

Rutin(std) 294.5 ± 1.5 SEMa is the standard error of the mean, (-) Not active, Rutin(std) is standard inhibitor for antiglycation activity

2.3.3 Di-peptidyl peptidase (DPP-IV) activity

Di-peptidyl peptidase is a proteolytic enzyme which belongs to the class exopeptidase, it is

present in endothelium of many organs. The enzyme is referred to as prolylamino

dipeptidase as it cleaves the peptide bond following the proline and thus it facilitate the

digestion of proline rich polypeptides and proteins present in wheat, rice, barley, and milk.

The DPP-IV enzyme inactivates incretins (GLP-1 and GIP) which are responsible for the

secretion of insulin depending upon the blood glucose levels, in addition to that GLP-1 also

suppresses the glucagon secretion from liver. DPP-IV inhibitors thus regulates the activation

of these incretin enzymes and thus used for the treatment of type-2 diabetes. The first

31

approves drug as the DPP-IV inhibitor is sitagliptin. (Thornberry and Gallwitz, 2009) (J. R.

White, 2008).

Dipeptidyl peptidase IV Inhibition assay

Sitagliptin is used as standard and (Gly-Pro-PNA) is used as substrate for this inhibitory

assay (Nongonierma, Mooney, Shields, and FitzGerald, 2013). In 96-well microtitre-plate

10 μL of test compounds, tris-HCl buffer of pH 8.0 and 0.025 U mL-1 of DPP-IV were added

and pre-incubated at 37 ºC for 20 min. The reaction was initiated by the addition of 0.2 mM

substrate. Microplate was then incubated at 37 ºC for 50 min. Absorbance (Spectramax 384,

Molecular Devices, CA, USA) was measured at 405 nm. Following formula was used for

the calculation of % inhibition:

Inhibition (%) = [(A negative control − A control) − (A sample − A sample control)]/ (A

negative control − A control) × 100%

All synthetic compound were evaluated for their DPP-IV inhibitory activity and found to be

inactive.

Table-5: DPP-IV inhibitory activity of Schiff bases of Tryptamine 60-85


60 - 73 -

61 - 74 -

62 - 75 -

63 - 76 -

64 - 77 -

65 - 78 -

66 - 79 -

67 - 80 -

68 - 81 -

69 - 82 -

70 - 83 -

71 - 84 -

72 - 85 -

Sitagliptin(std) 0.024 ± 0.4 SEMa is the standard error of the mean, (-) Not active, Sitagliptin(std)is standard inhibitor for DPP-IV inhibition activity

32

2.3.1 NTPDase (Nucleotide triphosphate diphospho hydrolase)

inhibitory activity

NTPDases are the ectoenzymes present in the central nervous system. It hydrolyzes ATP

and ADP into nucleoside monophosphates (AMP). These enzymes are localized on the cell

surface of many cells including endothelial cells, lining of blood vessels, etc. It inhibits the

activation and aggregation of platelets by converting ATP into ADP. This ectoenzyme exists

in various isoforms on the basis of functionality and molecularity including the

ectonucleoside triphosphate diphosphohydrolases (ENTPDase). The NTPDase family

contains eight members, namely NTPDase-1 to NTPDase-8, out of which four members

NTPDase-1, NTPDase-2, NTPDase-3 and NTPDase-8 exist in the plasma membrane having

active site outside of the cell. NTPDase1 hydrolyses ATP and ADP equally, while NTPDases

2, 3, and 8 preferentially hydrolyze ATP.

Role and inhibition of NTPDases

NTPDases reduce thrombogenecity by preventing the initiation of coagulation and thus are

prevailing and natural anti-platelet agents. These enzymes are involved in the immune

responses and are agonist of T-lymphocyte proliferation and humoral response. The

NTPDase inhibitors are so far derived from three different chemical classes including

nucleotides and their derivatives, sulfonated dyes such as reactive blue 2, suramin and its

analogues.

NTPDase inhibitors are used for the treatment of autoimmune disorders such as rheumatoid

arthritis and psoriasis and also for treating cancer, leukemia, lymphoma and various other

diseases. Inhibition of NTPDases promotes the immune response towards bacterial or viral

infections.

NTPDase inhibitory assay


Compounds 60-85 were categorized in three different categories in order to develop better

structure-activity relationship.

33

NTPDase-1 inhibitors

All compounds were subjected to NTPDase inhibitor activity, eighteen (18) compounds

were found to have good activity as compared to the standard suramin (IC50 = 16.1 ± 1.02

µM). The most active compounds of the series are 60 (IC50 = 0.27 ± 0.03 µM) and 68 (IC50

= 0.28 ± 0.01 µM). Compound 60 having hydroxy at ortho and two chloro substituents at

meta positions may be responsible for the activity of compound, however, compound 68

having hydroxy at para and two methoxy substituents at meta also showed similar activity.

The replacement of one chloro group to bromo showed some decreased activity as in

compound 64 (IC50 = 0.36 ± 0.01 µM). Compounds 61, 67, 70, and 71 have almost similar

activities with IC50 values of 0.43 ± 0.05, 0.43 ± 0.02, 0.43 ± 0.04, and 0.41 ± 0.03 µM,

respectively. All these compounds possess electron donating substituents, however,

compound 61 is selectively inhibits NTPDase-1. Compound 69 (IC50 = 0.53 ± 0.09 µM)

having two hydroxy substituents showed a decline in activity. Compounds 76 (IC50 = 0.64

± 0.04 µM) and 78 (IC50 = 0.65 ± 0.05 µM), respectively, showed similar activity.

Compound 75 (IC50 = 0.76 ± 0.06 µM) having two methoxy substituents showed similar

activity as compared to compound 77 (IC50 = 0.79 ± 0.08 µM) having ortho and meta

hydroxy substituents. Compound 82 selectively inhibits NTPDase-1 enzyme with IC50 value

of 1.01 ± 0.07 µM. All other compounds 63 (IC50 = 1.64 ± 0.17 µM), 81 (IC50 = 4.31 ± 0.98

µM), 83 (IC50 = 2.38 ± 0.56 µM), 84 (IC50 = 1.91 ± 0.56 µM), and 85 (IC50 = 4.51 ± 0.77

µM), respectively, were also found to possess good inhibitory activity as compared with

standard suramin (IC50 = 16.1 ± 1.02 µM).

34


Twelve (12) compounds showed good activity against NTPDase-3 enzyme. The most active

compound was 63 (IC50 = 0.26 ± 0.02 µM) having hydroxy and chloro group thus electron

donating effect may be contributing in the activity of compound. Compounds 60 and 68

showed similar activity with IC50 values of 0.39 ± 0.03, and 0.35 ± 0.03 µM, respectively.

Compound 64, 67, 70, 71, 77, 78, 81, and 83 possess electron donating substituents and were

found to have good activity with IC50 ranging between 0.49 - 3.89 µM.


Twenty one (21) compounds were found to have good to moderate activity as compared with

standard suramin (IC50 = 4.31 ± 0.41 µM). Compound 70 (IC50 = 0.26 ± 0.01 µM) was the

most active compound. Compound 65, 66, 72, and 73 were found to be selective inhibitors

of NTPDase-8 with IC50 values of 0.97 ± 0.05, 3.31 ± 0.56, 1.32 ± 0.02, and 1.71 ± 0.04

µM), respectively, Table-6.

35

Table-6: NTPDase inhibitory activity of Schiff bases 60-85

Compound NTPDase-1

IC50 ± SEM (μM)

NTPDase-3

IC50 ± SEM (μM)

NTPDase-8

IC50 ± SEM (μM)

60 0.27 ± 0.03 0.39 ± 0.04 0.79 ± 0.07

61 0.43 ± 0.05 - -

62 - - -

63 1.64 ± 0.17 0.26 ± 0.02 0.42 ± 0.03

64 0.36 ± 0.01 3.89 ± 0.76 1.57 ± 0.09

65 - - 0.97 ± 0.05

66 - - 3.31 ± 0.56

67 0.43 ± 0.02 0.75 ± 0.06 7.85 ± 0.97

68 0.28 ± 0.01 0.35 ± 0.03 1.07 ± 0.05

69 0.53 ± 0.09 - 4.55 ± 0.86

70 0.43 ± 0.04 3.88 ± 0.99 0.26 ± 0.01

71 0.41 ± 0.03 1.56 ± 0.14 1.06 ± 0.07

72 - - 1.32 ± 0.02

73 - - 1.71 ± 0.04

74 - - 2.12 ± 0.13

75 0.76 ± 0.06 - 1.82 ± 0.11

76 0.65 ± 0.05 - 13.8 ± 2.01

77 0.79 ± 0.08 0.51 ± 0.04 4.49 ± 0.89

78 0.64 ± 0.04 0.92 ± 0.06 7.15 ± 1.02

79 - - -

80 - - 0.69 ± 0.07

81 4.31 ± 0.98 0.49 ± 0.04 2.12 ± 0.13

82 1.01 ± 0.07 - -

83 2.38 ± 0.56 0.61 ± 0.03 2.48 ± 0.14

84 1.91 ± 0.56 1.01 ± 0.06 8.47 ± 1.02

85 4.51 ± 0.77 - 0.71 ± 0.02

Suramin 16.1 ± 1.02 24.1 ± 3.01 4.31 ± 0.41 SEMa is the standard error of the mean, (-) Not active, Suramin(std)is standard inhibitor for NTPDase inhibition activity

2.4 Conclusion

All synthetic compounds 60-85 were subjected for random biological activities, it was

observed that all compound were found to be inactive against DPP-IV enzyme. Fourteen

analogues were found to possess good to weak antiglycation activity. Compound 78 (IC50 =

206.9 ± 0.88 μM) was found to be the most potent compound of the series. While other

compounds 60, 62, 64, 67, 68, 69, 71, 76, 77, 79, 82, 83, and 84 showed good to moderate

antiglycation activity. Out of twenty six compounds, eight analogues 67, 68, 76, 77, 78, 79,

83, and 84 were found to be weakly active against leishmaniasis. Compound 60, 68, 63, and

36

70 were found to have good NTPDase activity against NTPDase-1, NTPDase-3, and

NTPDase-8 with IC50 values of 0.27 ± 0.03, 0.28 ± 0.01, 0.26 ± 0.02, and 0.26 ± 0.01 µM,

respectively.

General Procedure for the synthesis of Schiff bases (60-85)

Tryptamine (0.16 g, 1 mmol) was taken in a reaction flask in methanol then corresponding

benzaldehydes were added and reaction mixture was stirred for 24 h. The completion of

reaction was monitored through TLC and the solvent was dried under vacuum the residue

obtained was then washed with hexane. Pure products were obtained after crystallization

from methanol.

2.5 Physical Data for the Synthesized Compounds

2-((2-(1H-Indol-3-yl)ethylimino)methyl)-4,6-dichlorophenol (60)

Yield: 32%, M.p.: 160-162 ºC; 1H NMR (300 MHz, DMSO-d6): 14.61 (s, 1H, OH), 10.87

(s, 1H, NH), 8.45 (s, 1H, N=CH), 7.58 (d, 1H, J4,5 = 7.8 Hz, H-4), 7.54 (d, 1H, J4′,6′ = 2.7 Hz,

H-4′), 7.31 (ovp, 2H, H-7, H-2), 7.16 (d, 1H, J6′,4′ = 1.8 Hz, H-6′), 7.06 (t, 1H, J5,4/5,6 = 7.8

Hz, H-5), 6.96 (t, 1H, J6,5/6,7 = 7.8 Hz, H-6), 3.91 (t, 2H, J = 6.6 Hz, CH2), 3.09 (t, 2H, J =

6.9 Hz, CH2), 13C NMR (300 MHz, DMSO-d6): 169.1, 155.0, 136.0, 132.5, 127.2, 123.7,

120.9, 120.5, 118.6, 118.3, 113.7, 111.3, 108.6, 55.1, 33.6, EI MS m/z (% rel. abund.): 332

(M+, 16), 334 (M+2, 9), 130 (100), HREI-MS m/z: Calcd for C17H14Cl2N2O [332.0474],

Found [332.0483].

2-((2-(1H-Indol-3-yl)ethylimino)methyl)phenol (61)


(s, 1H, NH), 8.48 (s, 1H, N=CH), 7.56 (d, 1H, J7,6 = 7.8 Hz, H-4), 7.31 (ovp, 3H, H-7, H-4′,

H-6′), 7.13 (d, 1H, J = 2.1 Hz, H-2), 7.06 (t, 1H, J5,4/5,6 = 7.8 Hz, H-5),6.98 (t, 1H, J6,7/6,5 =

7.8 Hz, H-6), 6.84 (ovp, 2H, H-3′, H-5′), 3.88 (t, 2H, J = 6.6 Hz, CH2), 3.30 (t, 2H, J = 6.9

Hz, CH2), EI MS m/z (% rel. abund.): 264 (M+, 15), 130 (100.0), HREI-MS m/z: Calcd for

C17H16N2O [264.1269], Found [264.1263].

37

4-((2-(1H-Indol-3-yl)ethylimino)methyl)-2-bromo-6-methoxyphenol (62)


(s, 1H, NH), 8.10 (s, 1H, N=CH), 7.56 (ovp, 2H, H-2′, H-6′), 7.32 (d, 2H, J7,6/4,5 = 7.8 Hz,

H-7, H-4), 7.13 (s, 1H, H-2), 7.04 (t, 1H, J5,4/5,6 = 7.8 Hz, H-5), 6.96 (t, 1H, J6,5/6,7 = 7.8 Hz,

H-6), 3.77 (s, 5H, CH2, OCH3), 3.0 (s, 2H, CH2), EI MS m/z (% rel. abund.): 372 (M+, 7),

374 (M+2, 7), 163 (34), 130 (100), HREI-MS m/z : Calcd for C18H17BrN2O2 [372.0457],

Found [372.0473].

2-((2-(1H-Indol-3-yl)ethylimino)methyl)-4-chlorophenol (63)


(s, 1H, NH), 8.46 (s, 1H, N=CH), 7.56 (d, 1H, J4,5 = 7.8 Hz, H-4), 7.45 (d, 1H, J6′,3′ = 2.7 Hz,

H-6′), 7.31 (ovp, 2H, H-7, H-4′), 7.13 (d, 1H, J = 1.8 Hz, H-2), 7.06 (t, 1H, J5,4/5,6 = 7.8 Hz,

H-5), 6.95 (t, 1H, J6,5/6,7 = 7.8 Hz, H-6), 6.86 (d, 1H, J3′,4′ = 8.7 Hz, H-3′), 3.88 (t, 2H, J = 6.6

Hz, CH2), 3.05 (t, 2H, J = 6.9 Hz, CH2), 13C NMR (300 MHz, DMSO-d6): 164.6, 160.2,

136.1, 131.8, 130.4, 127.0, 122.9, 121.3, 120.9, 119.4, 118.7, 118.2, 118.2, 111.4, 111.3,

58.5, 26.3, EI MS m/z (% rel. abund.): 298 (M+, 44), 300 (M+2, 28), 143 (34), 130 (100),

HREI-MS m/z: Calcd for C17H15ClN2O [298.0866], Found [298.0873].

2-((2-(1H-Indol-3-yl)ethylimino)methyl)-6-bromo-4-chlorophenol (64)

Yield: 32%, M.p.: 94-96 ºC; 1H NMR (300 MHz, DMSO-d6): 14.65 (s, 1H, OH), 10.86 (s,

1H, NH), 8.43 (s, 1H, N=CH), 7.66 (d, 1H, J4′,6′ = 2.7 Hz, H-6′), 7.58 (d, 1H, J4,5 = 7.3 Hz,

H-4), 7.34 (ovp, 2H, H-7, H-2), 7.15 (d, 1H, J6′,4′ = 2.7 Hz, H-6′), 7.06 (t, 1H, J5,4/5,6 = 7.3

Hz, H-5), 6.96 (t, 1H, J6,5/6,7 = 7.3 Hz, H-6), 3.91 (t, 2H, J = 6.6 Hz, CH2), 3.0 (t, 2H, J = 6.9

Hz, CH2), 13C NMR (400 MHz, DMSO-d6): 165.2, 164.6, 136.2, 135.8, 130.9, 126.9, 123.2,

121.0, 118.3, 118.3, 117.0, 116.2, 115.4, 111.4, 110.5, EI MS m/z (% rel. abund.): 378 (M+,

19), 380 (M+2, 4), 130 (100), HREI-MS m/z: Calcd for C17H14BrClN2O [377.9881], Found

[377.9896].

2-((2-(1H-Indol-3-yl)ethylimino)methyl)-4-fluorophenol (65)


(s, 1H, NH), 8.45 (s, 1H, N=CH), 7.56 (d, 1H, J4,5 = 7.0 Hz, H-4), 7.33 (d, 1H, J7,6 = 7.0 Hz,

H-7), 7.26 (ovp, 1H, H-6′), 7.18 (ovp, 2H, H-4′, H-2), 7.05 (t, 1H, J5,4/5,6 = 7.0 Hz, H-5), 6.95

38

(t, 1H J 6,5/6,7 = 7.0 Hz, H-6), 6.88 (ovp, 1H, H-3′), 3.88 (t, 2H, J = 6.9 Hz, CH2), 3.05 (t, 2H,

J = 6.9 Hz, CH2), EI MS m/z (% rel. abund.): 282 (M+, 24), 130 (100), HREI-MS m/z: Calcd

for C17H15FN2O [282.1156], Found [282.1168].

N-(2-Bromo-4,5-dimethoxybenzylidene)-2-(1H-indol-3-yl)ethanamine (66)

Yield: 51%, M.p.: 220-222 ºC, 1H NMR (300 MHz, DMSO-d6): 7.89 (d, 2H, J4,5/7,6 = 7.6

Hz, H-4, H-7), 7.48 (s, 2H, H-2, H-3′), 7.33 (s, 1H, H-6′), 7.27 (ovp, 2H, H-5, H-6), 3.85 (d,

10H, J = 6.9 Hz, CH2, CH2, OCH3, OCH3), EI MS m/z (% rel. abund.): 387 (M+, 4), 130 (9).

4-((2-(1H-Indol-3-yl)ethylimino)methyl)-2-iodo-6-methoxyphenol (67)

Yield: 50%, M.p.: 225-228 ºC; 1H NMR (300 MHz, DMSO-d6): 10.79 (s, 1H, NH), 8.13

(s, 1H, N=CH), 7.55 (d, 1H, J4,5 = 7.8 Hz, H-4), 7.31 (d, 1H, J7,6 = 8.1 Hz, H-7), 7.13 (d, 1H,

J = 1.8 Hz, H-2), 7.04 (m, 1H, H-5), 6.99 (ovp, 2H, H-2′, H-6′), 6.95 (m, 1H, H-6), 3.81

(ovp, 5H, CH2, OCH3), 2.99 (t, 2H, J = 6.9 Hz, CH2), EI MS m/z (% rel. abund.): 420 (M+,

41), 163 (30), 130 (100), HREI-MS m/z: Calcd for C18H17IN2O2 [420.0342], Found

[420.0335].

4-((2-(1H-Indol-3-yl)ethylimino)methyl)-2,6-dimethoxyphenol (68)


(s, 1H, N=CH), 7.57 (d, 1H, J4,5 = 7.5 Hz, H-4), 7.31 (d, 1H, J7,6 = 7.2 Hz, H-7), 7.12 (s, 1H,

H-2), 7.06 (ovp, 4H, H-5, H-2′, H-6′, H-6), 3.77(ovp, 8H, CH2 , OCH3, OCH3), 3.01 (t, 2H,

J = 7.2 Hz, CH2), EI MS m/z (% rel. abund.): 324 (M+, 25), 130 (100), HREI-MS m/z: Calcd

for C19H20N2O3 [324.1482], Found [324.1474].

4-((2-(1H-Indol-3-yl)ethylimino)methyl)benzene-1,3-diol (69)



H-7), 7.10 (ovp, 3H, H-6′, H-2, H-5), 6.95 (t, 1H, J6,5/6,7 = 7.0 Hz, H-6), 6.20 (dd, 1H, J5′,6′ =

8.7 Hz, J5′,3′ = 2.4 Hz, H-5′), 6.10 (d, 1H, J3′,5′ = 2.1 Hz, H-3′), 3.78 (t, 2H, J = 6.9 Hz, CH2),

3.0 (t, 2H, J = 6.9 Hz, CH2), EI MS m/z (% rel. abund.): 280 (M+, 11), 151 (23), 130 (100),

HREI-MS m/z: Calcd for C17H16N2O2 [280.1206], Found [280.1212].

39

2-((2-(1H-Indol-3-yl)ethylimino)methyl)-4,6-di-tert-butylphenol (70)



H-7), 7.28 (d, 1H, J6′,4′ = 2.4 Hz, H-6′), 7.20 (d, 1H, J4′,6′ = 2.1 Hz, H-4′), 7.15 (d, 1H, J = 2.1

Hz, H-2), 7.06 (t, 1H, J5,4/5,6 = 7.8 Hz, H-5), 6.96 (t, 1H, J6,5/6,7 = 7.8 Hz, H-6), 3.86 (t, 2H J

= 7.2 Hz, CH2), 3.06 (t, 2H J = 7.2 Hz, CH2), 1.37 (s, 9H, tBu), 1.24 (s, 9H, tBu), EI MS m/z

(% rel. abund.): 376 (M+, 89), 361 (41), 130 (100), HREI-MS m/z: Calcd for C25H32N2O

[376.2509], Found [376.2515].

2-((2-(1H-Indol-3-yl)ethylimino)methyl)-3-bromo-6-methoxyphenol (71)


(s, 1H, NH), 8.49 (d, 1H, J = 5.7 Hz, N=CH), 7.59 (d, 1H, J4,5 = 7.5 Hz, H-4), 7.32 (d, 1H,

J7,6 = 7.5 Hz, H-7), 7.15 (s, 1H, H-2), 7.06 (t, 1H, J5,4/5,6 = 7.5 Hz, H-5), 6.96 (t, 1H, J6,5/6,7 =

7.5 Hz, H-6), 6.72 (ovp, 2H, H-4′, H-5′), 3.96 (ovp, 2H, CH2), 3.15 (s, 3H, OCH3), 3.07 (t,

2H, J = 6.9 Hz, CH2), EI MS m/z (% rel. abund.): 374 (M+, 2), 130 (100), HREI-MS m/z:

Calcd for C18H17BrN2O2 [372.0497], Found [372.0473].

N-(2-Fluoro-4-methoxybenzylidene)-2-(1H-indol-3-yl)ethanamine (72)


(s, 1H, N=CH), 7.87 (d, 1H, J6′,5′ = 8.4 Hz, H-6′), 7.56 (d, 1H, J4,5 = 7.5 Hz, H-4), 7.32 (d,

1H, J7,6 = 7.5 Hz, H-7), 7.12 (s, 1H, H-2), 7.06 (t, 1H, J5,4/5,6 = 7.5 Hz, H-5), 6.96 (t, 1H,

J6,5/6,7 = 7.5 Hz, H-6), 6.85 (ovp, 2H, H-3′, H-5′), 3.84 (ovp, 5H, CH2, OCH3), 3.06 (t, 2H, J

= 7.2 Hz, CH2), EI MS m/z (% rel. abund.): 296 (M+, 22), 166 (44), 130 (100), HREI-MS

m/z: Calcd for C18H17FN2O [296.1333], Found [296.1313].

2-(1H-Indol-3-yl)-N-(4-(methylthio)benzylidene)ethanamine (73)


(s, 1H, N=CH), 7.65 (d, 1H, J4,5 = 7.5 Hz, H-4), 7.56 (d, 1H, J2′,3′/6′,5′ = 7.8 Hz, H-2′, H-6′),

7.29 (ovp, 3H, H-3′, H-5′,H-7), 7.12 (d, 1H J = 1.5 Hz, H-2), 7.06 (t, 1H, J5,4/5,6 = 7.5 Hz, H-

5), 6.96 (t, 1H, J 6,5/6,7 = 7.5 Hz, H-6), 3.82 (t, 2H J = 7.2 Hz, CH2), 3.02 (t, 2H, J = 7.2 Hz,

CH2), EI MS m/z (% rel. abund.): 294 (M+, 36), 164 (73), 130 (100), HREI-MS m/z: Calcd

for C18H18N2S [294.1199], Found [294.1191].

40

N-(Furan-2-ylmethylene)-2-(1H-indol-3-yl)ethanamine (74)


(s, 1H, N=CH), 7.78 (s, 1H, H-5′), 7.55 (d, 1H, J4,5 = 7.8 Hz, H-4), 7.30 (d, 1H, J7,6 = 7.8 Hz,

H-7), 7.12 (d, 1H, J = 1.8 Hz, H-2), 7.04 (t, 1H, J5,4/5,6 = 7.8 Hz, H-5), 6.94 (ovp, 1H, H-6),

6.86 (d, 1H, J = 3.3 Hz, H-3′), 6.58 (ovp, 1H, H-4′), 3.80 (t, 2H, J = 7.2 Hz, 2H, CH2), 2.98

(t, 2H, J = 7.2 Hz, CH2), 13C NMR (300 MHz, DMSO-d6): 151.5, 149.8, 145.0, 136.1,

127.2, 122.7, 120.8, 118.4, 118.1, 113.7, 112.1, 111.8, 111.2, 61.3, 26.6, EI MS m/z (% rel.

abund.): 238 (M+, 73), 130 (100), HREI-MS m/z: Calcd for C15H14N2O [238.1114], Found

[238.1106].

N-(3,4-Dimethoxybenzylidene)-2-(1H-indol-3-yl)ethanamine (75)


(s, 1H, N=CH), 7.56 (d, 1H, J4,5 = 7.5 Hz, H-4), 7.36 (s, 1H, H-2′), 7.30 (d, 1H, J7,6 = 8.1 Hz,

H-7), 7.19 (d, 1H, J6′,5′ = 6.9 Hz, H-6′), 7.13 (s, 1H, H-2), 7.01 (ovp, 3H, H-5, H-6, H-5′),

3.78 (ovp, 8H, CH2, OCH3, OCH3), 3.00 (t, 2H, J = 7.2 Hz, CH2), EI MS m/z (% rel. abund.):

308 (M+, 88), 179 (95), 130 (100), HREI-MS m/z: Calcd for C19H20N2O2 [308.1507], Found

[308.1525].

4-((2-(1H-Indol-3-yl)ethylimino)methyl)-2-bromophenol (76)


(s, 1H, N=CH), 7.84 (s, 1H, H-2′), 7.54 (d, 2H, J4,5/6′,5′ = 8.1 Hz, H-4, H-6′), 7.30 (d, 1H, J7,6

= 8.1 Hz, H-7), 7.12 (s, 1H, H-2), 7.01 (t, 2H J5,4/6,7/5,6/6,5 = 8.1 Hz, H-5, H-6), 6.97 (d, 1H, J

5′,6′ = 7.2 Hz, H-5′), 3.75 (t, 2H, J = 7.2 Hz, CH2), 3.01 (m, 2H, CH2), EI MS m/z (% rel.

abund.): 343 (M+, 27), 130 (100), HREI-MS m/z: Calcd for C17H15BrN2O [342.0361], Found

[342.0368].

3-((2-(1H-Indol-3-yl)ethylimino)methyl)benzene-1,2-diol (77)


(s, 1H, N=CH), 7.63 (d, 1H, J4,5 = 7.8 Hz, H-4), 7.35 (d, 1H, J7,6 = 8.1 Hz, H-7), 7.17 (s, 1H,

H-2), 7.09 (ovp, 1H, H-5), 7.03 (ovp, 1H, H-6), 6.81 (dd, 1H, J6′,5′ = 7.8 Hz, J6′,4′ = 1.8 Hz,

H-6′), 6.74 (dd, 1H, J4′,5′ = 7.8 Hz, J4′,6′ = 1.5 Hz, H-4′), 6.55 (t, 1H, J5′,6′/5′,4′ = 7.8 Hz, H-5′),

3.97 (t, 2H, J = 6.9 Hz, CH2), 3.18 (t, 2H, J = 7.2 Hz, CH2), EI MS m/z (% rel. abund.): 280

41

(M+, 62), 184 (20), 130 (100), HREI-MS m/z: Calcd for C17H16N2O2 [280.1212], Found

[280.1212].

1-((2-(1H-Indol-3-yl)ethylimino)methyl)naphthalen-2-ol (78)


(s, 1H, NH), 9.0 (s, 1H, N=CH), 7.92 (d, 1H, J4′,3′ = 8.4 Hz, H-4′), 7.68 (ovp, 2H, H-5′, H-

8′), 7.59 (d, 1H, J4,5 = 7.8 Hz, H-4), 7.36 (ovp, 2H, H-7, H-7′), 7.20 (s, 1H, H-2), 7.16 (t, 1H

J5,4/5,6 = 7.8 Hz, H-5), 7.09 (t, 1H, J6,7/6,5 = 7.8 Hz, H-6), 7.01 (t, 1H, J6′,7′/6′,5′ = 7.0 Hz, H-6′),

6.67 (d, 1H, J3′,4′ = 9.3 Hz, H-3′), 3.94 (m, 2H, CH2), 3.12 (t, 2H, J = 6.6 Hz, CH2), EI MS

m/z (% rel. abund.): 314 (M+, 44), 157 (42), 130 (100), HREI-MS m/z: Calcd for C21H18N2O

[314.1425], Found [314.1419].

5-((2-(1H-Indol-3-yl)ethylimino)methyl)-2-methoxyphenol (79)


(s, 1H, N=CH), 7.54 (d, 1H, J4,5 = 7.8 Hz, H-4), 7.31 (d, 1H, J7,6 = 7.8 Hz, H-7), 7.22 (d, 1H,

J = 1.8 Hz, H-2′), 7.10 (s, 1H, H-2), 7.03 (ovp, 2H, H-5, H-6′), 6.94 (ovp, 2H, H-6, H-5′),

3.78 (ovp, 5H, CH2, OCH3), 2.97 (t, 2H, J = 7.5 Hz, CH2), EI MS m/z (% rel. abund.): 294


[294.1368].

2-(1H-Indol-3-yl)-N-(naphthalen-1-ylmethylene)ethanamine (80)


(s, 1H, N=CH), 7.98 (ovp, 3H, H-2′, H-4′, H-5′), 7.87 (d, 1H, J4,5 = 7.0 Hz, H-4), 7.58 (ovp,

4H, H-3′, H-6′, H-7′, H-8′), 7.32 (d, 1H, J7,6 = 7.0 Hz, H-7), 7.17 (d, 1H, J = 2.1 Hz, H2),

7.05 (t, 1H, J5,4/5,6 = 7.0 Hz, H-5), 6.96 (t, 1H, J6,5/6,7 = 7.0 Hz, H-6), 3.86 (t, 2H, J = 7.2 Hz,

CH2), 3.06 (t, 2H, J = 7.2 Hz, CH2), EI MS m/z (% rel. abund.): 298 (M+, 26), 181 (54), 130

(100), HREI-MS m/z: Calcd for C21H18N2 [298.1456], Found [298.1470].

N-(Anthracen-9-ylmethylene)-2-(1H-indol-3-yl)ethanamine (81)


(s, 1H, N=CH), 8.64 (s, 1H, H-10′), 8.24 (d, 2H, J1′,2′/4′,3′ = 8.4 Hz, H-1′, H-4′), 8.09 (d, 2H,

J5′,6′/8′,7′ = 8.1 Hz, H-5′, H-8′), 7.70 (d, 1H, J4,5 = 7.3 Hz, H-4), 7.53 (ovp, 4H, H-2′, H-3′, H-

6′, H-7′), 7.39 (d, 1H, J7,6 = 7.3 Hz, H-7), 7.22 (s, 1H, H2), 7.11 (t, 1H, J5,4/5,6 = 7.3 Hz, H-

42

5), 7.02 (t, 1H J6,5/6,7 = 7.3 Hz, H-6), 4.26 (t, 2H, J = 6.6 Hz, CH2), 3.26 (t, 2H, J = 6.9 Hz,


for C25H20N2 [348.1641], Found [348.1626].

4-((2-(1H-indol-3-yl)ethylimino)methyl)-2-chlorophenol (82)


(s, 1H, N=CH), 7.68 (s, 1H, H-2′), 7.55 (d, 1H, J4,5 = 7.5 Hz, H-4), 7.50 (ovp, 1H, H-6′), 7.32

(d, 1H, J7,6 = 8.1 Hz, H-7), 7.12 (s, 1H, H-2), 7.06 (t, 1H, J5,4/5,6 = 7.5 Hz, H-5), 6.96 (ovp,

2H, H-6, H-5′), 3.80 (t, 2H, J = 7.2 Hz, CH2), 3.00 (t, 2H, J = 7.2 Hz, CH2), EI MS m/z (%

rel. abund.): 298 (M+, 10), 130 (100), HREI-MS m/z: Calcd for C17H15ClN2O [298.0870],

Found [298.0868].

2-((2-(1H-Indol-3-yl)ethylimino)methyl)-4-nitrophenol (83)


(s, 1H, NH), 8.63 (s, 1H, N=CH), 8.32 (d, 1H, J6′,4′ = 2.4 Hz, H-6′), 8.01 (dd, 1H, J 4′,3′ = 5.1

Hz, J4′,6′ = 2.4 Hz, H-4′), 7.60 (d, 1H, J4,5 = 6.0 Hz, H-4), 7.34 (d, 1H, J7,6 = 6.0 Hz, H-7),

7.17 (d, 1H, J = 1.5 Hz, H-2), 7.08 (t, 1H J5,4/5,6 = 6.0 Hz, H-5), 6.98 (t, 1H, J6,5/6,7 = 6.0 Hz,

H-6), 6.57 (d, 1H, J3′,4′ = 7.2 Hz, H-3′), 3.94 (t, 2H, J = 5.1 Hz, CH2), 3.14 (t, 2H, J = 5.1 Hz,

CH2), 13C NMR (300 MHz, DMSO-d6): 177.8, 166.9, 136.2, 133.5, 132.5, 129.2, 126.8,


13), 130 (100), HREI-MS m/z: Calcd for C17H15N3O3 [309.1111], Found [309.1113].

2-((2-(1H-Indol-3-yl)ethylimino)methyl)-4-bromophenol (84)


(s, 1H, NH), 8.45 (s, 1H, N=CH), 7.57 (ovp, 2H, H-4, H-6′), 7.43 (dd, 1H, J4′,3′ = 6.4 Hz,

J3′,6′ = 2.8 Hz, H-4′), 7.33 (d, 1H, J7,6 = 7.6 Hz, H-7), 7.12 (d, 1H, J = 1.2 Hz, H-2), 7.07 (t,

1H, J5,4/5,6 = 7.6 Hz, H-5), 6.97 (t, 1H, J6,5/6,7 = 7.6 Hz, H-6), 6.81 (d, 1H, J3′,4′ = 8.8 Hz, H-

3′), 3.90 (t, 2H, J = 7.2 Hz, CH2), 3.07 (t, 2H, J = 7.2 Hz, CH2), 13C NMR (300 MHz, DMSO-

d6): 164.5, 160.7, 136.1, 134.6, 133.3, 127.0, 122.9, 120.8, 120.0, 119.2, 118.2, 118.2,

111.4, 111.3, 108.5, 58.4, 26.3, EI MS m/z (% rel. abund.): 342 (M+, 33), 344 (M+2, 35), 130

(100), HREI-MS m/z: Calcd for C17H15BrN2O [342.0367], Found [342.0368].

43

2-((2-(1H-Indol-3-yl)ethylimino)methyl)-5-methoxyphenol (85)



H-7), 7.12 (d, 1H, J = 2.1 Hz, H-2), 7.07 (t, 1H, J5,4/5,6 = 7.8 Hz, H-5), 6.97 (ovp, 3H, H-6,

H-6′, H-3′), 6.79 (d, 1H, J5′,6′ = 8.7 Hz, H-5′), 3.89 (t, 2H, J = 7.2 Hz, CH2), 3.68 (s, 3H,

OCH3), 3.07 (t, 2H, J = 7.2 Hz, CH2), EI MS m/z (% rel. abund.): 294 (M+, 54), 130 (100),


44

Part-B

3 Urea and Thiourea Derivatives of Tryptamine


3.1.1 Chemistry

Tryptamine was taken in a reaction flask in dichloromethane and triethylamine then different

substituted isocyanates or isothiocyanates were added to the reaction mixture and stirred at

room temperature for 2-24 h. The reaction progress was monitored by TLC, upon

consumption of starting materials the solvent was evaporated. The precipitates, thus obtained

were washed with hexane. The synthetic derivatives (87-111) were characterized by

different spectroscopic techniques such as EI-MS, HREI-MS, 1H NMR, and 13C NMR

Scheme-12, Figure-6. Fifteen compounds 91-99, 101, 105-107, 109, and 111 are new,

while, other ten compounds are already reported.

Scheme-12: Synthesis of Urea and Thiourea Analogues 87-111

Mechanism

Figure-6: Proposed mechanism for the formation of urea and thiourea derivatives of

Tryptamine

45

3.2 Characteristic Spectral Features of Representative

Compound 100


The 1H NMR was recorded in DMSO on a 400 MHz instrument. A broad singlet for NH,

the most downfield signal of the spectrum, appeared at δ 10.79. Another broad singlet

appeared at δ 9.07 representing the presence of another NH. The molecule comprises of nine

aromatic protons, H-4 appeared as doublet at δ 7.63 having coupling constant J4,5 = 5.7 Hz,

while H-7 appeared at δ 7.33 as doublet having coupling constant J7,6 = 5.7 Hz. H-3′, H-4′,

H-5′, and H-6′ appeared as overlapping multiplet at δ 7.21. A singlet for H-2 was appeared

at δ 7.11, while H-5 appeared as triplet and resonated at δ 7.07 having coupling constant J5,6

/5,4 = 5.7 Hz. H-6 of indole ring appeared as triplet at δ 6.98 having coupling constant J6,7/6,5

= 5.7 Hz. The signal for two methylene (CH2) protons adjacent to nitrogen atom appeared

as broad singlet at δ 3.71, while other two methylene protons were appeared at δ 2.91 as

triplet having coupling constant J = 5.4 Hz (Figure-7).

Figure-7: 1H NMR chemical shifts of compound 100

13C NMR broad-band decoupled spectrum (DMSO-d6) presented total 18 carbon signals

having one methyl, two methylene, nine methine, and six quaternary carbons. The most

downfield signal was of thiocarbonyl group appeared at C 180.8. Quaternary C-2′ and C-9

resonated at C 136.2, however, methine carbon C-3′ and C-4′ appeared at C 130.5 and

127.7, respectively. All remaining aromatic carbons signals appeared in the usual aromatic

range of C 111.3-136.2. The methylene adjacent to electronegative nitrogen atom appeared

at C 44.8 while another methylene resonated at C 24.7. The most up-field carbon signal was

of methyl appeared at C 17.6 Figure-8.

46

Figure-8: Representative 13C NMR signals for compound 100


The EI-MS spectrum of compound 100 showed the [M+] at m/z 309 for molecular formula

C18H19N3S. The significant fragment appeared at m/z 145 representing the 3-ethyl indole,

however, another fragment appeared at m/z 166 which showed the formation of an

isocyanate radical ion. The fragment appeared at m/z 131 confirmed formation of 3-methyl

indole while, another fragment appeared at m/z 152 formed by the loss of methyl group from

isocyanate radical ion. The key fragments are represented in Figure-9.

Figure-9: EI-MS fragmentation pattern of compound 100

47

Table-6: Urea and thiourea analogues of tryptamine 87-111


87

100

88

101

89

102

90

103

91

104

92

105

93

106

48

94

107

95

108

96

109

97

110

98

111

99

- -


3.3.1 Urease Inhibitory Activity

Urease is a nickel containing enzyme also commonly known as urea amidohydrolases that

hydrolysis the amide linkage of urea to produce ammonia and carbon dioxide (Mobley and

Hausinger, 1989). A wide variety of ureases are isolated from various creatures ranging from

human to bacteria possessing different protein structures, but they follow similar catalysis

49

pattern (Hanif et al., 2012). Urea was the first organic molecule to be synthesized, and urease

was the first enzyme which was crystallized (Sumner, 1926), (Dixon, Gazzola, Blakeley,

and Zerner, 1975). The family amidohydrolases is characterized by the presence of a metal

in the active site of enzyme. Urease catalyzes the hydrolysis of urea into ammonia and

carbamate which are proceeded for further hydrolysis to ammonia and carbonic acid Figure-

10 (Vassiliou et al., 2008). The resonance energy of the molecule is decreased up to 3040

kcal/mol due to the presence of non-bonding electrons of nitrogen (Gawley, 1999), that

results in further stability of molecule as a result an external source like urease is required

for its breakdown. The decomposition of urea in the absence of enzyme results in the

formation of ammonia with isocyanate. Primarily it is a source of nitrogen for organisms

required for their growth which it provides in the form of ammonia. However, increased

urease activity is responsible for release of unusually large amounts of ammonia leading to

various environmental and economic problems. The elevated levels of ammonia is

responsible for many pathological conditions including hepatic encephalopathy of human

and animal, gastric and peptic ulcers, hepatic coma urolithiasis, urinary catheter

encrustation, and pyelonephritis (Upadhyay, 2012).

Figure-10: Mechanism of action of urease enzyme


Whole synthetic library was evaluated for urease inhibitory activity. Out of twenty-five (25)

molecules ten (10) were found to be completely inactive. Five (5) compounds displayed a

comparable activity to standard thiourea, while other were also found to be quite active.

Compounds 87-111 were categorized in three different categories including methyl,

methoxy and halogen substituted compounds in order to develop a better structure-activity

relationship.

a) Methyl Substituted analogues

50

The most active compound of the series was compound 100 (IC50 = 11.4 ± 0.4 μM) having

methyl group at the ortho position, the activity of the compound may be due to the presence

of electron donating substituent. When compared with dimethyl substituted residue 98 (IC50

= 12.8 ± 0.5 μM) no profound increase or decrease is observed in case of di ortho substituted

analogue, but activity decreases when the position of these methyl substituents were

switched to meta, as in case of compound 108 (IC50 = 19.7 ± 0.7 μM) but yet better activity

than standard thiourea (IC50 = 21.2 ± 1.3 μM).

51

b) Methoxy substituted analogues

Compound 110 (IC50 = 14.2 ± 0.6 μM) having methoxy substituent at para position may be

responsible for the activity of compound. While compound 107 (IC50 = 23.0 ± 0.8 μM) and

104 (IC50 = 23.7 ± 0.22 μM) show slight decrease in activity only by switching the position

to meta and ortho position, respectively. The di-substituted methoxy analogue 103 (IC50 =

13.2 ± 0.9 μM), however, showed potent activity as compared to standard as well as most of

the other members of this class.

c) Halogen substituted analogues

Among halogen substituted compound 97 (IC50 = 14.5 ± 0.5 μM) was found to be active may

be due to fluorine atom at ortho position, however, switching the fluoro group to meta

position as in compound 94 (IC50 = 21.3 ± 1.5 μM) decreases the activity as compared to

compound 97. Compound 102 (IC50 = 13.7 ± 0.9 μM) possess chloro group at para position

showed better activity as compared to standard thiourea. Compound 105 (IC50 = 27.1 ± 0.6

μM) becomes less active as compared to compound 102 by just altering the position of

chlorine atom from para to ortho position. Nevertheless, compound 101 (IC50 = 17.1 ± 1.0

μM) having di-chloro substitution at ortho and meta positions also showed showed a better

activity than standard thiourea (IC50 = 21.2 ± 1.3 μM) Table-7.

52

Table-7: Urease Inhibitory Activity of Urea and Thiourea Derivatives of Tryptamine 87-

111

Compound IC50 ± SEMa (μM) Compound IC50 ± SEMa (μM)

87 - 100 11.4 ± 0.4

88 - 101 17.1 ± 1.0

89 - 102 13.7 ± 0.9

90 - 103 13.2 ± 0.9

91 - 104 23.7 ± 0.22

92 - 105 27.1 ± 0.64

93 - 106 -

94 21.3 ± 1.5 107 23.0 ± 0.83

95 - 108 19.7 ± 0.7

96 24.2 ± 1.5 109 -

97 14.5 ± 0.57 110 14.2 ± 0.6

98 12.8 ± 0.47 111 -

99 16.6 ± 1.4

Thiourea(std) 21.2 ± 1.3 SEMa is the standard error of the mean, (-) Not active, Thiourea (std) standard inhibitor for urease inhibitory activity.

3.3.2 Carbonic anhydrase inhibitory Activity

Carbonic anhydrase (CA) also belongs to the class of metalloenzyme and have zinc metal in

it. Carbonic anhydrase enzyme has been found in almost all living organisms including

humans and some invertebrates. The CAs are classified as α-CAs present predominantly in

53

animals, β-CAs are found commonly in plants, however, γ-CAs are predominantly found in

Archaea. In humans 15 different isoforms of α-CAs are present which differ in catalytic

rates, inhibitory activity, selectivity, cellular localization and tissue distribution (Christopher

et al., 2013). Living beings including humans possess carbon dioxide as a key molecule and

is found in equilibrium with bicarbonate. To maintain this equilibrium in living organism

there is a class of enzyme carbonic anhydrases that catalyzes the reversible hydration and

dehydration of carbon dioxide and bicarbonate (Esbaugh, and Tufts, 2006).


All synthetic derivatives were subjected to carbonic anhydrase inhibitory activity, out of

twenty-five (25), nine (9) compounds were found to have good inhibitory activity against

carbonic anhydrase as compared to standard acetazolamide (IC50 = 0.96 ± 0.18 μM). Six (6)

compounds were found to have weak activity, however, one was found to have comparable

activity with the standard. The synthesized compounds were divide into different categories

in order to develop a good structure activity relationship.

a) Urea derivatives

Among urea derivatives, only two compounds 87 (IC50 = 0.68 ± 0.02 μM) and 92 (IC50 =

1.06 ± 0.43 μM), were found to be active. Compound 87 possess naphthyl ring which is an

electron rich group and it may be responsible for the activity. On the other hand compound

92 having triflouro methyl group showed a decreased activity as compared to standard and

compound 87, so it can be concluded that the electron rich group is participating in the

activity of compounds.

b) Thiourea derivatives

54

In thiourea derivatives, fourteen (14) compounds were found to be good to moderately

active. Compound 88 (IC50 = 0.66 ± 0.05 μM) having phenyl ring as a substituent was found

to be more active as compared to standard acetazolamide (IC50 = 0.96 ± 0.18 μM). In case

of different substitutions at different positions on the phenyl ring variable pattern in the

activity was observed. Compound 93 (IC50 = 0.21 ± 0.02 μM) possess nitro group at para

position may contributed in the activity. Among halogen derivatives, compounds 96 and 102

were found to be most active with IC50 values of 0.28 ± 0.01 and 0.22 ± 0.04 μM,

respectively. Compound 102 possess a chloro group at para, again the inductive effect of

halogen may be responsible for the activity of compound, however in compound 96 the

position of chloro group was switched to meta and also the addition of methyl group showed

no profound effect on the activity and both compounds showed compareable activity.

Compound 101 (IC50 = 3.46 ± 0.22 μM) having two chloro groups at ortho and meta

positions resulted in decline in activity. In case of flouro substituents as in compounds 94

(IC50 = 0.38 ± 0.06 μM) and 106 (IC50 = 1.08 ± 0.15 μM) showed that the presence of one

flouro group was contributing in the activity and compound 94 was more active as compared

to standard, however in case of difluoro substituted compound the activity was decreased.

Compound 96 (IC50 = 0.52 ± 0.02 μM) having bromo group at ortho position showed good

activity, however, when the bromine group was switched from ortho to meta a sharp decline

in activity was observed as in compound 99 (IC50 = 1.89 ± 0.23 μM). Compound 108 (IC50

= 0.64 ± 0.03 μM) having two methyl groups at meta positions showed good activity in

comparison to standard but if compared with compound 88 (IC50 = 0.66 ± 0.05 μM), it was

observed that the addition of methyl groups were not contributing in the activity. Compounds

103 (IC50 = 0.51 ± 0.09 μM) and 104 (IC50 = 0.96 ± 0.08 μM) having two and one methoxy

groups, respectively, showed that 103 with two methoxy group was more active as compared

to 104 having one methoxy group Table-8.

55

Table-8: Carbonic anhydrase activity of urea and thiourea derivatives of tryptamine 86-

110


87 0.68 ± 100 1.84 ± 0.14

88 0.66 ± 0.05 101 3.46 ± 0.22

89 - 102 0.22 ± 0.04

90 - 103 0.51 ± 0.09

91 - 104 0.96 ± 0.08

92 1.06 ± 0.43 105 -

93 0.21 ± 0.02 106 1.08 ± 0.15

94 0.38 ± 0.06 107 -

95 0.28 ± 0.01 108 0.64 ± 0.03

96 0.52 ± 0.02 109 -

97 - 110 -

98 1.78 ± 0.12 111 -

99 1.89 ± 0.23

Acetzolamidestd) 0.96 ± 0.18 SEMa is the standard error of the mean, (-) Not active, Acetzolamide (std) standard inhibitor for urease inhibitory activity.

3.3.3 Bacterial multidrug resistance (MDR) activity

In last few decades, the reemergence of microbial infections has increased vastly. To treat

these microbial infections a wide variety of drugs have been introduced as a consequence of

which the various strains of microorganisms have developed resistant against these drugs.

The resistance developed against antimicrobial agents is known as multi-drug resistance

(MDR) (V. Singh, 2013). The reason for the sustainability and spreading of infections is that

56

the microorganisms are able to combat attack by antimicrobial drugs. Some of the common

factors for the development of MDR are considerable rise in the immuno-compromised

conditions, like diabetes, HIV-infections, organ transplantation, burn patients. Due to the

existence of such conditions the hospital acquired infections are spreading thereby

facilitating the spread of MDR. According to WHO report, high rates of resistance are

observed for cephalosporin and fluoroquinolones when administered against Escherichia

coli, similarly, Staphylococcus aureus against methicillin, Streptococcus pneumoniae

against penicillin, Klebsiella pneumoniae against cephalosporin and carbapenems, Shigella

species against fluoroquinolones, Nontyphoidal salmonella against fluoroquinolones,

Neisseria gonorrhoeae against cephalosporin, and Mycobacterium tuberculosis against

rifampicin, isoniazid, and fluoroquinolone causing common infection (Organization, 2014)

(Nikaido, 2009). There are various mechanisms involved in the development of MDR few

of them are, changes in bacterial cell wall and membrane permeability (Ferraro, 2002), active

efflux of the antimicrobial from the cell (Borges-Walmsley, McKeegan, and Walmsley,

2003), mutation in the active target sites (Woo, To, Lau, and Yuen, 2003), enzymatic

degradation or modification of the antibiotics (Paterson and Bonomo, 2005), (Wright, 1999),

microbes acquire an alternative metabolic pathways to escape drug inhibition (Klevens et

al., 2007).

Anti-MRSA Micro-Plate Alamar Blue Assay (MABA)

The organisms were grown in Mueller-Hinton medium and inoculums were adjusted to 0.5

McFarland turbidity index. The stock solutions of test compounds were prepared in DMSO

(1:1 concentration) which were then diluted serially up to 20 μg/mL (DMSO showed no

cidal influence on the bacterial cells culture) in Muller-Hinton broth. 5×106 bacterial cells

were added in a 96-well plate. The plate was then sealed and incubated at 37 °C for 18-20 h.

Test compound was not added in the control experiment. In each well Alamar blue dye

(10%) was added and placed in shacking incubator for 3-4 h at 37 °C and 80 rpm. The change

in colour of Alamar blue dye from blue to pink indicated the growth of bacterial strains. The

absorbance was measured at 570 and 600 nm (Pettit, Weber, Kean, Hoffmann, Pettit, Tan,

Franks, and Horton, 2005) by the ELISA reader (SpectraMax M2, Molecular Devices, CA,

USA).

57


All synthetic molecules 86-110 were subjected to bacterial multidrug resistance assay for

various strains including NCTC 13277, EMRSA-16, EMRSA-17, S.aureus, Klebsiella

clinical isolate and Kleibsiella ATCC 700603 strains. Out of 25 compounds only one

compound 92 was found to be active against all strains except Klebsiella. Compound 92

found to inhibit four strains with about 90% inhibition but was found to be inactive against

Klebsiella strains Table-9.

Table-9: Bacterial MDR activity of Urea and Thiourea derivatives of Tryptamine 86-110

Compound

NCTC-

13277

EMRSA-

16

EMRSA-

17

S.aureus

C isolate

Klebsiella

ATCC

700603

Klebsiella

C isolate

% Inhibition at 20 μg/mL (Solubility in DMSO)

87 - - - - - -

88 - - - - - -

89 - - - - - -

90 - - - - - -

91 - - - - - -

92 90.54 ±

0.60

90.99 ±

0.95

90.74 ±

1.05 90.26 ± 0.78 - -

93 - - - - - -

94 - - - - - -

95 - - - - - -

96 - - - - - -

97 - - - - - -

98 - - - - - -

99 - - - - - -

100 - - - - - -

101 - - - - - -

102 - - - - - -

103 - - - - - -

104 - - - - - -

105 - - - - - -

106 - - - - - -

58

107 - - - - - -

108 - - - - - -

109 - - - - - -

110 - - - - - -

111 - - - - - -

Oxacillinc

Streptomycinc

Clindamicinc

Vancomycinc 24 21 19 40 - -

No Inhibition (-); Standardsc Standards for for antibacterial activity



All synthetic compounds were evaluated for their antileishmanial activity, few compounds

showed good to moderate activity. Compounds 86-110 were categorized into two categories

including urea and thiourea derivatives of tryptamine

a) Urea analogues of tryptamine

Compound 92 (IC50 = 6.36 ± 0.5 μM) showed comparable activity to standard drug

pentamidine (IC50 = 5.09 ± 0.09 μM). The presence of triflouromethyl group at the meta

position of benzene ring may be responsible for the activity. Compound 90 (IC50 = 20.98 ±

0.9 μM) showed good activity the activity may be due to the presence of chlorine group,

while the presence of electron withdrawing nitro group in compound 89 (IC50 = 75.05 ± 0.9

μM) caused a sharp decline in the activity.

b) Thiourea analogues of tryptamine

Among thiourea derivatives the most active compound was found to be 88 (IC50 = 6.64 ± 0.4

μM) showed comparable activity to the standard drug pentamidine. The presence of sulfur

59

atom may be the responsible for activity, while presence of two methyl group on phenyl ring

lowers the activity up to two folds as in compound 108 (IC50 = 20.98 ± 0.7 μM). However,

presence of halogens results in further decrease in activity, as in compound 109 (IC50 = 22.5

± 0.5 μM) possessing two chlorine atoms showed slight decrease in activity, while in

compound 111 (IC50 = 26.19 ± 0.7 μM) the bromine atom further reduces the activity Table-

10.

Table-10: Antileishmanial activity of Urea and thiourea derivatives of tryptamine 87-111


87 - 100 -

88 6.64 ± 0.4 101 -

89 75.05 ± 0.9 102 -

90 20.98 ± 0.9 103 -

91 - 104 -

92 6.36 ± 0.5 105 -

93 - 106 -

94 - 107 -

95 - 108 20.98 ± 0.7

96 - 109 22.5 ± 0.5

97 - 110 -

98 - 111 26.19 ± 0.7

99 -

Amphotercin B(std) 0.29 ± 0.05 Pentamidine (std) 5.09 ± 0.09 SEMa is the standard error of the mean, (-) Not active, Amphotericin B (std) and Pentamidine (std) are standard inhibitor for


60

3.3.5 Antiepileptic Activity

Epilepsy is a neurological disorder characterized by seizures which are due to the abnormal

activity in brain. A human brain contains a millions of nerve cells commonly known as

neurons. These neurons are responsible for transferring messages from brain to different part

of body in the form of electrical messages. As we know, different parts of body are under

the influence of different regions of brain so any electrical imbalance in one such part of

brain may be responsible for these seizures. The seizures of an epileptic patients are

characterized by muscular fatigue, unconsciousness, however, behavior, emotions, and

sensations also get effected. The seizures may be of different types depending on which

portion of brain is effected by electrical imbalance, it may be motor seizures when abnormal

electrical discharge occurs in motor cortex, if it is sensory perception then sensory cortex is

involved. In case of lights, flashes or jagged lines visual cortex is effected, robotic behavior,

loss of memory, and seize of activities are the signs of deep temporal structure seizures.

However, if all the brain is effected then this type of seizures are characterized by jerking,

stiffening of muscles as well as body and finally the loss of consciousness (S. Sharma and

Dixit). The anticonvulsant agents are one of the common treatment for the cure of epilepsy,

however, some epileptic patients do not respond to these drugs while other may have minor

effects of these drugs resulting in less frequency of seizures.

61

All synthesized analogues were subjected to antiepileptic activity and all were found to be

inactive against epilepsy Table-11.

Table-11: Antiepileptic activity of urea and thiourea analogues of tryptamine 87-111


87 - 100 -

88 - 101 -

89 - 102 -

90 - 103 -

91 - 104 -

92 - 105 -

93 - 106 -

94 - 107 -

95 - 108 -

96 - 109 -

97 - 110 -

98 - 111 -

99 - Acetazolamide(std) 0.12 ± 0.009 SEMa is the standard error of the mean, (-) Not active, Acetazolamide (std) standard inhibitor for antiepileptic activity

3.4 Conclusion

Twenty five compounds 87-111 were synthesized by treating tryptamine with various

substituted isocyanates or isothiocyanates, triethyl amine was also added. The reaction

mixture was stirred at room temperature to afford a solid product which was purified by

crystallization or washing with hexane to afford pure products with good to moderate yields.

All synthesized molecules 87-111 were evaluated for their antibacterial, antileishmanial,

antiepileptic, and urease inhibitory activities. It was observed that only compound 91 was

found to be active against four strains of bacteria in MDR assay with 90% inhibition. On the

other hand seven compounds showed good to moderate activity against antileishmanial

activity, compounds 88 (IC50 = 6.64 ± 0.4 μM) and 92 (IC50 = 6.36 ± 0.5 μM) were found to

have a comparable activity to standard pentamidine (IC50 = 5.09 ± 0.09 μM), while all other

analogues showed either weak or no activity. All synthesized compounds were found to be

inactive against antiepileptic activity.

Out of twenty-five (25) compounds, ten (10) were found to be inactive against urease

inhibitory activity. Five (5) compounds 94, 96, 104, 105, and 107 showed IC50 values 21.3-

27.1 μM comparable activity to the standard thiourea. Nine compounds 97, 98, 99, 100, 101,

62

102, 103, 108, and 110 were found to be better active than standard IC50 values in the range

of 11.4-19.7 μM. Sixteen compounds showed good to moderate carbonic anhydrase

inhibitory activity, nine compounds 87, 88, 93, 94, 95, 96, 102, 103, and 108 showed good

activity as compared to standard acetazolamide (IC50 = 0.96 ± 0.18 μM).

General Procedure for the Synthesis of Urea and Thiourea analogues of Tryptamine

87-111

In a reaction flask tryptamine (0.160 g, 1 mmol), triethylamine in catalytic amount in

dichloromethane were taken then corresponding isocyanates and isothiocyanates were added

reaction mixture were kept on stirring for 24 h. The precipitates formed were filtered and

monitored with TLC. Pure products were obtained after washing with hexane.


1-(2-(1H-Indol-3-yl)ethyl)-3-(naphthalen-1-yl)urea (87)


(s, 1H, NH), 8.05 (ovp, 2H, NH, H-8′), 7.89 (m, 1H, H-5′), 7.59 (ovp, 2H, H-4′, H-4), 7.55

(ovp, 2H, H-6′, H-7′), 7.43 (ovp, 2H, H-2′, H-7), 7.20 (s, 1H, H-2), 7.09 (t, 1H, J5,6/5,4 = 7.2

Hz, H-5), 6.99 (t, 1H, J6,5/ 6,7 = 7.2 Hz, H-6), 6.61 (t, 1H, J3′,4′/3′,2′ = 5.3 Hz, H-3′), 3.49 (m,

2H, CH2), 2.91 (t, J = 6.9 Hz, 2H, CH2), EI MS m/z (% rel. abund.): 329 (M+, 9), 168 (46),

143 (100).

1-(2-(1H-Indol-3-yl)ethyl)-3-phenylthiourea (88)


(s, 1H, NH), 7.71 (br.s, 1H, NH), 7.63 (d, 1H, J4,5 = 7.8 Hz, H-4), 7.34 (ovp, 5H, H-7, H-2′,

H-3′, H-5′, H-6′), 7.16 (s, 1H, H-2), 7.10 (ovp, 2H, H-4′, H-5), 6.99 (t, 1H, J6,5/6,7 = 7.8 Hz,

H-6), 3.75 (d, 2H, J = 7.2 Hz, CH2), 2.99 (t, 2H, J = 7.2 Hz, CH2), EI MS m/z (% rel. abund.):

295 (M+, 3), 135 (34), 130 (100).

1-(2-(1H-Indol-3-yl)ethyl)-3-(4-nitrophenyl)urea (89)

Yield: 32%,1H NMR (300 MHz, DMSO-d6): 10.73 (s, 1H, NH), 8.20 (d, 2H, J3′,4′/5′,6′ = 9.3

Hz, H-3′, H-5′), 7.76 (d, 2H, J2′,3′/6′,5′ = 9.3 Hz, H-2′, H-6′), 7.51 (d, 1H, J4,5 = 7.5 Hz, H-4),

7.33 (d, 1H, J7,6 = 7.5 Hz, H-7), 7.11 (s, 1H, H-2), 7.06 (t, 1H, J5,6/5,4 = 7.5 Hz, H-5), 6.97 (t,

63

1H, J6,5/6,7 = 7.5 Hz, H-6), 2.82 (ovp, 4H, CH2), EI MS m/z (% rel. abund.): 324 (M+, 5), 160

(21), 130 (100).

1-(2-(1H-Indol-3-yl)ethyl)-3-(3-chlorophenyl)urea (90)


(s, 1H, NH), 7.67 (s, 1H, NH), 7.63 (d, 1H, J4,5 = 7.8 Hz, H-4), 7.34 (d, 1H, J7,6 = 7.8 Hz, H-

7), 7.24 (ovp, 3H, H-2′, H-6′, H-4′), 7.08 (t, 1H, J5,6/5,4 = 7.8 Hz, H-5), 6.99 (ovp, 2H, H-6,

H-2), 6.23 (t, 1H, J5′,6′/5′,4′ = 5.2 Hz, H-5′), 3.75 (ovp, 2H, CH2), 2.86 (t, 2H, J = 7.2 Hz, CH2),

EI MS m/z (% rel. abund.): 313 (M+, 6), 155 (30), 130 (100), HREI-MS m/z: Calcd for

C17H16NO3Cl [313.0991], Found [313.0982].

1-(2-(1H-Indol-3-yl)ethyl)-3-(2-(trifluoromethyl)phenyl)urea (91)

Yield: 22%, M.p.: 128-130 ºC; 1H NMR (400 MHz, CD3OD): 7.78 (d, 1H, J3′,4′ = 8.4 Hz,

H-3′), 7.60 (t, 2H, J4′,3′/5′,4′ = 8.4 Hz, H-4′, H-5′), 7.53 (d, 1H, J4,5 = 7.6 Hz, H-4), 7.33 (d, 1H,

J7,6 = 7.6 Hz, H-7), 7.21 (t, 1H, J5,4/5,6 = 7.6 Hz, H-5), 7.08 (ovp, 2H, H-2, H-6′), 7.07 (t, 1H,

J6,5/6,7 = 7.6 Hz, H-6), 3.51 (t, 2H, J = 7.2 Hz, CH2), 2.97 (t, 2H, J = 7.2 Hz, CH2), EI MS m/z

(% rel. abund.): 347 (M+, 2), 149 (24), 143 (100), HREI-MS m/z: Calcd for C21H15N3F2

[347.1240], Found [347.1234].

1-(2-(1H-Indol-3-yl)ethyl)-3-(3-(trifluoromethyl)phenyl)urea (92)


(s, 1H, NH), 8.02 (s, 1H, NH), 7.56 (d, 1H, J4,5 = 5.7 Hz, H-4), 7.44 (ovp, 2H, H-2′, H-4′),

7.34 (d, 1H, J7,6 = 5.7 Hz, H-7), 7.20 (ovp, 2H, H-2, H-6′), 7.07 (t, 1H, J5,6/5,4 = 5.7 Hz, H-

5), 6.98 (t, 1H, J6,5/6,7 = 5.7 Hz, H-6), 6.29 (t, 1H, J5′,6′/5′,4′ = 4.2 Hz, H-5′), 3.42 (ovp, 2H,

CH2), 2.87 (t, 2H, J = 5.4 Hz, CH2), EI MS m/z (% rel. abund.): 347 (M+, 7), 143 (46), 130

(100).

1-(2-(1H-Indol-3-yl)ethyl)-3-(4-nitrophenyl)thiourea (93)


(s, 1H, NH), 8.28 (br.s, 1H, NH), 8.14 (d, 2H, J3′,4′/5′,6′ = 6.9 Hz, H-3′, H-5′), 7.57 (d, 2H,

J2′,3′/6′,5′ = 6.9 Hz, H-2′, H-6′), 7.63 (d, 1H, J4,5 = 5.7 Hz, H-4), 7.35 (d, 1H, J7,6 = 5.7 Hz, H-

7), 7.20 (s, 1H, H-2), 7.08 (t, 1H, J5,6/5,4 = 5.7 Hz, H-5), 6.99 (t, 1H, J6,5/6,7 = 5.7 Hz, H-6),

3.80 (br.s, 2H, CH2), 3.02 (t, 2H, J = 5.4 Hz, CH2), 13C NMR (600 MHz, DMSO-d6): 179.8,

64

146.3, 141.7, 136.3, 127.2, 124.6, 122.9, 121.0, 120.3, 118.4, 118.3, 111.4, 111.3, 44.6, 24.1,


C17H16N4O2S [340.0992], Found [340.0994].

1-(2-(1H-Indol-3-yl)ethyl)-3-(3-fluorophenyl)thiourea (94)


(s, 1H, NH), 7.89 (br.s, 1H, NH), 7.63 (d, 1H, J4,5 = 5.7 Hz, H-4), 7.49 (d, 1H, J6′,5′ = 8.7 Hz,

H-6′), 7.34 (d, 1H, J7,6 = 5.7 Hz, H-7), 7.30 (ovp, 1H, H-5′), 7.17 (s, 1H, H-2), 7.11 (ovp,

2H, H-2′, H-4′), 6.99 (t, 1H, J5,6/5,4 = 5.7 Hz, H-5), 6.99 (ovp, 1H, H-6), 3.77 (d, 2H, J = 7.2

Hz, CH2), 2.99 (t, 2H, J = 7.2 Hz, CH2), EI MS m/z (% rel. abund.): 313 (M+, 6), 153 (16),

143 (100).

1-(2-(1H-Indol-3-yl)ethyl)-3-(5-chloro-2-methylphenyl)thiourea (95)


(s, 1H, NH), 7.69 (br.s, 1H, NH), 7.62 (d, 1H, J4,5 = 5.7 Hz, H-4), 7.35 (s, 1H, H-2′), 7.33 (d,

1H, J7,6 = 5.7 Hz, H-7), 7.25 (d, 1H, J5′,4′ = 6.0 Hz, H-5′), 7.19 (dd, 1H, J4′,5′ = 6.0 Hz, J4′,6′ =

1.5 Hz, H-4′), 7.14 (s, 1H, H-2), 7.07 (t, 1H, J5,6/5,4 = 5.7 Hz, H-5), 6.98 (t, 1H, J6,5/6,7 = 5.7

Hz, H-6), 3.72 (br.s, 2H, CH2), 2.97 (t, 2H, J = 5.4 Hz, CH2), EI MS m/z (% rel. abund.):

343 (M+, 4), 345 (M+2, 2), 130 (100), HREI-MS m/z: Calcd for C18H18N3ClS [343.0906],

Found [343.0910].

1-(2-(1H-Indol-3-yl)ethyl)-3-(2-bromophenyl)thiourea (96)


(s, 1H, NH), 7.90 (br.s, 1H, NH), 7.6 (ovp, 2H, H-4, H-5′), 7.56 (d, 1H, J3′,4′ = 5.4 Hz, H-3′),

7.35 (ovp, 2H, H-7, H-3′), 7.16 (ovp, 2H, H-2, H-4,), 7.07 (t, 1H, J5,6/5,4 = 5.5 Hz, H-5), 6.99

(t, 1H, J6,5/6,7 = 5.5 Hz, H-6), 3.72 (br.s, 2H, CH2), 2.97 (t, 2H, J = 5.4 Hz, CH2), 13C NMR

(300 MHz, DMSO-d6): 181.1, 137.4, 136.2, 132.6, 129.7, 127.7, 127.5, 127.2122.8, 120.9,

120.3, 118.5, 118.2, 111.5, 111.3, 44.8, 24.6, EI MS m/z (% rel. abund.): 373 (M+, .5), 375

(M+2, 5), 130 (100), HREI-MS m/z: Calcd for C17H16N3BrS [373.0285], Found [373.0284].

65

1-(2-(1H-Indol-3-yl)ethyl)-3-(2-fluorophenyl)thiourea (97)


(s, 1H, NH), 7.89 (s, 1H, NH), 7.66 (ovp, 2H, H-4, H-6′), 7.34 (d, 1H, J7,6 = 6.0 Hz, H-7),

7.23 (ovp, 2H, H-3′, H-5′), 7.15 (ovp, 2H, H-2, H-4′), 7.07 (t, 1H, J5,6/5,7 = 6.0 Hz, H-5), 6.99

(t, 1H, J6,5/6,7 = 6.0 Hz, 1H, H-6), 3.37 (br.s, 2H, CH2), 2.97 (t, 2H, J = 5.4 Hz, CH2), EI MS

m/z (% rel. abund.): 313 (M+, 1), 152 (19), 130 (100), HREI-MS m/z: Calcd for C17H16N3FS

[313.1034], Found [313.1049].

1-(2-(1H-Indol-3-yl)ethyl)-3-(2,6-dimethylphenyl)thiourea (98)


(s, 1H, NH), 7.63 (d, 1H, J4,5 = 6.0 Hz, H-4), 7.32 (d, 1H, J7,6 = 6.0 Hz, H-7), 7.07 (ovp, 5H,

H-3′, H-4′, H-5′, H-2, H-5), 6.97 (t, 1H, J6,5/6,7 = 5.4 Hz, H-6), 3.67 (s, 2H, CH2), 2.90 (s, 2H,

CH2), 2.48 (s, 6H, CH3), EI MS m/z (% rel. abund.): 323 (M+, 22), 181 (41), 143 (100),

HREI-MS m/z: Calcd for C19H21N3S [323.1434], Found [323.1456].



(s, 1H, NH), 7.91 (s, 1H, NH), 7.78 (s, 1H, H-6′), 7.63 (d, 1H, J4,5 = 5.5 Hz, H-4), 7.35 (d,

1H, J7,6 = 5.5 Hz, H-7), 7.29 (ovp, 3H, H-3′, H-4′, H-5′), 7.17 (s, 1H, H-2), 7.08 (t, 1H, J5,6/5,4

= 5.5 Hz, H-5), 6.99 (t, 1H, J6,5/6,7 = 5.5 Hz, H-6), 3.76 (br.s, 2H, CH2), 2.99 (t, 2H, J = 5.7

Hz, CH2), 13C NMR (300 MHz, DMSO-d6): 180.0, 141.0, 136.2, 130.3, 127.1, 126.2, 124.7,


2), 375 (M+2, 2), 130 (100), HREI-MS m/z: Calcd for C17H16N3BrS [373.0219], Found

[373.0248].

1-(2-(1H-Indol-3-yl)ethyl)-3-(o-tolyl)thiourea (100)

Yield: 62%, M.p.: 185-187ºC; 1H NMR (400 MHz, DMSO-d6): 10.79 (s, 1H, NH), 9.07

(s, 1H, NH), 7.63 (d, 1H, J4,5 = 5.7 Hz, H-4), 7.33 (d, 1H, J7,6 = 5.7 Hz, H-7), 7.21 (ovp, 4H,

H-2′, H-6′, H-3′, H-5′), 7.11 (s, 1H, H-2), 7.07 (t, 1H, J5,6/5,4 = 5.7 Hz, H-5), 6.98 (t, 1H,

J6,5/6,7 = 5.7 Hz, H-6), 3.71 (br.s, 2H, CH2), 2.95 (t, 2H, J = 5.4 Hz, CH2), 2.14 (s, 3H, OCH3),

13C NMR (300 MHz, DMSO-d6): 180.8, 136.2, 130.5, 127.7, 127.2, 126.5, 126.3, 122.7,

66

120.9, 118.5, 118.2, 111.6, 111.3, 44.8, 24.7, 17.6, EI MS m/z (% rel. abund.): 309 (M+, 21),

167 (35), 143 (100), HREI-MS m/z: Calcd for C18H19N3S [309.1286], Found [309.1300].

1-(2-(1H-Indol-3-yl)ethyl)-3-(2,5-dichlorophenyl)thiourea (101)


(s, 1H, NH), 8.21 (s, 1H, NH), 7.90 (s, 1H, H-6′), 7.62 (d, 1H, J4′,3′ = 6.3 Hz, H-4′), 7.51 (d,

1H, J4,5 = 6.0 Hz, H-4), 7.34 (d, 1H, J7,6 = 6.0 Hz, H-7), 7.26 (d, 1H, J3′,4′ = 6.3 Hz, H-3′),

7.17 (s, 1H, H-2), 7.06 (t, 1H, J5,6/5,4 = 6.0 Hz, H-5), 6.97 (t, 1H, J6,5/6,7 = 6.0 Hz, H-6), 3.76

(br.s, 2H, CH2), 2.99 (t, 2H, J = 5.4 Hz, CH2), 13C NMR (300 MHz, DMSO-d6): 180.8,

137.4, 136.2, 130.8, 130.6, 127.8, 127.2, 126.8, 126.1, 122.8, 120.9, 118.4, 118.2, 111.4,

111.3, 44.7, 24.3, FAB+ve m/z (% rel. abund.): 364 (M+).

1-(2-(1H-Indol-3-yl)ethyl)-3-(4-chlorophenyl)thiourea (102)


(s, 1H, NH), 7.83 (s, 1H, NH), 7.63 (d, 1H, J4,5 = 5.7 Hz, H-4), 7.40 (d, 2H, J3′,4′/5′,6′ = 6.6 Hz,

H-3′, H-5′), 7.34 (ovp, 3H, H-2′, H-6′, H-7), 7.17 (s, 1H, H-2), 7.08 (t, 1H, J5,6/5,4 = 5.7 Hz,

H-5), 6.97 (t, 1H, J6,5/6,7 = 5.7 Hz, H-6), 3.75 (br. s, 2H, CH2), 2.98 (t, 2H, J = 5.4 Hz, CH2),

EI MS m/z (% rel. abund.): 329 (M+, 4), 171 (53), 130 (100), 127 (64), HREI-MS m/z: Calcd

for C17H16N3SCl [329.0732], Found [329.0753].

1-(2-(1H-Indol-3-yl)ethyl)-3-(2,4-dimethoxyphenyl)thiourea (103)


(s, 1H, NH), 7.62 (d, 1H, J4,5 = 5.7 Hz, H-4), 7.41 (br. s, 1H, NH), 7.33 (ovp, 2H, H-7, H-

6′), 7.11 (s, 1H, H-2), 7.06 (t, 1H, J5,6/5,4 = 5.7 Hz, H-5), 6.97 (t, 1H, J6,5/6,7 = 5.7 Hz, H-6),

6.58 (d, 1H, J3′,5′ = 2.1 Hz, 1H, H-3′), 6.48 (dd, 1H, J5′,6′ = 4.5 Hz, J5′,3′ = 2.1 Hz, H-5′), 3.75

(ovp, 8H, CH2, OCH3, OCH3), 2.92 (t, 2H, J = 5.4 Hz, CH2), 13C NMR (400 MHz, DMSO-

d6): 180.7, 166.9158.2, 154.2, 136.2, 128.0, 127.2, 122.6, 120.9, 119.8, 118.5, 118.1, 111.6,

111.2, 104.3, 99.0, 55.4, 55.3, 44.6, 24.7, EI MS m/z (% rel. abund.): 355 (M+, 1), 290 (48),

144 (100), HREI-MS m/z: Calcd for C19H21O2N3S [355.1338], Found [355.1354].

67

1-(2-(1H-Indol-3-yl)ethyl)-3-(2-methoxyphenyl)thiourea (104)


(s, 1H, NH), 7.85 (s, 1H. NH), 7.77 (d, 1H, J3′,4′ = 5.4 Hz, H-3′), 7.63 (d, 1H, J4,5 = 5.7 Hz,

H-4), 7.34 (d, 1H, J7,6 = 5.7 Hz, H-7), 7.14 (ovp, 5H, H-5′, H-6′, H-4′, H-5, H-2), 6.90 (t, 1H,

J6,5/6,7 = 5.7 Hz, H-6), 3.77 (ovp, 5H, CH2, OCH3), 2.96 (t, 2H, J = 5.7 Hz, CH2), EI MS m/z

(% rel. abund.): 325 (M+, 1.4), 144 (55), 130 (100), HREI-MS m/z: Calcd for C18H19N3SO

[325.1227], Found [325.1249].

1-(2-(1H-Indol-3-yl)ethyl)-3-(2-chlorophenyl)thiourea (105)


(s, 1H, NH), 7.95 (s, 1H, NH), 7.63 (d, 2H, J4,5/3′,4′ = 5.7 Hz, H-4, H-3′), 7.48 (d, 1H, J7,6 =

5.7 Hz, H-7), 7.34 (ovp, 2H, H-5′, H-6′), 7.22 (t, 1H, J4′,5′/4′,3′ = 5.7 Hz, H-4′), 7.16 (s, 1H, H-

2), 7.07 (t, 1H, J5,6/5,7 = 5.7 Hz, H-5), 6.99 (t, 1H, J6,5/6,7 = 5.7 Hz, H-6), 3.75 (s, 2H, CH2),

2.98 (t, 2H, J = 5.4 Hz, CH2), EI MS m/z (% rel. abund.): 329 (M+, 2), 169 (64), 130 (100),

HREI-MS m/z: Calcd for C17H16N3SCl [329.0742], Found [329.0753].

1-(2-(1H-Indol-3-yl)ethyl)-3-(2,4-difluorophenyl)thiourea (106)

Yield: 51%, M.p.: 165-167 ºC; 1H NMR (400 MHz, CD3OD): 7.62 (d, 1H, J4,5 = 6.0 Hz,

H-4), 7.33 (d, 1H, J7,6 = 6.0 Hz, H-7), 7.09 (ovp, 2H, H-6′, H-3′), 6.97 (ovp, 2H, H-2, H-5),

6.95 (t, 1H, J6,5/6,7 = 6.0 Hz, H-6), 3.85 (br.s, 2H, CH2), 3.07 (t, 2H, J = 5.4 Hz, CH2), EI MS

m/z (% rel. abund.): 331 (M+, 8), 143 (100), 130 (86), HREI-MS m/z: Calcd for C17H15N3SF2

[331.0946], Found [331.0955].



H-4), 7.33 (d, 1H, J7,6 = 6.0 Hz, H-7), 7.15 (ovp, 3H, H-5, H-2, H-5′), 7.00 (t, 1H, J6,5/6,7 =

6.0 Hz, H-6), 6.75 (s, 1H, H-2′), 6.71 (d, 1H, J6′,5′ = 6.3 Hz, H-6′), 6.62 (d, 1H, J4′,5′ = 6.0 Hz,

H-4′), 3.87 (t, 2H, J = 5.1 Hz, CH2), 3.65 (s, 3H, OCH3), 3.08 (t, 2H, J = 5.1 Hz, CH2), EI

MS m/z (% rel. abund.): 325 (M+, 13), 143 (100), 130 (92).

68

1-(2-(1H-Indol-3-yl)ethyl)-3-(3,5-dimethylphenyl)thiourea (108)


H-4), 7.33 (d, 1H, J7,6 = 6.0 Hz, H-7), 7.09 (ovp, 2H, H-5, H-2), 7.00 (t, 1H, J6,5/6,7 = 6.0 Hz,

H-6), 6.78 (s, 1H, H-4′), 6.61 (s, 2H, H-2′, H-6′), 3.88 (t, 2H, J = 5.1 Hz, CH2), 3.07 (t, 2H,

J = 5.1 Hz, CH2), 2.15 (s, 6H, CH3), EI MS m/z (% rel. abund.): 323 (M+, 13), 163 (45), 143

(100), 130 (85).

1-(2-(1H-Indol-3-yl)ethyl)-3-(2,3-dichlorophenyl)thiourea (109)


H-4), 7.41 (ovp, 2H, H-4′, H-5′), 7.33 (d, 1H, J7,6 = 6.0 Hz, H-7), 7.10 (t, 1H, J5,6/5,4 = 6.0

Hz, H-5), 7.09 (ovp, 2H, H-2, H-6′), 7.01 (t, 1H, J6,5/6,7 = 6.0 Hz, H-6), 3.86 (s, 2H, CH2),

3.08 (t, 1H, J = 5.4 Hz, CH2), FAB+ m/z (% rel. abund.): 364 (M+, 10), 366 (6), 185 (100),

130 (3).



H-4), 7.33 (d, 1H, J7,6 = 6.0 Hz, H-7), 7.10 (t, 1H, J5,6/5,4 = 6.0 Hz, H-5), 7.02 (s, 1H, H-2),

6.98 (t, 1H, J6,7/6,5 = 6.0 Hz, H-6), 6.93 (d, 2H, J3′,2′/5′,6′ = 8.4 Hz, H-3′, 5′), 6.79 (d, 2H, J2′,3′/6′,5′

= 8.4 Hz, H-2′, H-6′), 3.83 (t, 2H, J = 6.4 Hz, CH2), 3.75 (s, 1H, OCH3), 3.03 (t, 2H, J = 6.4

Hz, CH2), 2.15 (s, 6H, CH3), EI MS m/z (% rel. abund.): 325 (M+, 13), 165 (45), 143 (100),

130 (85).



H-4), 7.35 (ovp, 3H, H-7, H-3′, H-5′), 7.11 (ovp, 4H, H-5, H-2, H-2′, H-6′), 7.01 (t, 1H,

J6,5/6,7 = 5.5 Hz, H-6), 3.86 (s, 2H, CH2), 3.08 (t, 2H, J = 5.4 Hz, CH2), EI MS m/z (% rel.

abund.): 372 (M+, 1), 374 (M+2, 1), 130 (100).

69

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75

Chapter 2

Synthesis of Indole Derivatives and Their Structure-

Activity Relationship Studies

76

Summary

This chapter includes the synthesis of indole -3-acetamides via a facile and novel approach

and synthesis of indole acrylonitriles. The synthetic compounds were evaluated for various

biological activities including urease inhibition, MCF-7 assay, antiglycation, bacterial

MDR assay, and antileisminicidal activity.

4 Introduction

4.1 General introduction to indoles

Indole (1) is one of the aromatic heterocyclic compound and has come into consideration in

mid nineteenth century with the investigations of indigo which was used as a dye. In 1841,

this dye was reduced to isatin which was then reduced to oxindole in 1866 which upon

pyrolysis with zinc dust reduced to indole. Indole chemistry was considered to be important

and fascinating at that time due its wide use in dyes but at the beginning of twentieth century

when new dyes were introduced indole has lost its importance. Later in 1930s indole

chemistry again came into revival when some naturally isolated alkaloids were found to have

indole ring in their core nucleus possessing various biological activities. Indole ring was

synthesized by the fusion of a benzene ring with pyrrole ring at α,ß-position and is composed

of 10 electrons system from four double bonds and one nitrogen atom lone pair Figure-1.

Figure-1: Systematic IUPAC numbering of indole 1

Indole exists in solid colorless crystals which melts at 52 °C. It is soluble in benzene, ether,

and alcohol and can be recrystallized from water. Its resonance energy is 47-49 Kcal/mole.

It is a very weak base with pKa value 3.63. Tryptophan (2) which is an essential amino acid

also composed of indole ring and is component of various proteins. Similarly, indole is a

basic functional unit in plants as an auxin which promotes the growth of plants (Epstein,

77

Chen, and Cohen, 1989) as well as animals as its fecal discharge contains skatole (3) which

also an indole based compound (Cheeke and Dierenfeld, 2010) Figure-2.

Figure-2: Indole based natural compounds

Indole is a precursor for the synthesis of many pharmaceuticals and prevalent element of

fragrances (Barden, 2010). It is also found in brain and in the content of intestine (D. Kumar,

Kumar, Kumar, Singh, and Singh, 2010). Indole has also been found in coal tar (Yamamoto

et al., 1991) and molasses tar has also yielded some of its bases (Shaikh and Chen, 2011).

Indole comprises as a core skeleton of many synthetic drugs e.g. indomethacin (4), it also

provides the framework of indole alkaloids that are biologically active natural products and

extracted from plants like strychnine (5) Figure-3.

Figure-3: Synthetic and naturally isolated drugs comprising indole nucleus

Thus a wide range of medicinally important moieties were incorporated with indole nucleus

which were widely accepted as a pharmacophores and are a versatile heterocycles possessing

wide spectrum of biological activities (V. Sharma, Kumar, and Pathak, 2010). Indole ring is

a part of wide variety of substances that commonly exist in nature and are principal building

blocks of numerous agrochemicals and pharmaceuticals. In addition, more than 200

78

compounds that are currently marked as drugs or undergoing clinical trials are comprised of

indole nucleus (Liu et al., 2015). Indole and its derivatives are broadly distributed among

nature and are part of natural products, found in variety of plants like orange blossoms,

Robinia pseudacacia, jasmines, and various citrus plants (Ali, Ali, Ahmad Dar, Pradhan,

and Farooqui, 2013).

4.1.1 Reactivity of Indole

Indole is non-basic as compared to most of the amines, the bonding situation is comparable

to that of pyrrole. Protonation of indole requires very strong acids such as hydrochloric acid

which has pKa of -3.6 and this protonation is responsible for the sensitivity of many indole

compounds (e.g. tryptamine) (Dhani et al., 2011). Resonance hybrids of indole are as follows

Figure-4.

Figure-4: Resonance stabilized hybrids of indole

Indoles readily undergoes electrophilic substitution. Substitution takes place preferentially

at pyrrole ring as it is more electron rich as compared to the benzene. Pyrrole ring provides

three sites for the ring including reaction at ring nitrogen, at C-2 position and at C-3 position.

The electrophilic attack at ring nitrogen will result in the loss of aromaticity of five

membered ring by generating a localized citation, so it is eliminated. The C-2 and C-3

positions are readily available for the electrophilic attack but the substitution takes place

preferentially at C-3 Figure-5.

Figure-5: Electrophilic attack at C-3

79

Figure-6: Electrophilic attack at C-2

The attack at C-3 position is favored due the formation of most stable intermediate 10 in

which the charged is located at the carbon adjacent to nitrogen and so lone pair of nitrogen

stabilizes this charge through resonance but the attack at C-2 position results in the formation

of benzylic cation which is also stable but it cannot derive assistance from nitrogen without

disrupting the benzenoid resonance as shown in Figure-6 (Joule and Mills, 2008).

4.2 Biological activities of Indole

Indoles and its analogues have captured the extensive attention of synthetic chemists due to

the wide range of biological activities. They are found in abundance in compounds such as

agrochemicals, alkaloids, and pharmaceuticals (Ali et al., 2013).

NSAIDs are drugs used for the treatment of pain and inflammation. NSAIDs possess a wide

variety of classes and functional groups. Indomethacin and tenidap possessing indole

nucleus and are most commonly used as NSAIDs. The inhibition of selective

cyclooxygenase enzymes are of greater concern. Among cyclooxygenases COX-1 is known

to be the house keeping enzyme while COX-2 enzyme is responsible for the inflammation

and to reduce the effect the expression of this enzyme must be selectively inhibited.

Indomethacin selectively inhibits COX-1 enzyme, the selectivity towards COX-2 enzyme is

achieved by the structural modifications in the indole nucleus (Mitchell, Larkin, and

Williams, 1995).

The modifications at C-3 and N-1 positions of indole ring alters the selectivity of

cyclooxygenase enzyme which results in selective inhibition against COX-2 enzyme. The

compound 14 exhibited significant COX-2 inhibition and selectivity (COX-1 IC50 > 100 μM,

COX-2 IC50 = 0.32 μM) in the in vitro studies. The energy calculations for intermolecular

interactions (Eintermolecular = -12.50) against COX-1/COX-2 active site were also supported by

docking studies (Kaur, Bhardwaj, Huang, and Knaus, 2012).

80

Indole substituted at the position 3 with numerous pyrazolines, and chalcones were estimated

for antiinflammatory activity. The compound 15 showed good antiinflammatory activity

(Rani, Srivastava, and Kumar, 2004).

Radwan group synthesized and evaluated 3-substituted indole analogues as potential

analgesic and antiinflammatory agents, according to their report 3-indolyl thiophene

analogue 16 exhibited potential antiinflammatory activity, however, thiazolidine-4-one

derivative showed analgesic activity (Radwan, Ragab, Sabry, and El-Shenawy, 2007).

Isatin and indole based oximes 17 and 18 were found to exhibit potential fungicidal activity

(Przheval Skii, Magedov, and Drozd, 1997).

81

Various 3-(aryl) and 3-(heteroaryl) indoles were found to possess antibacterial activity

among them most active compound was reported 19 which exhibits MIC ≈ 7 μg/cm3 against

Escherichia coli and Staphylococcus aureus (Panwar, Verma, Srivastava, and Kumar, 2006).

Sharma et al. synthesized novel analogues of indole and investigated the insecticidal activity

of these synthesized compound, derivatives 20 and 21 showed good activity against Jeliothis

armigera and Spodoptera liture (K. Sharma, Jain, and Joshi, 1992).

Yohimbine (22) used as drug for the male impotency (A. Kumar and Arora, 2013).

82

Compound 23 found to possess cardiovascular activity at the dose of 2.5 mg/Kg (N. Singh,

Agarwal, and Singh).

Compounds 24 and 25 exhibited anticancer activity against (HONE-1) cell lines used for

human nasopharyngeal carcinoma against (NUGC-3) cell lines and also used for gastric

adenocarcinoma (Hong, Jiang, Chang, and Lee, 2006).

Indole containing compounds also possess antiviral activity and are also used as drugs.

Arbidol (26) is one of the broad spectrum antiviral agent (Boriskin, Leneva, Pecheur, and

Polyak, 2008).

83

4.3 Synthetic strategies of indole

4.3.1 Sigmatropic rearrangements

4.3.1.1 The Fischer Indole Synthesis

First synthesis of indole was carried out by Emil Fischer and Friedrich Jourdan in 1883.

They treated aryl hydrazones in the presence of an acid catalyst to form indoles. The reaction

was also carried out under microwave irradiation in the presence of ZnCl2 along with

montmorillonite clay which afforded 2-(2-pyridyl) indole (28) in solvent free conditions and

reaction was carried out at low temperatures (Lipinska, Guibe-jampel, Petit, and Loupy,

1999) Scheme-1.

Scheme-1: Microwave assisted synthesis of 2-(2-pyridyl) indoles

4.3.1.2 Gassman Indole synthesis

The scheme proceeds through a series of reactions and results in the formation of substituted

indoles by reacting aniline with ketone in the presence of trimethylamine and benzoyl

chloride resulting in the formation of product which bears thioether substituent, upon

desulfurization with Raney-nickel afforded compound 31 ( Gassman, Van Bergen, Gilbert,

and Cue Jr, 1974) Scheme-2.

84

Scheme-2: Synthesis of 2-substituted indoles through Gassman indole synthesis

4.3.1.3 Bartoli indole synthesis

It is another method for the synthesis of substituted indoles. The reaction of ortho-substituted

nitroarenes with vinyl Grignard reagents in THF affords corresponding indole (Bartoli,

Bosco, Dalpozzo, Palmieri, and Marcantoni, 1991) Scheme-3.

Scheme-3: Synthesis of indole through Grignard reagent

4.3.1.4 Thyagarajan indole synthesis

This method involves the [3,3]- and [2,3]- sigmatropic rearrangements of aryl

propynylamine to afford indole ring (Thyagarajan, Hillard, Reddy, and Majumdar, 1974)

Scheme-4.

Scheme-4: Synthesis of indoles through sigmatropic rearrangements

4.3.1.5 Julia indole synthesis

The synthetic strategy involves the [3,3]-sigmatropic rearrangement of sulfinamides which

are readily available (Baudin and Julia, 1986) Scheme-5.

85

Scheme-5: Synthesis of indoles through the sigmatropic rearrangement of sulfonamides

4.3.2 Nucleophilic cyclization

4.3.2.1 Madelung indole synthesis

The Madelung synthesis is a chemical reaction that produces (substituted or unsubstituted)

indoles when N-phenylamides cyclize intramolecularly using strong base at high

temperature (Houlihan, Parrino, and Uike, 1981) Scheme-6.

Scheme-6: Intramolecular cyclization of N-phenylamides to afford indoles

4.3.2.2 Nenitzescu indole synthesis

In this scheme 5-hydroxyindole derivatives were synthesized from benzoquinone and β-

aminocrotonic esters (Velezheva et al., 2006) Scheme-7.

Scheme-7: Synthesis of 5-hydroxy indole derivatives

86

4.3.3 Electrophilic cyclization

4.3.3.1 Bischler indole synthesis

The synthesis of 2-aryl-indole was carried out by reacting α-bromo-acetophenone with

excess aniline (Bashford et al., 2002) Scheme-8.

Scheme-8: Formation of 2-aryl indoles through Bischler indole synthesis

4.3.4 Nitrene Cyclization

4.3.4.1 Sundberg indole synthesis

The thermal decomposition of o-azidostyrenes followed by cyclization of nitrene affords 2-

(2-azidoethyl) indole (Molina and Lo, 1996) Scheme-9.

Scheme-9: Synthesis of 2-(2-azidoethyl) indole via nitrene cyclization

4.3.5 Oxidative cyclization

4.3.5.1 Watanabe indole synthesis

The reaction of glycols, anilines or ethanolamines catalyzed by metal results in the formation

of alcohol intermediate which upon intramolecular cyclization affords indole (Aoyagi,

Mizusaki, and Ohta, 1996) Scheme-10.

Scheme-10: Oxidative cyclization to afford indoles

87

PART-A

5 Synthesis of Indole-3-acetamides


5.1.1 Chemistry

Twenty-seven (27) indole-3-acetamides 55-81 were synthesized from commercially

available indole-3-acetic acid via condensation with substituted anilines. The reaction was

carried out in acetonitrile along with pyridine and carbonyldiimidazole. The reaction was

performed in two steps, in the first step coupling was carried out by reacting indole-3-acetic

acid with carbonyldiimidazole in the presence of pyridine, the reaction was kept on stirring

for 45 minutes in the second step aniline was added to the reaction pot and mixture was kept

on stirring for 24 h. Products were then isolated and purified through liquid extraction and

pure products were obtained on evaporation of solvent on a rotary evaporator under reduced

pressure Scheme-11, Table-1.

The structure of the synthetic compounds 55-81 were determined by 1H, and 13C NMR, and

EI-MS, HREI-MS. Out of twenty seven compounds eighteen compounds 56-59, 61-66, 68,

70-71, 73, 75, 78-80 are new, while, others are reported.

Scheme-11: Synthesis of indole-3-acetamides via CDI

88

5.1.2 Plausible mechanism for the synthesis of indole-3-acetamides

Reaction starts with the abstraction of acidic proton via base (pyridine) which results in the

formation of carboxylate ion which then attacks the carbonyl carbon of 1,1-

carbonyldiimidazol forming an intermediate with the elimination of first imidazol anion.

Evolution of CO2 results in the formation of stable intermediate. The lone pair of nitrogen

atom of aniline then attacks the carbonyl carbon and second imidazole ion leaves with the

formation of desired amide Figure-7.

Figure-7: A plausible reaction mechanism for the formation of indole-3-acetamides via

CDI

5.2 Spectral Characterization of Representative Analogue 55

5.2.1 1H NMR Spectroscopic characterization

The 1H NMR of compound 55 was recorded in deuterated acetone on 300 MHz instrument.

The most downfield signal of the spectrum appeared at δ 12.20 justifying the presence of

amide group. However, another singlet appeared at δ 10.1 for NH as a singlet. There are ten

aromatic protons present, the signals for three of them H-2′, H-6′, and H-4′ appeared at δ

7.62 as an overlapping multiplet. H-4 of indole ring appeared as doublet at δ 7.38 which is

coupled with H-5 with a coupling constant J 4,5 = 8.1 Hz, H-2 appeared as doublet at δ 7.32

with coupling a constant J = 2.1 Hz. A triplet of H-3′ and H-5′, was observed at δ 7.23

showing coupling with H-2′, H-4′, and H-6′, respectively, with a coupling constant J 3′,4′/3′,5′

89

= J 5′,4′/5′,6′ = 7.5 Hz, another triplet justified the presence of H-6 at δ 7.09 showing a coupling

with H-5 and H-7, while H-5 and H-7 appeared as an overlapping multiplet at δ 7.01. The

structure carries two methylene protons, which appeared at δ 3.80 as a sharp singlet Figure-

8.


13C NMR broad-band decoupled spectrum (DMSO-d6) showed total 14 carbon signals

including five quaternary, eight methine, and one methylene. The quaternary carbon of

carbonyl (amide) was the most downfield signal resonated at C 169.7. Quaternary carbons

C-9 and C-1′ adjacent to nitrogen atom appeared at C 139.4 and C 136.1, respectively. C-

3′ and C-5′ appeared at C 128.6 as a signal. Quaternary C-4 resonated at C 127.1 while, C-

4′ resonated at C 123.8, respectively. C-2ʹ and C-6′ resonated at C 150.26 and 131.85,

respectively. Signals of all remaining aromatic carbons appeared in the usual aromatic range

of C 116.9-134.5. Methylene carbon was the most up-field one and appeared at C 33.77

Figure-9.

Figur-9: Representative 13C NMR signals for compound 55


The EI-MS spectral data of 55 represented the [M+] at m/z 250 confirming the molecular

formula C16H14N2O. The formation of acylium ion was justified by the presence of

90

significant fragment which appeared at m/z 157 which upon loss of CO carbon monoxide

resulted the formation of indole methylium ion which appeared at m/z 130. While the

formation of ((phenylamino)methylidyne)oxonium ion appears at m/z 120 which upon losing

CO appeared at m/z 92. The prominent fragmentations are presented in Figure-10.

Figure-10: Representative fragmentation pattern of analogue 55

91

Table-1: Synthetic derivatives of indole-3-acetamides 55-81

Compound Structures Compound Structures

55

69

56

70

57

71

58

72

59

73

60

74

92

61

75

62

76

63

77

64

78

65

79

66

80

93

67

81

68

- -


5.3.1 Urease Inhibitory Activity

All synthetic compounds 55-81 were evaluated for their urease inhibitory activity, and were

found to be inactive Table-2.

Table-2: Urease inhibitory activity of indole-3-acetamides 55-81.

Compound IC50 ± SEMa( μM) Compound IC50 ± SEMa ( μM)

55 - 69 -

56 - 70 -

57 - 71 -

58 - 72 -

59 - 73 -

60 - 74 -

61 - 75 -

62 - 76 -

63 - 77 -

64 - 78 -

65 - 79 -

66 - 80 -

67 - 81 -

68 - Thiourea(std) 21.2 ± 1.3 SEMa is the standard error of the mean, (-) Not active, Thiourea (std) standard inhibitor for urease inhibitory activity

94


Structural-Activity Relationship

All synthetic derivatives 55-81 were screened for their antileishmanial activity. Except few

compounds majority of the analogues were found to be inactive against leishmaniasis.

Compound 59 (IC50 = 47.4 ± 0.4 μM) showed some activity several folds less than the

standards, the compound possess two donating substituents methoxy and methyl on the

benzene ring thus this activity may be due to the presence of these electron donating

substituents. Compound 61 (IC50 = 94.4 ± 0.5 μM) was found to be weakly active. Compound

62 (IC50 = 47.2 ± 0.2 μM) and 73 (IC50

= 47.2 ± 0.2 μM), however, showed activity similar

to compound 59, the compound 62 possess a bromine atom at para position so the activity

would be due to the mesomeric effect of bromine, while compound 73 possess a methoxy

substituent, thus the presence of electron donating substituent might be the reason for the

activity of this compound. The standards used for this activity are amphotericin B (IC50 =

0.29 ± 0.05 μM) and pentamidine (IC50 = 5.09 ± 0.09 μM). Rest of the compounds were


95

Table-3: Antileishmanial activity of indole-3-acetamides 55-81

Compound IC50 ± SEMa ( μM) Compound IC50 ± SEMa ( μM)

55 - 69 -

56 - 70 -

57 - 71 -

58 - 72 -

59 47.4 ± 0.4 73 47.2 ± 0.2

60 - 74 -

61 94.4 ± 0.5 75 -

62 47.2 ± 0.2 76 -

63 - 77 -

64 - 78 -

65 - 79 -

66 - 80 -

67 - 81 -

68 -

Amphotericin B(std) 0.29 ± 0.05 Pentamidine (std) 5.09 ± 0.09 SEMa is the standard error of the mean, (-) Not active, Amphotericin B(std) and Pentamidine(std) are standard inhibitor for


5.3.3 Anticancer Activity

Cancer has become the major cause of death in many countries globally (Wang et al., 2012).

Cancer is one of the non-communicable disease characterized by formation of tumors which

are lumps or masses of tissue formed mainly due to the uncontrolled growth of damaged

cells. These tumors can interfere with digestive system, nervous system, and they can also

alter the body function as they are capable of releasing hormones. Tumors can be localized

with limited growth but it can be invasive when it goes through the process of invasion and

angiogenesis. In these processes the healthy tissues are destroyed by the formation of

damaged cells which spread throughout the body by division accompanied with the growth

of these cell and formation of new blood vessels, respectively (Steeg, 2006). The most

common pathway to eliminate these tumors is to induce apoptosis of these damaged cells.

Apoptosis is a natural process through which unwanted cells are eliminated from body and

is important in animal development as well as in homeostasis (Reed and Tomaselli, 2000).

However, many pathological conditions may appear if process of apoptosis becomes

abnormal. One of the common pathological condition is cancer which is characterized by

the cell accumulation due to unchecked cell proliferation (Reed, 1999). Chemotherapy,

radiation, TNFα and FAS ligand are some of the initiators of apoptosis. The mechanism

96

proceeds sequential activation of initiators and effector caspases. Caspases belongs to the

family of cysteine proteases that are cleaved in the presence of aspartic acid residues at the

P1 position of substrates (Leung, Abbenante, and Fairlie, 2000). The activation of caspases

is triggered by two main pathways, extrinsic and intrinsic. Recent reports showed that

various anticancer agents possess apoptosis inducing ability, such as imatinib, sorafenib, and

lapatinib (Wang et al., 2012). On the basis of WHO reports the most common types of cancer

are lung cancer, breast cancer, stomach, liver, and colorectal cancer. MCF-7 cell line are

being used as breast cancer cell lines. It is a cultured cell line and was originally derived at

Michigan Cancer Foundation from a malignant pleural effusion from a postmenopausal

woman with metastatic breast cancer who had been previously treated with radiation therapy

and hormonal manipulation (Soule, Vazquez, Long, Albert, and Brennan, 1973) Table-4.

Table-4: Anticancer activity (MCF-7) of indole-3-acetamides 55-81

Compound IC50 ± SEMa ( μM) Compound IC50 ± SEMa ( μM)

55 - 69 -

56 - 70 -

57 - 71 -

58 - 72 -

59 - 73 -

60 - 74 -

61 - 75 -

62 - 76 -

63 - 77 -

64 - 78 -

65 - 79 -

66 - 80 -

67 - 81 -

68 - Doxorubicin(std) 1.56 ± 0.05

SEMa is the standard error of the mean, (-) Not active Doxorubicin (std) is standard inhibitor for MCF-7 anti-

cancer activity.

5.3.4 Bacterial Multi Drug Resistance

All synthetic molecules were evaluated for multidrug resistance (MDR) assay and showed

weak inhibition against various strands of bacteria including EMRSA-17, EMRSA-16,

MRSA-252, clinical isolates of S. aureus and P. aeruginosa and NCTC strain of P.

aeruginosa. Out of twenty-seven compounds, only one compound 73 was found to be

97

completely inactive against all strains of bacteria. All other derivatives showed weak

inhibition ranging from 1% to 37% against various strains of bacteria Table-5.

Table-5: Bacterial MDR activity of indole-3-acetamides 55-81

Compound

EMRSA

-17 EMRSA-16 MRSA-252

S.aureus

Clinical

isolate

P.aeruginosa

Clinical

isolate

P.aeruginosa

NCTC

% Inhibition at 20 μg/mL (Solubility in DMSO)

55 3 - 7 19 27 12

56 12 10 3 26 26 24

57 9 10 9 29 26 24

58 11 13 16 21 27 17

59 6 10 11 20 28 18

60 5 7 8 17 21 9

61 1 8 10 8 24 6

62 5 - - 2 22 1

63 12 - 15 36 28 13

64 13 6 - 23 20 16

65 13 5 16 36 28 16

66 16 12 10 33 29 22

67 11 7 13 34 21 17

68 9 6 12 25 29 11

69 3 6 - 11 20 12

70 8 - 5 14 21 2

71 10 7 11 27 16 21

72 15 8 14 25 17 16

73 - - - 1 - -

74 17 7 15 20 - 5

75 - 7 6 20 3 3

76 3 13 15 - 3 5

77 2 21 37 - 3 8

78 - 10 10 1 - -

79 24 4 - 12 4 4

80 - - - - - 2

81 14 3 24 - - 5

Oxacillinc

Streptomycinc

Clindamicinc

Vancomycinc 24 21 19 40 - - No Inhibition (-); Standardc (Inhibitor for antibacterial activity).

98

5.3.5 Antiglycation activity

All synthetic derivatives were evaluated for antiglycation activity, and were found to be in

active Table-6.

Table 6: Antiglycation activity of indole-3-acetamides 55-81


55 - 69 -

56 - 70 -

57 - 71 -

58 - 72 -

59 - 73 -

60 - 74 -

61 - 75 -

62 - 76 -

63 - 77 -

64 - 78 -

65 - 79 -

66 - 80 -

67 - 81 -

68 - Rutin(std) 294.5 ± 1.50 SEMa is the standard error of the mean, (-) Not active Rutin (std) is standard inhibitor for antiglycation.

5.4 Conclusion

Twenty-seven (27) analogues of indole-3-acetamides 55-81 were synthesized by newly

developed methodology via CDI (1,1-carbonyldiimidazole) using pyridine as a base

minimizes the coupling time and formation of intermediate takes place efficiently which was

consumed without isolating and reacted with substituted anilines to produce Indole-3-

acetamides. A plausible mechanism is also proposed. In addition, all the compounds 55-81

were evaluated for their various biological activities. Out of twenty-seven compounds 55-

81 were found to be inactive against urease, anticancer (MCF-7), and antiglycation activities.

Few compounds showed very weak inhibition in multidrug resistance assay (MDR). Only

four compounds 59 (IC50 = 47.4 ± 0.4 μM), 61 (IC50

= 94.4 ± 0.5 μM), 62 (IC50 = 47.2 ± 0.2

μM), and 73 (IC50 = 47.2 ± 0.2 μM) were found to be weakly active for antileishmanial

activity.

General Procedure for the synthesis of indole-3-acetamides (55-81)

99

In a reaction flask indole-3-acetic acid (0.175 g, 1 mmol), pyridine (0.8 mL) and CDI

equivalent (0.168 g) were taken along with acetonitrile (20 mL) and reaction mixture was

stirred for 45 minutes at room temperature. Then corresponding anilines (1 mmol) were

added in the reaction flask followed by further stirring for 2-24 h. The completion of reaction

was monitored with TLC and products were extracted with dichloromethane. Pure solid

products were obtained after washing with hexane.


2-(1H-Indol-3-yl)-N-phenylacetamide (55)

Yield: 78%, M.p.: 186-188 ºC, 1H NMR (300 MHz, Acetone-d6): 10.14 (s, 1H, NH), 9.04

(s, 1H, NH), 7.62 (ovp, 3H, H-4′, H-2′, H-6′), 7.38 (d, J4,5 = 8.1 Hz, 1H, H-4), 7.32 (s, 1H,

H2), 7.24 (t, J3′,2′/3′,4′/5′,6′/5′,4′ = 7.9 Hz, 2H, H-3′, H-5′), 7.11 (ovp, 1H, H-7), 7.03 (ovp, 2H, H-

5, H-6), 3.80 (s, 2H, H-2″), 13C NMR (300 MHz, DMSO-d6): 169.6, 139.3, 136.0, 128.6,

127.1, 123.8, 122.9, 120.9, 119.0, 118.6, 118.3, 111.3, 108.5, 33.7, EI MS m/z (% rel.

abund.): 250 (M+, 82.6), 157 (22), 130 (100), 103 (57), 93 (54), 77 (70), HREI-MS m/z:

Calcd for C16H14N2O [250.1106], Found [250.1095].

N-(4-Bromophenyl)-2-(1H-indol-3-yl) acetamide (56)


(s, 1H, NH), 7.60 (ovp, 3H, H-4, H-3′, H-5′), 7.41 (ovp, 3H, H-2′, H-6′, H-7), 7.32 (s, 1H,

H-2), 7.08 (ovp, 1H, H-5), 7.01 (ovp, 1H, H-6), 3.80 (s, 2H, H-2″), EI MS m/z (% rel.

abund.): 328 (M+, 39), 330 (M+2, 7), 130 (100), 103 (11.2), 77 (9), HREI-MS m/z: Calcd for

C16H13BrN2O [328.0211], Found [328.0202].

N-(2,5-Dimethoxyphenyl)-2-(1H-indol-3-yl) acetamide (57)


(s, 1H, NH), 8.08 (d, J6′,4′ = 3.0 Hz, 1H, H-6′), 7.63 (d, J3′,4′ = 8.1 Hz, 1H, H-3′), 7.45 (s, 1H,

H-2), 7.42 (ovp, 1H, H-4), 7.16 (ovp, 1H, H-5), 7.05 (ovp, 1H, H-6), 6.77 (d, J7,6 = 8.7 Hz,

1H, H-7), 6.48 (dd, J4′,6′ = 3.0 Hz, J4′,3′ = 3 Hz, 1H, H-4′), 3.87 (s, 2H, H-2″), 3.69 (s, 3H,

OCH3), 3.49 (s, 3H, OCH3), EI MS m/z (% rel. abund.): 310 (M+, 61), 153 (24), 138 (25),

130 (100), HREI-MS m/z: Calcd for C18H18N2O3 [310.1317], Found [310.1314].

100

N-(4-Ethylphenyl)-2-(1H-indol-3-yl) acetamide (58)


(s, 1H, NH), 7.63 (d, J4,5 = 7.8 Hz, 1H, H-4), 7.50 (d, J2′,3′/6′,5′ = 8.4 Hz, 2H, H-2′,H-6′), 7.38

(d, J7,6 = 7.8 Hz, 1H, H-7), 7.32 (s, 1H, H-2), 7.08 (ovp, 3H, H-3′, H-5′, H-5), 7.01 (t, J6,5/6,7

= 7.8 Hz, 1H, H-6), 3.78 (s, 2H, H-2″), 2.54 (ovp, 2H, CH2), 1.15 (t, J = 7.5 Hz, 3H, CH3),


C18H18N2O [278.1419], Found [278.1400].

2-(1H-Indol-3-yl)-N-(3-methoxy-4-methylphenyl) acetamide (59)


(s, 1H, NH), 7.64 (d, J4,5 = 7.8 Hz, 1H, H-4), 7.38 (d, J7,6/6′,5′ = 7.5 Hz, 2H, H-7, H-6′), 7.32

(s, 1H, H-2), 7.11 (t, J5,4/5,6 = 7.8 Hz, 1H, H-5), 7.02 (ovp, 3H, H-2′, H-5′, H-6), 3.78 (s, 2H,

H-2″), 3.73 (s, 3H, OCH3), 2.04 (ovp, 3H, CH3), 13C NMR (300 MHz, DMSO-d6): 169.5,

157.1, 138.5, 136.0, 130.0, 127.2, 123.8, 120.9, 119.9, 118.6, 118.3, 111.3, 110.5, 108.5,

101.8, 54.9, 33.8, 15.5, EI MS m/z (% rel. abund.): 294 (M+, 34), 157 (5), 130 (100), 77 (8),


N-(4-Fluorophenyl)-2-(1H-indol-3-yl) acetamide (60)


(s, 1H, NH), 7.69 (ovp, 1H, H-3′), 7.64 (m, 1H, H-5′), 7.39 (d, J4,5 = 8.5 Hz, 1H, H-4), 7.32

(s, 1H, H-2), 7.69 (ovp, 2H, H-2′, H-6′), 7.12 (ovp, 1H, H-5), 7.02 (ovp, 1H, H-6), 6.77 (ovp,

1H, H-7), 3.78 (s, 2H, H-2″), EI MS m/z (% rel. abund.): 268 (M+, 68), 157 (2), 130 (100),

HREI-MS m/z: Calcd for C16H13FN2O [268.1012], Found [268.1003].

N-(3-Fluorophenyl)-2-(1H-indol-3-yl) acetamide (61)


(s, 1H, NH), 7.61 (ovp, 3H, H-2′, H-4′, H-4), 7.38 (d, J7,6 = 8.1 Hz, 1H, H-7), 7.31 (s, 1H,

H-2), 7.09 (t, J5,6 /5,4 = 7.8 Hz, 1H, H-5), 7.02 (ovp, 3H, H-6, H-5′, H-6′), 3.79 (s, 2H, H-2″),


C16H13FN2O [268.1012], Found [268.1022].

101

N-(3-Bromophenyl)-2-(1H-indol-3-yl) acetamide (62)


(s, 1H, NH), 8.03 (s, 1H, H-2′), 7.62 (d, J4′,5′ = 7.8 Hz, 1H, H-4′), 7.49 (ovp, 1H, H-6′), 7.38

(d, J7,6 = 8.1 Hz, 1H, H-4), 7.32 (s, 1H, H-2), 7.19 (ovp, 2H, H-7, H-5′), 7.10 (t, J5,6/5,4 = 8.0

Hz, 1H, H-5), 7.01 (t, J6,5/6,7 = 8.1 Hz, 1H, H-6), 3.81 (s, 2H, H-2″), 13C NMR (300 MHz,

DMSO-d6): 170.0, 140.9, 136.0, 130.6, 127.1, 125.5, 123.8, 121.4, 121.3, 120.9, 118.5,


(100), 103 (17), HREI-MS m/z: Calcd for C16H13BrN2O [328.0211], Found [328.0233].

N-(4-Butylphenyl)-2-(1H-indol-3-yl) acetamide (63)


(s, 1H, NH), 7.63 (d, J4,5 = 7.5 Hz, 1H, H-4), 7.50 (d, J2′,3′/6′,5′ = 8.7 Hz, 2H, H-2′, H-6′), 7.38

(d, J7,6 = 7.5 Hz, 1H, H-7), 7.32 (s, 1H, H-2), 7.06 (ovp, 4H, H-5, H-6, H-3′,H-5′), 3.78 (s,

2H, H-2″), 2.53 (t, J = 7.5 Hz, 2H, CH2), 1.52 (ovp, 2H, CH2), 1.29 (ovp, 2H, CH2), 0.88 (t,

J = 7.5 Hz, 3H, CH3), EI MS m/z (% rel. abund.): 306 (M+, 86), 130 (100), 106 (33). HREI-

MS m/z: Calcd for C20H22N2O [306.1732], Found [306.1699].

2-(1H-Indol-3-yl)-N-(3-(methylthio) phenyl) acetamide (64)


(s, 1H, NH), 7.66 (ovp, 1H, H-6′), 7.63(d, J4,5 = 8.1 Hz, 1H, H-4), 7.38 (d, J7,6 = 8.1 Hz, 1H,

H-7), 7.35 (s, 1H, H-2), 7.31(ovp, 1H, H-2′), 7.18 (t, J5′,6′/5′,4′ = 7.9 Hz 1H, H-5′), 7.07 (ovp,

1H, H-5), 7.01 (ovp, 1H, H-6), 6.91 (ovp, 1H, H-4′), 3.80 (s, 2H, H-2″), 2.42 (s, 3H, SCH3),


C17H16N2OS [296.0983], Found [296.0970].

2-(1H-Indol-3-yl)-N-(4-(methylthio) phenyl) acetamide (65)


(s, 1H, NH), 7.63 (d, J4,5 = 7.8 Hz, 1H, H-4), 7.58 (d, J2′,3′/6′,5′ = 8.7 Hz, 2H, H-2′, H-6′),

7.38(d, J7,6 = 7.9 Hz, 1H, H-7), 7.31 (s, 1H, H-2), 7.18 (ovp, 2H, H-3′, H-5′), 7.07 (ovp, 1H,

H-5), 7.01 (ovp, 1H, H-6), 3.79 (s, 2H, H-2″), 2.42 (s, 3H, SCH3), EI MS m/z (% rel. abund.):

296 (M+, 11), 130 (100), 77 (27), HREI-MS m/z: Calcd for C17H16N2OS [296.0983], Found

[296.0976].

102

N-(5-Chloro-2-methylphenyl)-2-(1H-indol-3-yl) acetamide (66)


(s, 1H, NH), 8.01 (s, 1H, H-6′), 7.64 (d, J4′,3′ = 7.8 Hz, 1H, H-4′), 7.43 (ovp, 2H, H-4, H-2),

7.10 (ovp, 3H, H-5, H-6, H-3′), 6.98 (ovp, 1H, H-7), 3.88 (s, 2H, H-2″), 1.88 (s, 3H, CH3),

13C NMR (300 MHz, DMSO-d6): 169.9, 137.7, 136.1, 131.6, 129.7, 129.4, 127.1, 124.2,

124.0, 123.4, 121.0, 118.5, 118.4, 111.3, 108.5, 33.1, 17.1, EI MS m/z (% rel. abund.): 298

(M+, 7), 300 (M+2, 2), 130 (100), 76 (56), HREI-MS m/z : Calcd for C17H15ClN2O

[298.0873], Found [298.0873].

2-(1H-Indol-3-yl)-N-(4-iodophenyl) acetamide (67)


(s, 1H, NH), 7.60 (ovp, 3H, H-6′, H-2′, H-4), 7.46 (d, J3′,2′/5′,6′ = 9.0 Hz, 2H, H-3′, H-5′), 7.39

(d, J7,6 = 7.5 Hz 1H, H-7), 7.31 (s, 1H, H-2), 7.09 (t, J5,4/5,6 = 7.5 Hz, 1H, H-5), 7.01 (t, J6,5/6,7

= 7.5 Hz, 1H, H-6), 3.80 (s, 2H, H-2″), EI MS m/z (% rel. abund.): 376 (M+, 10), 218 (3),

130 (100), HREI-MS m/z: Calcd for C16H13IN2O [376.0073], Found [376.0052].

N-(2,4-Difluorophenyl)-2-(1H-indol-3-yl) acetamide (68)


(s, 1H, NH), 8.14 (ovp, 1H, H-5′), 7.64 (d, J6′,5′ = 7.8 Hz, 1H, H-6′), 7.38 (ovp, 2H, H-2, H-

4), 7.10 (t, J5,6 /5,4 = 7.5 Hz, 1H, H-5), 7.04 (ovp, 1H, H-6), 6.95 (ovp, 2H, H-3′, H-7), 3.89

(s, 2H, H-2″), EI MS m/z (% rel. abund.): 286 (M+, 17), 130 (100), 77 (18), HREI-MS m/z:

Calcd for C16H12FN2O [286.0918], Found [286.0912].

N-(4-Chlorophenyl)-2-(1H-indol-3-yl) acetamide (69)


(s, 1H, NH), 7.64 (ovp, 3H, H-5′, H-3′, H-4), 7.38 (d, J7,6 = 7.2 Hz, 1H, H-7), 7.32 (s, 1H,

H-2), 7.25 (d, J2′,3′/6′,5′ = 9.0 Hz, 2H, H-2′, H-6′), 7.10 (t, J5,6/5,4 = 7.2 Hz, 1H, H-5), 7.02 (t,

J6,5/6,7 = 7.2 Hz, 1H, H-6), 3.80 (s, 2H, H-2″), EI MS m/z (% rel. abund.): 283 (M+, 16), 285

(M+2, 4), 130 (100), 77(23), HREI-MS m/z: Calcd for C16H13ClN2O [284.0716], Found

[284.0724].

103

2-(1H-Indol-3-yl)-N-(4-(octyloxy) phenyl) acetamide (70)


(s, 1H, NH), 7.63 (d, J4,5 = 7.8 Hz 1H, H-4), 7.49 (d, J2′,3′/6′,5′ = 8.1 Hz, 2H, H-2′, H-6′), 7.38

(d, J7,6 = 7.8 Hz, 1H, H-7), 7.31 (s, 1H, H-2), , 7.10 (t, J5,6/5,4 = 7.8 Hz, 1H, H-5), 7.00 (t,

J6,5/6,7 = 7.8 Hz, 1H, H-6), 6.79 (d, J3′,2′/5′,6′ = 9.0 Hz, 2H, H-3′, H-5′), 3.91 (t, J = 6.6 Hz, 2H,

OCH2), 3.76 (s, 2H, H-2″), 1.71 (ovp, 2H, CH2), 1.41 (ovp, 2H,CH2), 1.29 (ovp, 8H, CH2,

CH2, CH2, CH2), 0.85 (m, 3H, CH3), EI MS m/z (% rel. abund.): 378 (M+, 74), 131 (71), 130

(100), 109 (65), HREI-MS m/z: Calcd for C16H13BrN2O [328.0211], Found [328.0233].

N-(4-Butoxyphenyl)-2-(1H-indol-3-yl) acetamide (71)



(d, J7,6 = 7.2 Hz, 1H, H-7), 7.31 (s, 1H, H-2), 7.10 (t, J5,6/5,4 = 7.2 Hz, 1H, H-5), 7.01 (t, J6,5/6,7

= 7.2 Hz, 1H, H-6), 6.80 (d, J3′,2′/5′,6′ = 9.0 Hz, 2H, H-3′, H-5′), 3.91 (t, J′ = 6.3 Hz, 2H, OCH2),

3.76 (s, 2H, CH2), 1.71 (ovp, 2H, CH2), 1.45 (ovp, 2H, CH2), 0.92 (t, J = 7.5 Hz 3H, CH3),

EI MS m/z (% rel. abund.): 322 (M+, 96), 131 (60), 130 (100), 109 (54), HREI-MS m/z:

Calcd for C20H22N2O2 [322.1681], Found [322.1703].

2-(1H-Indol-3-yl)-N-(4-methoxyphenyl) acetamide (72)



(d, J7,6 = 7.2 Hz, 1H, H-7), 7.31 (s, 1H, H-2), 7.10 (t, J5,6/5,4 = 7.2 Hz, 1H, H-5), 7.00 (t, J6,5/6,7

= 7.2 Hz, 1H, H-6), 6.80 (d, J3′,2′/5′,6′ = 9.0 Hz, 2H, H-3′, H-5′), 3.76 (s, 2H, H-2″), 3.72 (s,

3H, OCH3), 13C NMR (300 MHz, DMSO-d6): 169.1, 155.0, 136.0, 132.5, 127.2, 123.7,

120.9, 120.5, 118.6, 118.3, 113.7, 111.3, 108.6, 55.1, 33.6, EI MS m/z (% rel. abund.): 280


[280.1194].

2-(1H-Indol-3-yl)-N-(3-iodophenyl) acetamide (73)


(s, 1H, NH), 8.18 (s, 1H, H-2′), 7.63 (d, J4′,5′ = 7.8 Hz 1H, H-4′), 7.54 (d, J6′,5′ = 7.5 Hz, 1H,

H-6′), 7.38 (d, J4,5 = 7.8 Hz, 1H, H-4), 7.32 (s, 1H, H-2), 7.07 (ovp, 4H, H-5′, H-5, H-6, H-

104

7), 3.81 (s, 2H, H-2″), EI MS m/z (% rel. abund.): 376 (M+, 95), 131 (42), 130 (100), HREI-

MS m/z: Calcd for C16H13IN2O [376.0073], Found [376.0032].

N-(3,4-Dimethylphenyl)-2-(1H-indol-3-yl) acetamide (74)


(s, 1H, NH), 7.63 (d, J4′,5′ = 8.1 Hz 1H, H-5′), 7.36 (ovp, 4H, H-4, H-6′, H-2, H-7), 7.10 (t,

J5,4/5,6 = 7.2 Hz 1H, H-5), 7.01 (ovp, 2H, H-6, H-2′), 3.77 (s, 2H, H-2″), 2.14 (s, 6H, CH3),

13C NMR (300 MHz, DMSO-d6): 169.3, 137.0, 136.1, 136.0, 130.6, 129.4, 127.1, 123.7,

120.9, 120.3, 118.6, 118.3, 116.5, 111.2, 108.6, 33.7, 19.5, 18.6, EI MS m/z (% rel. abund.):

278 (M+, 57), 131 (34), 130 (100), 121 (25), HREI-MS m/z: Calcd for C18H18N2O

[278.1419], Found [278.1431].

N-(3,4-Difluorophenyl)-2-(1H-indol-3-yl) acetamide (75)


(s, 1H, NH), 7.84 (m, 1H, H-5′), 7.62 (d, J4,5 = 7.8 Hz, 1H, H-4), 7.38 (d, J7,6 = 7.8 Hz, 1H,

H-7), 7.31 (s, 1H, H-2), 7.24 (ovp, 1H, H-2′), 7.18 (ovp, 1H, H-6′), 7.14 (ovp, 1H, H-5), 7.01

(t, J6,7/6,5 = 7.8 Hz, 1H, H-6), 3.80 (s, 2H, H-2″), EI MS m/z (% rel. abund.): 286 (M+, 92),

131 (63), 130 (100), HREI-MS m/z: Calcd for C16H12F2N2O [286.0918], Found [286.0943].



(s, 1H, NH), 7.68 (d, J4,5 = 7.8 Hz, 1H, H-4), 7.44 (ovp, 3H, H-2, H-5′, H-7), 7.12 (t, J5,4/5,6

= 7.8 Hz, 1H, H-5), 7.01 (ovp, 2H, H-6, H-3′), 6.90 (d, J6′,5′ = 7.2 Hz, 1H, H-6′), 3.84 (s, 2H,

H-2″), 2.82 (s, 3H, CH3), 1.84 (s, 3H, CH3), EI MS m/z (% rel. abund.): 278 (M+, 93), 131

(52), 130 (100), 121 (25), HREI-MS m/z: Calcd for C18H18N2O [278.1419], Found

[278.1422].



(s, 1H, NH), 7.65 (ovp, 2H, H-6′, H-2), 7.42 (d, J7,6 /4,5 = 6.3 Hz, 2H, H-7, H-4), 7.12 (t,

J5,4/5,6 = 6.3 Hz, 1H, H-5), 7.04 (t, J6,5/6,7 = 6.3 Hz, 1H, H-6), 6.94 (d, J3′,4′ = 7.5 Hz, 1H, H-

3′), 6.78 (d, J4′,3′ = 7.5 Hz, 1H, H-4′), 3.84 (s, 2H, H-2″), 2.23 (s, 3H, CH3), 1.85 (s, 3H, CH3),

105

EI MS m/z (% rel. abund.): 278 (M+, 58), 131 (22), 130 (100), 121 (11), HREI-MS m/z:

Calcd for C18H18N2O [278.1419], Found [278.1408].

N-(5-Chloro-2,4-dimethoxyphenyl)-2-(1H-indol-3-yl)acetamide (78)

Yield: 10%, 1H NMR (300 MHz, Acetone-d6): 10.26 (s, 1H, NH), 8.38 (s, 1H, NH), 8.28

(s, 1H, H-6′), 7.64 (d, J4,5 = 7.3 Hz, 1H, H-4), 7.41 (ovp, 2H, H-2, H-7), 7.14 (t, J5,4/5,6 = 7.3

Hz 1H, H-5), 7.04 (t, J6,5/6,7 = 7.3 Hz 1H, H-6), 6.73 (s, 1H, H-3′), 3.85 (s, 2H, H-2″), 3.82

(s, 3H, OCH3), 3.65 (s, 3H, OCH3), EI MS m/z (% rel. abund.): 344 (M+, 74), 346 (M+2, 50),

172 (61), 130 (100), 103 (22).

2-(1H-Indol-3-yl)-N-(3-nitrophenyl) acetamide (79)


(s, 1H, NH), 8.69, (s, 1H, H-2′), 7.94 (d, J4′,5′ = 8.1 Hz, 1H, H-4′), 7.88 (ovp, 1H, H-6′), 7.64

(d, J4,5 = 7.8 Hz, 1H, H-4), 7.54 (t, J5′,4′/5′,6′ = 8.1 Hz 1H, H-5′), 7.38 (ovp, 2H, H-7, H-2),

7.10 (t, J5,6/5,4 = 7.2 Hz 1H, H-5), 7.02 (t, J6,5/6,7 = 7.2 Hz 1H, H-6), 3.87 (s, 2H, H-2″), 13C

NMR (300 MHz, DMSO-d6): 170.4, 147.9, 140.4, 136.0, 130.1, 127.1, 124.9, 123.9, 121.0,

118.5, 118.4, 117.5, 113.0, 111.3, 107.9, 33.8, EI MS m/z (% rel. abund.): 295 (M+, 18), 130

(100), HREI-MS m/z: Calcd for C16H13N3O3 [295.0957], Found [295.0949].

2-(1H-Indol-3-yl)-N-(2-methoxyphenyl) acetamide (80)


(ovp, 2H, NH, H-6′), 7.64 (d, J4,5 = 7.5 Hz, 1H, H-4), 7.42 (ovp, 2H, H-2, H-7), 7.14 (t, J5,4/5,6

= 7.5 Hz, 1H, H-5), 7.05 (t, J6,5/6,7 = 7.5 Hz, 1H, H-6), 6.96 (ovp, 1H, H-5′), 6.85 (ovp, 2H,

H-3′, H-4′), 3.87 (s, 2H, H-2″), 3.58 (s, 3H, OCH3), EI MS m/z (% rel. abund.): 280 (M+,

57), 131 (28), 130 (100), 123 (17), HREI-MS m/z: Calcd for C17H16N2O2 [280.1212], Found

[280.1200].

2-(1H-Indol-3-yl)-N-(p-tolyl) acetamide (81)


(s, 1H, NH), 7.64 (d, J4,5 = 7.8 Hz, 1H, H-4), 7.47 (d, J2′,3′/6′,5′ = 8.4 Hz, 2H, H-2′, H-6′), 7.38

(d, J7,6 = 7.8 Hz, 1H, H-7), 7.31 (s, 1H, H-2), 7.04 (ovp, 4H, H-3′, H-5′, H-5, H-6), 3.78 (s,

2H, H-2″), 2.22 (s, 3H, CH3), EI MS m/z (% rel. abund.): 264 (M+, 80), 131 (53), 130 (100).

107

Part-B

6 Synthesis of Indole acrylonitriles


6.1.1 Chemistry

Cyanoacetic acid and indole were taken in a reaction flask along with acetic anhydride and

reaction mixture was kept under reflux with stirring for 10 minutes. The reaction progress

was monitored via TLC, precipitates formed were filtered and washed with water to afford

cyanoacetyl indole in good yields. Cyanoacetyl indole was then taken in a reaction flask in

ethanol and catalytic amount of trimethylamine was added. Corresponding benzaldehydes

were then added and mixture was stirred at 60 ˚C for 1-2 h. Progress of the reaction was

monitored by TLC. The formation of precipitates indicates the completion of reaction which

were filtered and washed with hexane to afford the desired product in good to excellent yield.

The structure determination of all synthetic molecules (83-120) was done by different

spectroscopic techniques such as 1H-, 13C-NMR and EI-MS, and HREI-MS studies Scheme-

12 and Scheme-13. Thirty two compounds 83-84, 86-91, 93-96, 98-115, and 117-118 are

newly synthesized compounds, however, only compounds 85, 92, 97, and 116 are already

reported in the literature.

Scheme-12: Synthesis of cyanoacetyl indole 82

108

Scheme 13: Synthesis of indole acrylonitriles 83-120

Mechanism

The overall reaction involves two mechanistic steps, first step is the formation of

intermediate 1 through the attack of cyanoacetic acid on carbonyl carbon of acetic anhydride.

Indole will then attack on carbonyl carbon adjacent to the cyano group thus result in the

formation of cyanoacetyl indole Figure-11.

Figure-11: Mechanism for the formation of cyanoacetyl indole 82

The base abstracted the acidic proton resulting in the formation of enolate which then

condensed with aldehyde thus resulting in the formation of an aldol product which upon

subsequent elimination of water affords α,ß unsaturated product Figure-12.

109

Figure-12: Mechanism for the formation of indole acrylonitriles 83-120

6.2 Representative structure elucidation by 1H NMR and mass

spectroscopy of compound 113


The 1H NMR was recorded in DMSO on a 400 MHz instrument. A sharp singlet for NH of

indole appeared at δ 12.25 the most downfield signal of the spectrum. There are 8 aromatic

protons present, one of them H-1″ appeared at δ 8.43 as a singlet. H-2 of indole ring appeared

as doublet at δ 8.34 showing resonance with NH having coupling constant J = 2.0 Hz, H-2′

appeared at δ 8.17 as a sharp singlet. Two protons H-4, H-6′ appeared at δ 8.13 as an overlap.

H-7 of indole ring appeared at δ 7.53 as doublet having coupling constant J 7, 6 = 7.2 Hz, H-

5′, appeared as doublet at δ 7.35 showing coupling with H-6′ having coupling constant J 5′,6′

= 8.8 Hz. H-5 and H-6 appeared as an overlap at δ 7.25. A sharp singlet for three protons of

OCH3 appears at δ 3.96 Figure-13.

110

Figure-13: Representative 1H NMR signals for compound 113


including nine quaternary, nine methine, and one methoxy. The quaternary carbon of

carbonyl (α,ß unsaturated ketone) was the most downfield signal appeared at C 181.3. C-4′

appeared at C 158.4 due to directly attached electronegative oxygen. Quaternary C-8 and C-

9 resonated at C 136.5 and 126.1, respectively. Quaternary nitrile carbon appeared at C

117.9. The quaternary carbon at α position to carbonyl appeared C 109.6, while, quaternary

carbon C-1′ resonated at C 126.3. The quaternary carbon C-3′ attached to bromine atom

appeared at C 111.1. C-2ʹ and C-6′ resonated at C 150.3 and 131.9, respectively. The

methine carbon C-2 attached next to the nitrogen resonated at C 135.0. Signals of all

remaining aromatic carbons appeared in the usual aromatic range of C 116.9-134.5.

Methoxy carbon was the most up-field one and appeared at C 56.8 Figure-14.

Figure-14: Representative 13C NMR signals for compound 113

111

The 13C/1H NMR chemical shifts assigned on the basis of HSQC and HMBC correlations.

The α,ß unsaturated double bond was found to have (Z) stereochemistry after observing the

NOESY interaction of H-1″ with H-2′ and H-2 with H-2′ of the benzene ring.


The EI-MS spectra of compound 113 showed the [M+] at m/z 380 for molecular formula

C19H13BrN2O2. The significant fragment appears at m/z 355 by the loss of CN. The fragment

which showed loss of bromine appears at m/z 301. Formation of ((1H-indol-3-yl)

methylidyne) oxonium ion appears at m/z 144 while the formation of indolium ion appears

at m/z 116. The key fragments are represented in Figure-15.

Figure-15: EI-MS fragmentation pattern of compound 113

112

Table-7: Synthetic derivatives of indole acrylonitriles 83-120


83

101

84

102

85

103

86

104

87

105

113

88

106

89

107

90

108

91

109

92

110

114

93

111

94

112

95

113

96

114

97

115

115

98

116

99

117

100

118


6.3.1 Antiepileptic Activity


All synthetic compounds were evaluated for their antiepileptic activity only eight

compounds were found to exhibit very weak activity almost inactive, if compared with

standard acetazolamide (IC50 = 0.12 ± 0.009 μM), while other analogues were completely in

active.

Compound 118 (IC50 = 53.9 ± 1.30 μM) was found to be the most active among these

analogues as the compound possess methyl, methoxy, and chloro substituents at different

position of the molecule so the combination of these electron donating substituents may be

responsible for the activity of compound. Analogue 115 (IC50 = 67.3 ± 3.81 μM) was the

116

second active among eight analogues possessing a naphthyl group as substituent which is

again an electron donating substituent, while compound 88 (IC50 = 81.0 ± 1.29 μM) having

two methoxy substituent along with bromine which may results in the activity of compound,

when compared with compound 118, it shows that the absence of methyl substituents and

replacing bromine with chlorine had a wide impact on the activity by decreasing the activity

up to one fold. Compound 111 (IC50 = 90.7 ± 3.29 μM) having anthracene ring as substituent

showed decrease in activity when compared with compound 115 having naphthyl group,

compound 85 (IC50 = 113.5 ± 4.16 μM) possessing two indole ring also showed weak activity

and also showed that the addition another indole ring results in the decreased activity,

compound 84 (IC50 = 122.3 ± 2.25 μM) having two chloro substituents when compared with

118 indicates that methoxy group are participating in enhancing the activity of compounds

and replacing the methoxy substituent with chlorine results in the decreased activity, while

94 (IC50 = 159.5 ± 5.38 μM) possessing only methoxy substituent also showed decrease in

activity indicating that chloro and methoxy substituents are together playing a role in the

activity of analogue 118, and compound 106 (IC50 = 183.1 ± 1.26 μM) having one bromo

and fluoro substituents was found to be the least active among all eight active analogues

which may be due to the inductive effect of both these substituents.

117

Table-8: Antiepileptic activity of indole acrylonitriles 83-118


83 - 101 -

84 122.3 ± 2.25 102 -

85 113.5 ± 4.16 103 -

86 - 104 -

87 - 105 -

88 81.0 ± 1.29 106 183.1 ± 1.26

89 - 107 -

90 - 108 -

91 - 109 -

92 - 110 -

93 - 111 90.7 ± 3.29

94 159.5 ± 5.38 112 -

95 - 113 -

96 - 114 -

97 - 115 67.3 ± 3.81

98 - 116 -

99 - 117 -

118

100 - 118 53.9 ± 1.30

Acetazolamide 0.12 ± 0.009 - - SEMa is the standard error of the mean, (-) Not active Acetazolamide(std) is standard drug used for antiepilepsy.

6.3.2 Anticancer Activity (HeLa cancer cell lines)


Compounds 83-118 were categorized in four different categories including, alkoxy, halogen,

and nitro substituted and ring size effect to develop a better structure-activity relationship.

a) Alkoxy Substituted Analogues

The most active compound of the series was compound 83 (IC50 = 4.03 ± 0.05 μM), it possess

two methoxy substituents at ortho and meta positions which may contribute in the activity

of this compound by their donating effect, compound 83 when compared with compound

102 (IC50 = 20.51 ± 0.04 μM) showed several folds decrease in activity by just adding two

methyl groups at indole ring which showed that indole ring plays a major contribution in

activity. Compound 99 (IC50 = 6.28 ± 0.05 μM) having three methoxy substituents at

adjacent carbons again contributing in activity, however, also indicates that addition of

another methoxy substituents decreases the activity of compound as compare to 83. The

variation in positions of methoxy substituent effects the activity of compounds, such as

compound 94 (IC50 = 7.19 ± 0.05 μM) also possess two methoxy substituent but the variation

of position decreased the activity. Compound 87 (IC50 = 27.12 ± 0.05 μM) showed a several

folds decrease in activity as compared with compound 83 just by shifting one of the methoxy

substituent from ortho to para which showed that ortho substituent must be a contributing

factor in the anticancer activity. Compound 100 (IC50 = 26.14 ± 0.04 μM) having only one

methoxy substituent showed a weak activity.

119

b) Halogen substituted analogues

Compound 84 (IC50 = 10.71 ± 0.03 μM) having two chloro substituents at ortho and para

positions contributing in the activity, replacement of chloro to fluoro and methoxy

substituent as in compound 114 (IC50 = 11.81 ± 0.02 μM) showed a comparable activity

which indicates that mesomeric effect of halogen substituent is contributing in the activity

of these compounds. Compound 98 (IC50 = 10.38 ± 0.02 μM) having two methoxy groups

along with bromine and substituted indole ring also showed same activity pattern.

Compound 95 (IC50 = 19.89 ± 0.01 μM) a closely related compound to 98 without methyl

groups at indole ring resulted a decreased activity. Similarly compound 88 (IC50 = 25.05 ±

0.02 μM) with a slight change of position bromine as compare to 98 showed a decreased

activity. However, compound 96 (IC50 = 22.46 ± 0.04 μM) having one methoxy and one

bromine substituent found to be less active.

120

c) Nitro substituted Analogues

Compound 91 (IC50 = 4.89 ± 0.05 μM) is the second most active compound of series having

an electron donating hydroxyl group and an electron withdrawing nitro group but as we had

encountered earlier that all electron donating substituents are contributing in the anticancer

activity so it can be concluded that electron withdrawing substituent does not alter the

activity of compounds. Compound 93 (IC50 = 21.94 ± 0.05 μM) however showed three folds

decrease in activity as compared to compound 91 by replacement of hydroxyl group to

chloro group.

121

d) Ring size effect

Compound 97 (IC50 = 10.59 ± 0.03 μM) with a phenyl ring showed anticancer activity,

however, variation in the ring size as in compound 115 (IC50 = 9.35 ± 0.5 μM) having a

naphthyl ring does not have any profound effect on the activity.

Table-9: Anticancer (HeLa cell lines) activity of indole acrylonitriles 83-118


83 4.03 ± 0.05 101 -

84 10.71 ± 0.03 102 20.51 ± 0.04

85 - 103 -

86 - 104 -

87 27.12 ± 0.05 105 -

88 25.05 ± 0.02 106 -

89 - 107 -

90 - 108 -

91 4.89 ± 0.05 109 -

92 - 110 -

93 21.94 ± 0.05 111 -

94 7.19 ± 0.05 112 -

95 19.89 ± 0.01 113 -

96 22.46 ± 0.04 114 11.81 ± 0.02

97 10.59 ± 0.03 115 9.35 ± 0.5

98 10.38 ± 0.02 116 -

99 6.28 ± 0.05 117 -

100 26.14 ± 0.6 118 -

Doxorubicin 0.2 ± 0.03 SEMa is the standard error of the mean, (-) Not active Doxorubicin (std) is standard drug used for anticancer activity.

6.3.3 Antiinflammatory activity

Inflammation refers to a normal biological response to any virulent stimuli such as any

physical trauma, harmful chemicals or microbiological agents (Ashley, 2012).

122

Inflammations may be categorized as acute and chronic depending on the response against

stimuli. The four basic signs of inflammation are heat, redness, swelling, and pain. It is one

of the protective measure taken by the individual organism in order to remove harmful

stimuli and begin the healing process. Acute inflammation is a regulated response and

characterized by increase in capillary infiltration, emigration of leukocytes, and vascular

permeability and to the site of infection However, chronic inflammation is a dysregulated

response and is characterized by proliferation, invasion of mononuclear immune cells,

monocytes, neutrophils, fibrosis, and macrophages thus indicating the presence of persistent

noxious stimuli and results in tissue malfunction (de Sousa, 2014). This persistent

inflammatory condition indicates the presence of any chronic human disorders, including

allergy, cancer, atherosclerosis, arthritis, and autoimmune diseases. The release of chemical

agents such as mediators indicates the start of an inflammatory response in recognition with

any infectious agent. Among these mediators are some amines like histamine and 5-

hydroxytryptamine, bradykinin, interleukin-1 (IL-1) (Vandekerckhove, 1991), lipids such as

prostaglandins (PGs) and leukotrienes (LTs), and enzymes.

The alkaloids are the largest source of plants secondary metabolites. These isolated alkaloids

have been found to possess a significant range of pharmacological activity, and are also

sometimes found to be toxic to man. Many alkaloids are used in therapeutics and as

pharmacological tools (Souto, 2011). The alkaloids family have been reported for wide range

of biological activities, including emetic, antitumor, diuretic, antiviral, antihypertensive,

hypnoanalgesic, antidepressant, antimicrobial and antiinflammatory activities. Indole and its

derivatives comprises of the alkaloid family and are also reported as anti-inflammatory

agents (Rani, 2004). NSAIDs are the drugs used for the treatment of inflammation and pain.

Indomethacin is one of the indole containing compound also found to possess activity against

COX-1 enzyme.


All synthesized compounds 83-118 were evaluated for their antiinflammatory activity and

only four compounds showed activity, while rest of them were found to be inactive.

123

The most active member of this library was compound 90 (IC50 = 1.3 ± 0.01 μM) having an

electron rich ring having hydroxyl, iodo, and methoxy substituent as electron donating

groups which may contribute in the activity. Compound 91 (IC50 = 5.38 ± 1.4 μM) is the

second most potent compound having hydroxyl and nitro substituents, the presence of an

electron withdrawing group resulted in decreased activity. Compound 111 (IC50 = 6.01 ± 0.9

μM) having anthracene ring having electron donating effect but less active as compared to

compound 90. Compound 98 (IC50 = 9.4 ± 1.9 μM) possessing methoxy and bromo

substituents also found to be active.

Table-10: Antiinflammatory activity of compounds 83-118

Compound (IC50 ± SEM)a

µM

Compound (IC50 ± SEM)a

µM

Compound (IC50 ±

SEM)a µM

83 >100 95 - 107 -

84 - 96 - 108 -

85 - 97 - 109 -

86 - 98 9.4 ± 1.9 110 -

87 - 99 - 111 6.01 ± 0.9

88 - 100 - 112 -

89 - 101 - 113 -

90 1.3 ± 0.01 102 - 114 -

91 5.38 ± 1.4 103 - 115 -

92 - 104 - 116 -

93 - 105 - 117 -

94 - 106 - 118 -

Ibuprofen(std) 11.2 ± 1.9 SEMa is the standard error of the mean, (-) Not active Ibuprofen (std) is standard drug used for antiinflammatory activity.

124

6.4 Conclusion

Random screening of compounds 83-118 in different bioassays showed very weak activity

against epilepsy, Hela cancer cell lines, and good antiinflammatory activity. Out of thirty six

analogues only eight compounds 84, 85, 88, 94, 106, 111, 115, and 118 were found to be

weakly active in antiepileptic assay almost inactive as compared to the standard

acetazolamide (IC50 = 0.12 ± 0.009 μM). Sixteen compounds were found to be active against

anticancer activity. Compound 83 and 91 were found to be the most active compounds of

the series while other compound 84, 87, 88, 93, 94, 95, 96, 97, 98, 99, 100, 102, 114, and

115 showed weak activities. However, four synthesized molecules showed good

antiinflammatory activity.

General Procedure for the synthesis of indole acrylonitriles (83-120)

In a reaction flask cyanoacetyl indole (0.184 g, 1 mmol), triethylamine (0.8 ml) and

benzaldehyde (1 mmol) were dissolved in ethanol (20 mL) and refluxed for 2 h at 80 ˚C. The

precipitates formed were filtered and washed with hexane and purity was examined via TLC.


3-(2,3-Dimethoxyphenyl)-2-(1H-indole-3-carbonyl)acrylonitrile (83)

Yield: 45%, M.p.: 211-213 ºC, 1H NMR (300 MHz, DMSO-d6): 12.30 (s, 1H, NH), 8.44

(s, 1H, H-2), 8.33 (s, 1H, H-1″), 8.18 (ovp, 1H, H-4), 7.77 (d, 1H, J6′,5′ = 7.2 Hz, H-6′), 7.54

(d, 1H, J7,6 = 6.9 Hz, H-7), 7.27 (ovp, 4H, H-6, H-5, H-5′, H-4′), 3.86 (s, 3H, 2′OCH3), 3.84

(s, 3H, 3′OCH3), FAB+: 333 (M+1), FAB-: 331 (M-1), HREI-MS m/z: Calcd for C20H16N2O3

[332.1168], Found [332.1161].

3-(2,4-Dichlorophenyl)-2-(1H-indole-3-carbonyl)acrylonitrile (84)


(s, 1H, H-2), 8.25 (s, 1H, H-1″), 8.19 (ovp, 2H, H-6′, H-4), 7.89 (d, 1H, J3′,5′ = 1.5 Hz, H-3′),

7.70 (dd, 1H, J5′,6′ = 6.3 Hz, J5′,3′ = 1.5 Hz, H-5′), 7.55 (d, 1H, J7,6 = 5.4 Hz, H-7), 7.29 (ovp,

2H, H-6, H-5), EI MS m/z (% rel. abund.): 340 (M+, 6), 342 (M+2, 4), 306 (23), 144 (100),

HREI-MS m/z: Calcd for C18H10N2OCl2 [340.0159], Found [340.0170].

125

3-(1H-Indol-3-yl)-2-(1H-indole-3-carbonyl)acrylonitrile (85)


(s, 1H, NH), 8.62 (s, 1H, H-2), 8.60 (s, 1H, H-2′), 8.47 (s, 1H, H-1″), 8.20 (d, 1H, J4,5 = 5.3

Hz, H-4), 7.93 (d, 1H, J4′,5′ = 5.7 Hz, H-4), 7.57 (d, 1H, J7,6 = 5.3 Hz, H-7), 7.53 (d, 1H, J7′,6′

= 5.4 Hz, H-7′), 7.25 (ovp, 4H, H-6, H-5, H-5′, H-6′), 13C NMR (500 MHz, DMSO-d6):

180.8, 145.1, 136.4, 136.2, 133.9, 131.4, 127.3, 126.4, 123.4, 123.2, 122.0, 121.8, 121.5,

120.7, 118.6, 114.3, 112.8, 112.3, 110.4, 101.73, EI MS m/z (% rel. abund.): 311 (M+, 100),

282 (18), 144 (89), HREI-MS m/z: Calcd for C20H13N3O [311.1050], Found [311.1059].

3-(4-Ethoxy-3-methoxyphenyl)-2-(1H-indole-3-carbonyl)acrylonitrile (86)


(s, 1H, H-2), 8.17 (ovp, 2H, H-1″, H-4), 7.90 (d, 1H, J2′,6′ = 1.2 Hz, H-2′), 7.70 (dd, 1H, J6′,5′

= 6.3 Hz, J6′,2′ = 1.2 Hz, H-6′), 7.55 (d, 1H, J7,6 = 5.4 Hz, H-7), 7.27 (ovp, 2H, H-6, H-5),

7.17 (d, 1H, J5′,6′ = 6.3 Hz, H-5′), 4.12 (ovp, 2H, CH2), 3.82 (s, 3H, 3′OCH3), 1.38 (t, 3H, J

= 7.2 Hz, OCH2CH3), EI MS m/z (% rel. abund.): 346 (M+, 100), 317 (41), 143 (100), HREI-

MS m/z: Calcd for C21H18N2O3 [346.1319], Found [346.1317].



(s, 1H, H-2), 8.16 (ovp, 2H, H-4, H-1″), 7.79 (d, 1H, J2′,6′ = 1.6 Hz, H-2′), 7.71 (dd, 1H, J6′,5′

= 6.8 Hz, J6′,2′ = 1.6 Hz, H-6′), 7.54 (ovp, 1H, H-7), 7.24 (ovp, 2H, H-5, H-6), 7.19 (d, 1H,

J5′,6′ = 8.4 Hz, H-5′), 3.87 (s, 3H, 3′OCH3), 3.81 (s, 3H, 4′OCH3), EI MS m/z (% rel. abund.):

332 (M+, 92), 317 (26), 144 (100), 116 (39), 89 (22), HREI-MS m/z: Calcd for C20H16N2O3

[332.1157], Found [332.1161].

3-(2-Bromo-4,5-dimethoxyphenyl)-2-(1H-indole-3-carbonyl)acrylonitrile (88)


(s, 1H, H-2), 8.26 (s, 1H, H-1″), 8.19 (ovp, 1H, H-4), 7.92 (s, 1H, H-3′), 7.55 (d, 1H, J7,6 =

6.9 Hz, H-7), 7.41 (s, 1H, H-6′), 7.31 (ovp, 2H, H-5, H-6), 3.87 (s, 3H, OCH3), 3.85 (s, 3H,

OCH3), EI MS m/z (% rel. abund.): 410 (M+, 4), 412 (M+2, 4), 331 (100), 144 (18), HREI-

MS m/z: Calcd for C20H15N2O3Br [410.0265], Found [410.0266].

126



(s, 1H, H-2), 8.17 (dd, 1H, J4,5 = 4.8 Hz, J4,6 = 2.4 Hz, H-4), 8.07 (s, 1H, H-1″), 7.55 (d, 1H,

J7,6 = 6.8 Hz, H-7), 7.48 (t, 1H, J4′,5′ = J4′,3′ = 8.4 Hz, H-4′), 7.30 (ovp, 2H, H-5, H-6), 6.80

(d, 2H, J (3′,4′), (5′,4′) = 8.4 Hz, H-3′, H-5′), EI MS m/z (% rel. abund.): 332 (M+, 5), 301 (100),

144 (99), 116 (25), HREI-MS m/z: Calcd for C20H16N2O3 [332.1165], Found [332.1161].

3-(4-Hydroxy-3-iodo-5-methoxyphenyl)-2-(1H-indole-3-carbonyl)acrylonitrile (90)


(s, , 1H, OH), 8.39 (ovp, 1H, H-6′), 8.16 (d, 1H, J4,5 = 7.2 Hz, H-4), 8.07 (s, 1H, H-2), 7.77

(s, 1H, H-2), 7.53 (d, 1H, J7,6 = 7.6 Hz, H-7), 7.28 (ovp, 2H, H-5, H-6), 3.84 (s, 3H, OCH3),


C19H13N2O3I [443.9961], Found [443.9971].

3-(5-Hydroxy-2-nitrophenyl)-2-(1H-indole-3-carbonyl)acrylonitrile (91)


(s, 1H, H-2), 8.44 (s, 1H, H-1″), 8.22 (ovp, 2H, H-3′, H-4), 7.55 (d, 1H, J7,6 = 6.8 Hz, H-7),

7.32 (ovp, 2H, H-5, H-6), 7.16 (d, 1H, J = 2 Hz, H-6′), 7.48 (t, 1H, J4′,5′ = J4′,3′ = 8.4 Hz, H-

4′), 7.30 (ovp, 2H, H-5, H-6), 6.80 (d, 2H, J (3′,4′), (5′,4′) = 8.4 Hz, H-3′, H-5′), EI MS m/z (%

rel. abund.): 333 (M+, 5), 287 (49), 144 (100), 116 (18), HREI-MS m/z: Calcd for

C18H11N3O4 [333.0756], Found [333.0750].

2-(1H-Indole-3-carbonyl)-3-(3,4,5-trimethoxyphenyl)acrylonitrile (92)


(s, 1H, H-2), 8.18 (ovp, 2H, H-1″, H-4), 7.55 (d, 1H, J7,6 = 7.2 Hz, H-7), 7.49 (s, 2H, H-2′,

H-6′), 7.30 (ovp, 2H, H-6, H-5), 3,83 (s, 6H, OCH3), 3.77 (s, 3H, OCH3), EI MS m/z (% rel.

abund.): 362 (M+, 89), 331 (15), 144 (100), HREI-MS m/z: Calcd for C21H18N2O4

[362.1258], Found [362.1267].

3-(2-Chloro-5-nitrophenyl)-2-(1H-indole-3-carbonyl)acrylonitrile (93)


(d, 1H, J6′,4′ = 2.4 Hz, H-6′), 8.56 (s, 1H, H-2), 8.42 (dd, 1H, J4′,3′ = 8.8 Hz, J4′,6′ = 2.4 Hz, H-

127

4′), 8.29 (s, 1H, H-1″), 7.99 (d, 1H, J3′,4′ = 8.8 Hz, H-3′), 7.57 (ovp, 1H, H-7), 7.33 (ovp, 2H,

H-6, H-5), 13C NMR (400 MHz, DMSO-d6): 179.9, 146.3, 146.0, 140.4, 137.0, 136.8,

132.4, 131.4, 126.9, 125.9, 124.6, 123.8, 122.7, 121.3, 117.7, 116.0, 113.4, 112.6, EI MS

m/z (% rel. abund.): 351 (M+, 52), 316 (31), 144 (100), 116 (30), 89 (18), HREI-MS m/z:

Calcd for C18H10N3O3Cl [351.0419], Found [351.0411].



(s, 2H, H-2, H-1″), 8.26 (d, 1H, J6′,5′ = 6.6 Hz, H-6′), 8.15 (d, 1H, J4,5 = 5.4 Hz, H-4), 7.54

(d, 1H, J7,6 = 5.4 Hz, H-7), 7.30 (ovp, 2H, H-6, H-5), 6.78 (dd, 1H, J5′,6′ = 6.6 Hz, J5′,3′ = 1.5

Hz, H-5′), 6.72 (d, 1H, J3′,5′ = 1.8 Hz, H-5′), 3.89 (s, 6H, 2′OCH3, 4′ OCH3), 13C NMR (300

MHz, DMSO-d6): 181.2, 164.9, 160.8, 146.2, 136.4, 134.8, 129.8, 126.2 123.4, 122.2,

121.4, 118.7, 113.8, 113.6, 112.4, 107.1, 106.7, 98.3, 56.2, 55.8, EI MS m/z (% rel. abund.):

332 (M+, 36), 300 (78), 144 (100), 116 (56), HREI-MS m/z: Calcd for C20H16N2O3

[332.1170], Found [332.1161].

3-(3-Bromo-4, 5-dimethoxyphenyl)-2-(1H-indole-3-carbonyl)acrylonitrile (95)


(s, 1H, H-2), 8.15 (ovp, 2H, H-1″, H-4), 7.93 (d, 1H, J2′,6′ = 1.2 Hz, H-2′), 7.82 (d, 1H, J6′,2′

= 1.2 Hz, H-6′), 7.55 (dd, 1H, J7,6 = 4.8 Hz, J7,5 = 1.2 Hz, H-7), 7.27 (ovp, 2H, H-6, H-5),

3.89 (s, 3H, 4′ OCH3), 3.84 (s, 3H, 5′ OCH3), EI MS m/z (% rel. abund.): 410 (M+, 31), 412

(M+2, 39), 331 (18), 144 (100), HREI-MS m/z: Calcd for C20H15N2O3Br [410.0256], Found

[410.0266].

3-(5-Bromo-2-methoxyphenyl)-2-(1H-indole-3-carbonyl) acrylonitrile (96)


(s, 1H, H-1″), 8.24 (ovp, 2H, H-6′, H-2), 8.17 (d, 1H, J4,5 = 6.8 Hz, H-4), 7.77 (dd, 1H, J4′,3′

= 8.8 Hz, J4′,6′ = 2.4 Hz, H-4′), 7.55 (d, 1H, J7,6 = 7.2 Hz, H-7), 7.31 (ovp, 2H, H-6, H-5),

7.19 (d, 1H, J 3′,4′ = 8.8 Hz, H-3′), 3.87 (s, 3H, OCH3), EI MS m/z (% rel. abund.): 380 (M+,

23), 382 (M+2, 22), 144 (10), HREI-MS m/z: Calcd for C19H13N2O2Br [380.0171], Found

[380.0160].

128

2-(1H-Indole-3-carbonyl)-3-phenylacrylonitrile (97)


(s, 1H, H-2), 8.23 (s, 1H, H-1″), 8.18 (dd, 1H, J4,5 = 6.8 Hz, J4,6 = 2.4 Hz, H-4), 8.05 (dd, 2H,

J (2′,3′),(6′,5′) = 5.2 Hz, J(2′,4′),(6′,4′) = 1.6 Hz, H-2′, H-6′), 7.60 (ovp, 3H, H-3′, H-4′, H-5′), 7.55

(dd, 1H, J7,6 = 6.4 Hz, J7,5 = 1.6 Hz, H-7), 7.30 (ovp, 2H, H-6, H-5), EI MS m/z (% rel.

abund.): 272 (M+, 60), 144 (100), 89 (59), HREI-MS m/z: Calcd for C18H12N2O [272.0964],

Found [272.0950].

3-(3-Bromo-4,5-dimethoxyphenyl)-2-(1,2-dimethyl-1H-indole-3-

carbonyl)acrylonitrile (98)

Yield: 63%, M.p.: 167-169 ºC, 1H NMR (300 MHz, DMSO-d6): 7.93 (s, 1H, H-1″), 7.89

(s, 1H, H-2′), 7.78 (s, 1H, H-6′), 7.74 (d, 1H, J4,5 = 7.6 Hz, H-4), 7.59 (d, 1H, J7,6 = 7.6 Hz,

H-7), 7.26 (t, 1H, J5,6 = J5,4 = 7.6 Hz, H-5), 7.19 (t, 1H, J6,5 = J6,7 = 7.6 Hz, H-6), 3.86 (s,

6H, OCH3), 3.78 (s, 3H, CH3), 2.62 (s, 3H, CH3), 13C NMR (300 MHz, DMSO-d6): 183.6,

153.2, 150.7, 148.8, 146.0, 136.5, 129.3, 126.5, 126.0, 122.3, 121.9, 119.8, 117.0, 116.8,

114.1, 112.9, 110.7, 110.4, 60.4, 56.2, 29.9, 12.7, EI MS m/z (% rel. abund.): 438 (M+, 56),

440 (M+2, 78), 172 (100), HREI-MS m/z: Calcd for C22H19N2O3Br [438.0594], Found

[438.0579].

2-(1H-Indole-3-carbonyl)-3-(2,3,4-trimethoxyphenyl)acrylonitrile (99)


(s, 1H, H-2), 8.30 (s, 1H, H-1″), 8.18 (d, 1H, J4,5 = 6.8 Hz, H-4), 8.09 (d, 1H, J6′,5′ = 8.8 Hz,

H-6′), 7.55 (d, 1H, J7,6 = 7.2 Hz, H-7), 7.30 (ovp, 2H, H-5, H-6), 7.11 (d, 1H, J5′,6′ = 9.2 Hz,

H-5′), 3.91 (s, 3H, OCH3), 3.87 (s, 3H, OCH3), 3.78 (s, 3H, OCH3), 13C NMR (400 MHz,

DMSO-d6): 181.2, 157.5, 153.7, 146.2, 141.5, 136.5, 135.1, 126.1, 123.8, 123.5, 122.3,

121.3, 118.6, 118.2, 113.7, 112.4, 109.0, 108.4, 61.8, 60.5, 56.3, EI MS m/z (% rel. abund.):


[362.1267].

2-(1H-Indole-3-carbonyl)-3-(3-methoxyphenyl) acrylonitrile (100)


(s, 1H, H-2), 8.18 (ovp, 2H, H-4, H-1″), 7.64 (d, 2H, J7,6 = J6′,5′ = 6.8 Hz, H-7, H-6′), 7.53

129

(ovp, 2H, H-5, H-5′), 7.28 (ovp, 2H, H-6, H-2′), 7.19 (d, 1H, J4′,5′ = 7.2 Hz, H-4′), 3.82 (s,

3H, OCH3), EI MS m/z (% rel. abund.): 302 (M+, 82), 144 (100), 116 (16), HREI-MS m/z:


3-(2-Chloro-3,4-dimethoxyphenyl)-2-(1H-indole-3-carbonyl)acrylonitrile (101)


(s, 1H, H-2), 8.30 (s, 1H, H-1″), 8.19 (ovp, 1H, H-4), 8.08 (d, 1H, J6′,5′ = 8.7 Hz, H-6′), 7.56

(ovp, 1H, H-7), 7.35 (d, 1H, J5′,6′ = 8.7 Hz, H-5′), 7.29 (ovp, 2H, H-5, H-6), 3.95 (s, 3H,

OCH3), 3.79 (s, 3H, OCH3), 13C NMR (400 MHz, DMSO-d6): 180.7, 156.4, 147.6, 145.2,

136.6, 135.7, 129.1, 126.0, 125.4, 123.6, 123.2, 122.4, 121.3, 117.3, 113.6, 112.5, 112.3,

111.8, 60.2, 56.5, FAB+: 367 (M+1), FAB-: 365 (M-1),

3-(2,3-Dimethoxyphenyl)-2-(1,2-dimethyl-1H-indole-3-carbonyl)acrylonitrile (102)


(dd, 1H, J4,5 = 7.2 Hz, J4,6 = 1.2 Hz, H-4), 7.71 (d, 1H, J6′,5′ = 8.0 Hz, H-6′), 7.60 (d, 1H, J7,6

= 8.0 Hz, H-7), 7.33 (ovp, 2H, H-5′, H-4′), 7.24 (ovp, 1H, H-6), 7.19 (t, 1H, J5,6 = 7.6 Hz,

H-5), 3.84 (s, 3H, OCH3), 3.79 (s, 3H, OCH3), 3.65 (s, 3H, CH3), 2.65(s, 3H, CH3), EI MS

m/z (% rel. abund.): 360 (M+, 69), 329 (100), 144 (13), HREI-MS m/z: Calcd for C22H20N2O3

[360.1483], Found [360.1474].

3-(2,4-Dimethoxyphenyl)-2-(1,2-dimethyl-1H-indole-3-carbonyl)acrylonitrile (103)

Yield: 72%, M.p.: 165-167 ºC, 1H NMR (400 MHz, DMSO-d6): 8.25 (d, 1H, J6′,5′ = 6.8

Hz, H-6′), 8.17 (s, 1H, H-1″), 7.67 (d, 1H, J4,5 = 7.6 Hz, H-4), 7.57 (d, 1H, J7,6 = 7.6 Hz, H-

7), 7.24 (t, 1H, J5,6 = J5,4 = 7.6 Hz, H-5), 7.16 (t, 1H, J6,5 = J6,7 = 7.6 Hz, H-6), 6.80 (dd, 1H,

J5′,6′ = 6.8 Hz, J5′,3′ = 2.0 Hz, H-5′), 6.86 (d, 1H, J3′,5′ = 2.0 Hz, H-3′), 3.88 (s, 3H, OCH3),

3.78 (s, 6H, OCH3, CH3), 2.61 (s, 3H, CH3), EI MS m/z (% rel. abund.): 360 (M+, 62), 172

(100), 144 (24), HREI-MS m/z: Calcd for C22H20N2O3 [360.1475], Found [360.1474].

2-(1,2-Dimethyl-1H-indole-3-carbonyl)-3-(2-fluoro-4-methoxyphenyl)acrylonitrile

(104)

Yield: 82%, M.p.: 146-148 ºC, 1H NMR (400 MHz, DMSO-d6): 8.31 (ovp, 1H, H-6′), 7.96

(s, 1H, H-1″), 7.69 (d, 1H, J4,5 = 7.2 Hz, H-4), 7.59 (d, 1H, J7,6 = 7.2 Hz, H-7), 7.25 (t, 1H,

130

J5,6 = J5,4 = 7.2 Hz, H-5), 7.17 (t, 1H, J6,5 = J6,7 = 7.2 Hz, H-6), 7.08 (ovp, 2H, H-5′,H-3′),

3.87 (s, 3H, OCH3), 3.78 (s, 3H, CH3), 2.63 (s, 3H, CH3), EI MS m/z(% rel. abund.): 348

(M+, 56), 172 (100).

3-(4-Bromo-2-fluorophenyl)-2-(1,2-dimethyl-1H-indole-3-

carbonyl)acrylonitrile(105)

Yield: 78%, M.p.: 191-193 ºC, 1H NMR (400 MHz, DMSO-d6): 8.31 (ovp, 1H, H-4), 7.95

(s, 1H, H-1″), 7.82 (dd, 1H, J5′,6′ = 8.4 Hz, J5′,4′ = 1.6 Hz, H-5′), 7.75 (ovp, 2H, H-7, H-3′),

7.60 (d, 1H, J6′/5′ = 8.4 Hz, H-6′), 7.26 (t, 1H, J5,6 = J5,4 = 7.3 Hz, H-5), 7.17 (t, 1H, J6,5 = J6,7

= 7.3 Hz, H-6), 3.87 (s, 3H, OCH3), 3.78 (s, 3H, CH3), 2.65 (s, 3H, CH3), EI MS m/z (% rel.

abund.): 396 (M+, 25), 398 (M+2, 28), 172 (100), HREI-MS m/z: Calcd for C20H14N2OBrF

[396.0289], Found [396.0274].

3-(4-Bromo-2-fluorophenyl)-2-(1H-indole-3-carbonyl)acrylonitrile (106)


(s, 1H, H-2), 8.18 (ovp, 3H, H-1″, H-4, H-6′), 7.84 (dd, 1H, J5′,6′ = 8.4 Hz, J5′,4′ = 1.6 Hz, H-

5′), 7.71 (d, 1H, J = 8.0 Hz, H-3′), 7.56 (ovp, 1H, H-7), 7.31 (ovp, 2H, H-5, H-6), EI MS m/z

(% rel. abund.): 368 (M+, 16), 370 (M+2, 16), 144 (100), HREI-MS m/z: Calcd for

C18H10N2OBrF [367.9943], Found [367.9961].

3-(5-Bromo-2-methoxyphenyl)-2-(1,2-dimethyl-1H-indole-3-carbonyl)acrylonitrile

(107)

Yield: 68%, M.p.: 188-190 ºC, 1H NMR (400 MHz, DMSO-d6): 8.31 (m, 1H, H-6′), 7.95

(s, 1H, H-1″), 7.82 (dd, 1H, J4′,3′ = 8.4 Hz, J4′,6′ = 1.6 Hz, H-4′), 7.75 (ovp, 2H, H-4, H-7),

7.60 (d, 1H, J3′,4′ = 8.4 Hz, H-3′), 7.26 (t, 1H, J5,6 = J5,4 = 7.3 Hz, H-5), 7.17 (t, 1H, J6,5 = J6,7

= 7.3 Hz, H-6), 3.87 (s, 3H, OCH3), 3.78 (s, 3H, CH3), 2.65 (s, 3H, CH3), 13C NMR (400

MHz, DMSO-d6): 183.3, 157.4, 146.2, 145.7, 136.5, 136.3, 130.6, 125.8, 122.8, 122.3,

121.8, 119.9, 116.3, 114.9, 114.4, 112.0, 110.5, 110.4, 56.3, 29.9, 12.67, EI MS m/z (% rel.

abund.): 408 (M+, 57), 410 (M+2, 60), 223 (27), 172 (100), HREI-MS m/z : Calcd for

C21H17N2O2Br [408.0477], Found [408.0473].

131

3-(3-Bromo-4-methoxyphenyl)-2-(1,2-dimethyl-1H-indole-3-carbonyl)acrylonitrile

(108)

Yield: 73%, M.p.: 183-186 ºC, 1H NMR (400 MHz, DMSO-d6): 8.30 (d, 1H, J2′,6′ = 2.0

Hz, H-2′), 8.09 (dd, 1H, J6′,5′ = 8.8 Hz, J6′,2′ = 2.0 Hz, H-6′), 7.92 (s, 1H, H-1″), 7.71 (d, 1H,

J4,5 = 8.0 Hz, H-4), 7.59 (d, 1H, J7,6 = 8.0 Hz, H-7), 7.35 (d, 1H, J5′,6′ = 8.8 Hz, H-5′), 7.25

(t, 1H, J5,6 = J5,4 = 8.0 Hz, H-5), 7.17 (t, 1H, J6,5 = J6,4 = 8.0 Hz, H-6), 3.95 (s, 3H, OCH3),

3.77 (s, 3H, CH3), 2.61 (s, 3H, CH3), 13C NMR (400 MHz, DMSO-d6): 183.9, 158.7, 150.9,

145.7, 136.5, 134.8, 131.9, 126.1, 126.0, 122.3, 121.8, 119.8, 117.1, 113.2, 111.3, 111.0,


(100), 143 (11), HREI-MS m/z: Calcd for C21H17N2O2Br [408.0477], Found [408.0473].

2-(1,2-Dimethyl-1H-indole-3-carbonyl)-3-(3,4,5-

trimethoxyphenyl)acrylonitrile (109)


(d, 1H, J4,5 = 7.5 Hz, H-4), 7.59 (d, 1H, J7,6 = 7.5 Hz, H-7), 7.44 (s, 2H, H-2′, H-6′), 7.24 (t,

1H, J5,6 = J5,4 = 7.5 Hz, H-5), 7.17 (t, 1H, J6,5 = J6,4 = 7.5 Hz, H-6), 3.80 (ovp, 12H, OCH3,

OCH3, OCH3, CH3 ), 2.62 (s, 3H, CH3), EI MS m/z (% rel. abund.): 390 (M+, 29), 388 (73),


3-(2-Bromo-5-fluorophenyl)-2-(1H-indole-3-carbonyl) acrylonitrile (110)

Yield: 65%, M.p.: 277-279 ºC, 1H NMR (400 MHz, CD3OD-): 8.43 (s, 1H, H-2), 8.32 (s,

1H, H-1″), 8.29 (d, 1H, J4,5 = 8.8 Hz, H-4), 7.93 (ovp, 1H, H-3′), 7.82 (ovp, 1H, H-4′), 7.52

(d, 1H, J7,6 = 8.0 Hz, H-7), 7.30 (ovp, 3H, H-5, H-6, H-6′), EI MS m/z (% rel. abund.): 368

(M+, 13), 370 (M+2, 13), 144 (100), HREI-MS m/z: Calcd for C18H10N2OBrF [367.9958],

Found [367.9961].

3-(Anthracen-9-yl)-2-(1H-indole-3-carbonyl) acrylonitrile (111)


(s, 1H, H-2), 8.81 (s, 1H, H-10′), 8.59 (s, 1H, H-1″), 8.31 (ovp, 1H, H-4), 8.22 (d, 2H, J(1′,2′),

(8′,9′) = 8.0 Hz, H-1′, H-8′), 8.14 (d, 2H, J(4′,3′),(5′,6′) = 8.4 Hz, H-4′, H-5′), 7.63 (ovp, 5H, H-2′,

H-3′, H-6′, H-7′, H-7), 7.32 (ovp, 2H, H-5, H-6), EI MS m/z (% rel. abund.): 372 (M+, 46),


132

3-(3-Bromo-4-methoxyphenyl)-2-(1H-indole-3-carbonyl) acrylonitrile (112)


(s, 1H, H-1″), 8.34 (d, 1H, J = 2.0 Hz, H-2), 8.17(s, 1H, H-2′), 8.13 (ovp, 2H, H-4, H-6′),

7.53 (d, 1H, J7,6 = 7.2 Hz, H-7), 7.35 (d, 1H, J5′,6′ = 8.8 Hz, H-5′), 7.25 (ovp, 2H, H-5, H-6),

3.96 (s, 3H, OCH3). EI MS m/z (% rel. abund.): 380 (M+, 9), 382 (M+2, 8), 144 (100), 89

(33), HREI-MS m/z: Calcd for C19H13N2O2Br [380.0134], Found [380.0160].

2-(1,2-Dimethyl-1H-indole-3-carbonyl)-3-phenylacrylonitrile (113)

Yield: 76%, M.p.: 167-169 ºC, 1H NMR (400 MHz, DMSO-d6): 8.00 (ovp, 3H, H-2′, H-

6′, H-1″), 7.73 (d, 1H, J4,5 = 7.3 Hz, H-4), 7.59 (ovp, 4H, H-7, H-3′, H-4′, H-5′), 7.25 (t, 1H,

J5,6 = J5,4 = 7.3 Hz, H-5), 7.17 (t, 1H, J6,5 = J6,4 = 7.3 Hz, H-6), 3.78 (s, 3H, CH3), 2.64 (s,

3H, CH3), EI MS m/z (% rel. abund.): 300 (M+, 77), 172 (100), 143 (9), HREI-MS m/z: Calcd

for C20H16N2O [300.1250], Found [300.1263].

3-(4-Fluoro-3-methoxyphenyl)-2-(1H-indole-3-carbonyl) acrylonitrile (114)

Yield: 70%, M.p.: 210-212 ºC, 1H NMR (400 MHz, CD3OD-d): 8.39 (s, 1H, H-2), 8.27 (d,

1H, J4,5 = 6.8 Hz, H-4), 8.17 (s, 1H, H-1″), 7.93 (dd, 1H, J6′,5′ = 8.0 Hz, J6′,2′ = 2.0 Hz, H-6′),

7.60 (ovp, 1H, H-5′), 7.51 (d, 1H, J6,7 = 7.2 Hz, H-7), 7.27 (ovp, 3H, H-5, H-6, H-2′), EI MS

m/z (% rel. abund.): 320 (M+, 60), 144 (100), 116 (15), HREI-MS m/z: Calcd for

C19H13N2O2F [320.0965], Found [320.0961].

2-(1,2-Dimethyl-1H-indole-3-carbonyl)-3-(naphthalen-2-yl) acrylonitrile (115)

Yield: 75%, M.p.: 207-210 ºC, 1H NMR (400 MHz, DMSO-d6): 8.47 (s, 1H, H-1′), 8.21

(d, 1H, J4,5 = 8.4 Hz, H-4), 8.12 (ovp, 2H, H-1″, H-3′), 8.00 (ovp, 2H, H-8′, H-5′), 7.75 (d,

1H, J7,6 = 8.0 Hz, H-7), 7.68 (t, 1H, J6′,7′ = 7.2 Hz, H-6′), 7.62 (ovp, 2H, H-7′, H-4′), 7.25 (t,

1H, H-5), 7.17 (t, 1H, J6,7 = 7.2 Hz, H-6), EI MS m/z (% rel. abund.): 350 (M+, 94), 172

(100), 144 (5), HREI-MS m/z: Calcd for C24H18N2O [350.1410], Found [350.1419].

3-(4-Bromophenyl)-2-(1H-indole-3-carbonyl) acrylonitrile (116)

Yield: 79%, M.p.: 221-223 ºC, 1H NMR (400 MHz, DMSO-d6): 8.39 (s, 1H, H-2), 8.26

(dd, 1H, J4,5 = 6.4 Hz, J4,6 = 1.6 Hz, H-4), 8.15 (s, 1H, H-1″), 7.96 (d, 2H, J(3′,2′),(5′,6′) = 8.4

Hz, H-3′, H-5′), 7.73 (d, 2H, J(2′,3′),(6′,5′) = 8.8 Hz, H-2′ H-6′), 7.50 (dd, 1H, J7,6 = 6.4 Hz, J7,5

133

= 1.6 Hz, H-7), 7.28 (ovp, 2H, H-5, H-6), EI MS m/z (% rel. abund.): 350 (M+, 48), 144 (100),

116 (22).

3-(2-Bromo-4,5-dimethoxyphenyl)-2-(1,2-dimethyl-1H-indole-3-


Yield: 72%, M.p.: 228-230 ºC, 1H NMR (400 MHz, DMSO-d6): 8.06 (s, 1H, H-3′), 7.98

(s, 1H, H-1″), 7.74 (d, 1H, J4,5 = 8.0 Hz, H-4), 7.58 (d, 1H, J7,6 = 8.4 Hz, H-7), 7.37 (s, 1H,

H-6′), 7.24 (t, 1H, J5,6 = 7.2 Hz, H-5), 7.16 (t, 1H, J6,7 = 8.4 Hz, H-6), 7.28 (ovp, 2H, H-5,

H-6), EI MS m/z (% rel. abund.): 438 (M+, 23), 440 (M+2, 25), 359 (100), 172 (77), HREI-

MS m/z: Calcd for C22H19N2O3Br [438.0565], Found [438.0579].

3-(2-Chloro-3,4-dimethoxyphenyl)-2-(1,2-dimethyl-1H-indole-3-


Yield: 70%, M.p.: 183-185 ºC, 1H NMR (400 MHz, DMSO-d6): 8.11 (d, 1H, J4,5 = 7.4 Hz,

H-4), 8.07 (s, 1H, H-1″), 7.72 (d, 1H, J7,6 = 7.4 Hz, H-7), 7.58 (d, 1H, J5′,6′ = 8.0 Hz, H-5′),

7.34 (d, 1H, J6′,5′ = 8.8 Hz, H-6′), 7.24 (t, 1H, J5,6/5,4 = 7.4 Hz, H-5), 7.16 (t, 1H, J6,7/6,4 = 7.4

Hz, H-7), EI MS m/z (% rel. abund.): 394 (M+, 28), 396 (M+2, 12), 359 (100), 172 (68),

HREI-MS m/z: Calcd for C22H19N2O3Cl [394.1085], Found [394.1084].

134

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Chapter-3

Synthesis and biological activities of Bis(indolyl)

methanes

140

Summary

This chapter includes the synthesis of an efficient acid catalyzed synthesis of bisindolyl

methanes. The synthesized analogues were evaluated for various biological activities

including anticancer, antiglycation and antiinflammatory activities.

7 General Introduction

7.1 Introduction

Heterocyclic compounds are found to exhibit a wide variety of biological activities, among

them nitrogen containing heterocycles are one of the most common class having a vast range

of biological activities. Bis(indolyl)methanes (BIMs) belongs to the family of alkaloids

comprising the basic skeleton of two indole atoms bridged by single methyl carbon; they can

be differentiated into various compounds on the basis of substituents attached to the bridging

methyl carbon. They are widely distributed in natural resources and are found commonly in

marine and terrestrial organisms. Bis(indolyl)methanes are of been great importance for

chemist due to their wide application in pharmaceutical as well as in agrochemicals. The

symmetric skeleton of the BIMs allows some simple and efficient approaches to synthesize

them from environmental friendly, cost effective, and green synthesis (J. Hong, 2006; Kamal

et al., 2012, Khan et al., 2012, Khan et al., 2014). Marine sources have provide a large

number and variety of bis(indolyl)methanes and bis(indolyl)ethanes such as vibrindole A is

isolated from marine bacterium, Vibrio parahaemalyticus Ostracioncubicus (Pablo Garcia

Merinos, Lopez Ruiz, Lopez, and Rojas Lima, 2015; Veluri, Oka, Wagner-Dobler, and

Laatsch, 2003). Bisindole and trisindoles are derived from the basic unit of indole, and are

biologically active pharmacophores (Pal, 2013). Various bisindole derivatives had been

isolated from numerous marine and terrestrial plants, possessing a variety of biological

activities such as parasitic bacteria, tunicates, and sponge are found as possible antibacterial,

anticarcinogenic, genotoxic, and DNA damaging agents (Andreani et al., 2008;

Kochanowska-Karamyan and Hamann, 2010), (Osawa and Namiki, 1983). In addition, the

141

discovery of potent anticarcinogenic properties of bisindole has attracted a major attention

of many research groups. Bisindole is the most active cruciferous substance for promoting

beneficial estrogen metabolism in women and men, and may have useful application as

breast cancer preventive agent (Andreani et al., 2008), (Ge, Yannai, Rennert, Gruener, and

Fares, 1996), (Mulla et al., 2012). BIMs are useful in the treatment of fibromyalgia, chronic

fatigue and irritable bowel syndrome. Diindolyl methane and its derivatives are used as

dietary supplements and helps in preventing from various types of cancer and are found in

cruciferous vegetables like cabbage, broccoli etc (Maciejewska, Rasztawicka, Wolska,

Anuszewska, and Gruber, 2009), (Kaishap and Dohutia, 2013).

7.2 Biological activities of Bisindolyl methanes

Many marketed drugs contains bisindoles such as vincristine (1) which is used as anticancer

drug (Ishikawa et al., 2009).

Bisindolylmethanes found to exhibit anticancer activity against breast cancer through cell

proliferation (Marques et al., 2014, C. Hong, Kim, Firestone, and Bjeldanes, 2002).

Bisindoles 2 and 3 were found to exhibit activity against HeLa cell lines (Jamsheena et al.,

2016).

142

Bisindolyl methane moderates the risk of developing thyroid proliferative disease through

enhancing estrogen metabolism (Rajoria et al., 2011).

Bisindolylmethane Schiff bases also possess antibacterial activity against some selected

strains of Gram-positive and Gram-negative bacteria such as Salmonella typhi, S. paratyphi

A, and S. paratyphi B (Imran et al., 2014).

143

Vinblastine, vinorelbine, and vindesine are other marketed drugs that are used to treat cancer

and possess two to three indole rings in their core structure (Biswal, Sahoo, Sethy, Kumar,

and Banerjee, 2012).

Bisindolyl methane derivatives 9 and 10 also found to possess potent ß-glucuronidase

inhibitory activity with IC50 values in the range of 1.62-22.30 μM (Khan et al., 2014).

7.3 Synthesis of Bisindolyl methanes and its derivatives

Bisindolyl methanes are synthesized by fruit juice catalyzed reactions a green chemistry

approach. Indole was treated with various aryl substituted aldehydes in the presence of grape

juice which was used as catalyst, reaction was carried out in aqueous medium under

refluxing and the products were obtained in good to moderate yields (Sheikh, and

Nazeruddin, 2016) Scheme-1.

Scheme-1: Synthesis of bisindolyl methane via grape juice catalyzed reaction a greener

approach

Bis(indolyl)methanes and other derivatives of indole can be prepared via the condensation

of indole with different aldehydes and ketones under various conditions by using (PCBS)

poly (N,N′-dichloro-N-ethyl-benzene-1,3-disulfonamide) and (TCBDA) N,N,N′,N′-

tetrachlorobenzene-1,3-disulfonamide as novel catalysts (Ghorbani-Vaghei and Veisi, 2010)

Scheme-2.

144

Scheme-2: TCBDA and PCBS catalyzed synthesis of bisindolyl methane derivatives

Solid phase synthesis of bisindolyl methane derivatives was carried out using stannous

chloride dihydrate as a catalyst via electrophilic substitution reaction of indole with carbonyl

compounds (aldehydes and ketones) (Shaikh, Mohammed, Patel, Syed, and Patil, 2010)

Scheme-3.

Scheme-3: Stannous chloride mediated solid phase synthesis of bisindolyl methanes

Aryl Schiff bases of bisindolyl methane were prepared in two steps. First step comprises of

acid catalyzed condensation of indole and 4-nitroaldehyde in the next step the nitro

substituent at the aryl part is first reduced to amino group and then treated with various

substituted benzaldehydes to afford Schiff bases (Imran et al., 2014) Scheme-4.

145

Scheme-4: Synthesis of bisindolyl methane Schiff bases

Infrared irradiated synthesis of bisindolyl methane via condensation of indole with

benzaldehydes in the presence of bentonitic clay which is used as catalyst. The reaction was

completed in 15 minutes and product was purified by recrystallization (Velasco-Bejarano et

al., 2008) Scheme-5.

Scheme-5: Bentonite clay catalyzed synthesis of bisindolyl methanes

Hasaninejad et al reported an efficient method for condensation of indoles with carbonyl

compounds in the presence of catalytic amount of picric acid at room temperature in aqueous

medium (A Hasaninejad, Mohammadizadeh, and Babamiri, 2008) Scheme-6.

146

Scheme-6: Picric acid catalyzed reaction of indole with benzaldehydes

Another method reported by Hasaninejad et al which involves the condensation of indole

and carbonyl compounds aldehydes and ketones in the presence of PCl5 (Alireza

Hasaninejad, Zare, Sharghi, Khalifeh, and Zare, 2008) Scheme-7.

Scheme-7: PCl5 mediated synthesis of indoles

Bisindolyl methanes can also be prepared in the presence of catalytic amount of iodine via

electrophilic substitution of aldehydes and ketones with indole in aprotic solvent in good to

high yields (Bandgar and Shaikh, 2003) Scheme-8.

Scheme-8: Iodine catalyzed synthesis of di indolyl methanes.

147

Microwave-assisted synthesis of bisindolyl methane and its derivatives is also reported by

Vijaykumar et al which utilizes the catalytic amount of CeCl3.7H2O to afford desired product

(Vijay kumara and Shakthi, 2013) Scheme-9.

Scheme-9: Microwave-assisted synthesis in the presence of CeCl3.7H2O.

Another efficient method reported by our research group for the synthesis of bisindolyl

methanes in which sodium bromate and sodium hydrogen sulfite are used as catalyst in

aqueous medium (Khan et al., 2012) Scheme-10.

Scheme-10: An eco-friendly and efficient method for the synthesis of di indolyl methanes

148


7.4.1 Chemistry

In the recent past our group had synthesized analogues of bisindolyl methanes that were

found to be biologically active. This prompted us to synthesize few more analogues in order

to obtain the lead molecule for better results. We had synthesized fifty-five analogues of

bisindolyl methane in our recent work using commercially available reagents. In typical

experimental procedure indole was treated with different substituted benzaldehyde catalytic

amount of citric acid was also added along with ethanol. The reaction mixture was kept

under reflux for 2-3 h, the reaction progress was monitored via TLC. Precipitates formed

were filtered, dried and crystallized from ethanol to afford the desired products in good to

excellent yields. The structure of the compounds 38-92 were determined by 1H, 13C-NMR,

and EI mass spectrometry given below in Scheme-11, Table-1. Forty four compounds 39-

40, 43-45, 47-48, 50, 55-64, 67-92 are new, however, eleven compounds 38, 41-42, 46, 49,

51-54, 65, and 66 are already known in literature.

Scheme-11: Citric acid catalyzed synthesis of bisindolyl methanes 38-92

Mechanism

The reaction starts with the protonation of aldehyde thus making carbonyl carbon more

electrophilic, indole ring will then attack on electrophilic center thus resulting in the

formation of an intermediate. Another indole ring then attacks on this intermediate thus

resulting in the formation of product Figure-1.

149

Figure-1: Plausible mechanism for the synthesis of bisindolyl methanes.

7.5 Spectral Characterization of Representative Compound 44


The 1H NMR was recorded in deuterated DMSO on a 400 MHz instrument. The most

downfield signal of the spectrum appeared at δ 10.7 for two NH of indole rings as a singlet.

There are fourteen aromatic protons present in the structure, two of them H-4, H-4a appeared

at δ 7.31 as a doublet with coupling constant J4,5/4a,5a = 8.4 Hz. H-7 and H-7a also appeared

as doublet at δ 7.20 having coupling constant J7,6/7a,6a = 8.0 Hz. H-5 and H-5a of indole rings

resonated at δ 7.01 as triplet with coupling constant J5,6/5a,6a/5,4/5a,4a = 7.4 Hz showing coupling

with H-4, H-6, H-4a, and H-6a, respectively. H-3′ of benzene ring appeared at δ 6.93 as

doublet having coupling constant J3′,4, = 9.2 Hz, while, H-6 and H-6a appeared at δ 6.84 as

triplet showing coupling with H-5, H-6, H-5a, and H-6a, respectively, with a coupling

constant J6,5/6a,5a/6,7/6a,7a = 7.4 Hz. A singlet of H-2 and H-2a was appeared at δ 6.73 while,

H-4′ resonated at δ 6.71 as an overlapping multiplet. H-6′ appeared as doublet showing meta

coupling with H-4′ having coupling constant J = 3.2 Hz. The methylene proton appeared as

most up field signal at δ 6.14 Figure-2.

150



including six quaternary, nine methine, and two methoxy. The quaternary carbon C-5′ and

C-2′ were the most downfield signal appeared at C 152.9 and 150.6, respectively.

Quaternary carbons C-9 and C-8 of indole resonated at C 136.5 and C 134.1, however, C-

1′ appeared at C 126.7. The methine C-2, C-6, C-5, C-4, and C-7 of indole ring appeared at

C 123.5, 120.8, 118.8, 118.1, and 110.3, respectively, while, quaternary carbon C-3

appeared at C 117.6. The methine C-3′, C-4′, and C-6′of benzene ring appeared at C 111.4,

111.8, and 116.1, respectively. The two methoxy resonated at C 56.2 and 55.0, however, the

methine carbon outside the ring was the most up field one and resonated at C 31.7 Figure-

3.

Figure-3: Representative 13C-NMR signals of compound 44


The EI-MS spectral data of compound 44 represented the [M+] at m/z 322 confirming the

molecular formula C25H22N2O2. The presence of significant fragment which appeared at m/z

246 represents the formation of bisindolyl methane fragment. The loss of one of the indole

group was confirmed by the presence of fragment which appeared at m/z 207. While the

151

formation of indole ion appears at m/z 117.0. The prominent fragmentations are represented

in Figure-4.

Figure-4: Representative fragmentation pattern of compound 44

Table-1: Synthesized compounds of Bisindolylmethanes


38

66

39

67

152

40

68

41

69

42

70

43

71

44

72

153

45

73

46

74

47

75

48

76

49

77

154

50

78

51

79

52

80

53

81

54

82

155

55

83

56

84

57

85

58

86

59

87

156

60

88

61

89

62

90

63

91

64

92

65

- -

157


7.6.1 Anticancer Activity

Structure activity relationship

All synthetic compounds were evaluated for their anticancer activity against HeLa cell lines

seventeen compounds were found to be weakly active while others showed no activity.

Compound 56 was found to be the most active analogue having (IC50 = 8.51 ± 0.04 M)

possessing pyrene ring having electron donating effect which may be responsible for the

activity. Compound 59 (IC50 = 9.09 ± 0.01 M) and 48 (IC50 = 9.44 ± 0.02 M) having

methoxy and halogen substituents also showed some weak activity. However, other

compounds 78 (IC50 = 9.72 ± 0.01 M), 71 (IC50 = 13.79 ± 0.01 M), 38 (IC50 = 14.31 ±

0.04 M), 69 (IC50 = 15.8 ± 0.56 M), 68 (IC50 = 15.82 ± 0.05 M), 60 (IC50 = 16.49 ± 0.4),

43 (IC50 = 16.78 ± 0.05 M), 39 (IC50 = 17.36 ± 0.01 M), 54 (IC50 = 17.55 ± 0.01 M), 41

(IC50 = 20.72 ± 0.04 M), 67 (IC50 = 18.10 ± 0.01 M), 53 (IC50 = 29.54 ± 0.04 M), and

52 (IC50 = 29.84 ± 0.03 M) showed very weak almost inactive as comparable to the standard

doxorubicin (IC50 = 0.2 ± 0.03 M) Table-2.

Table-2: Anticancer activity of Bisindolylmethanes 38-92


38 14.13 ± 0.04 66 -

39 17.36 ± 0.01 67 18.10 ± 0.01

40 - 68 15.82 ± 0.05

41 20.72 ± 0.04 69 15.8 ± 0.56

42 - 70 -

43 16.78 ± 0.05 71 13.79 ± 0.01

44 29.13 ± 0.04 72 -

45 - 73 -

46 - 74 -

47 - 75 -

48 9.44 ± 0.02 76 -

49 - 77 -

50 - 78 9.72 ± 0.01

51 - 79 -

52 29.84 ± 0.03 80 -

53 29.54 ± 0.04 81 -

54 17.55 ± 0.01 82 -

55 - 83 -

158

56 8.51 ± 0.04 84 -

57 - 85 -

58 - 86 -

59 9.09 ± 0.01 87 -

60 16.49 ± 0.4 88 -

61 - 89 -

62 - 90 -

63 - 91 -

64 - 92 -

65 - - -

Doxorubicin 0.2 ± 0.03 SEMa is the standard error of the mean, (-) Not active Doxorubicin (std) is standard inhibitor for anti-cancer activity

7.6.2 Antiglycation Activity

All synthetic compounds 38-92 were evaluated for their antiglycation activity and were


Table-3: Antiglycation activity of Bisindolylmethanes 38-92


38 - 66 -

39 - 67 -

40 - 68 -

41 - 69 -

42 - 70 -

43 - 71 -

44 - 72 -

45 - 73 -

46 - 74 -

47 - 75 -

48 - 76 -

49 - 77 -

50 - 78 -

51 - 79 -

52 - 80 -

53 - 81 -

54 - 82 -

55 - 83 -

56 - 84 -

57 - 85 -

58 - 86 -

59 - 87 -

60 - 88 -

61 - 89 -

62 - 90 -

159

63 - 91 -

64 - 92 -

65 - - -

Rutin(std) 294.5 ± 1.50 SEMa is the standard error of the mean, (-) Not active Ibuprofen (std) is standard drug used for antiinflammatory activity

7.6.3 Antiinflammatory Activity

All synthetic derivatives 38-92 were subjected for antiinflammatory activity. Out of fifty

five compounds, 5 analogues were found to be active as compared to the standard ibuprofen

(IC50 = 11.2 ± 1.9 μM), two of them were found to be weakly active, while others were

found to be inactive.

Compound 64 (IC50 = 2.7 ± 0.1 μM) was the most potent compound of the series having

fluoro and hydroxyl substituents along with two indole rings which may be responsible for

the activity of compound. Compound 47 (IC50 = 5.4 ± 0.8 μM) is the second most potent

compound possessing iodo, hydroxyl, and methoxy substituents which showed that the

addition of one methoxy substituent and replacement of fluoro to iodo results in the decline

of activity. On the other hand, compound 52 (IC50 = 6.7 ± 0.6 μM) having an electron

withdrawing substituent nitro group showed further decrease in activity. Compounds 78

(IC50 = 7.1 ± 1.3 μM) and 42 (IC50 = 7.2 ± 1.2 μM) were also potent compounds as compared

to the standard and are structurally similar compound 78 have two methyl substituents in

indole ring while both compounds have hydroxyl and methoxy substituents as electron

donating substituents which indicated that addition of methyl substituents does not effect

on activity.

Compounds 45 (IC50 = 17.1 ± 0.2 μM) and 41 (IC50 = 26.6 ± 2.4 μM) having very weak

activity as compared to standard ibuprofen Table-4.

160

Table-4: Antiinflammatory activity of Bisindolylmethanes 38-92


38 >100 66 >100

39 - 67 -

40 - 68 -

41 26.6 ± 2.4 69 >100

42 7.2 ± 1.2 70 >100

43 - 71 -

44 - 72 >100

45 17.1 ± 0.2 73 >100

46 - 74 -

47 5.4 ± 0.8 75 -

48 - 76 -

49 - 77 -

50 - 78 7.1 ± 1.3

51 - 79 -

52 6.7 ± 0.6 80 -

53 - 81 -

54 >100 82 -

55 - 83 -

161

56 - 84 -

57 - 85 -

58 - 86 -

59 - 87 -

60 - 88 >100

61 - 89 >100

62 - 90 -

63 - 91 -

64 2.7 ± 0.1 92 >100

65 - - -

Ibuprofen(std) 11.2 ± 1.9 SEMa is the standard error of the mean, (-) Not active Ibuprofen (std) is standard drug used for antiinflammatory activity.

7.7 Conclusion

Fifty-five bisindolyl methanes 38-92 were synthesized by acid catalyzed reaction of

aldehyde and indole. All the synthetic derivatives were evaluated for their various biological

activities. Out of fifty-six compounds 38-92 all were found to be inactive against

antiglycation activity. Only seventeen compounds were found to be exhibit weak activity as

compared to the standard doxorubicin (IC50 = 0.2 ± 0.03 μM). The synthesized analogues

were also evaluated for their antiinflammatory activity out of which five analogues 64 (IC50

= 2.7 ± 0.1 μM), 47 (IC50 = 5.4 ± 0.8 μM), 52 (IC50 = 6.7 ± 0.6 μM), 78 (IC50 = 7.1 ± 1.3

μM), and 42 (IC50 = 7.2 ± 1.2 μM) were found to have potent activity as compared to the

standard ibuprofen (IC50 = 11.2 ± 1.9 μM), while compounds 45 (IC50 = 17.1 ± 0.2 μM) and

41 (IC50 = 26.6 ± 2.4 μM) showed weak antiinflammatory activity.

General Procedure for the Synthesis of Bis(indolyl) methanes 38-92

In a reaction flask indole (0.234 g, 2 mmol) was dissolved in ethanol then citric acid and

various substituted benzaldehyde (1 mmol) were added and reaction mixture was refluxed

for 2-3 h. The reaction progress was monitored by TLC, reaction mixture was poured into

water, precipitates were formed which were filtered and purified by washing with hexane.


3,3′-(Phenylmethylene)bis(1H-indole) (38)

Yield: 78%, M.p.: 91-93 °C; 1H NMR (400 MHz, DMSO-d6): 10.77 (s, 2H, NH), 7.33

(ovp, 4H, H-4, H-4a, H-2′, H-6′), 7.24 (ovp, 4H, H-7, H-7a, H-3′, H-5′), 7.17 (m, 1H, H-4′),

162

7.01 (t, 2H, J = 7.6 Hz, H-5, H-5a), 6.83 (t, 2H, J = 7.6 Hz, H-6, H-6a), 6.80 (s, 2H, H-2, H-

2a), 5.80 (s, 1H, CH), 13C NMR (300 MHz, DMSO-d6): 144.9, 136.5, 128.2, 127.9, 126.5,


100), 245 (72), 204 (27), HREI-MS m/z: Calcd for C23H18N2 [322.1455], Found [322.1470].

3,3′-(Naphthalen-2-ylmethylene)bis(1H-indole) (39)


(ovp, 4H, H-4, H-4a, H-5′, H-8′), 7.54 (d, 1H, J4′,3′ = 8.4 Hz, H-4′), 7.42 (ovp, 2H, H-6′, H-

7′), 7.32 (ovp, 4H, H-7, H-7a, H-3′, H-1′), 7.01 (t, 2H, J = 7.4 Hz, H-5, H-5a), 6.83 (ovp, 4H,

H-6, H-6ª, H-2, H-2a), 5.99 (s, 1H, CH), EI MS m/z (% rel. abund.): 372 (M+, 100), 254 (74),

245 (80), HREI-MS m/z: Calcd for C27H20N2 [372.1641], Found [372.1626].

3,3′-((2,3-Dimethoxyphenyl)methylene)bis(1H-indole) (40)


(d, 2H, J4,5/4a,,5a = 8.0 Hz, H-4, H-4a), 7.23 (d, 2H, J7,6/7a,6a = 8.0 Hz, H-7, H-7a), 7.01 (t, 2H,

J = 7.2 Hz, H-5, H-5a), 6.86 (ovp, 4H, H-6, H-6a, H-5′, H-6′), 6.74 (ovp, 3H, H-4′, H-2, H-

2a), 6.16 (s, 1H, CH), 3.79 (s, 3H, OCH3), 3.60 (s, 3H, OCH3), EI MS m/z (% rel. abund.):


[382.1681].

3,3′-((2-Methoxyphenyl)methylene)bis(1H-indole) (41)


(d, 2H, J4,5/4a,5a = 8.0 Hz, H-4, H-4a), 7.19 (d, 2H, J7,6/7a,6a = 8.0 Hz, H-7, H-7a), 7.13 (ovp,

2H, H-6′, H-4′), 7.00 (ovp, 3H, H-5, H-5a, H-5′), 6.81 (ovp, 3H, H-6, H-6a, H-3′), 6.70 (s,

2H, H-2, H-2a), 6.19 (s, 1H, CH), 3.79 (s, 3H, OCH3), EI MS m/z (% rel. abund): 352 (M+,

100), 243 (39), 220 (46), HREI-MS m/z: Calcd for C24H20N2O [352.1576], Found

[352.1576].

4-(Di(1H-Indol-3-yl)methyl)-2-methoxyphenol (42)


(s, 1H, OH), 7.32 (d, 2H, J4,5/4a,,5a = 8.0 Hz, H-4, H-4a), 7.27 (d, 2H, J7,6/7a,6a = 8.0 Hz, H-7,

H-7a), 7.00 (t, 2H, J = 7.4 Hz, H-5, H-5a), 6.93 (s, 1H, H-2′), 6.83 (t, 2H, J = 7.4 Hz, H-6,

163

H-6a), 6.78 (s, 2H, H-2, H-2a), 6.70 (d, 1H, J5′,6′ = 8.4 Hz, H-5′), 6.64 (d, 1H, J6′,5′ = 8.0 Hz,

H-6′), 5.69 (s, 1H, CH), 3.64 (s, 3H, OCH3), EI MS m/z (% rel. abund.): 368 (M+, 100), 245


3,3′-((2-Bromo-5-fluorophenyl)methylene)bis(1H-indole) (43)


(m, 1H, H-6′), 7.35 (d, 2H, J4,5/4a,,5a = 8.0 Hz, H-4, H-4a), 7.21 (d, 2H, J7,6/7a,6a = 8.0 Hz, H-

7, H-7a), 7.05 (t, 3H, J = 8.0 Hz, H-5, H-5a, H-3′), 6.96 (dd, 1H, J4′,3′ = 7.2 Hz, J4′,6′ = 2.8 Hz,

H-3′), 6.88 (t, 2H, J = 7.4 Hz, H-6, H-6a), 6.78 (d, 2H, J = 1.6 Hz, H-2, H-2a), 6.12 (s, 1H,

CH), EI MS m/z (% rel. abund.): 418 (M+, 86), 420 (M+2 , 81), 245 (100), 222 (81), HREI-

MS m/z: Calcd for C23H16BrN2F [418.0509], Found [418.0481].



(d, 2H, J4,5/4a,,5a = 8.4 Hz, H-4, H-4a), 7.20 (d, 2H, J7,6/7a,6a = 8.0 Hz, H-7, H-7a), 7.01 (t, 2H,

J = 7.6 Hz, H-5, H-5a), 6.93 (d, 1H, J3′,4′ = 9.2 Hz, H-3′), 6.84 (t, 2H, J = 7.4 Hz, H-6, H-6a),

6.71 (ovp, 3H, H-2, H-2a, H-4′), 6.65 (d, 1H, J6′,4′ = 3.2 Hz, H-6′), 6.14 (s, 1H, CH), 13C

NMR (400 MHz, DMSO-d6): 152.8, 150.5, 136.5, 134.1, 126.6, 123.5, 120.7, 118.8, 118.0,


100), 245 (53), 117 (32), HREI-MS m/z: Calcd for C23H22N2O2 [382.1729], Found

[382.1681].

2,4-Dichloro-6-(di(1H-indol-3-yl)methyl)phenol (45)


(s, 1H, OH), 7.35 (ovp, 3H, H-4, H-4a, H-4′), 7.23 (d, 2H, J7,6/7a,6a = 8.0 Hz, H-7, H-7a ),

7.03 (t, 2H, J = 8.0 Hz, H-5, H-5a), 6.93 (d, 1H, J6′,4′ = 2.4 Hz, H-6′), 6.87 (t, 2H, J = 7.4 Hz,

H-6, H-6a), 6.77 (d, 2H, J = 1.6 Hz, H-2, H-2a), 6.22 (s, 1H, CH), EI MS m/z (% rel. abund.):

406 (M+, 25), 408 (M+2 , 16), 289 (100), 245 (47), HREI-MS m/z: Calcd for C23H16Cl2N2O

[406.0669], Found [406.0640].

164

3,3′-((2-Nitrophenyl)methylene)bis(1H-indole) (46)

Yield: 85%, M.p.: 142-143 °C, 1H NMR (400 MHz, DMSO-d6): 10.88 (s, 2H, NH), 7.86

(d, 1H, J3′,4′ = 7.6 Hz, H-3′), 7.53 (ovp, 1H, H-4′), 7.44 (t, 1H, J = 7.6 Hz, H-5′), 7.38 (d, 1H,

J6′,5′ = 7.2 Hz, H-6′), 7.34 (d, 2H, J4,5/4a,5a = 8.4 Hz, H-4, H-4a), 7.19 (d, 2H, J7,6/7a,6a = 8.0 Hz,

H-7, H-7a ), 7.04 (t, 2H, J = 7.4 Hz, H-5, H-5a), 6.87 (t, 2H, J = 7.4 Hz, H-6, H-6a), 6.75 (d,

2H, J = 1.6 Hz, H-2, H-2a), 6.39 (s, 1H, CH), EI MS m/z (% rel. abund.): 367 (M+, 24), 319


4-(Di(1H-indol-3-yl)methyl)-2-iodo-6-methoxyphenol (47)


(s, 1H, OH), 7.32 (d, 2H, J4,5/4a,5a = 8.4 Hz, H-4, H-4a), 7.27 (d, 2H, J7,6/7a,6a = 8.0 Hz, H-7,

H-7a), 7.14 (s, 1H, H-6′), 7.02 (ovp, 3H, H-5, H-5a, H-2′), 6.85 (t, 2H, J = 7.4 Hz, H-6, H-

6a), 6.80 (d, 2H, H-2, H-2a), 5.71 (s, 1H, CH), 3.68 (s, 3H, OCH3), EI MS m/z (% rel.

abund.): 494 (M+, 100), 245 (75), 183 (5), HREI-MS m/z: Calcd for C24H19IN2O2

[494.0493], Found [494.0491].

3,3′-((5-Bromo-2-methoxyphenyl)methylene)bis(1H-indole) (48)


(d, 3H, J4,5/4a,5a/4′,3′ = 8.0 Hz, H-4, H-4a, H-4′), 7.18 (d, 2H, J7,6/7a,6a = 8.0 Hz, H-7, H-7a), 7.14

(d, 1H, J6′,4′ = 2.0 Hz, H-6′), 7.02 (ovp, 3H, H-5, H-5a, H-3′), 6.85 (t, 2H, J = 7.4 Hz, H-6,

H-6a), 6.74 (s, 2H, H-2, H-2a), 6.15 (s, 1H, CH), EI MS m/z (% rel. abund.): 430 (M+, 62),

432 (M+2, 100), 245 (95), 130 (26).

3,3′-((3,4,5-Trimethoxyphenyl)methylene)bis(1H-indole) (49)


(ovp, H-4, H-4a, H-7, H-7a), 7.01 (t, 2H, J = 7.4 Hz, H-5, H-5a), 6.86 (ovp, 4H, H-6, H-6a,

H-2′, H-6′), 6.96 (s, 2H, H-2, H-2a), 5.75 (s, 1H, CH), 3.63 (ovp, 9H, OCH3), EI MS m/z (%

rel. abund.): 412 (M+, 100), 381 (25), 280(39), 245 (81), HREI-MS m/z: Calcd for

C26H24N2O3 [412.1788], Found [412.1787].

165



(ovp, 4H, H-4, H-4a, H-7, H-7a), 7.01 (t, 2H, J = 7.4 Hz, H-5, H-5a), 6.85 (ovp, 4H, H-6, H-

6a, H-2′, H-6′), 6.49 (d, 2H, J = 1.6 Hz, H-2, H-2a), 6.31 (s, 1H, H-4′), 5.73 (s, 1H, CH),

3.64 (s, 6H, OCH3), EI MS m/z (% rel. abund.): 382 (M+, 100), 245(76), 243 (18), HREI-

MS m/z: Calcd for C25H22N2O2 [382.1662], Found [382.1681].

3,3′-((2,3,4-Trimethoxyphenyl)methylene)bis(1H-indole) (51)


(d, 2H, J4,5/4a,5a = 8.0 Hz, H-4, H-4a), 7.23 (d, 2H, J7,6/7a,6a = 8.0 Hz, H-7, H-7a), 7.01 (t, 2H,

J = 7.4 Hz, H-5, H-5a), 6.85 (t, 2H, J = 7.4 Hz, H-6, H-6a), 6.79 (d, 2H, J6′,5′ = 8.8 Hz, H-6′),

6.71 (d, 2H, J = 1.2 Hz, H-2, H-2a), 6.65 (d, 1H, J5′,6′ = 8.4 Hz, H-5′), 6.04 (s, 1H, CH), 3.75

(s, 3H, OCH3), 3.72 (s, 3H, OCH3), 3.63 (s, 3H, OCH3), EI MS m/z (% rel. abund.): 412 (M+,

100), 381 (20), 282 (45), 245 (45), HREI-MS m/z: Calcd for C26H24N2O3 [412.1780], Found

[412.1787].

3,3′-((4-Nitrophenyl)methylene)bis(1H-indole) (52)


(d, 2H, J3′,2′/5′,6′ = 8.4 Hz, H-3′, H-5′), 7.60 (d, 2H, J2′,3′/6′,5′ = 8.8 Hz, H-2′, H-6′), 7.35 (d, 2H,

J4,5/4a,5a = 8.0 Hz, H-4, H-4a), 7.27 (d, 2H, J7,6/7a,6a = 8.0 Hz, H-7, H-7a), 7.04 (t, 2H, J = 7.4

Hz, H-5, H-5a), 6.86 (ovp, 4H, H-6, H-6a, H-2, H-2a), 6.01 (s, 1H, CH), 13C NMR (400

MHz, DMSO-d6): 153.0, 145.7, 136.5, 129.4, 126.3, 123.8, 123.3, 121.0, 118.8, 118.3,

116.6, 111.5, 39.4, EI MS m/z (% rel. abund.): 367 (M+, 100), 320 (24), 245 (96), HREI-MS

m/z: Calcd for C23H17N3O2 [367.1328], Found [367.1321].




3H, H-5, H-5a, H-6′), 6.82 (t, 2H, J = 7.4 Hz, H-6, H-6a), 6.67 (d, 2H, J = 1.6 Hz, H-2, H-

2a), 6.57 (d, 1H, J3′,5′ = 2.4 Hz, H-3′), 6.38 (dd, 1H, J5′,3′ = 2.4 Hz, J5′,6′ = 8.4 Hz, H-5′), 6.07

(s, 1H, CH), 3.77 (s, 3H, OCH3), 3.70 (s, 3H, OCH3), EI MS m/z (% rel. abund.): 382 (M+,

166

100), 252 (22), 243 (24), HREI-MS m/z: Calcd for C25H22N2O2 [382.1672], Found

[382.1681].

3,3′-((4-Bromophenyl)methylene)bis(1H-indole) (54)


(d, 2H, J4,5/4a,5a = 8.4 Hz, H-4, H-4a), 7.34 (d, 2H, J3′,2′/5′,6′ = 8.0 Hz, H-3′, H-5′), 7.26 (ovp,

4H, H-7, H-7a, H-2′, H-6′), 7.02 (t, 2H, J = 7.2 Hz, H-5, H-5a), 6.85 (t, 2H, J = 7.4 Hz, H-6,

H-6a), 6.81 (d, 2H, J = 1.6 Hz, H-2, H-2a), 5.81 (s, 1H, CH), EI MS m/z (% rel. abund.): 400

(M+, 86), 402 (M+2, 84), 245 (100), 204 (75), HREI-MS m/z: Calcd for C23H17N2Br

[400.0573], Found [400.0575].

3,3′-((3-Methylthiophen-2-yl)methylene)bis(1H-indole) (55)


(ovp, 4H, H-4, H-4a, H-7, H-7a), 7.14 (d, 1H, J5′,4′ = 5.2 Hz, H-5′), 7.02 (t, 2H, J = 7.4 Hz,

H-5, H-5a), 6.84 (ovp, 4H, H-6, H-6a, H-2, H-2a, H-4′), 6.03 (s, 1H, CH), 2.15 (s, 3H, CH3),


C22H18N2S [342.1200], Found [342.1191].

3,3′-(Pyren-1-ylmethylene)bis(1H-indole) (56)


(d, 1H, J6′,7′ = 9.6 Hz, H-6′), 8.25 (ovp, 2H, H-4′, H-5′), 8.13 (ovp, 4H, H-3′, H-7′, H-8′, H-

9′), 8.05 (ovp, 1H, H-2′), 7.88 (d, 1H, J10′,9′ = 8.0 Hz, H-10′), 7.34 (d, 2H, J4,5/4a,5a = 8.0 Hz,

H-4, H-4a), 7.24 (d, 2H, J7,6/7a,6a = 8.0 Hz, H-7, H-7a), 7.02 (t, 2H, J5,6/5a,6a = 7.6 Hz H-5, H-

5a), 6.94 (s, 1H, H-1”), 6.81 (t, 2H, J = 7.6 Hz, H-6, H-6a), 6.76 (d, 2H, J = 1.2 Hz, H-2, H-

2a), EI MS m/z (% rel. abund.): 446 (M+, 89), 328 (100), 243 (43), HREI-MS m/z: Calcd for

C33H22N2 [446.1784], Found [446.1783].

3,3′-((2-Fluoro-4-methoxyphenyl)methylene)bis(1H-indole) (57)



3H, H-5, H-5a, H-3′), 6.85 (t, 2H, J = 7.4 Hz, H-6, H-6a), 6.79 (ovp, 3H, H-2, H-2a, H-6′),

6.64 (dd, 1H, J3′,5′ = 2.4 Hz, J5′,6′ = 6.4 Hz, H-5′), 5.98 (s, 1H, CH), 3.71 (s, 3H, OCH3), EI

167

MS m/z (% rel. abund.): 370 (M+, 100), 253 (40), 245 (51), HREI-MS m/z: Calcd for

C24H19N2OF [370.1454], Found [370.1481].

4-(Di(1H-indol-3-yl)methyl)-2-methoxyphenyl acetate (58)


(ovp, 4H, H-4, H-4a, H-7, H-7a), 7.16 (s, 1H, H-2′), 7.02 (t, 2H, J = 7.4 Hz, H-5, H-5a), 6.93

(d, 1H, J5′,6′ = 8.4 Hz, H-5′), 6.85 (ovp, 5H, H-6, H-6a, H-2, H-2a, H-6′), 5.83 (s, 1H, CH),

3.65 (s, 3H, OCH3), 2.20 (s, 3H, OCOCH3), EI MS m/z (% rel. abund.): 410 (M+, 97), 368




(d, 2H, J4,5/4a,5a = 8.0 Hz, H-4, H-4a), 7.27 (d, 2H, J7,6/7a,6a = 8.0 Hz, H-7, H-7a), 7.18 (d, 1H,

J6′,5′ = 7.6 Hz, H-6′), 7.04 (ovp, 3H, H-5, H-5a, H-5′), 6.84 (ovp, 5H, H-6, H-6a, H-2, H-2a,

H-2′), 5.81 (s, 1H, CH), 3.72 (s, 3H, OCH3), EI MS m/z (% rel. abund.): 370 (M+, 100), 254

(6), 185 (6), HREI-MS m/z: Calcd for C24H19N2OF [370.1487], Found [370.1481].

3,3′-((4-Bromo-2-fluorophenyl)methylene)bis(1H-indole) (60)


(d, 1H, J5′,6′ = 8.0 Hz, H-5′), 7.34 (d, 2H, J4,5/4a,5a = 8.0 Hz, H-4, H-4a), 7.28 (s, 1H, J6′,5′ = 8.4

Hz, H-6′), 7.22 (d, 2H, J7,6/7a,6a = 7.6 Hz, H-7, H-7a), 7.13 (t, 1H, J = 8.4 Hz, H-3′), 7.03 (t,

2H, J = 7.6 Hz, H-5, H-5a), 6.87 (t, 2H, J = 7.4 Hz, H-6, H-6a), 6.82 (d, 2H, J = 1.6 Hz, H-

2, H-2a), 6.03 (s, 1H, CH), EI MS m/z (% rel. abund.): 418 (M+, 90), 420 (M+2, 100), 231

(43), 205 (18), HREI-MS m/z: Calcd for C23H16N2BrF [418.0461], Found [418.0481].

2-Bromo-4-(di(1H-indol-3-yl)methyl)phenol (61)


(s, 1H, OH), 7.33 (ovp, 3H, H-4, H-4a, H-2′), 7.25 (d, 2H, J7,6/7a,6a = 8.0 Hz, H-7, H-7a), 7.13

(d, 1H, J5′,6′ = 7.2 Hz, H-5′), 7.02 (t, 2H, J = 7.4 Hz, H-5, H-5a), 6.85 (t, 2H, J = 7.6 Hz, H-

6, H-6a), 6.79 (d, 2H, J = 1.6 Hz, H-2, H-2a), 5.72 (s, 1H, CH), EI MS m/z (% rel. abund.):

416 (M+, 91), 418 (M+2, 100), 245 (65), HREI-MS m/z: Calcd for C23H17N2BrO [416.0534],

Found [416.0524].

168

2-Bromo-4-(di(1H-indol-3-yl)methyl)-6-methoxyphenol (62)



H-7a), 7.02 (ovp, 3H, H-5, H-5a, H-2′), 6.93 (s, 1H, H-6′), 6.86 (t, 2H, J = 7.6 Hz, H-6, H-

6a), 6.81 (s, 2H, H-2, H-2a), 5.74 (s, 1H, CH), 3.70 (s, 3H, OCH3), EI MS m/z (% rel.

abund.): 446 (M+, 100), 448 (M+2, 92), 245 (58), HREI-MS m/z: Calcd for C24H19N2O2Br

[446.0644], Found [446.0630 ].




J6′,5′ = 7.2 Hz, H-6′), 7.03 (ovp, 3H, H-5, H-5a, H-5′), 6.83 (ovp, 5H, H-2, H-2a, H-6, H-6a,

H-2′), 5.81 (s, 1H, CH), 3.72 (s, 3H, OCH3), FAB+ve: 370 (M+1).

2-(Di(1H-indol-3-yl)methyl)-5-fluorophenol (64)



H-7a), 7.01 (t, 2H, J = 7.6 Hz, H-5, H-5a), 6.85 (t, 2H, J = 7.4 Hz, H-6, H-6a), 6.80 (ovp,

2H, H-3′,H-4′), 6.75 (ovp, 3H, H-2, H-2a, H-6′), 6.13 (s, 1H, CH), EI MS m/z (% rel. abund.):

456 (M+, 28), 239 (90), 238 (100), HREI-MS m/z: Calcd for C23H17N2OF [356.1334], Found

[356.1325].

3,3′-((2-Chlorophenyl)methylene)bis(1H-indole) (65)


(ovp, 1H, H-3′), 7.34 (d, 2H, J4,5/4a,5a = 8.4 Hz, H-4, H-4a), 7.25 (ovp, 1H, H-5′), 7.22 (ovp,

4H, H-4′, H-2′, H-7, H-7a), 7.03 (t, 2H, J = 7.4 Hz, H-5, H-5a), 6.86 (t, 2H, J = 7.4 Hz, H-6,

H-6a), 6.73 (d, 2H, J = 1.6 Hz, H-2, H-2a), 6.19 (s, 1H, CH), 6.19 (s, 1H, CH), EI MS m/z

(% rel. abund.): 356 (M+, 100), 358 (M+2 ,34), 245 (58), HREI-MS m/z: Calcd for

C23H17N2Cl [356.1078], Found [356.1080].

169

3,3′-(Furan-2-ylmethylene)bis(1H-indole) (66)


(br.s, 1H, H-3′), 7.37 (d, 2H, J4,5/4a,5a = 8.0 Hz, H-4, H-4a), 7.32 (d, 2H, J7,6/7a,6a = 8.4 Hz, H-

7, H-7a), 7.01 (ovp, 4H, H-5, H-5a, H-2, H-2a), 6.86 (t, 2H, J = 7.4 Hz, H-6, H-6a), 6.34 (m,

1H, H-4′), 6.07 (d, 1H, J5′,4′ = 2.8 Hz, H-5′), 5.87 (s, 1H, CH), EI MS m/z (% rel. abund.):

312 (M+, 100), 283 (52), 167 (18), HREI-MS m/z: Calcd for C21H16N2O [312.1264], Found

[312.1263].

3,3′-((1H-Indol-2-yl)methylene)bis(1H-indole) (67)

Yield: 79%, M.p.: 235-237 °C; 1 H NMR (400 MHz, DMSO-d6): 10.67 (s, 3H, NH), 7.36

(d, 3H, J4,5/4a,5a/4′,5′ = 7.6 Hz, H-4, H-4a, H-4′), 7.30 (d, 3H, J7,6/7a,6a/7′,6′ = 8.4 Hz, H-7′, H-7,

H-7a), 6.99 (t, 3H, J = 7.6 Hz, H-5, H-5a, H-5′), 6.91 (d, 3H, J = 1.6 Hz, H-2′, H-2, H-2a),

6.82 (t, 3H, J = 7.4 Hz, H-6, H-6a, H-6′), 6.02 (s, 1H, CH), EI MS m/z (% rel. abund.): 361

(M+, 100), 245 (18), 243 (50), HREI-MS m/z: Calcd for C25H19N3 [361.1580], Found

[361.1579].

3,3′-(Naphthalen-1-ylmethylene)bis(1H-indole) (68)


(d, 1H, J5′,6′ = 8.0 Hz, H-5′), 7.91 (d, 1H, J8′,7′ = 7.6 Hz, H-8′), 7.76 (d, 1H, J4′,3′ = 8.0 Hz, H-

4′), 7.43 (ovp, 2H, H-6′, H-7′), 7.35 (ovp, 3H, H-4, H-4a, H-3′), 7.24 (d, 3H, J7,6/7a,6a/2′,3′ =

8.0 Hz, H-2′, H-7, H-7a), 7.01 (t, 2H, J = 7.6 Hz, H-5, H-5a), 6.83 (t, 2H, J = 7.4 Hz, H-6,

H-6a), 6.71 (d, 2H, J = 2.0 Hz, H-2, H-2a), 6.61 (s, 1H, CH), EI MS m/z (% rel. abund.): 372

(M+, 100), 254 (41), 245 (30), HREI-MS m/z: Calcd for C22H18N2S [342.1200], Found

[342.1191].

3,3′-(o-Tolylmethylene)bis(1H-indole) (69)

Yield: 65%, M.p.: 202-204 ˚C; 1H NMR (400 MHz, DMSO-d6): 10.76 (s, 2H, NH), 7.33


J6′,5′ = 7.2 Hz, H-6′), 7.07 (ovp, 1H, H-4′), 7.03 (ovp, 4H, H-5, H-5a, H-5′, H-2′), 6.83 (t, 2H,

J6,5/6,7/6a,5a/6a,7a = 7.4 Hz, H-6, H-6a), 6.65 (d, 2H, J = 2.0 Hz, H-2, H-2a), 5.93 (s, 1H, CH),

2.32 (s, 3H, CH3), EI MS m/z (% rel. abund.): 336 (M+, 100), 245 (46), 218 (54), HREI-MS

m/z: Calcd for C24H20N2 [336.1643], Found [336.1626].

170

3,3′-((4-Bromo-3,5-dimethoxyphenyl)methylene)bis(1H-indole) (70)

Yield: 81%, M.p.: 207-209 ˚C; 1H NMR (400 MHz, DMSO-d6): 10.80 (s, 2H, NH), 7.32

(ovp, 4H, H-4, H-4a, H-7, H-7a), 7.03 (t, 2H, J = 7.4 Hz, H-5, H-5a), 6.86 (ovp, 4H, H-2′,

H-5′, H-6, H-6a), 6.77 (s, 2H, H-2, H-2a), 5.83 (s, 1H, CH), 2.49 (s, 3H, OCH3), EI MS m/z

(% rel. abund.): 460 (M+, 95), 462 (M+2, 100), 245 (96), HREI-MS m/z: Calcd for

C25H21N2O2Br [460.0784], Found [460.0786].

3,3′-((4-Nitrophenyl)methylene)bis(1H-indole-5-carbonitrile) (71)


(d, 2H, J3′,2′/6′,5′ = 8.7 Hz, H-3′, H-5′), 7.86 (br.s, 2H, H-2′, H-6′), 7.63 (d, 2H, J7,6/7a,6a = 8.7

Hz, H-7, H-7a), 7.53 (d, 2H, J4,5/4a,5a = 8.4 Hz, H-4, H-4a), 7.42 (m, 2H, H-6, H-6a), 7.20 (d,

2H, J = 2.1 Hz, H-2, H-2a), 6.24 (s, 1H, CH), EI MS m/z (% rel. abund.): 417 (M+, 100), 415


3,3′-((2,4-Dimethoxyphenyl)methylene)bis(1,2-dimethyl-1H-indole) (72)

Yield: 77%, M.p.: 170-172 °C; 1H NMR (400 MHz, DMSO-d6): 7.28 (d, 2H, J4,5/4a,5a = 8.0

Hz, H-4, H-4a), 6.90 (ovp, 3H, H-5, H-5a, H-6′), 6.74 (d, 2H, J7,6/7a,6a = 8.0 Hz, H-7, H-7a),

6.66 (t, 2H, J6,5/6,7/6a,5a/6a,7a = 7.4 Hz, H-6, H-6a), 6.54 (d, 1H, J3′,5′ = 2.0 Hz, H-3′), 6.38 (dd,

1H, J5′,3′ = 2.4 Hz, J5′,6′ = 8.8 Hz, H-5′), 6.02 (s, 1H, CH), 3.72 (s, 3H, OCH3), 3.61 (s, 3H,

OCH3), 3.60 (s, 6H, NCH3), 2.05 (s, 6H, CH3), EI MS m/z (% rel. abund.): 438 (M+, 63),

423 (100), 293 (18).



Hz, H-4, H-4a), 6.94 (t, 2H, J5,4/5,6/5a,4a/5a,6a = 7.4 Hz, H-5, H-5a), 6.84 (d, 2H, J7,6/7a,6a = 8.0

Hz, H-7, H-7a), 6.70 (t, 2H, J6,5/6,7/6a,5a/6a,7a = 7.6 Hz, H-6, H-6a), 6.31 (ovp, 3H, H-2′, H-6′,

H-4′), 5.89 (s, 1H, CH), 3.62 (s, 6H, OCH3), 3.59 (s, 6H, NCH3), 2.16 (s, 6H, CH3), EI MS

m/z (% rel. abund.): 438 (M+, 89), 423 (100), 301 (89), HREI-MS m/z: Calcd for C29H30N2O2

[438.2323], Found [438.2307].

171

3,3′-((4-Bromo-3,5-dimethoxyphenyl)methylene)bis(1,2-dimethyl-1H-indole) (74)



Hz, H-7, H-7a), 6.73 (t, 2H, J6,5/6,7/6a,5a/6a,7a = 7.4 Hz, H-6, H-6a), 6.60 (s, 2H, H-2′, H-6′),

5.99 (s, 1H, CH), 3.63 (s, 6H, OCH3), 3.54 (s, 6H, NCH3), 2.18 (s, 6H, CH3), EI MS m/z (%

rel. abund.): 516 (M+ , 90), 518 (M+2, 97), 301 (100), 277 (27), HREI-MS m/z: Calcd for

C29H29N2O2Br [516.1398], Found [516.1412].

3,3′-((2-Bromo-4,5-dimethoxyphenyl)methylene)bis(1,2-dimethyl-1H-indole) (75)


Hz, H-4, H-4a), 7.13 (s, 1H, H-3′), 6.96 (ovp, 2H, H-7, H-7a), 6.88 (s, 1H, H-6′), 6.72 (ovp,

4H, H-5, H-5a, H-6, H-6a), 5.98 (s, 1H, CH), 3.76 (s, 3H, OCH3), 3.62 (s, 6H, NCH3), 3.36

(s, 3H, OCH3), 2.11 (s, 6H, CH3), EI MS m/z (% rel. abund.): 516 (M+, 100), 518 (M+2, 96),

501 (71), 292 (100), HREI-MS m/z: Calcd for C29H29N2O2Br [342.1200], Found [342.1191].

3,3′-(Naphthalen-2-ylmethylene)bis(1,2-dimethyl-1H-indole) (76)

Yield: 77%, M.p.: 214-216 °C; 1H NMR (400 MHz, DMSO-d6): 7.86 (d, 1H, J8′,7′ = 7.6 Hz,

H-8′), 7.77 (d, 1H, J5′,6′ = 8.4 Hz, H-5′), 7.65 (d, 1H, J4′,3′ = 8.0 Hz, H-4′), 7.54 (s, 1H, H-1′),

7.41 (ovp, 3H, H-3′, H-6′, H-7′), 7.34 (d, 2H, J4,5/4a,5a = 8.0 Hz, H-4, H-4a), 6.94 (t, 2H,

J5,4/5,6/5a,4a/5a,6a = 7.4 Hz, H-5, H-5a), 6.78 (d, 2H, J7,6/7a,6a = 7.6 Hz, H-7, H-7a), 6.65 (t, 2H,

J6,5/6,7/6a,5a/6a,7a = 7.4 Hz, H-6, H-6a), 6.16 (s, 1H, CH), 3.65 (s, 6H, NCH3), 2.18 (s, 6H, CH3),


C31H28N2 [428.2253], Found [428.2252].

3,3′-((3,4,5-Trimethoxyphenyl)methylene)bis(1,2-dimethyl-1H-indole) (77)



Hz, H-7, H-7a), 6.72 (t, 2H, J6,5/6,7/6a,5a/6a,7a = 7.4 Hz, H-6, H-6a), 6.52 (s, 2H, H-2′, H-6′),

5.93 (s, 1H, CH), 3.64 (s, 9H, OCH3), 3.62 (s, 6H, NCH3), 2.16 (s, 6H, CH3), EI MS m/z (%

rel. abund.): 468 (M+, 94), 453 (100), 301 (44), HREI-MS m/z: Calcd for C30H32N2O3

[468.2418], Found [468.2413].

172

4-(Bis(1,2-dimethyl-1H-indol-3-yl)methyl)-2-methoxyphenol (78)

Yield: 74%, M.p.: 165-167 °C; 1H NMR (400 MHz, DMSO-d6): 8.70 (s, 1H, OH), 7.30 (d,

2H, J4,5/4a,5a = 8.0 Hz, H-4, H-4a), 6.93 (t, 2H, J5,4/5,6/5a,4a/5a,6a = 7.6 Hz, H-5, H-5a), 6.80 (d,

2H, J 7,6/7a,6a = 8.0 Hz, H-7,H-7a), 6.78 (s, 1H, H-2′), 6.69 (t, 2H, J6,5/6,7/6a,5a/6a,7a = 7.4 Hz, H-

6, H-6a), 6.61 (d, 1H, J5′,6′ = 8.0 Hz, H-5′), 6.47 (d, 1H, J6′,5′ = 8.0 Hz, H-6′), 5.88 (s, 1H,

CH), 3.61 (s, 6H, NCH3), 3.52 (s, 3H, OCH3), 2.14 (s, 6H, CH3), 13C NMR (300 MHz,

DMSO-d6): 147.3, 144.6, 136.1, 134.7, 133.3, 127.2, 120.7, 119.5, 118.7, 118.0, 114.8,

113.4, 112.8, 108.7, 55.7, 38.4, 29.2, 10.3, EI MS m/z (% rel. abund.): 424 (M+, 82), 409


3,3′-((4-Bromophenyl)methylene)bis(1,2-dimethyl-1H-indole) (79)

Yield: 78%, M.p.: 155-157 °C; 1H NMR (400 MHz, DMSO-d6): 7.42 (d, 2H, J3′,2′/5′,6′ = 8.4

Hz, H-3′, H-5′), 7.32 (d, 2H, J′4,5/4a,5a = 8.0 Hz, H-4, H-4a), 7.07 (d, 2H, J2′,3′/6′,5′ = 8.4 Hz, H-

2′, H-6′), 6.95 (t, 2H, J5,4/5,6/5a,4a/5a,6a = 7.4 Hz, H-5, H-5a), 6.77 (d, 2H, J7,6/7a,6a = 8.0 Hz, H-

7, H-7a), 6.70 (t, 2H, J6,5/6,7/6a,5a/6a,7a = 7.4 Hz, H-6, H-6a), 5.97 (s, 1H, CH), 3.63 (s, 6H,

NCH3), 2.17 (s, 6H, CH3), 13C NMR (400 MHz, DMSO-d6): 143.6, 136.2, 133.8, 130.8,

130.7, 126.9, 119.6, 118.6, 118.5, 118.2, 111.6, 108.9, 38.4, 29.2, 10.2, EI MS m/z (% rel.

abund.): 456 (M+, 94), 458 (M+2, 100), 441 (87), 312 (41), HREI-MS m/z: Calcd for

C27H25N2Br [456.1207], Found [456.1201].

3,3′-((5-bromo-2-fluorophenyl)methylene)bis(1,2-dimethyl-1H-indole) (80)

Yield: 85%, M.p.: 196-198 °C; 1H NMR (400 MHz, DMSO-d6): 7.50 (m, 1H, H-2′), 7.35

(d, 2H, J4,5/4a,5a = 8.0 Hz, H-4, H-4a), 7.13 (ovp, 2H, H-4′, H-5′), 6.98 (ovp, 2H, H-7, H-7a),

6.73 (ovp, 4H, H-5, H-5a, H-6, H-6a), 6.05 (s, 1H, CH), 3.64 (s, 6H, NCH3), 2.16 (s, 6H,

CH3), EI MS m/z (% rel. abund.): 474 (M+, 97), 476 (M+2, 100), 461 (81), 301 (68), HREI-

MS m/z: Calcd for C27H24N2BrF [474.1108], Found [474.1107].



Hz, H-4, H-4a), 6.91 (ovp, 3H, H-5, H-5a, H-5′), 6.77 (dd, 1H, J4′,5′ = 7.2 Hz, J4′,2′ = 3.2 Hz,

H-4′), 6.74 (d, 2H, J7,6/7a,6a = 7.6 Hz, H-7, H-7a), 6.67 (t, 2H, J6,5/6,7/6a,5a/6a,7a = 7.2 Hz, H-6,

H-6a), 6.60 (d, 1H, J2′,4′ = 2.8 Hz, H-2′), 6.07 (s, 1H, CH), 3.61 (s, 6H, NCH3), 3.56 (s, 3H,

173

OCH3), 3.53 (s, 3H, OCH3), 2.07 (s, 6H, CH3), EI MS m/z (% rel. abund.): 438 (M+, 75),

423 (100), 293 (30), 262 (31), HREI-MS m/z: Calcd for C29H30N2O2 [438.2295], Found

[438.2307].

3,3′-((4-Nitrophenyl)methylene)bis(1,2-dimethyl-1H-indole) (82)


Hz, H-3′, H-5′), 7.36 (ovp, 4H, H-4, H-4a, H-2′,H-6′), 6.97 (t, 2H, J5,4/5,6/5a,4a/5a,6a = 7.2 Hz,

H-5, H-5a), 6.73 (ovp, 4H, H-6, H-6a, H-7, H-7a), 6.15 (s, 1H, CH), 3.64 (s, 6H, NCH3),

2.20 (s, 6H, CH3), 13C NMR (400 MHz, DMSO-d6): 152.6, 145.7, 136.2, 134.2, 129.7,

126.7, 123.2, 119.8, 118.5, 118.4, 110.9, 109.1, 38.8, 29.3, 10.3, EI MS m/z (% rel. abund.):


[423.1947].

2-(Bis(1,2-dimethyl-1H-indol-3-yl)methyl)-3-bromo-6-methoxyphenol (83)


2H, J4,5/4a,5a = 8.0 Hz, H-4, H-4a), 7.04 (d, 1H, J3′,4′ = 8.8 Hz, H-3′), 6.93 (ovp, 4H, H-7, H-

7a, H-5, H-5a), 6.79 (d, 1H, J 4′,3′ = 8.8 Hz, H-4′), 6.70 (ovp, 2H, H-6, H-6a), 6.36 (s, 1H,

CH), 3.71 (s, 3H, OCH3), 3.59 (s, 6H, NCH3), 1.95 (s, 6H, CH3), EI MS m/z (% rel. abund.):

502 (M+ , 29), 504 (M+2, 27), 342 (100), 278 (39), HREI-MS m/z: Calcd for C28H27N2O2Br

[502.1251], Found [502.1256].

3,3′-(Phenylmethylene)bis(1,2-dimethyl-1H-indole) (84)

Yield: 79%, M.p.: 210-212 ˚C; 1H NMR (400 MHz, DMSO-d6): 7.31 (d, 2H, J4,5/4a,5a = 8.0

Hz, H-4, H-4a), 7.21 (ovp, 3H, H-3′, H-4′, H-5′), 7.14 (d, 2H, J2′,3′/6′,5′ = 7.2 Hz, H-2′, H-6′),

6.94 (t, 2H, J5,4/5,6/5a,4a/5a,6a = 7.6 Hz, H-5, H-5a), 6.75 (d, 2H, J7,6/7a,6a = 8.0 Hz, H-7, H-7a),

6.67 (t, 2H, J6,5/6,7/6a,5a/6a,7a = 7.4 Hz, H-6, H-6a), 5.99 (s, 1H, CH), 3.62 (s, 6H, NCH3), 2.15

(s, 6H, CH3), EI MS m/z (% rel. abund.): 378 (M+, 100), 363 (86), 301 (52), 232 (79), HREI-

MS m/z: Calcd for C27H26N2 [378.2101], Found [378.2096].



Hz, H-4, H-4a), 6.94 (t, 2H, J5,4/5,6/5a,4a/5a,6a = 7.6 Hz, H-5, H-5a), 6.80 (ovp, 4H, H-7, H-7a,

174

H-6′, H-2′), 6.69 (t, 2H, J6,5/6,7/6a,5a/6a,7a = 7.4 Hz, H-6, H-6a), 6.58 (d, 1H, J5′,6′ = 8.0 Hz, H-

5′), 5.92 (s, 1H, CH), 3.70 (s, 3H, OCH3), 3.62 (s, 6H, NCH3), 3.30 (s, 3H, OCH3), 2.15 (s,

6H, CH3), EI MS m/z (% rel. abund.): 438 (M+, 75), 423 (100), 291 (37), HREI-MS m/z:


4-(Bis(1,2-dimethyl-1H-indol-3-yl)methyl)-2-iodo-6-methoxyphenol (86)


2H, J4,5/4a,5a = 8.1 Hz, H-4, H-4a), 6.95 (t, 3H, J5,4/5,6/5a,4a/5a,6a = 7.2 Hz, H-5, H-5a, H-6′), 6.84

(d, 3H, J7,6/7a,6a = 7.5 Hz, H-7, H-7a, H-2′), 6.69 (t, 2H, J6,5/6,7/6a,5a/6a,7a = 7.3 Hz, H-6, H-6a),

5.90 (s, 1H, CH), 3.70 (s, 3H, OCH3), 3.62 (s, 6H, NCH3), 3.55 (s, 3H, OCH3), 2.16 (s, 6H,


for C28H27N2O2I [550.1125], Found [550.1117].

3,3′-((4-(Benzyloxy)phenyl)methylene)bis(1,2-dimethyl-1H-indole) (87)

Yield: 77%, M.p.: 172-174 °C; 1H NMR (300 MHz, DMSO-d6): 7.41 (ovp, 3H, H-3”, H-

4”, H-5”), 7.34 (ovp, 4H, H-4, H-4a, H-6, H-6a), 7.03 (d, 2H, J2′,3′/6′,5′ = 8.7 Hz, H-2′, H-6′),

6.96 (ovp, 2H, H-2”, H-6”), 6.88 (d, 2H, J7,6/7a,6a = 8.7 Hz, H-7, H-7a), 6.78 (d, 2H, J3′,2′/5′,6′

= 7.8 Hz, H-3′, H-5′), 6.68 (t, 2H, J5,6/5,4/5a,6a/5a,4a = 7.0 Hz, H-5, H-5a), 5.92 (s, 1H, CH), 5.05

(s, 2H, CH2), 3.62 (s, 6H, NCH3), 2.15 (s, 6H, CH3), EI MS m/z (% rel. abund.): 484 (M+,

89), 469 (72), 301 (35), 248 (100), HREI-MS m/z: Calcd for C34H32N2O [484.2516], Found

[484.2515].

4-(Bis(1,2-dimethyl-1H-indol-3-yl)methyl)-2-bromo-6-methoxyphenol (88)


Hz, H-4, H-4a), 6.95 (t, 2H, J5,6/5a,6a/5,4/5a,4a = 7.0 Hz, H-5, H-5ª), 6.83 (d, 3H, J7,6/7a,6a = 7.8

Hz, H-7, H-7ª, H-2′), 6.72 (t, 3H, J6,5/6a,5a/6,7/6a,7a = 7.0 Hz, H-6, H-6ª, H-6′), 5.91 (s, 1H, CH),

3.62 (s, 6H, NCH3), 3.57 (s, 3H, OCH3), 2.20 (s, 6H, CH3), FAB- : (M+, 501), HREI-MS

m/z: Calcd for C28H27N2O2Br [502.1257], Found [502.1256].

3,3′-((2-Fluoro-4-methoxyphenyl)methylene)bis(1,2-dimethyl-1H-indole) (89)


Hz, H-4, H-4a), 6.95 (d, 2H, J7,6/7a,6a = 7.2 Hz, H-7, H-7a), 6.92 (ovp, 1H, H-6′), 6.75 (ovp,

175

3H, H-5, H-5a, H-3′), 6.70 (ovp, 2H, H-6, H-6a), 6.66 (dd, 1H, J5′,6′ = 6.4 Hz, J5′,3′ = 2.4 Hz,

H-5′), 5.96 (s, 1H, CH), 3.73 (s, 3H, OCH3), 3.62 (s, 6H, NCH3), 2.13 (s, 6H, CH3), EI MS

m/z (% rel. abund.): 426 (M+, 95), 410 (100), 301 (16), 144 (16), HREI-MS m/z: Calcd for

C28H27N2OF [426.2103], Found [426.2107].

3,3′-((4-Methoxyphenyl)methylene)bis(1,2-dimethyl-1H-indole) (90)


Hz, H-2′, H-6′), 7.21 (t, 1H, J5,4/5,6 = 7.8 Hz, H-5), 7.04 (d, 1H, J4,5 = 7.6 Hz, H-4), 6.97 (d,

1H, J4a,5a = 8.4 Hz, H-4a),6.92 (t, 2H, J6,7/6,5/6a,7a/6a,5a = 7.4 Hz, H-6, H-6a), 6.79 (t, 1H,

J5a,4a/5a,6a = 7.4 Hz, H-5a), 6.70 (d, 2H, J3′,2′/5′6′ = 7.6 Hz, H-3′, H-5′), 6.66 (d, 2H, J7,6/7a,6a =

7.2 Hz, H-7, H-7a), 6.12 (s, 1H, CH), 3.62 (s, 3H, OCH3), 3.60 (s, 6H, NCH3), 2.05 (s, 6H,


for C28H28N2O [408.2204], Found [408.2202].

3,3′-((5-Methylfuran-2-yl)methylene)bis(1,2-dimethyl-1H-indole) (91)


Hz, H-4, H-4a), 6.96 (ovp, 4H, H-7, H-7a, H-5, H-5a), 6.76 (t, 2H, J6,7/6,5/6a,7a/6a,5a = 6.7 Hz

H-6, H-6a), 5.95 (m, 1H, H-5′), 5.82 (s, 1H, CH), 5.61 (m, 1H, H-4′), 3.61 (s, 6H, NCH3),

2.20 (s, 6H, CH3), 2.18 (s, 3H, CH3), EI MS m/z (% rel. abund.): 382 (M+, 100), 367 (87),

237 (77), 194 (46), HREI-MS m/z: Calcd for C26H26N2O [382.2058], Found [382.2045].

3,3′-(Thiophen-3-ylmethylene)bis(1,2-dimethyl-1H-indole) (92)

Yield: 83%, M.p.: 199-201 °C; 1H NMR (400 MHz, DMSO-d6): 7.43 (m, 1H, H-2′), 7.30

(d, 2H, J4,5/4a,5a = 8.1 Hz, H-4,H-4a), 6.95 (ovp, 2H, H-7, H-7a), 6.88 (ovp, 3H, H-5, H-5a,

H-4′), 6.78 (m, 1H, H-5′), 6.72 (t, 2H, J6,5/6a,5a/6,7/6a,7a = 7.2 Hz, H-6, H-6a), 5.94 (s, 1H, CH),

3.62 (s, 6H, NCH3), 2.20 (s, 6H, CH3), EI MS m/z (% rel. abund.): 384 (M+, 100), 301 (19),

239 (82), HREI-MS m/z: Calcd for C25H24N2S [384.1671], Found [384.1660].

176

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synthesis of indole and tryptamine derivatives and their

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