inis.iaea.org · 2012. 8. 17. · sulfonamides and quinoline derivatives regarding their mechanisms of action, and the rationale for combining chemotherapy and radiotherapy. In addition,

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Thesis presented by ��

M.Sc. in Pharmaceutical Sciences (Pharmaceutical Chemistry)

Faculty of Pharmacy Cairo University

(2007)

Submitted for the fulfillment of Ph.D. Degree in Pharmaceutical Sciences

(Pharmaceutical Chemistry)

Under the supervision of

Prof. Dr. Fatma Abd El-Fattah Ragab

Professor of Pharmaceutical Chemistry Pharmaceutical Chemistry Department


Prof. Dr. Mostafa Mohamed Ghorab

Professor of Applied Organic Chemistry Drug Radiation Research Department

National Centre for Radiation Research and Technology

Atomic Energy Authority

Dr. Helmy Ismail Heiba

Assoc. Professor of Applied Organic Chemistry

Drug Radiation Research Department National Centre for Radiation Research and

Technology Atomic Energy Authority

Dr. Reem Khidr Arafa

Lecturer of Pharmaceutical Chemistry Pharmaceutical Chemistry Department


Faculty of Pharmacy

Cairo University (2010)

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Synthesis of some new heterocyclic

compounds bearing a sulfonamide moiety and studying their combined anticancer

effect with �-radiation Presented by: Ebaa Mostafa Mohamed El-Hossary Approved by: Prof. Dr. Fatma Abdel-Fattah Ragab .................................... Professor of Pharmaceutical Chemistry Faculty of Pharmacy, Cairo University Dr. Helmy Ismail Heiba .................................... Assoc. Professor of Applied Organic Chemistry National Centre for Radiation Research & Technology Prof. Dr. Hassan Hassan Farag .................................... Professor of Pharmaceutical Chemistry Faculty of Pharmacy, Assuit University Prof. Dr. Kamelia Mahmoud Amin .................................... Professor of Pharmaceutical Chemistry Faculty of Pharmacy, Cairo University

AcknowledgementAcknowledgementAcknowledgementAcknowledgement Firstly, I want to thank Allah Allah Allah Allah who gave me the patience and support to achieve my goal. Many thanks to Prof. Dr. Fatma Abd ElProf. Dr. Fatma Abd ElProf. Dr. Fatma Abd ElProf. Dr. Fatma Abd El----Fattah RagabFattah RagabFattah RagabFattah Ragab, Prof. of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University, for her encouragement and kind supervision. Words couldn’t help me in expressing my deep appreciation to Prof. Prof. Prof. Prof. Dr.Dr.Dr.Dr. Mostafa MMostafa MMostafa MMostafa Mohamedohamedohamedohamed Ghorab, Ghorab, Ghorab, Ghorab, Professor of Applied Organic Chemistry, Drug Radiation Research Department, National Centre for Radiation Research and Technology, for suggesting the research project and his great support, faithful advice and unlimited efforts in this research. My special thanks to Dr. Helmy Ismail HeibaDr. Helmy Ismail HeibaDr. Helmy Ismail HeibaDr. Helmy Ismail Heiba, Assoc. Prof. of Applied Organic Chemistry, National Centre for Radiation Research and Technology, for his great support, helpful suggestions and advice which helped me to reach this work in its final form. I am also grateful to Dr. Reem Khidr Arafa, Dr. Reem Khidr Arafa, Dr. Reem Khidr Arafa, Dr. Reem Khidr Arafa, Lecturer of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University, for her helpful suggestions and advice which helped me to finalize this work. I would like to give my special thanks to my whole famfamfamfamilyilyilyily for the patience and moral support they provided through out my research work. Finally, my deep thanks to my colleagues my colleagues my colleagues my colleagues and to all who gave me help and support for the fulfillment of this work.

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Contents

Abstract……………………………………...…………………... I - VII

1. Introduction…………………………………………...……… 1

1.1. Sulfonamides as anticancer agents………………………… 1

1.2. Quinolines as anticancer agents…….……………………… 16

1.3. Chemoradiotherapy………………………………………… 19

1.4. Synthesis of quinolines………………………...….….......... 25

1.5. Synthesis of pyrimido[4,5-b]quinolines…………………… 34

2. Aim of the present investigation……………………...…....... 38

3. Theoretical Discussion……………………………...……....... 43

4. Experimental…………………………………………………. 75

5. Biological Activity………………………………...………….. 117

5.1. In vitro anticancer screening……………………………….. 117

5.2. Radiosensitizing evaluation……………...…….…………... 138

6. Molecular Docking………………………………….....……... 142

7. References…………………………………...……………....... 150

Arabic summary………………………………..………………. �

List of Figures

Figure (1): Schematic representation of the catalytic mechanism for the �-CA catalysed CO2 hydration… … … … … … … … … .. 3

Figure (2): Zinc binding groups… … … … … … … … … … … … … … … 4 Figure (3): Structural elements of CA inhibitors in the CA enzymatic

active site… … … … … … … … … … … … … … … … … … … . 4 Figure (4): The cell cycle… … … … … … … … … … … … … … … … … .. 11 Figure (5): Dose-response curves for tumor control and normal tissue

damage… … … … … … … … … … … … … … … … … … … … . 19 Figure (6): Representative examples of the synthesized compounds

showing great compliance to the general pharmacophore of CA inhibitors… … … … … … … … … … … … … … … … ... 40

Figure (7): Survival curve of Doxorubicin… … … … … … … … … … … 119 Figure (8): Survival curve of compound 6a… … … … … … … … … … .. 119 Figure (9): Survival curve of compound 6b… … … … … … … … … … .. 119 Figure (10): Survival curve of compound 7a… … … … … … … … … … 120 Figure (11): Survival curve of compound 7b… … … … … … … … … … 120 Figure (12): Survival curve of compound 8a… … … … … … … … … … 120 Figure (13): Survival curve of compound 8b… … … … … … … … … … 121 Figure (14): Survival curve of compound 9a… … … … … … … … … … 121 Figure (15): Survival curve of compound 9b… … … … … … … … … … 121 Figure (16): Survival curve of compound 10a… … … … … … … … … .. 122 Figure (17): Survival curve of compound 10b… … … … … … … … … .. 122 Figure (18): Survival curve of compound 11a… … … … … … … … … .. 122 Figure (19): Survival curve of compound 11b… … … … … … … … … .. 123 Figure (20): Survival curve of compound 12a… … … … … … … … … .. 123 Figure (21): Survival curve of compound 12b… … … … … … … … … .. 123 Figure (22): Survival curve of compound 13a… … … … … … … … … .. 124 Figure (23): Survival curve of compound 13b… … … … … … … … … .. 124 Figure (24): Survival curve of compound 14a… … … … … … … … … .. 124 Figure (25): Survival curve of compound 14b… … … … … … … … … .. 125 Figure (26): Survival curve of compound 15a… … … … … … … … … .. 125 Figure (27): Survival curve of compound 15b… … … … … … … … … .. 125 Figure (28): Survival curve of compound 16a… … … … … … … … … .. 126 Figure (29): Survival curve of compound 16b… … … … … … … … … .. 126 Figure (30): Survival curve of compound 17a… … … … … … … … … .. 126

Figure (31): Survival curve of compound 17b… … … … … … … … … .. 127 Figure (32): Survival curve of compound 18a… … … … … … … … … .. 127 Figure (33): Survival curve of compound 18b… … … … … … … … … .. 127 Figure (34): Survival curve of compound 19a… … … … … … … … … .. 128 Figure (35): Survival curve of compound 19b… … … … … … … … … .. 128 Figure (36): Survival curve of compound 20a… … … … … … … … … .. 128 Figure (37): Survival curve of compound 20b… … … … … … … … … .. 129 Figure (38): Survival curve of compound 21a… … … … … … … … … .. 129 Figure (39): Survival curve of compound 21b… … … … … … … … … .. 129 Figure (40): Survival curve of compound 22a… … … … … … … … … .. 130 Figure (41): Survival curve of compound 22b… … … … … … … … … .. 130 Figure (42): Survival curve of compound 23a… … … … … … … … … .. 130 Figure (43): Survival curve of compound 23b… … … … … … … … … .. 131 Figure (44): Survival curve of compound 24a… … … … … … … … … .. 131 Figure (45): Survival curve of compound 24b… … … … … … … … … .. 131 Figure (46): Survival curve of compound 25a… … … … … … … … … .. 132 Figure (47): Survival curve of compound 25b… … … … … … … … … .. 132 Figure (48): Survival curve for MCF7 cell line for compound 8b

alone or in combination with �-irradiation (8 Gy)… … … 140 Figure (49): Survival curve for MCF7 cell line for compound 11a

alone or in combination with �-irradiation (8 Gy)… … … 140 Figure (50): Survival curve for MCF7 cell line for compound 12a

alone or in combination with �-irradiation (8 Gy)… … … 141 Figure (51): Survival curve for MCF7 cell line for compound 18b

alone or in combination with �-irradiation (8 Gy)… … … 141 Figure (52): Superimposition of hCA II-inhibitor adducts… … … … ... 142 Figure (53): CA inhibition mechanism by sulfonamides… … … … … .. 143 Figure (54): Interaction map of N-(2,3,4,5,6-pentafluorobenzyl)-4-

sulfamoyl-benzamide with hCA II… … … … … … … … … 144 Figure (55): Interaction map of E7070 with the active site of hCA II.. 145 Figure (56): Interaction map of compound 8b with the active site of

hCA II… … … … … … … … … … … … … … … … … … … ... 146 Figure (57): Interaction map of compound 11a with the active site of

hCA II… … … … … … … … … … … … … … … … … … … .. 147 Figure (58): Interaction map of compound 12a with the active site of

hCA II… … … … … … … … … … … … … … … … … … … ... 147 Figure (59): Interaction map of compound 18b with the active site of

hCA II… … … … … … … … … … … … … … … … … … … ... 148 Figure (60): Superimposition of E7070 and compound 11a in the

active site of hCA II… … … … … … … … … … … … … … . 149

List of Tables

Table (1): Physical data and microanalysis of compounds 6a & 6b… … … . 77 Table (2): Physical data and microanalysis of compounds 7a & 7b… … … . 79 Table (3): Physical data and microanalysis of compounds 8a & 8b… … … . 81 Table (4): Physical data and microanalysis of compounds 9a & 9b… … … . 83 Table (5): Physical data and microanalysis of compounds 10a & 10b… … . 85 Table (6): Physical data and microanalysis of compounds 11a & 11b… … . 87 Table (7): Physical data and microanalysis of compounds 12a & 12b… … . 89 Table (8): Physical data and microanalysis of compounds 13a & 13b… … . 91 Table (9): Physical data and microanalysis of compounds 14a & 14b… … . 93 Table (10): Physical data and microanalysis of compounds 15a & 15b… ... 95 Table (11): Physical data and microanalysis of compounds 16a & 16b… ... 97 Table (12): Physical data and microanalysis of compounds 17a & 17b… ... 99 Table (13): Physical data and microanalysis of compounds 18a & 18b… ... 101 Table (14): Physical data and microanalysis of compounds 19a & 19b… ... 103 Table (15): Physical data and microanalysis of compounds 20a & 20b… ... 105 Table (16): Physical data and microanalysis of compounds 21a & 21b… ... 107 Table (17): Physical data and microanalysis of compounds 22a & 22b… ... 109 Table (18): Physical data and microanalysis of compounds 23a & 23b… ... 111 Table (19): Physical data and microanalysis of compounds 24a & 24b… ... 113 Table (20): Physical data and microanalysis of compounds 25a & 25b… ... 115 Table (21): In vitro anticancer screening of the synthesized compounds

against human breast cancer cell line (MCF7)… … .… … … … ... 133 Table (22): In vitro anticancer screening of compounds 8b, 11a, 12a and

18b against human breast cancer cell line (MCF7) in combination with �-radiation… … … ...… … … … … … … … … ... 139

Abbreviations

• ANOVA: Analysis of variance.

• CA: Carbonic anhydrase.

• CAI: Carbonic anhydrase inhibitor.

• CARP: Carbonic anhydrase related protein.

• CDK: Cyclin dependent kinase.

• CNS: Central nervous system.

• Conc.: concentrated.

• CRT: Chemoradiotherapy.

• CT: Chemotherapy.

• Cys: Cysteine.

• dil.: diluted.

• DMF: dimethyl formamide.

• DMSO: Dimethyl sulfoxide.

• DNA: Deoxyribonucleic acid.

• EDTA: Ethylene diamine tetra-acetic acid.

• EGFR: Epidermal growth factor receptor.

• EI/MS: Electron impact mass spectrometry.

• ELISA: Enzyme-linked immunosorbent assay.

• 5-FU: 5-fluorouracil.

• Gln: Glutamine.

• Glu: Glutamic acid.

• h: hour.

• hCA: human carbonic anhydrase.

• His: Histidine.

• 1H-NMR: Proton nuclear magnetic resonance.

• IR: Infrared.

• Leu: Leucine.

• MDR: Multi-drug resistant.

• min: minute.

• MMFF94x: Merck Molecular Force Field 94x.

• MMP: Matrix metalloproteinase.

• MMPIs: Matrix metalloproteinase inhibitors.

• MOE: Molecular operating environment.

• MWI: Microwave irradiation.

• NSCLC: Non-small cell lung cancer.

• OTT: Overall treatment time.

• Phe: Phenylalanine.

• PI3: Phosphoionositide 3.

• pRb: Retinoblastoma protein.

• Pro: Proline.

• PTSA: p-Toluene sulfonic acid.

• RNA: Ribonucleic acid.

• RT: Radiotherapy.

• SAR: Structure activity relationship.

• SE: Standard error.

• SRB: Sulfo-rhodamine B.

• TCA: Trichloroacetic acid.

• TEA: Triethylamine.

• TFAE: Trifluoro-acetaldehyde ethyl hemiacetal.

• Thr: Threonine.

• TLC: Thin layer chromatography.

• TMS: Tetramethylsilane.

• UK: United Kingdom.

• USA: United States of America.

• UV: Ultraviolet.

• Val: Valine.

• ZBG: Zinc binding group.

Abstract

I

In search for new cytotoxic agents with improved anticancer profile, some

new halogen-containing quinoline and pyrimido[4,5-b]quinoline derivatives

bearing a free sulfonamide moiety were synthesized. All the newly synthesized

target compounds were subjected to in vitro anticancer screening against human

breast cancer cell line (MCF7). The most potent compounds, as concluded from

the in vitro anticancer screening, were selected to be evaluated again for their in

vitro anticancer activity in combination with �-radiation. Also, the newly

synthesized compounds were docked in the active site of the carbonic anhydrase

enzyme.

The thesis includes the following parts:

1. Introduction

This part includes a brief literature review on anticancer activity of

sulfonamides and quinoline derivatives regarding their mechanisms of action, and

the rationale for combining chemotherapy and radiotherapy. In addition, the

different methods for the synthesis of quinoline derivatives and pyrimido[4,5-

b]quinoline derivatives are discussed.

2. Aim of the present investigation

The rationale upon which synthesis of the new compounds, evaluation of their

anticancer activity alone or in combination with �-irradiation, and suggestion of

their mechanism of action through a docking study, is presented in this part.

3. Theoretical discussion

This part deals with the discussion of the experimental methods adopted for

the synthesis of the synthesized compounds, as well as different analytical

methods adopted for the identification and the verification of the structures of the

Abstract

II

synthesized compounds. Schemes (1-5) illustrate the synthetic pathways adopted

in the preparation of the designed compounds.

4. Experimental

This part describes the practical procedures used for the synthesis of forty

new final compounds and one known intermediate, with their elemental analyses

and spectral data (IR, 1H-NMR and mass spectroscopy).

Known intermediate:

• 4-(5,5-Dimethyl-3-oxocyclohex-1-enylamino)benzenesulfonamide (3)

New final compounds:

• 4-[2-Amino-3-cyano-4-(4-fluorophenyl)-7,7-dimethyl-5-oxo-5,6,7,8-

tetrahydroquinolin-1(4H)-yl]benzenesulfonamide (6a)

• 4-[2-Amino-4-(4-chlorophenyl)-3-cyano-7,7-dimethyl-5-oxo-5,6,7,8-

tetrahydroquinolin-1(4H)-yl]benzenesulfonamide (6b)

• 4-[5-(4-Fluorophenyl)-8,8-dimethyl-4,6-dioxo-3,4,6,7,8,9-hexahydro-

pyrimido[4,5-b]quinolin-10(5H)-yl]benzenesulfonamide (7a)

• 4-[5-(4-Chlorophenyl)-8,8-dimethyl-4,6-dioxo-3,4,6,7,8,9-

hexahydropyrimido[4,5-b]quinolin-10(5H)-yl]benzenesulfonamide (7b)

• N-[3-Cyano-4-(4-fluorophenyl)-7,7-dimethyl-5-oxo-1-(4-sulfamoyl-

phenyl)-1,4,5,6,7,8-hexahydroquinolin-2-yl]acetamide (8a)

Abstract

III

• N-[4-(4-Chlorophenyl)-3-cyano-7,7-dimethyl-5-oxo-1-(4-

sulfamoylphenyl)-1,4,5,6,7,8-hexahydroquinolin-2-yl]acetamide (8b)

• N-Acetyl-N-[3-cyano-4-(4-fluorophenyl)-7,7-dimethyl-5-oxo-1-(4-

sulfamoylphenyl)-1,4,5,6,7,8-hexahydroquinolin-2-yl]acetamide (9a)

• N-Acetyl-N-[4-(4-chlorophenyl)-3-cyano-7,7-dimethyl-5-oxo-1-(4-

sulfamoyl-phenyl)-1,4,5,6,7,8-hexahydroquinolin-2-yl]acetamide (9b)

• 4-[5-(4-Fluorophenyl)-2,8,8-trimethyl-4,6-dioxo-3,4,6,7,8,9-hexahydro-


• 4-[5-(4-Chlorophenyl)-2,8,8-trimethyl-4,6-dioxo-3,4,6,7,8,9-

hexahydropyrimido[4,5-b]quinolin-10(5H)-yl]benzenesulfonamide (10b)

• 4-[4-Amino-5-(4-fluorophenyl)-8,8-dimethyl-6-oxo-6,7,8,9-tetrahydro-


• 4-[4-Amino-5-(4-chlorophenyl)-8,8-dimethyl-6-oxo-6,7,8,9-tetrahydro-

pyrimido[4,5-b]quinolin-10(5H)-yl]benzenesulfonamide (11b)

• Ethyl N-[3-cyano-4-(4-fluorophenyl)-7,7-dimethyl-5-oxo-1-(4-sulfamoyl-

phenyl)-1,4,5,6,7,8-hexahydroquinolin-2-yl]formimidate (12a)

• Ethyl N-[4-(4-chlorophenyl)-3-cyano-7,7-dimethyl-5-oxo-1-(4-

sulfamoylphenyl)-1,4,5,6,7,8-hexahydroquinolin-2-yl]formimidate (12b)

Abstract

IV

• 2-Chloro-N-[3-cyano-4-(4-fluorophenyl)-7,7-dimethyl-5-oxo-1-(4-

sulfamoylphenyl)-1,4,5,6,7,8-hexahydroquinolin-2-yl]acetamide (13a)

• 2-Chloro-N-[4-(4-chlorophenyl)-3-cyano-7,7-dimethyl-5-oxo-1-(4-

sulfamoyl-phenyl)-1,4,5,6,7,8-hexahydroquinolin-2-yl]acetamide (13b)

• 4-[2-(Chloromethyl)-5-(4-fluorophenyl)-8,8-dimethyl-4,6-dioxo-

3,4,6,7,8,9-hexahydropyrimido[4,5-b]quinolin-10(5H)-

yl]benzenesulfonamide (14a)

• 4-[2-(Chloromethyl)-5-(4-chlorophenyl)-8,8-dimethyl-4,6-dioxo-


yl]benzenesulfonamide (14b)

• 4-[2-(Cyanomethyl)-5-(4-fluorophenyl)-8,8-dimethyl-4,6-dioxo-


yl]benzenesulfonamide (15a)

• 4-[5-(4-Chlorophenyl)-2-(cyanomethyl)-8,8-dimethyl-4,6-dioxo-



• 4-[3-Cyano-2-(3-ethylthioureido)-4-(4-fluorophenyl)-7,7-dimethyl-5-oxo-

5,6,7,8-tetrahydroquinolin-1(4H)-yl]benzenesulfonamide (16a)

• 4-[4-(4-Chlorophenyl)-3-cyano-2-(3-ethylthioureido)-7,7-dimethyl-5-oxo-

5,6,7,8-tetrahydroquinolin-1(4H)-yl]benzenesulfonamide (16b)

Abstract

V

• 4-[3-Cyano-2-(2,5-dioxopyrrolidin-1-yl)-4-(4-fluorophenyl)-7,7-dimethyl-

5-oxo-5,6,7,8-tetrahydroquinolin-1(4H)-yl]benzenesulfonamide (17a)

• 4-[4-(4-Chlorophenyl)-3-cyano-2-(2,5-dioxopyrrolidin-1-yl)-7,7-dimethyl-

5-oxo-5,6,7,8-tetrahydroquinolin-1(4H)-yl]benzenesulfonamide (17b)

• 2-Amino-4-(4-fluorophenyl)-7,7-dimethyl-5-oxo-1-(4-sulfamoyl-phenyl)-

1,4,5,6,7,8-hexahydroquinoline-3-carboxamide (18a)

• 2-Amino-4-(4-chloro-phenyl)-7,7-dimethyl-5-oxo-1-(4-sulfamoylphenyl)-

1,4,5,6,7,8-hexahydro-quinoline-3-carboxamide (18b)

• 4-[2-Amino-3-(4,5-dihydro-1H-imidazol-2-yl)-4-(4-fluorophenyl)-7,7-

dimethyl-5-oxo-5,6,7,8-tetrahydroquinolin-1(4H)-yl]benzenesulfonamide

(19a)

• 4-[2-Amino-4-(4-chlorophenyl)-3-(4,5-dihydro-1H-imidazol-2-yl)-7,7-

dimethyl-5-oxo-5,6,7,8-tetrahydroquinolin-1(4H)-yl]benzenesulfonamide

(19b)

• N-[3-Cyano-4-(4-fluorophenyl)-7,7-dimethyl-5-oxo-1-(4-sulfamoyl-

phenyl)-1,4,5,6,7,8-hexahydroquinolin-2-yl]-3-oxobutanamide (20a)

• N-[4-(4-chlorophenyl)-3-cyano-7,7-dimethyl-5-oxo-1-(4-

sulfamoylphenyl)-1,4,5,6,7,8-hexahydroquinolin-2-yl]-3-oxobutanamide

(20b)

Abstract

VI

• 4-[5-(4-Fluorophenyl)-8,8-dimethyl-6-oxo-4-thioxo-3,4,6,7,8,9-

hexahydropyrimido[4,5-b]quinolin-10(5H)-yl]benzenesulfonamide (21a)

• 4-[5-(4-Chlorophenyl)-8,8-dimethyl-6-oxo-4-thioxo-3,4,6,7,8,9-

hexahydro-pyrimido[4,5-b]quinolin-10(5H)-yl]benzenesulfonamide (21b)

• 4-[4-Chloro-5-(4-fluorophenyl)-8,8-dimethyl-6-oxo-6,7,8,9-tetrahydro-


• 4-[4-Chloro-5-(4-chlorophenyl)-8,8-dimethyl-6-oxo-6,7,8,9-tetrahydro-


• Ethyl 2-[5-(4-fluorophenyl)-8,8-dimethyl-4,6-dioxo-10-(4-sulfamoyl-

phenyl)-6,7,8,9-tetrahydropyrimido[4,5-b]quinolin-3(4H,5H,10H)-

yl]acetate (23a)

• Ethyl 2-[5-(4-chlorophenyl)-8,8-dimethyl-4,6-dioxo-10-(4-

sulfamoylphenyl)-6,7,8,9-tetrahydropyrimido[4,5-b]quinolin-

3(4H,5H,10H)-yl]acetate (23b)

• 4-(5-[4-Fluorophenyl)-4-hydrazinyl-8,8-dimethyl-6-oxo-6,7,8,9-

tetrahydro-pyrimido[4,5-b]quinolin-10(5H)-yl]benzenesulfonamide (24a)

• 4-[5-(4-Chlorophenyl)-4-hydrazinyl-8,8-dimethyl-6-oxo-6,7,8,9-

tetrahydropyrimido[4,5-b]quinolin-10(5H)-yl]benzenesulfonamide (24b)

• 4-[5-(4-Fluorophenyl)-4-isothiocyanato-8,8-dimethyl-6-oxo-6,7,8,9-

tetrahydropyrimido[4,5-b]quinolin-10(5H)-yl]benzenesulfonamide (25a)

Abstract

VII

• 4-[5-(4-Chlorophenyl)-4-isothiocyanato-8,8-dimethyl-6-oxo-6,7,8,9-

tetrahydro-pyrimido[4,5-b]quinolin-10(5H)-yl]benzenesulfonamide (25b)

5. Biological activity

Forty new synthesized compounds were evaluated for their in vitro

anticancer activity against human breast cancer cell line (MCF7), alone or in

combination with �-irradiation. The results are presented and discussed.

6. Molecular Docking

This part includes the docking of the synthesized compounds in the active

site of carbonic anhydrase enzyme to give an idea if these compounds could act

as carbonic anhydrase inhibitors or not as this may have a role in their anticancer

activity.

7. References

This part includes 137 references.

INTRODUCTION

Introduction

1

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1.1. Sulfonamides as anticancer agents

Sulfonamides constitute an important class of drugs, with several types of

pharmacological activities including antibacterial,1 anti-carbonic anhydrase,2

diuretic,3 hypoglycemic4 and antithyroid activity.5 Also, some structurally novel

sulfonamide derivatives have recently been reported to show substantial

antitumor activity in vitro and/or in vivo.

E7010 I, ER-34410 II and E7070 (Indisulam) III are examples for

antitumor sulfonamides in advanced clinical trials.6

There are a variety of mechanisms describing the antitumor action of

sulfonamides, such as carbonic anhydrase (CA) inhibition, cell cycle arrest in the

G1 phase, disruption of microtubules and angiogenesis (matrix metalloproteinase,

MMP) inhibition. The most prominent mechanism was the inhibition of carbonic

anhydrase isozymes (CAs).6

Introduction

2

1.1.1. Sulfonamides as carbonic anhydrase inhibitors

1.1.1.1. Carbonic anhydrases

The carbonic anhydrases are metalloenzymes containing zinc ion in their

active site. Carbonic anyhdrases are present in prokaryotes and eukaryotes, and

are encoded by four distinct gene families: the �-CAs, �-CAs, �-CAs and �-CAs.

In mammals the �-CA is present and 16 different �-CA isozymes or CA-related

proteins (CARP) were described with different subcellular localization and tissue

distribution.7, 8 Basically there are several cytosolic forms (CA I-III, CA VII and

CA XIII), five membrane-bound isozymes (CA IV, CA IX, CA XII, CA XIV and

CA XV), two mitochondrial isoforms (CA VA & VB) and CA VI is secreted in

the saliva and milk. Three cytosolic acatalytic forms are also known (CARP VIII,

CARP X and CARP XI).9

All these CAs are able to catalyze the hydration of CO2 to bicarbonate at

physiological pH (Figure 1).2, 9, 10 This chemical interconversion is crucial since

bicarbonate is the substrate for several carboxylation steps in a number of

fundamental metabolic pathways such as gluconeogenesis, biosynthesis of

several amino acids, lipogenesis, ureagenesis and pyrimidine synthesis.11 Apart

from these biosynthetic reactions, some of the CAs are involved in many

physiological processes related to respiration and transport of CO2/bicarbonate

between metabolizing tissues and the lungs, pH homeostasis and electrolyte

secretion in a variety of tissues/organs.2, 10

Introduction

3

Figure (1): Schematic representation of the catalytic mechanism for the �-CA

catalysed CO2 hydration. The hydrophobic pocket for the binding of substrate(s) is shown schematically at step (B)

1.1.1.2. Carbonic anhydrases inhibition

CA inhibitors are mainly used as antiglaucoma agents,3, 10, 12 anti-thyroid

drugs,12 hypoglycemic agents4 and, ultimately, some novel types of anticancer

agents.13 Sulfonamides are known to possess high affinity for CAs as such

compounds possess a zinc binding group (ZBG) by which they interact with the

metal ion in the active site of the enzyme and the residues Thr 199 and Glu 106

in its neighborhood.14 In recent years, an entire range of new ZBGs were reported

as shown in figure 2. These new ZBGs include, in addition to the classical

sulfonamides, sulfamates, sulfamides, substituted sulfonamide, Schiff’s base,

urea and hydroxyurea derivatives, as well as hydroxamates.8, 14

Introduction

4

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Figure (2): Zinc binding groups: sulfonamides, sulfamates, sulfamides, substituted sulfonamides, Schiff’s base, urea, hydroxyurea and hydroxamates.

A general pharmacophore (Figure 3)15 for the sulfonamide compounds

acting as carbonic anhydrase inhibitors has been reported by Thiry et al. This

pharmacophore originated from the analysis of the CAs active site and from the

structure of inhibitors described in literature.16

Figure (3): Structural elements of CA inhibitors in the CA enzymatic active site.

Introduction

5

1.1.1.3. Role of carbonic anhydrases in cancer

The importance of this family of enzymes for the uptake of bicarbonate by

many organisms, and the presence of a large number of isoforms (which can be

distinguished from each other in activity and are located in different areas inside

the cell) make the CAs undoubtedly involved in cell growth. Furthermore, at least

three CA isozymes (CA IX, CA XII and CA XIV) have close connections with

tumors.11, 17 The role of CAs in cancer can be explained in light of the metabolic

processes required by growing cancer cells that develop with a higher rate of

replication than normal cells. Such a circumstance requires a high flux of

bicarbonate into the cell in order to provide substrate for the synthesis of either

nutritionally essential components (nucleotides) or cell structural components

(membrane lipids).11

1.1.1.4. The action of sulfonamides as anticancer agents through CA inhibition

Sulfonamides CA inhibitors reduce the provision of bicarbonate for the

synthesis of nucleotides (mediated by carbamoyl phosphate synthetase II) and

other cell components such as membrane lipids (mediated by pyruvate

carboxylase). Such mechanism would likely involve CA II and CA V.18

An alternative, or additional mechanism, may involve the acidification of

intracellular milieu as a consequence of CA inhibition by these potent CA

inhibitors.19 It is also possible that the sulfonamides interfere with the activity of

the CA isozymes known to be present predominantly in tumor cells, CA IX, XII

and XIV.2, 10, 17 A combination of these mechanisms proposed above is also

possible.

Introduction

6

1.1.1.5. Examples of sulfonamides acting as anticancer agents by CA inhibition

Several potent, clinically used sulfonamide CA inhibitors such as

acetazolamide IV, methazolamide V, or ethoxzolamide VI have shown to inhibit

the growth of human lymphoma cells.18

A program of screening several hundred sulfonamide CA inhibitors (both

aromatic as well as heterocyclic derivatives) for their tumor cell growth

inhibitory effects against the panel of 60 cancer cell lines of the National Cancer

Institute of the USA, has identified derivatives VII-XI as interesting leads. These

derivatives showed potent activities, against a wide variety of cancer cell lines

including leukemia, non-small cell lung cancer, ovarian, melanoma, colon, CNS,

renal, prostate and breast cancer cell lines.20, 21

Introduction

7

1.1.1.6. Examples of Sulfonamides acting as selective CA inhibitiors

Several miscellaneous sulfonamides have been synthesized and screened by

Vullo et al.22 in an attempt to obtain selective CA inhibitors especially towards

the tumor-associated transmembrane CA IX and CA XII. From these compounds,

compounds XII-XIV were found to behave as very potent CA IX inhibitors,

although they are weak or medium-weak inhibitors of CA I, II and IV.

Casey et al.23 prepared a series of positively charged, membrane-

impermeant sulfonamide CA inhibitors XV-XVIII and showed high affinity for

the cytosolic isozymes CA I and CA II, as well as for the membrane-bound ones

CA IV and CA IX. As for the aminobenzolamide derivatives XV, it was seen that

the best CA IX inhibitors should contain either only small, compact aliphatic

Introduction

8

moieties substituting the pyridinium ring or a 4-phenyl moiety. On the other

hand, the SAR of the derivatives of compounds XVI-XVIII has shown that for a

given substitution pattern of the pyridinium ring, the activity decreases by

decreasing the spacer (n) between the pyridinium and the benzenesulfonamide

moieties. Compounds XIX and XX are the most potent CA IX inhibitors in this

group of compounds.23, 24

Ex vivo studies showed that this new class of inhibitors is able to

discriminate between the membrane-bound versus the cytosolic isozymes. These

compounds were proved to be unable to penetrate through the membranes,

obviously due to their cationic nature. These compounds constitute the basis of

selectively inhibiting only the target, tumor-associated CA IX in vivo, whereas

the cytosolic isozymes would remain unaffected.23

Polyfluorinated CAIs have shown very good inhibitory properties against

different CA isozymes,25 but such compounds have not been tested for their

interaction with the transmembrane, tumor associated isozyme CA IX. Also there

Introduction

9

was a problem related to the pentafluorophenyl-containing CAIs previously

reported,25 which is the high reactivity of the fluorine atom in para to the

sulfonamide/carboxamido moiety, which was shown to covalently bind to thiol

reagents, such as Cys 239 of �-tubulin, glutathione, cysteine itself, etc., leading

to modification of the thiol reagent/protein.26-28

In order to prepare fluorine-containing CAIs devoid of enhanced reactivity,

miscellaneous derivatives lacking the para-fluorine reactive group, with two

types of such derivatives being prepared: the 2,3,5,6-tetrafluorophenyl-

carboxamides, and the 2,3,5,6-tetrafluoro-phenyl-sulfonamides. All these

compounds showed potent CA II and CA IX inhibition than CA I. Among these,

the first subnanomolar and rather selective CA IX inhibitor has been discovered,

as the 2,3,5,6-tetrafluorobenzoyl derivative of metanilamide XXI showing an

inhibition constant of 0.8nM against hCA IX, and a selectivity ratio of 26.25

against CA IX over CA II.29

The inhibition of a newly cloned hCA XII – the second tumor-associated

CA isozyme described, after CA IX – has been investigated by Vullo et al.29 with

a miscellaneous series of sulfonamides. CA XII is present in a rather wide range

of tumors and it appears probable that inhibition of this isozyme with potent and

possibly specific inhibitors may have clinical relevance for the development of

novel antitumor therapies. The most selective hCA XII over hCA II inhibitors

were compounds XII, XXII and XXIII.

Introduction

10

A series of indanesulfonamide derivatives XXIV and XXV were

synthesized by Thiry et al.15 and tested for their inhibitory activity against CA IX,

and against CA I and II and two other physiologically relevant CA isozymes, to

measure the selectivity of the compounds. Nearly all the derivatives show a high

inhibitory potency against CA IX and CA II and a weak inhibition against CA I.

Compounds XXVI-XXIX are selective against CA IX with reference to CA I,

while they preferentially inhibit CA IX with reference to CA II.

Introduction

11

1.1.2. Sulfonamides targeting G1 phase of cell cycle

G1 phase of the cell cycle is an important period where various complex

signals interact to decide a cell’s fate: proliferation, quiescence, differentiation, or

apoptosis (Figure 4).30

Figure (4): The cell cycle

It is now well-recognized that malfunctioning of cell cycle control in G1

phase is among the most critical molecular bases for tumorigenesis and tumor

progression. Thus, there is a growing possibility that a small molecule targeting

the control machinery in G1 phase can be a new type of drug efficacious against

refractory clinical cancers.31

E7070 III was found to block the entry of human NSCLC A549 cells into

the S phase, leading to the accumulation of cells in the late G1 phase. In addition,

treatment of A549 cells with E7070 resulted in the inhibition of pRb

phosphorylation, a crucial step in the G1/S transition. Down regulation of CDK2

and cyclin A expressions as well as suppression of CDK2 catalytic activity with

Introduction

12

the induction of p53 and p21, may account for the inhibition of pRb

phosphorylation by E7070.32

In addition, E7070 was shown to inhibit the phosphorylation of CDK2 itself,

leading to the inhibiton of CDK2 catalytic activity. These results suggest that

E7070 may target the late G1 phase of the cell cycle and restore the pRb-

dependent growth-inhibitory pathway disrupted in human NSCLC cells. This is

accomplished by inhibiting CDK2 catalytic activity.32

1.1.3. Sulfonamides causing disruption of microtubules

The effects of E7010 on microtubule structure in colon 38 cells were

examined, and E7010 was shown to cause the disappearance of cytoplasmic

microtubules and mitotic spindles. The experiments clearly demonstrated that the

growth-inhibitory activity of E7010 is caused by the inhibition of microtubule

assembly.33

A novel series of 7-aroylaminoindoline-1-benzenesulfonamides have been

identified by Chang et al.34 as a novel class of highly potent antitubulin agents.

The lead compounds XXX and XXXI exhibit antiproliferative activity, with IC50 values ranging from 8.6 to 11.1 nM in a variety of human cancer cell lines from

different organs, including the MDR-positive cell line. The SAR information of

the 7-aminoindoline-substitution pattern revealed that the 7-amide bond

formation in the indoline-1-sulfonamides contributed to a significant extent for

maximal activity rather than the carbamate, carbonate, urea, alkyl, and

sulfonamide linkers.

Introduction

13

Hu et al.35 have synthesized two series of carbazole sulfonamide

compounds. Compounds such as XXXII and XXXIII exhibit strong activities

against human leukemia cells. Preliminary mode of action studies demonstrated

that the lead compound XXXII arrests tumor cell cycle at M-phase and induces

apoptotic cell death by increasing expression of p53 and promoting bcl-2

phosphorylation. Lead compounds such as XXXII and XXXIII merit further

studies as novel promising antimitotic agents against solid tumors.

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1.1.4. Sulfonamides as matrix metalloproteinase (MMP) inhibitors

The matrix metalloproteinases (MMPs), a family of zinc-containing

endopeptidases, were shown to play a central role in several physiological and

physiopathological processes and in our main interest regards the involvement of

these enzymes in angiogenesis and tumor invasion.36, 37

Introduction

14

At least 20 members of this enzyme family, sharing significant sequence

homology, have been reported. They consist of a Zn(II) ion coordinated by three

histidines, with the fourth ligand being a water molecule/hydroxide ion which is

the nucleophile intervening in the catalytic cycle of the enzyme.38

Inhibition of MMPs is correlated with the coordination of the inhibitor

molecule (in neutral or ionized state) to the catalytic metal ion, with or without

replacement of the metal-bound water molecule.10, 37, 39 Thus, MMP inhibitors

(MMPIs) must contain a zinc-binding function attached to a scaffold that will

interact with other binding regions of the enzymes.38

Sulfonylated amino acid hydroxamates were only recently discovered to act

as efficient MMPIs.40, 41 The first compounds from this class to be developed for

clinical trials are (CGS 27023A) XXXIV and (CGS 25966) XXXV.40 Further

developments in this field have led to some strong and relatively selective

inhibitors of this type such as XXXVI.41 Then a large number of arylsulfonyl

hydroxamates derived from glycine, L-alanine, L-valine and L-leucine possessing

N-benzyl- or N-benzyl-substituted moieties, of types XXXVII, were reported.42

Also, some hydroxamates structurally related to the MMPIs were shown to act as

CAIs XXXVIII.38

Introduction

15

Introduction

16

1.2. Quinolines as anticancer agents

Quinolines and fused quinoline derivatives are known to possess several

biological activities43-49 including anticancer activity.50-57 There are a variety of

mechanisms for the antitumor action of quinolines and fused quinoline

derivatives such as DNA intercalation,58, 59 topoisomerase inhibition,60, 61 cell

cycle arrest,62 and several other mechanisms.

Recently, some quinoline derivatives such as compound XXXIX have been

found to act as PI3 kinase inhibitors. The PI3 kinases are a family of enzymes

displaying a protein kinase activity which mediate signaling pathways having a

central role in a number of cell processes including proliferation and survival.

Deregulation of these pathways is a causative factor in a wide spectrum of human

cancers and other diseases.63

The development of cancer can depend on the accumulation of specific

genetic alterations that allow aberrant cell proliferation, including growth of

tumor cells. Protection from such aberrant growth is provided by several

mechanisms that work by inducing apoptotic cell death in cells undergoing

oncogenic changes. Therefore, for a tumor cell to survive, it must acquire genetic

alterations that perturb the link between abnormal growth and cell death. The p53

tumor suppressor protein can induce apoptotic cell death and plays a pivotal role

Introduction

17

in tumor suppression. The pyrimidoquinoline derivatives such as compound XL

have shown significant antitumor activity by modulating or stabilizing p53

activity.64

Also some reduced quinoline derivatives have shown anticancer activity as

that reported by Liou et al.65 who synthesized tetrahydroquinoline derivatives

such as compound XLI which were evaluated for their antiproliferative activities

against oral epidermoid carcinoma KB cells, non-small lung carcinoma H460

cells, and stomach carcinoma MKN45 cells, as well as one type of MDR-positive

cell line, KB-vin 10 cells. Compound XLI showed significant antiproliferative

activities against the previously mentioned four cell lines, and acting through the

inhibition of tubulin polymerization.

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Introduction

18

Some other tetrahydroisoquinoline derivatives were also found to act as

antiestrogens and antiandrogens such as compound XLII used for treatment of

breast and prostate cancer. It is commonly acknowledged that estrogen and the

estrogen receptors play essential roles in the development of breast tumors,

although the precise mechanisms involved have not been determined. Estrogen

receptors also play a role in prostate cancer, thus agents that modulate estrogen

receptors may also be useful in treatment of prostate cancer. Also in the early

stage of prostate cancer, its growth highly relies on the androgen and the use of

antiandrogen deprivation therapy can strongly slow down the growth rate of

prostate cancer.66

Introduction

19

1.3. Chemoradiotherapy

Chemoradiotherapy (CRT) represents definite progress in clinical oncology.

Recently, the concurrent use of chemotherapy (CT) and radiation therapy (RT)

has become a standard treatment for many types of cancer.

1.3.1. Therapeutic ratio

In general, tumor response and normal tissue damage are positively

correlated with the dose of radiation, and this relationship is commonly described

by a sigmoid curve (Figure 5). The therapeutic ratio is defined as the ratio of the

dose that produces a given probability (50% is most commonly used in

experimental studies) of normal tissue damage and the dose that produces the

same probability of tumor control. When CT is combined with RT, the tumor

control curve shifts to the left, along with the response curve for normal tissue

damage. The goal of combining CT and RT is to obtain a positive therapeutic

ratio, and thus to enhance the antitumor effect while minimizing toxicity to

critical normal tissues.67

Figure (5): Dose-response curves for tumor control and normal tissue damage.

When chemotherapy (CT) is combined with radiotherapy (RT), the tumor control curve shifts to the left (long arrow), and the response curve for normal tissue

damage also shifts in the same direction, as indicated by the short arrow.

Introduction

20

1.3.2. Rationale for combining chemotherapy and radiotherapy

The rationale for combining CT and RT is mainly based on two ideas,68-70

one being spatial cooperation, and the other the enhancement of radiation effects.

Spatial cooperation is effective if CT is sufficiently active to eradicate subclinical

metastases and if the primary local tumor is effectively treated by RT. In this

regard, no interaction between RT and CT is required, but differing toxicities are

needed so that both modalities can be used at effective dosages.

A major limitation is the relatively poor efficacy of anticancer drugs against

common solid tumors in adults. It is often difficult to eradicate even small

subclinical metastases by CT. Also, local failure rates of a primary tumor

following RT are high for many tumor sites.

To decrease the local failure rate, the enhancement of RT effects is

necessary. In the presence of chemotherapeutic drugs, an increased response such

as enhancement occurs within the irradiated volume. However, virtually all

chemotherapeutic agents enhance radiation damage to normal tissues as well.

Consequently, a therapeutic benefit is only achieved if enhancement of the tumor

response is greater than that for normal tissues.

Among the many chemotherapeutic agents used, cisplatin is one of the best

agents for yielding a therapeutic benefit. An enhancing effect by the additional

use of daily cisplatin before each RT fraction was observed in an in vivo animal

study.71

Introduction

21

1.3.3. Mechanisms responsible for CT-RT interactions

Recent clinical trials, including metaanalyses, have shown that CT given

concurrently with RT results in improved local control and survival,72-76 implying

interactions between CT and RT. Five major mechanisms responsible for CT-RT

interactions are discussed in the following paragraphs68-70;

1.3.3.1. Initial radiation damage

The first mechanism responsible for CT-RT interaction is the direct

enhancement of the initial radiation damage, resulting from the incorporation of

the chemotherapeutic drugs into DNA. The primary target for radiation injury is

DNA, where halogenated pyrimidines such as 5-fluorouracil (5-FU) are

incorporated, making the DNA more susceptible to RT. Cisplatin interacts with

nucleophilic sites on DNA or RNA to form intra- and interstrand cross-links.

When cisplatin-DNA cross-links are formed during RT, radioenhancement by

cisplatin may occur. This has been observed in both hypoxic and oxygenated

cells.70

1.3.3.2. Inhibition of radiation damage repair

Secondly, the inhibition of cellular repair increases radiation damage. Cells

have the ability to repair sublethal and potentially lethal radiation damage.68

Halogenated pyrimidines, nucleoside analogs, and cisplatin interfere with cellular

repair mechanisms. This inhibition of cellular repair can be effective when drugs

are administered following fractionated RT. In general, nucleoside analogs such

as fludarabine and gemcitabine are potent radiosensitizers. In animal

experiments, the effect of fludarabine on radiocurability was greater when

fludarabine was combined with fractionated RT than when it was combined with

single-dose RT.77 This implies that the inhibition of sublethal or potentially lethal

Introduction

22

damage repair is a significant mechanism responsible for the enhancement of the

tumor radioresponse to fludarabine.

1.3.3.3. Cell-cycle effects

The third mechanism focuses on a cell-cycle effect. The cytotoxicity of most

chemotherapeutic agents and that of radiation is highly dependent on the phase of

the cell cycle. Both chemotherapeutic agents and radiation are more effective

against proliferating cells than against nonproliferating cells. Among

proliferating cells, cells in the G2 and M phases are the most radiosensitive, and

the cells in the S phase are the most radioresistant.68 Based on this variation in

radiosensitivity over the cell cycle, there exist two strategies for CRT, the use of

chemotherapeutic agents that accumulate cells in a radiosensitive phase or those

that eliminate radioresistant S-phase cells. The latter strategy is related to the

mode of action of nucleoside analogs. Fludarabine and gemcitabine are

incorporated into radioresistant S-phase cells, many of which die by apoptosis.

This preferential removal of S-phase cells therefore contributes to the

radioenhancement effects.

1.3.3.4. Hypoxic cells

Hypoxic cells are 2.5–3.0 times less sensitive to radiation than well-

oxygenated cells.68, 69 Tumors often include hypoxic areas, which is a cause of

radioresistance. Chemotherapeutic agents can improve the RT effect by

eliminating well-oxygenated tumor cells, which leads to tumor reoxygenation,

selectively eliminating hypoxic cells, or sensitizing the hypoxic cells to radiation.

1.3.3.5. Repopulation of tumor cells

The importance of the overall treatment time (OTT) for local tumor control

by RT has been documented in a number of studies of head and neck cancers,

uterine cervical cancer, and esophageal cancer.78-80 Withers and colleagues80

Introduction

23

found that the TCD50 (the radiation dose which yields local control in 50% of

tumors) progressively increased over time if the OTT was prolonged beyond 30

days.80 Their analysis of esophageal and laryngeal squamous cell carcinomas

treated by RT alone showed that the prolongation of OTT significantly reduced

the local control rate.78, 79 One mechanism responsible for this may be the

accelerated repopulation of tumor cells during fractionated RT.

Any approach that reduces or eliminates the accelerated repopulation of

tumor cells improves the efficacy of RT. This is likely to be one of the major

mechanisms by which CT improves local tumor control when given concurrently

with RT. Even a small decrease in repopulation between radiation fractions can

significantly improve the tumor response to fractionated RT. However, most

chemotherapeutic drugs inhibit repopulation not only in the tumor, but also in the

compensatory cell regeneration of normal tissues that occurs during fractionated

RT. Thus, a therapeutic benefit is expected if drugs are tumorselective or if

repopulation is faster in the tumor.

Recently, various molecular targeting drugs have become clinically

available. Several drugs, such as epidermal growth factor receptor (EGFR)

inhibitors, block the membrane receptors of growth factors or interfere with the

signaling pathways involved in cell proliferation. These agents offer another

possible method for inhibiting the accelerated repopulation of tumor cells during

fractionated RT.81, 82

1.3.4. Sequencing of CT and RT

According to the sequencing of CT and RT, CT is designated as either

induction (neoadjuvant) CT, concurrent CT, or adjuvant CT, when it is given

before, during, or after the course of RT, respectively. As clinical trials of

Introduction

24

adjuvant CT following RT have not been systematically studied, the aims and

clinical results of induction CT and concurrent CT are described in the following

paragraphs;

1.3.4.1. Induction chemotherapy

Induction CT has two main objectives,68, 69 one being the eradication of

micrometastases while they contain small numbers of tumor cells, and the other

being to reduce the size of the primary tumor that is to be irradiated. Reducing

the number of clonogenic cells in the tumor increases the probability of tumor

control by RT. In addition, CT induced tumor shrinkage may provide a smaller

target volume for RT, thereby limiting normal tissue damage.

1.3.4.2. Concurrent chemotherapy

For concurrent CRT, CT can act on both systemic and primary lesions.

However, the main objective of concurrent CT is to use CT-RT interactions to

maximize the antitumor effect, even though it inevitably increases the acute

toxicity of the treatment.68-70 Therefore, it should be remembered that the

therapeutic benefit of concurrent CRT only occurs when enhancement of the

tumor response is greater than the toxic effects on critical normal tissues.

For one mode of concurrent CRT, an alternating schedule of CT and RT can

be used.75, 83 For this combination, RT and CT are given alternatively, without a

treatment gap, to minimize excessive toxic effects on normal tissues and to

enhance the tumor response by perturbing cell cycling or reoxygenation. Several

clinical trials involving alternating schedules have yielded promising results.83

Introduction

25

1.4. Synthesis of quinolines

Several synthetic pathways were reported in the literature for the synthesis

of quinoline derivatives;

1.4.1. From aniline derivatives

1.4.1.1. From m-phenylenediamine

7-Amino-4-methyl-quinoline-2-one XLIV was prepared from m-phenylene

diamine XLIII and ethyl acetoacetate.84

1.4.1.2. From substituted anilines

Substituted 2,4-dimethoxyquinolines XLVII were synthesized by cyclo-

condensation of the appropriate substituted anilines XLV with malonic acid and

phosphorus oxychloride to give the 2,4-dichloroquinolines XLVI, followed by

displacement by methoxide ion to give the required quinolines XLVII.85

Introduction

26

1.4.1.3. From acetanilide derivatives

Treatment of acetanilide derivatives XLVIII with Vilsmeier reagent

(DMF/POCl3) gave 2-chloro-3-formylquinolines XLIX in excellent yield 86.

1.4.1.4. From 2-aminobenzonitrile

Warshakoon et al. synthesized the quinoline derivative LI via a Gould-

Jacobs condensation of 2-amino-benzonitrile L with diethyl ethoxymethylene-

malonate. The resulted quinolone LI was then converted to the quinoline

derivative LII.87

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Introduction

27

1.4.1.5. From o-isopropenylaniline

The reaction of o-isopropenylaniline LIII with trifluoro-acetaldehyde ethyl

hemiacetal (TFAE) was carried out in toluene to give 2-trifluoromethylquinoline

derivative LIV and 2-trifluoro-methyl-1,2-dihydroquinoline derivative LV.88

1.4.2. From vanillin

It was reported that alkylation of vanillin LVI with 2-chloroethyl methyl

ether followed by nitration provided compound LVII. Condensation of LVII

with methyl cyanoacetate and subsequent reduction with iron in acetic acid

provided the quinoline derivative LVIII.89

Introduction

28

Also, compound LVII was reacted with malononitrile, followed by

reduction with iron in acetic acid provided the quinoline derivative LIX.89

1.4.3. From 2-aminobenzophenone

Condensation of 2-aminobenzophenone LX with ethyl acetoacetate in the

presence of a catalytic amount of yttrium triflate [Y(SO3CF3)3] at room temp-

erature results in the formation of ethyl 2-methyl-4-phenylquinoline-3-

carboxylate LXI in 92% yield. Similarly, various cyclic ketones such as

cyclopentanone, cyclohexanone and dimedone reacted with 2-aminoaryl ketones

to afford the respective tricyclic quinolines LXII, LXIII and LXIV.90, 91

Introduction

29

1.4.4. Via Diels-Alder reaction

Quinolinediones LXVII were prepared via the Diels-Alder reactions of the

corresponding azadienes LXVI with the desired dienophiles LXV.92

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1.4.5. From cyclohexanone

Ghorab et al. reported that the reaction of aromatic aldehydes with ethyl

cyanoacetate and cyclohexanone in the presence of ammonium acetate yielded

the corresponding tetrahydroquinoline derivatives LXVIII.93

Introduction

30

In addition 2-amino-4-(2-bromophenyl)-5,6,7,8-tetrahydro-quinoline-3-

carbonitrile LXIX was obtained via reaction of 2-bromobenzaldehyde with

malononitrile and cyclohexanone in presence of ammonium acetate.94

1.4.6. From enaminones

The hexahydroquinoline derivative95 LXXI was obtained through reaction of

enaminone LXX with activated cyano olefins in refluxing ethanolic piperidine.96

Also, it was mentioned that the treatment of 5,5-dimethyl-3-(naphthalene-1-

ylamino)-cyclohex-2-enone LXXII with 2-arylidene-malononitriles in the

presence of a catalytic amount of triethylamine resulted in cycloaddition

affording the hexahydroquinolines LXXIII.97

Introduction

31

Additionally, it was reported that the reaction of enaminone LXXII with

ethyl �-cyano-�-ethoxyacrylate by refluxing in ethanol afforded a high yield of 2-

cyano-3-[4,4-dimethyl-2-(naphthalen-1-ylamino)-6-oxo-cyclohex-1-enyl]-acrylic

acid ethyl ester LXXIV which upon heating in glacial acetic acid, afforded the

corresponding quinoline derivative LXXV.97

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Introduction

32

1.4.7. From 3,1-Benzoxazines

Ghorab et al. reported that the reaction of 2-methyl-6-iodo-3,1-benzoxazine

LXXVI with active methylene compounds, namely malononitrile, acetylacetone

or diethylmalonate, gave the corresponding quinoline derivatives LXXVII,

LXXVIII and LXXIX, respectively.98

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1.4.8. From 3-aminocyclohex-2-en-1-one

It has been reported that the 3-aminocyclo-hex-2-en-1-one LXXX99 readily

reacted with 2-benzylidenemalononitrile, in the presence of catalytic amounts of

piperidine, to afford the hexahydroquinoline derivative LXXXI.100

Introduction

33

1.4.9. From Dimedone

The quinoline derivatives LXXXII were prepared via reaction of 2-

arylidenemalononitrile with dimedone and excess of ammonium acetate in acetic

acid as a solvent.101

On the other hand, hexahydroquinoline derivatives LXXXIIIa and

LXXXIIIb were synthesized through condensation of dimedone and aromatic

aldehydes with either methyl 3-aminocrotonate or ethyl acetoacetate in the

presence of ammonium acetate, respectively.102, 103

Introduction

34

1.5. Synthesis of pyrimido[4,5-b]quinolines

Several synthetic pathways were reported in the literature for the synthesis

of pyrimido[4,5-b]quinoline derivatives;

1.5.1. From quinolines

1.5.1.1. From 2-chloro-3-formylquinolines

The 2-oxo-pyrimido[4,5-b]quinoline derivative LXXXV was synthesized by

the condensation reaction of 2-chloro-3-formylquinoline LXXXIV with urea in

the presence of p-toluene sulfonic acid, using microwave irradiation for 5 min.104

1.5.1.2. From 2-aminoquinoline-3-carboxylic acid methyl ester

Cyclization of the quinoline derivative LVIII with formamide at high

temperature gave the corresponding pyrimido[4,5-b]quinoline derivative

LXXXVI.89

Introduction

35

1.5.1.3. From 2-amino-3-cyanoquinolines

Several cyclization reactions were reported for the synthesis of

pyrimido[4,5-b]quinoline derivatives from the 2-amino-3-cyano-quinoline

derivative LXXXVII. When compound LXXXVII was refluxed with either

acetic anhydride, formic acid or formamide, the corresponding pyrimido[4,5-

b]quinoline derivatives LXXXVIII, LXXXIX and XC were obtained,

respectively.97

The 2-thioxo-pyrimido[4,5-b]quinoline XCI was obtained by the reaction of

compound LXXXVII with phenyl isothiocyanate in pyridine.97

Introduction

36

It has been reported that the reaction of compound LXXXVII with

diethyloxalate in ethanol containing sodium ethoxide furnished the 4-ethoxy-

pyrimido[4,5-b] quinoline-2-carboxylic acid ethyl ester XCII.97

The reaction of the quinoline derivatives LXXXVII with benzoylchloride

gave the corresponding 2-phenyl-pyrimido[4,5-b]quinoline derivative XCIII.97

Introduction

37

1.5.2. From pyrimidines

1.5.2.1. From 2,4,6-triaminopyrimidine

Reaction of guanidine nitrate with malononitrile in the presence of sodium

alkoxide in dry ethanol or methanol yielded 2,4,6-triaminopyrimidine XCIV.

Reaction of the pyrimidine derivative XCIV with 2,4-dichlorobenzoic acid, in the

presence of activated copper bronze powder at 180-190°C yielded N-(2,4-

diamino-6-pyrimidino)-4-chloroanthranilic acid XCV. The 2,4-diaminopyrimi-

do[4,5-b]quinoline derivative XCVI was then obtained upon cyclization of XCV

using concentrated sulfuric acid.105

1.5.2.2. From 6-aminopyrimidines

A simple and efficient approach to prepare the pyrimido[4,5-b]quinolines

XCVIII was described in a three components reaction from 6-aminopyrimidines

XCVII, dimedone and aromatic aldehydes.106, 107

AIM OF THE PRESENT INVESTIGATION

Aim of the present investigation

38

� � ��

Several structurally novel sulfonamide derivatives have been recently

reported to show substantial anticancer activity in vitro and/or in vivo 22, 108-117. In

order to explain this antitumor activity, several mechanisms were adopted

including carbonic anhydrase inhibition, cell cycle arrest at G1 phase, disruption

of microtubules, and angiogenesis inhibition. The most prominent among these

mechanisms was carbonic anhydrase inhibition.6

On the other hand, quinolines and fused quinoline derivatives are known to

possess several biological activities43-49 including anticancer activity.50-57

Furthermore, it has been reported that some quinoline and pyrimidoquinoline

derivatives containing a sulfonamide moiety exhibit certain anticancer

activity.118, 119

Also, the special properties of the fluorine atom, such as strong

electronegativity, small size and the low polarisability of the C–F bond, can have

considerable impact on the behavior of a molecule in a biological environment.120

The incorporation of fluorine into a drug allows simultaneous modulation of

electronic, lipophilic and steric parameters, all of which can critically influence

both the pharmacodynamic and pharmacokinetic properties of drugs. Bioisosteric

substitution for hydrogen by fluorine is, therefore, an important strategy for

incorporation of a group capable of reinforcing drug–receptor interactions

(electronic modulation), aiding translocation across lipid bilayers or absorption

(lipophilic modulation) and inducing conformational change/blocking

metabolism (steric parameters).121


39

Based on the above information, and as a continuation of our previously

reported work,122 the present investigation deals with the design, synthesis and in

vitro anticancer evaluation of some new 4-haloarylquinolines and pyrimido[4,5-

b]quinolines, having a free sulfonamide moiety. Recently, some newly

synthesized sulfonamide derivatives were reported to exhibit promising in vitro

cytotoxic activity against human breast cancer cell line (MCF7), in comparison

with doxorubicin and other anticancer drugs.26, 119, 123, 124

As the carbonic anhydrase inhibition is the most prominent mechanism of

the antitumor activity of sulfonamide derivatives, the synthesized compounds

were designed to comply with the previously mentioned pharmacophore of

compounds that may act as CA inhibitors (Figure 3), as this may have a role in

their anticancer activity together with the other anticancer mechanisms of

sulfonamides.

This pharmacophore includes mainly the presence of a sulfonamide moiety

which coordinates with the zinc ion of the active site and the sulfonamide is

attached to an aryl scaffold which is usually a benzene ring. The side chain might

posses a hydrophilic link able to interact with the hydrophilic part of the active

site and a hydrophobic moiety which can interact with the hydrophobic part of

the CA active site.

Figure 6 includes representative examples of the designed compounds,

showing the compliance with the general pharmacophore.


40

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�� Figure (6): Representative examples of the synthesized compounds showing

great compliance to the general pharmacophore of CA inhibitors

Docking of the synthesized compounds will be done on hCA in order to give

an idea if these compounds may act as carbonic anhydrase inhibitors which could

have a role, at least in one part, to their anticancer activity.

The present work reports the design, synthesis, and anticancer evaluation of

the following classes:


41

i. Hexahydroquinoline derivatives 6, 8, 9, 12, 13 and 16-20, bearing a free

sulfonamide moiety

ii. Pyrimido[4,5-b]quinoline derivatives 7, 10, 11, 14, 15 and 21-25, bearing a

free sulfonamide moiety


42

On the other hand, the rationale for combining chemotherapy and

radiotherapy is based mainly on two ideas, one being spatial cooperation, which

is effective if chemotherapy is sufficiently active to eradicate subclinical

metastases and if the primary local tumor is effectively treated by radiotherapy.

In this regard, no interaction between radiotherapy and chemotherapy is required.

The other idea is the enhancement of radiation effects by direct enhancement of

the initial radiation damage by incorporating drugs into DNA, inhibiting cellular

repair, accumulating cells in a radiosensitive phase or eliminating radioresistant

phase cells, eliminating hypoxic cells, or inhibiting the accelerated repopulation

of tumor cells. Virtually, all chemotherapeutic agents have the ability to sensitize

cancer cells to the lethal effects of ionizing radiation.67

Consequently, the most two active fluorinated compounds and the most two

active chlorinated compounds, will be selected to be evaluated for their ability to

enhance the cell killing effect of �-irradiation.

THEORETICAL DISCUSSION

Theoretical discussion

43

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Schemes 1-5 illustrate the pathways for the synthesis of the target

compounds.

Scheme 1


44

Scheme 2


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Scheme 5


48

4-(5,5-Dimethyl-3-oxocyclohex-1-enylamino)benzenesulfonamide (3)

Compound 3 was prepared according to the published procedure.122

4-[2-Amino-3-cyano-4-(4-fluorophenyl)-7,7-dimethyl-5-oxo-5,6,7,8-

tetrahydroquinolin-1(4H)-yl]benzenesulfonamide (6a) and 4-[2-amino-4-(4-

chlorophenyl)-3-cyano-7,7-dimethyl-5-oxo-5,6,7,8-tetrahydroquinolin-1(4H)-


Treatment of enaminone 3 with 2-(4-fluorobenzylidene)malononitrile 4a or

2-(4-chlorobenzylidene)malononitrile 4b in presence of a catalytic amount of

triethylamine (TEA), as a basic catalyst, yielded the corresponding

hexahydroquinolines 6a and 6b respectively, via the formation of the

intermediates 5a,b, followed by intramolecular cyclization. The arylidenes 4a

and 4b were prepared by just stirring the corresponding aldehyde with

malononitrile for about 20 min in ethanol containing few drops of TEA at room

temperature.122


49

The N-aryl substituent (benzenesulfonamide) decreases the nucleophilicity

of the enaminone 3 toward 2-arylidene-malononitrile 4a and 4b. The basic

catalyst, TEA; was required to generate the anion of the enaminone 3, thus

facilitating the addition to the unsaturated nitrile 4a and 4b.

The formation of compounds 6a and 6b was proved from their

microanalytical and spectral data. IR spectra of compounds 6a and 6b showed

bands in the range of 3467-3199 cm-1 for (NH2), sharp bands at 2176 & 2174

cm-1 corresponding to the cyano group (C�N) and bands at 1652 & 1645 cm-1 for

(C=O).

1H-NMR spectra of the quinoline derivatives 6a and 6b showed the presence

of singlets at 4.4 & 4.5 ppm for CH, singlets at 5.5 & 5.5 ppm for NH2, and

multiplets for aromatic prortons and SO2NH2 in the range of 7.1-8.0 ppm.


50

Additionally, mass spectra of compound 6a and 6b exhibited molecular ion

peaks at m/z 466 (M+, 39.87%) & m/z 482 (M+, 27.47), with a base peaks at m/z

371, respectively.

4-[5-(4-Fluorophenyl)-8,8-dimethyl-4,6-dioxo-3,4,6,7,8,9-hexahydro-

pyrimido[4,5-b]quinolin-10(5H)-yl]benzenesulfonamide (7a) and 4-[5-(4-

chlorophenyl)-8,8-dimethyl-4,6-dioxo-3,4,6,7,8,9-hexahydropyrimido[4,5-

b]quinolin-10(5H)-yl]benzenesulfonamide (7b)

The pyrimido[4,5-b]quinoline derivatives 7a and 7b were obtained by

refluxing compound 6a or 6b in formic acid. This reaction proceeded via

condensation followed by elimination of two moles of water to give the

pyrimido[4,5-b]quinoline derivatives 7a and 7b, as previously reported in the

literature .125


51

IR spectrum of compound 7a revealed the absence of the band

corresponding to the cyano group and the presence of two bands at 1714 cm-1

and 1647 cm-1, attributed to two C=O of the 4- and 6-oxo groups, respectively.

Additionally, mass spectrum of compound 7a exhibited a molecular ion peak at

m/z 495 (M+1, 0.16%), with a base peak at m/z 101.

Also, IR spectrum of compound 7b revealed the absence of the band

corresponding to the cyano group and the presence of two bands at 1708 cm-1

and 1644 cm-1, corresponding to two C=O of the 4-oxo and 6-oxo groups,

respectively. Additionally, 1H-NMR spectrum of 7b showed the presence of

singlet at 7.9 ppm for NH, singlet at 8.0 ppm for CH=N, and the absence of the

signal corresponding to the 2-amino group.

N-[3-cyano-4-(4-fluorophenyl)-7,7-dimethyl-5-oxo-1-(4-sulfamoyl-phenyl)-

1,4,5,6,7,8-hexahydroquinolin-2-yl]acetamide (8a) and N-[4-(4-

chlorophenyl)-3-cyano-7,7-dimethyl-5-oxo-1-(4-sulfamoylphenyl)-1,4,5,6,7,8-

hexahydroquinolin-2-yl]acetamide (8b)

The starting compounds 6a and 6b were refluxed with acetic anhydride.

Different products were obtained according to the time of the reaction. When

compounds 6a or 6b were refluxed in acetic anhydride for 5 hours, acetylation of

the 2-amino group was carried out affording the corresponding monoacetyl

derivatives 8a and 8b, respectively, instead of the fused pyrimido[4,5-b]quinoline

systems 10a and 10b as previously reported.126


52

The structure of compound 8a was established on the basis of elemental

analysis and spectral data. IR spectrum of compound 8a showed a band at 2212

cm-1 assigned to the cyano group, and two bands at 1725 cm-1 and 1652 cm-1,

attributed to two C=O of the acetyl group and the 5-oxo group, respectively. 1H-

NMR spectrum of compound 8a revealed the acetyl protons as one singlet at 1.5

ppm corresponding to three protons. Furthermore, mass spectrum of compound

8a exhibited a molecular ion peak at m/z 510 (M+2, 1.24%), with a base peak at

m/z 90.

Also, IR spectrum of compound 8b showed a band at 2213 cm-1 assigned to

the cyano group, and two bands at 1720 cm-1 and 1649 cm-1, corresponding to

2C=O of the acetyl group and the 5-oxo group, respectively. In addition, 1H-

NMR spectrum of compound 8b revealed the acetyl protons as one singlet at 1.5

ppm corresponding to three protons.


53

N-Acetyl-N-[3-cyano-4-(4-fluorophenyl)-7,7-dimethyl-5-oxo-1-(4-

sulfamoylphenyl)-1,4,5,6,7,8-hexahydroquinolin-2-yl]acetamide (9a) and N-

acetyl-N-[4-(4-chlorophenyl)-3-cyano-7,7-dimethyl-5-oxo-1-(4-sulfamoyl-

phenyl)-1,4,5,6,7,8-hexahydroquinolin-2-yl]acetamide (9b)

When compounds 6a or 6b was refluxed in acetic anhydride for 10 hours,

acetylation of the 2-amino group was carried out affording the corresponding

diacetyl derivatives 9a and 9b, respectively, instead of the expected fused

pyrimido[4,5-b]quinoline systems 10a and 10b, as previously reported in the

literature.127

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The structure of compound 9a was established on the basis of elemental

analysis and spectral data. IR spectrum of compound 9a showed a band at 2214


54

cm-1 assigned to the cyano group, and two bands at 1727 cm-1 and 1655 cm-1,

corresponding to the carbonyl groups. 1H-NMR spectrum of compound 9a

revealed the acetyl protons as one singlet at 2.4 ppm corresponding to six

protons, and the absence of the signal corresponding to the 2-amino group.

Also, IR spectrum of compound 9b showed a band at 2213 cm-1 assigned to

the cyano group, and two bands at 1728 cm-1 and 1655 cm-1, attributed to the

carbonyl groups. In addition, 1H-NMR spectrum of compound 9b revealed the

acetyl protons as one singlet at 2.4 ppm corresponding to six protons.

Furthermore, mass spectrum of compound 9a exhibited a molecular ion peak at

m/z 566 (M-1, 0.27%), with a base peak at m/z 90.

4-[5-(4-Fluorophenyl)-2,8,8-trimethyl-4,6-dioxo-3,4,6,7,8,9-hexahydro-

pyrimido[4,5-b]quinolin-10(5H)-yl]benzenesulfonamide (10a) and 4-[5-(4-

chlorophenyl)-2,8,8-trimethyl-4,6-dioxo-3,4,6,7,8,9-hexahydropyrimido[4,5-

b]quinolin-10(5H)-yl]benzenesulfonamide (10b)


refluxing compounds 6a or 6b in acetic anhydride for 15 hours, respectively.97


55

The structure of compound 10a was confirmed by the absence of the cyano

group, and the presence of two bands at 1720 cm-1 and 1656 cm-1 for two

carbonyl groups. Additionally, mass spectrum of compound 10a exhibited a

molecular ion peak m/z 508 (M+, 9.66%) and a base peak at m/z 456.

Also, IR spectrum of compound 10b revealed the absence of the band

corresponding to cyano group, and the presence of two bands corresponding to

two carbonyl groups at 1724 cm-1 and 1656 cm-1. Furthermore, 1H-NMR


56

spectrum of compound 10b revealed the 2-methyl group protons as one singlet at

2.4 ppm, corresponding to three protons.

4-[4-Amino-5-(4-fluorophenyl)-8,8-dimethyl-6-oxo-6,7,8,9-tetrahydro-

pyrimido[4,5-b]quinolin-10(5H)-yl]benzenesulfonamide (11a) and 4-[4-

amino-5-(4-chlorophenyl)-8,8-dimethyl-6-oxo-6,7,8,9-tetrahydro-



reaction of compound 6a or 6b with formamide, where cyclization occurred

through elimination of one molecule of water, followed by intramolecular

cyclization.128

The structures of compounds 11a and 11b were established on the basis of

elemental analysis and spectral data. IR spectra of compounds 11a and 11b

revealed the absence of (C�N) bands, which confirms the cyclization and the

formation of the pyrimido[4,5-b]quinoline systems.


57

Also, mass spectra of compounds 11a and 11b showed molecular ion peaks

at m/z 494 (M+1, 0.16%) & m/z 510 (M+, 0.46%) with base peaks at m/z 90 &

87, respectively.

Ethyl N-[3-cyano-4-(4-fluorophenyl)-7,7-dimethyl-5-oxo-1-(4-sulfamoyl-

phenyl)-1,4,5,6,7,8-hexahydroquinolin-2-yl]formimidate (12a) and Ethyl N-

[4-(4-chlorophenyl)-3-cyano-7,7-dimethyl-5-oxo-1-(4-sulfamoylphenyl)-

1,4,5,6,7,8-hexahydroquinolin-2-yl]formimidate (12b)

The quinoline derivatives 12a and 12b were obtained by refluxing

compound 6a or 6b in triethylorthoformate in the presence of few drops of acetic

anhydride, respectively. The reaction proceeded via the elimination of two moles

of ethanol.119


58

The formation of compounds 12a and 12b was supported by their

microanalytical and spectral data. 1H-NMR spectra showed the presence of a

triplet at 1.2 ppm for the CH3, a quartet at 4.3 ppm of CH2 of the ethyl group and

a singlet corresponding to one proton of the N=CH at 8.7 ppm.

Additionally, mass spectrum of compound 12a showed a molecular ion

peak at m/z 539 (M+, 0.52%) with base peak at m/z 90.

2-Chloro-N-[3-cyano-4-(4-fluorophenyl)-7,7-dimethyl-5-oxo-1-(4-

sulfamoylphenyl)-1,4,5,6,7,8-hexahydroquinolin-2-yl]acetamide (13a) and 2

Documents

inis.iaea.org · 2012. 8. 17. · sulfonamides and quinoline derivatives regarding their mechanisms of action, and the rationale for combining chemotherapy and radiotherapy. In addition,