An-Najah National University Faculty of Graduate Studies Synthesis and Biological Activities of Distamicin A Analogues By Somoud Mohammed Ahmed Yasin Supervisor Dr. Waheed Jondi Co-Supervisor Dr. Hassan Alnees This Thesis is Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Chemistry, Faculty of Graduate Studies, An- Najah National University, Nablus, Palestine. 2015 III Dedication Submitted with grateful love, recognition and appreciatıon to my great husbund Nasry Yasin. To my father, mother, brothers and sisters. To all teachers in my entire life. To my dearest friends. I dedicate this work. IV Acknowledgements After thanking God, who granted me the ability to finish this work, I would like to express my sincere gratitude to my supervisors: Dr. Waheed Jondi for all his guidance, understanding, support and advices in all of my research work and Dr. Hassan Alnees for his scientific support and constructive advice. Also my special thanks go to my doctors Dr. Ahmad Husein for his endless encouragment,scientific support and constructıve advices, Dr.İsmail Warrad and Dr. Mohammed Al-Nuri for their help and support during my study. I appreciate help and support from all members of the Chemistry Department at An-Najah National University especially laboratory technicians, in particular Mr. Omair Nabulsi and Mr. Nafiz Dweikat (BERC). Thanks to Biological and Enviromental Research Center-Til- Nablus for providing us with the biological isolates and doing biological activity tests. Thanks to my family with all my love, especially my husbund, who stood with me throughout my study and provided me with psychological support and encouragement. VI Table of Contents No. Subject Page Dedication III Acknowledgements IV Declaration V Table of Content VI List of Figures VIII List of Tabels IX Appendix X Abstract XII Chapter1: Introduction 1 1.1 Distamycin A 1 1.2 Distamycin A Structure and Binding 2 1.3 Distamycin A Analogues 4 1.4 Discovery and Properties of DNA 9 1.5 Natural Compounds That Bind to the Minor Groove 13 1.5.1 Netropsin 14 1.5.2 Thiazotropsin C 15 1.6 Antimicrobial Activity of Distamycin A Analogues 16 1.7 Proposed Pathway for Synthesis of the Distamycin A 17 Analogues 1.8 Aim of the Project 18 Chapter 2: Chemical Synthesis 19 2.1 Materials and Chemicals 19 2.2 Equipments and Devices 19 2.3 Chemical Synthesis 19 2.3.1 Preparation of Primary Compounds 19 2.3.1.1 Preparation of Trichloroacetyl Chloride.Scheme1 19 2.3.1.2 Preparation of 2,2,2-Trichloro-1-(1-methyl-1H-pyrrol-2-yl) 20 ethanone. Scheme2 2.3.1.3 Preparation of 2,2,2-Trichloro-1-(1-methyl-4-nitrio-1H- 21 pyrrole-2-yl)ethanone.Scheme3 2.3.1.4 Preparation of 1-Methyl-4-nitro-1H-pyrrole-2-carboxylic acid (2-(4-hydroxy-phenyl)-ethyl)-amide . 22 Scheme4 2.3.1.5 Preparation of Methanesulfonic cid4-(2-((1-methyl-4-nitro- 23 1H-pyrrole-2- carbonyl)-ethyl)-phenyl ester . Scheme5 2.3.1.6 Preparation of 1-Methyl-4-nitro-1H-pyrrole-2-carboxylic acid 24 (3-dımethylamino-propyl)-amide . Scheme6 2.3.1.7 Preparation of 1-Methyl-4-nitro-1H-pyrrole-2-carboxylic acid 24 (2-(sulfonyl-phenyl)-ethyl)-amide. Scheme7 2.3.2 Preparation of Distamycin A Analogues 25 2.3.2.1 Preparation of Methanesulfonicacid 4-(2-((1-methyl-4- 25 VII (pyridin-3- carbonyl)-amino)-1H-pyrrol-2-carbonyl)-amino)- ethyl)-phenyl ester.Scheme8 2.3.2.2 Preparationof Methanesulfonicacid 4-(2-((4- benzoylamino- 1-methyl-1H-pyrrole-2-carbonyl)amino)-ethyl)-phynelester. 26 Scheme9 2.3.2.3 N(-( (3-(dimethylamino)propyl)amino)carbonyl)-1-methyl-1- 27 H-pyrrole-3-yl)nicotinamide.Scheme10 2.3.2.4 4-((benzoylamino)methyl)-N-(3-(dimethylamino)propyl)- 29 N,1dimethyl-1H-pyrrole-2-carboxamide.Scheme11 2.3.2.5 Preparation of N-(1-Methyl-5-(2-(4-sulfamoyl-phenyl)- 30 ethylcarbamoyl)-1H-pyrrol-3-yl)-nicotinamide. Scheme12 Chapter 3: Biological Activities 31 3.1 Antioxidant Activity 31 3.1.1 Chemicals 31 3.1.2 DPPH Assay 31 3.2 Antibacterial Activity 32 3.2.2 Microorganisms Used and Growth Conditions 32 3.2.3 Disk Diffusion Method 32 3.3 Antifungal Activity 33 3.3.1 Microorganisms 33 3.3.2 Antifungal Testing 33 Chaptre 4: Results and Discussion 36 4.1 Synthesis of Distamycin A Analogues 36 4.2 Synthesis of Minor Groove Binders Monomers 36 4.3 Synthesis of Methanesulfonic acid 4-(2-((1-methyl-4(pyridin- 3-carbonyl)-amino)-1H-pyrrol-2-carbonyl)-amino)-ethyl)- phenyl ester.and synthesis of Methanesulfonic acid 4-(2-((4- 37 benzoylamino-1-methyl-1H-pyrrole-2-carbonyl)amino)- ethyl)-phynel ester.Scheme13 4.4 Synthesis of N-(5-((3-dimethylamino-propylcarbamoyl)-1- methyl-1H-pyrrol-3-yl)nicotinamide and Synthesis of 4- 39 benzoylamino-1-methyl-1H-pyrrole-2-carboxylic acid(3- dimethylamino-propyl)-amide.Scheme14 4.5 Preparation of N-(1-Methyl-5-(2-(4-sulfamoyl-phenyl)- ethylcarbamoyl)-1H-pyrrol-3-yl)-nicotinamide. 40 Scheme15 4.6 Biological Activity Result 42 4.6.1 Antioxidant Activity Test 42 4.6.2 Antibacterial Activity Test 43 4.6.3 Antifungal Activity Test 44 4.7 Conclusion and Future Work 46 References 48 صخلولا ب VIII List of Figures No. Subject Page 1.1 Structure of Distamycin A. 2 1.2 Binding of Distamycin in the Minor Groove 4 1.3 DNA Structure 10 1.4 Structural Formulas of the Constituents of DNA 11 1.5 Chemical Constituents of DNA. 11 1.6 Deoxyribose And Ribose Sugars, Deoxyribose Ribose 15 1.7 Hydrogen Bonds Formed in Base Pairing 13 1.8 Netropsin Structure 15 1.9 Netropsin Bound in the Minor Groove 1:1 15 1.10 Thiazotropsin A and its Analogue Thiazotropsin C 16 1.11 Retrosynthetic Analysis of Distamysin A analogues 17 4.1 Structure of Some Monomers Used in the Synthesis of 37 Distamycin A analogues. 4.2 Antioxidant Effect of Synthesized Compound 43 4.3 Antibacterial Activities of Synthesized Compound 44 4.4 Antifungal Activity of Tested Compounds 45 IX List of Tabels No. Subject Page 3.1 Types and names of investigated microorganisms 33 3.2 Types of investigated fungi in the test 34 4.1 Frequencies of main functional groups of Distamycine A 42 analogues in cm-1 4.2 Antifungal activity of the compounds against Trychophyton 44 Rubrum 4.3 Antifungal activity of the compounds against Microsporum 45 canis 4.4 Antifungal activity of the compounds against 45 Epidermophyton floccosum 4.5 structure of distamycine analogues and other compounds 46 used in biological activity test. X Table of Appendix No. Appendix Page Figure 1 FT-IR spectrum of 2-trichloroacetyl-N-methylpyrrole 54 Figure 2 FT-IR spectrum of 2,2,2-trichloro-1-(1-methyl-4-nitro-1H- 54 pyrrol-2-yl)ethanone Figure 3 FT-IR spectrum of 1-Methyl-4-nitro-1H-pyrrole-2- 55 carboxylic acid(2-(4-hydroxy-phenyl)-ethyl)-amide 4 Figure 4 FT-IR spectrum of Methanesulfoniccid4-(2-((1-methyl-4- 55 nitro-1H-pyrrole-2-carbonyl)-ethyl)-phenyl ester Figure 5 FT-IR spectrum of Methanesulfonicacid 4-(2-((1-methyl-4- 56 (pyrıdin-3-carbonyl)-amino)-1H-pyrrol-2-carbonyl)-amino)- ethyl)-phenyl ester Figure 6 FT-IR spectrum of Methanesulfonic acid 4-(2-((4- 56 benzoylamino-1-methyl-1H-pyrrole-2-carbonyl)amino)- ethyl)-phynel ester Figure 7 FT-IR spectrum of 1-Methyl-4-nitro-1H-pyrrole-2- 57 carboxylic acid(3-dimethylamino-propyl)-amide Figure 8 FT-IR spectrum of N(-( (3- 57 (dimethylamino)propyl)amino)carbonyl)-1-methyl-1-H- pyrrole-3-yl)nicotinamide Figure 9 4-((benzoylamino)methyl)-N-(3-(dimethylamino)propyl)- 58 N,1dimethyl-1H-pyrrole-2-carboxamide Figure 10 FT-IR spectrum of 1-Methyl-4-nitro-1H-pyrrole-2-carboxylic 58 acid(2-(sulfonyl-phenyl)-ethyl)-amide Figure 11 FT-IR spectrum of N-(1-Methyl-5-(2-(4-sulfamoyl-phenyl)- 59 ethylcarbamoyl)-1H-pyrrol-3-yl)-nicotinamide Figure 12 1HNMR spectra of Methanesulfonic acid 4-(2-((1-methyl-4- 59 (pyridin-3-carbonyl)-amino)-1H-pyrrol-2-carbonyl)-amino)- ethyl)-phenyl ester Figure 12.1 1HNMR spectra of Methanesulfonic acid 4-(2-((1-methyl-4- 60 (pyridin-3-carbonyl)-amino)-1H-pyrrol-2-carbonyl)-amino)- ethyl)-phenyl ester Figure 12.2 1HNMR spectra of Methanesulfonic acid 4-(2-((1-methyl-4- 60 (pyridin-3-carbonyl)-amino)-1H-pyrrol-2-carbonyl)-amino)- ethyl)-phenyl ester 6 . Figure 12.3 1HNMR spectra of Methanesulfonic acid 4-(2-((1-methyl-4- 61 (pyridin-3-carbonyl)-amino)-1H-pyrrol-2-carbonyl)-amino)- ethyl)-phenyl ester Figure 13 1HNMR spectrum of Methanesulfonic acid 4-(2-((4- 61 benzoylamino-1-methyl-1H-pyrrole-2-carbonyl)amino)- ethyl)-phynel ester 7 Figure 13.1 1HNMR spectrum of Methanesulfonic acid 4-(2-((4- 62 benzoylamino-1-methyl-1H-pyrrole-2-carbonyl)amino)- ethyl)-phynel ester 7 Figure 14 1HNMR spectrum of N(-((3-(dimethylamino) propyl)amino) 62