Phytochemical Investigation on the Chemical Constituents of Zygophyllum eurypterum and Synthesis with Biological Evaluation of Schiff Bases of Substituted Phenyl Hydrazine and Benzimidazole Derivatives Thesis submitted For the partial fulfillment of the degree of DOCTOR OF PHILOSOPHY BY Mr. ZARBAD SHAH H.E.J. RESEARCH INSTITUTE OF CHEMISTRY INTERNATIONAL CENTER FOR CHEMICAL AND BIOLOGICAL SCIENCES, UNIVERSITY OF KARACHI KARACHI-75270, PAKISTAN 2011 i, Dedicated to parents especially my mother (late) & My beloved teachers, Prof. Dr. Viqar Uddin Ahmad, H.I., S.I. & Prof. Dr. Khalid Mohammed Khan, T.I., S.I. ii, iii, iv, LIST OF CONTENTS Acknowledgements xiii Personal Introduction xiv Summary xvi Khulasa xix Abbreviations xxi Part- A CHAPTER-1 1. Family Zygophyllaceae 2 1.1. The genus Zygophyllum 2 1.1.1. Zygophyllum eurypterum 2 1. 1.2. Zygophyllum megacarpum 2 1.1.3. Zygophyllum simplex 3 1.1.4. Zygophyllum propinquum 3 1.1.5. Zygophyllum fabago 3 1.2. Medicinal importance of the genus Zygophyllum 4 1.3. Literature review of the genus Zygophyllum 6 CHAPTER-2 2. Stigmasterol (1) 28 2.1. β-Sitosterol (2) 29 2.2. β-Sitosterol glucoside (3) 30 2.3. Oleanolic acid (4) 31 2.4. Harmine (5) 33 2.5. 7,3',4'-Trimethoxyflavone (6) 34 2.6. 5-Methylflavanone (7) 35 v, CHAPTER-3 3. Stigmasterol (1) 37 3.1. β-Sitosterol (2) 38 3.2. β-Sitosterol glucoside (3) 39 3.3. Oleanolic acid (4) 40 3.4. Harmine (5) 41 3.5. 7,3',4' -Trimethoxyflavone (6) 42 3.6. 5-Methylflavanone (7) 43 3.7. General notes 44 3.8. Instrumentation 44 3.9. Chromatography 44 3.10. Spray reagents 44 3.10.1. Ceric sulphate 45 3.10.2. Aniline phathalate 45 3.11. Zygophyllum eurypterum (Z. atriplicoides) 45 3.11.1. Plant collection 45 3.11.2. Isolation procedure 45 3.12. Isolation scheme of Zygophyllum eurypterum 47 CHAPTER-4 4. References 49 Part- B CHAPTER-5 5. Introduction to Schiff bases 55 5.1.1. Synthetic approaches to Schiff bases 58 5.1.2. Synthesis of hydrazones by using ethanol and triethylamine 58 5.1.3. Synthesis of azines using solvent free condition 58 vi, 5.1.4. Synthesis of hydrazones by using methanol and sulphuric acid 59 5.1.5. Synthesis of hydrazones by using DMF 59 5.1.6. Synthesis of aryl-hydrazones using ultrasound irradiation 60 5.2. Results and Discussion 61 5.2.1 Chemistry 61 5.3. General structure elucidation of compounds by spectroscopic techniques 64 5.4. Biological Studies 66 5.5. Conclusion 85 5.6. General instrumentation 86 5.6.1. Synthetic procedure of compounds 28-62 87 5.6.1.1. 3, 4, 5-Trimethoxybenzaldehyde N-(2, 4, 6-trichlorophenyl) hydrazone (28) 87 5.6.1.2. 2-Hydroxy-3-methoxybenzaldehyde N-(2, 4, 6-trichlorophenyl) hydrazone: (29) 87 5.6.1.3. Benzaldehyde N-(2, 4, 6-trichlorophenyl)hydrazone (30) 87 5.6.1.4. 2-Fluorobenzaldehyde N-(2, 4, 6-trichlorophenyl) hydrazone (31) 88 5.6.1.5. 4-(Methylsulfanyl)benzaldehyde N-(2, 4, 6-trichlorophenyl) hydrazone (32) 88 5.6.1.6. 4-(Dimethylamino)benzaldehyde N-(2, 4, 6-trichlorophenyl) hydrazone (33) 88 5.6.1.7. 2-Hydroxy-5-methylbenzaldehyde N-(2, 4, 6-trichlorophenyl) hydrazone (34) 88 5.6.1.8. 2, 5-Dihydroxybenzaldehyde N-(2, 4, 6-trichlorophenyl) hydrazone (35) 88 5.6.1.9. 2, 4-Dichlorobenzaldehyde N-(2, 4, 6-trichlorophenyl) hydrazone (36) 89 5.6.1.10. 4-Chlorobenzaldehyde N-(2, 4, 6-trichlorophenyl) hydrazone (37) 89 5.6.1.11. 2-Chlorobenzaldehyde N-(2, 4, 6-trichlorophenyl) hydrazone (38) 89 vii, 5.6.1.12. 3, 5-Dichloro-2-hydroxybenzaldehyde N-(2, 4, 6-trichlorophenyl) hydrazone (39) 89 5.6.1.13. 3, 4-Dichlorobenzaldehyde N-(2, 4, 6-trichlorophenyl) hydrazone (40) 89 5.6.1.14. 3, 4-Dihydroxybenzaldehyde N-(2, 4, 6-trichlorophenyl) hydrazone (41) 90 5.6.1.15. 4-Hydroxybenzaldehyde N-(2, 4, 6-trichlorophenyl) hydrazone (42) 90 5.6.1.16. 3, 4-Dimethoxybenzaldehyde N-(2, 4, 6-trichlorophenyl) hydrazone (43) 90 5.6.1.17. 2, 3, 4-Trihydroxybenzaldehyde N-(2, 4, 6-trichlorophenyl) hydrazone (44) 90 5.6.1.18. 2, 3-Dihydroxybenzaldehyde N-(2, 4, 6-trichlorophenyl) hydrazone (45) 91 5.6.1.19. 3-Thiophenecarbaldehyde N-(2, 4, 6-trichlorophenyl) hydrazone (46) 91 5.6.1.20. Isonicotinaldehyde N-(2, 4, 6-trichlorophenyl) hydrazone (47) 91 5.6.1.21. 2-Bromobenzaldehyde N-(2, 4, 6-trichlorophenyl) hydrazone (48) 91 5.6.1.22. 2-Hydroxybenzaldehyde N-(2, 4, 6-trichlorophenyl) hydrazone (49) 92 5.6.1.23. 3-Chlorobenzaldehyde N-(2, 4, 6-trichlorophenyl) hydrazone (50) 92 5.6.1.24. 1-Phenanthrenecarbaldehyde N-(2, 4, 6-trichlorophenyl) hydrazone (51) 92 5.6.1.25. Nicotinaldehyde N-(2, 4, 6-trichlorophenyl) hydrazone (52) 92 5.6.1.26. 2-Naphthaldehyde N-(2, 4, 6-trichlorophenyl) hydrazone (53) 93 5.6.1.27. 2-Methylbenzaldehyde N-(2, 4, 6-trichlorophenyl) hydrazone (54) 93 viii, 5.6.1.28 2, 4, 6-Trihydroxybenzaldehyde N-(2, 4, 6-trichlorophenyl) hydrazone (55) 93 5.6.1.29. 2, 4-Dihydroxybenzaldehyde N-(2, 4, 6-trichlorophenyl) hydrazone (56) 93 5.6.1.30. 4-Methylbenzaldehyde N-(2, 4, 6-trichlorophenyl) hydrazone (57) 93 5.6.1.31. 3, 5-Dibromo-2-hydroxybenzaldehyde N-(2, 4, 6-trichlorophenyl) hydrazone (58) 94 5.6.1.32. 2-chloro-5-nitrobenzaldehyde N-(2, 4, 6-trichlorophenyl) hydrazone (59) 94 5.6.1.33. 4-Nitrobenzaldehyde N-(2, 4, 6-trichlorophenyl) hydrazone (60) 94 5.6.1.34. 5-Bromo-2-hydroxybenzaldehyde N-(2, 4, 6-trichlorophenyl) hydrazone (61) 94 5.6.1.35. 2-Nitrobenzaldehyde N-(2, 4, 6-trichlorophenyl) hydrazone (62) 95 5.7. References 96 CHAPTER-6 6. Introduction to Benzimidazole 110 6.1. Synthetic approaches to Benzimidazoles 115 6.1.1. Synthesis of benzimidazole from triethoxy methane and o-alkylated oximes 115 6.1.2. Photolysis of protected Benzimidazole 115 6.1.3. Synthesis of 2-substituted benzimidazole by using Lewis acids 116 6.1.4. Synthesis of 2-substituted benzimidazole via clay and IR 116 6.1.5. Microwave assisted synthesis of substituted benzimidazoles 116 6.1.6.. Synthesis of substituted benzimidazoles by using esters 117 6.1.7. Synthesis of substituted benzimidazoles via reductive cyclization 117 6.1.8. Synthesis of substituted benzimidazoles by using polyphosphoric acids 117 ix, 6.1.9. Synthesis of benzimidazoles by using 12-tungstophosphoricacid as a catalyst 118 6.2. Results and discussion 119 6.2.1 Chemistry 119 6.3. General structure elucidation of compound by spectroscopic techniques 122 6.4. Biological studies 124 6.5. Conclusion 137 6.6. General instrumentation 138 6.6.1 Synthetic procedure of compounds 37-66 138 6.6.1.1. 4-(6-Nitro-1H-benzimidazol-2-yl)phenol (37) 139 6.6.1.2. 2, 4-Dibromo-6-(6-nitro-1H-benzimidazol-2-yl)phenol (38) 139 6.6.1.3. 2-(3, 4-Dimethoxyphenyl)-6-nitro-1H-benzimidazole (39) 139 6.6.1.4. 2-(2-Chlorophenyl)-6-nitro-1H-benzimidazole (40) 139 6.6.1.5. 2, 4-Dichloro-6-(6-nitro-1H-benzimidazol-2-yl)phenol (41) 140 6.6.1.6. 6-Nitro-2-(4-nitrophenyl)-1H-benzimidazole (42) 140 6.6.1.7. 2-(4-Methylphenyl)-6-nitro-1H-benzimidazole (43) 140 6.6.1.8. 2-(3, 4-Dichlorophenyl)-6-nitro-1H-benzimidazole (44) 140 6.6.1.9. 4-(6-Nitro-1H-benzimidazol-2-yl)-1, 3-benzenediol (45) 141 6.6.1.10. 2-(4-Chlorophenyl)-6-nitro-1H-benzimidazole (46) 141 6.6.1.11. 4-(6-Nitro-1H-benzimidazol-2-yl)-1, 2-benzenediol (47) 141 6.6.1.12. 2-Methoxy-6-(6-nitro-1H-benzimidazol-2-yl)phenol (48) 141 6.6.1.13. 2-(6-Nitro-1H-benzimidazol-2-yl)-1, 3, 5-benzenetriol (49) 142 6.6.1.14. 4-(6-Nitro-1H-benzimidazol-2-yl)-1, 2, 3-benzenetriol (50) 142 6.6.1.15. N, N-dimethyl-4-(6-nitro-1H-benzimidazol-2-yl)aniline (51) 142 6.6.1.16. 2-Ethoxy-6-(6-nitro-1H-benzimidazol-2-yl)phenol (52) 142 6.6.1.17. 4-Chloro-2-(6-nitro-1H-benzimidazol-2-yl)phenol (53) 143 6.6.1.18. 2-(6-Nitro-1H-benzimidazol-2-yl)phenol (54) 143 6.6.1.19. 2-(2, 4-Dichlorophenyl)-6-nitro-1H-benzimidazole (55) 143 6.6.1.20. 2-(2-Chloro-5-nitrophenyl)-6-nitro-1H-benzimidazole (56) 143 6.6.1.21. 2-(2-Fluorophenyl)-6-nitro-1H-benzimidazole (57) 144 x, 6.6.1.22. 2-(4-Bromophenyl)-6-nitro-1H-benzimidazole (58) 144 6.6.1.23. 6-Nitro-2-(3-thienyl)-1H-benzimidazole (59) 144 6.6.1.24. 6-Nitro-2-(1-phenanthryl)-1H-benzimidazole (60) 144 6.6.1.25. 6-Nitro-2-phenyl-1H-benzimidazole (61) 145 6.6.1.26. Methyl 4-(6-nitro-1H-benzimidazol-2-yl)phenyl sulfide (62) 145 6.6.1.27. 6-Nitro-2-(4-pyridinyl)-1H-benzimidazole (63) 145 6.6.1.28. 2-(6-Nitro-1H-benzimidazol-2-yl)-1, 4-benzenediol (64) 145 6.6.1.29. 2-(9-Anthryl)-6-nitro-1H-benzimidazole (65) 146 6.6.1.30. 3-(6-Nitro-1H-benzimidazol-2-yl)-1, 2-benzenediol (66) 146 6.7. References 147 CHAPTER-7 7.1 In vitro antiglycation bioassay 159 7.2. In vitro carbonic anhydrase bioassay 159 7.3. In vitro α-chymotrypsin bioassay 160 7.4.