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Synthesis and Bioactivity Study of 4,6-Diamino-1,3,5-Triazines

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Synthesis and Bioactivity Study of 4,6-Diamino-1,3,5-Triazines View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by ScholarBank@NUS SYNTHESIS AND BIOACTIVITY STUDY OF 4,6-DIAMINO-1,3,5-TRIAZINES MA XIANG NATIONAL UNIVERSITY OF SINGAPORE 2006 SYNTHESIS AND BIOACTIVITY STUDY OF 4,6-DIAMINO-1,3,5-TRIAZINES MA XIANG (B. Sc. (Pharm.)), Shenyang Pharm. Univ. (M. Sc. (Pharm.)), Shanghai Inst. Pharm. Indust. A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF PHARMACY NATIONAL UNIVERSITY OF SINGAPORE 2006 ACKNOWLEDGEMENTS I can hardly express my sincere thanks and gratitude enough to Dr. Chui Wai Keung, for his patient guidance, enormous support and continuous encouragement throughout the period of this research. His invaluable advice and constructive ideas are immeasurable and have benefited me. Special thanks are extended to Assoc. Prof. Chan Sun Yung, Head, Department of Pharmacy, for providing the necessary facilities for this project, and the National University of Singapore for providing the research scholarship. I also wish to thank A/P Prof. Ho Chi Lui, Paul, A/P Prof. Go Mei Lin, Dr. Koh Hwee Lin, Dr. Lam Yu Lin, and Dr. Bong Yong Koy for their precious guidance and help. I am also grateful to Miss Wu Jinzhu for her help and advice in the cell culture work. My gratitude also goes to Miss Ng Sek Eng, Mdm Wong Mei Yin, Miss Ang Li Kiang and Miss Dyah Nanik Irawati for providing the technical support for my project. I also wish to thank all the friends in the Department of Pharmacy in National University of Singapore for their help, encouragement and friendship. I would like to thank my parents and sister for their understanding and support. Last but not least, my most sincere thanks go to my wife Pan Mengfei, for her invaluable emotional support as well as help and encouragement in the preparation of this manuscript. i CONTENTS PAGE ACKNOWLEDGEMENTS i CONTENTS ii SUMMARY v ABBREVIATIONS viii LIST OF SCHEMES xi LIST OF FIGURES xv LIST OF TABLES xvii INTRODUCTION 1. 1,3,5-Triazines 1 1.1. Synthesis of 1,3,5-Triazines 2 1.1.1. Synthesis of Fully Unsaturated 1,3,5-Triazines 2 1.1.1.1. Synthesis from 1,3,5-Triazines 2 1.1.1.2. Synthesis from Cyclization of Six-atom Urea Derivatives 5 1.1.1.3. Synthesis from Five-atom Biguanides 6 1.1.1.4. Synthesis from Dicyandiamide (Cyanoguanidine) 8 1.1.1.5. Synthesis from Amidines and Guanidine Derivatives 10 1.1.1.6. Synthesis from Nitriles, Isocyanates, Imidates and Carbodiimides 11 1.1.2. Synthesis of Dihydro-1,3,5-Triazines 12 1.1.2.1. Synthesis from 1,3,5-Triazines 12 1.1.2.2. Synthesis from Biguanides 14 1.1.2.3. Synthesis from Dicyandiamide (Cyanoguanidine) 15 1.1.2.4. Synthesis from Amidines or Their Derivatives 16 1.1.2.5. Synthesis from Nitriles 17 1.1.3. Synthesis of Fused 1,3,5-Triazines 18 1.2. Pharmaceutical Applications and Important Compounds 21 1.2.1. Fully Unsaturated 1,3,5-Triazines 21 1.2.2. Dihydro-1,3,5-Triazines 24 1.2.3. Fused 1,3,5-Triazines 26 1.3. Conclusion 27 2. Dihydrofolate Reductase (DHFR) Inhibitors 28 2.1. Biochemistry of Dihydrofolate Reductase 28 2.2. Structure and Ligand Interactions 32 ii 2.2.1. Escherichia coli DHFR 32 2.2.2. Chicken Liver DHFR 34 2.2.3. Mouse L1210 DHFR 37 2.2.4. Human DHFR 38 2.2.5. Mycobacterium tuberculosis DHFR 39 2.2.6. Lactobacillus casei DHFR and Neisseria gonorrhoeae DHFR 40 2.3. DHFR Inhibitors as Antimetabolites 42 2.3.1. DHFR Inhibitors as Cancer Chemotherapeutic Agents 43 2.3.1.1. New Classical Antifolates 48 2.3.1.2. New Non-Classical Antifolates 49 2.3.2. DHFR Inhibitors as Antibacterial Agents 53 2.3.3. DHFR Inhibitors as Antimalarials and Other Antiprotozoal Agents 55 2.4. Antifolates Used as Antiepileptic Sodium Channel Blockers 57 2.5. Pharmacological Evaluations 60 2.5.1. DHFR Inhibitory Activity by Enzyme Kinetics Assay 60 2.5.2. Antiproliferative Assay 62 2.5.3. Antibacterial Assay 63 2.5.4. Sodium Channel Blockade Assay 66 2.5.5. in vivo Anticonvulsant Experiment 69 3. Rationale and Objectives 72 RESULTS AND DISCUSSION 4. 2-Amino-4-(Substituted Phenylamino)-1,3,5-Triazine (M-I) 81 4.1. Chemistry 82 4.2. Pharmacological Evaluations 84 4.2.1. Antifolate Activity of Compounds M-I against Bovine DHFR 84 4.2.2. Antimicrobial Activity of M-I 85 4.2.3. Sodium Channel Blockade Activity of Compounds M-I 89 4.3. Conclusion 91 5. 4-Amino-6-(Substituted Anilino)-1,2-Dihydro-2,2-Dimethyl-1,3,5- Triazine (M-II) 92 5.1. Chemistry 93 5.1.1. The Synthesis of Triazines I 93 5.1.2. The Synthesis of Target Compounds M-II via a Dimroth Rearrangement 95 5.2. Pharmacological Evaluations 98 5.2.1. Antifolate Activity of Compounds M-II against Bovine DHFR 98 5.2.2. Antimicrobial Activity of M-II 100 5.2.3. Sodium Channel Blockade Activity of Compounds M-II 102 5.2.4. in vivo Anticonvulsant Activity and Sedative Action 105 5.3. Conclusion 107 6. 4,6-Diamino-1-Benzyl-1,2-Dihydro-2,2-Disubstituted-1,3,5- Triazine (M-III) 108 iii 6.1. Chemistry 109 6.2. Pharmacological Evaluations 116 6.2.1. Antifolate Activity of Compounds M-III against Bovine DHFR 116 6.2.2. Antimicrobial Activity of M-III 118 6.3. Conclusion 120 7. 4,6-Diamino-1-Phenalkyloxy-1,2-Dihydro-1,3,5-Triazines (M-IV) 122 7.1. Chemistry 123 7.2. Pharmacological Evaluations 126 7.2.1. Antifolate Activity of Compounds M-IV against Bovine DHFR 126 7.2.2. Antiproliferative Activity of Compounds M-IV against the MCF-7 Breast Cancer Cell Line 130 7.3. Conclusion 131 8. 4,6-Diamino-1-((4’-Substituted Phenyloxy)Alkyloxy)-1,2-Dihydro- 1,3,5-Triazines (M-V) 132 8.1. Chemistry 133 8.2. Pharmacological Evaluations 139 8.2.1. Antifolate Activity of Compounds M-V against Bovine DHFR 139 8.2.2. Antiproliferative Activity 143 8.2.3. Rescue Experiments to Investigate the Mechanism of Inhibition of Cancer Cells A549 Growth 153 8.3. Conclusion 157 CONCLUSION and FUTURE WORK 9. Concluding Remarks 160 10. Potential Future Development 164 MATERIALS AND METHODS 11. Chemistry 167 11.1. Materials and Equipments 167 11.2. Procedures for the Synthesis 167 12. Pharmacological Evaluations 202 12.1. DHFR Enzyme Assay 202 12.2. Antiproliferative Assay 204 12.3. Antimicrobial Assay 208 12.4. Sodium Channel Assay 210 12.5. Rotorod Test 212 12.6. Anticonvulsant Assay 212 BIBLIOGRAPHY 214 iv SUMMARY The 2-amino-1,3-diaza structural motif is a common feature found in many pharmaceutically useful agents. For instance, the structural motif is embedded in the structure of 4,6-diamino-1,3,5-triazine. Studies have shown that some of these triazines are found to be potent DHFR inhibitors. Such DHFR inhibitors have been noted for their therapeutic value as anticancer, antimalarial, antibacterial and anti-protozoal agents. As an extension to these studies, it was the intent of this dissertation to explore further if 4,6- diamino-1,3,5-triazines would also possess other forms of pharmacological activities. Therefore, the first objective of this dissertation was to design and synthesize five libraries of 4,6-diamino-1,3,5-triazine and its analogues; while the second objective was to investigate whether the triazines exhibit inhibitory activity against various microorganisms, cancer cells and neuronal sodium channels in addition to the antifolate activity. A small library M-I consisting of seven aromatic 1,3,5-triazines was synthesized by an alkoxide-catalyzed condensation of biguanide hydrochlorides with suitably substituted carboxylic acid esters at room temperature. Another fourteen 1,2-dihydro-1,3,5-triazines in library M-II were prepared based on a two-step reaction: the first step involved the preparation of 4,6-diamino-2,2-dimethyl-1-phenyl-1,2-dihydro-1,3,5-triazine (I), then the target compounds were derived via a base-catalyzed Dimroth rearrangement of I. In the synthesis of library M-III, a two-component reaction was carried out between benzylbiguanide and ketones or aldehyde with the use of water scavengers. The synthesis of M-IV involved a three-component synthesis that employed cyanoguanidine, O- v aminophenylalkyl alcohol hydrochloride and various ketones. The fifth library M-V was produced using fragment coupling synthesis strategy by alkylation with substituted phenoxyalkyl bromide of an N-hydroxytriazines. In the first two libraries (M-I and M-II), all the compounds were screened for inhibitory activity against bovine DHFR, neuronal sodium channels and an array of microorganisms. Limited bovine DHFR inhibitory activity was observed in both libraries. However, both libraries exhibited moderate to potent neuronal sodium channel blockade activity in vitro; among them, M-II-12 showed the best blockade activity with an IC50 value of 12.57 µM, which was ten times lower than positive control, phenytoin. While M-II exhibited limited antibacterial activity, some compounds in M-I library demonstrated relatively good antifungal activity against C. albicans and A. niger. The M- III library was designed based on the same side chain found in TMP. Twenty-three 4,6- diamino-1,2-dihydro-1,3,5-triazines in M-III were synthesized and screened against S. aureus and M. smegmatis for their antibacterial activity. All compounds in this library did not exhibit activity against S.
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