Derivatives of pyrazolo1,5 a1,3,5triazines as enzyme inhibitors with potential therapeutic value

DERIVATIVES OF PYRAZOLO[1,5-a][1,3,5]TRIAZINES AS ENZYME INHIBITORS WITH POTENTIAL THERAPEUTIC VALUE SUN LINGYI (B. Sc. (Pharm.)), Shanghai Jiao Tong Univ. A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF PHARMACY NATIONAL UNIVERSITY OF SINGAPORE 2013 ACKNOWLEDGEMENTS I wish to express my heartfelt gratitude to my supervisor Assoc. Prof. Chui Wai Keung, Head, Department of Pharmacy, for his enormous and continuous support throughout my PhD study. I sincerely appreciate him for granting me such a great freedom to work independently while at the same time providing valuable suggestions. The most important thing I have learned from him is how to think critically when setting up the experiments, which would benefit me a lot in my future career. This project was supported by NMRC Grant R-148-000-102-275. Thanks to Assoc. Prof. Chan Sun Yung, who was the head of department during large portion of my PhD study, for providing me the necessary facilities to finish this project. Thanks to National University of Singapore for providing me the research scholarship. Thanks to Dr. Anton Dolzhenko for his guidance and precious advice in my chemistry work. My gratitude also goes to Dr. Gigi Chiu Ngar Chee, Ms. Tan Bee Jen and Ms. Gan Fei Fei for their kind help in my cell work. Their precious advice helped me solve lots of problems encountered in the cell work. Special thanks are extended to lab technologists Ms. Ng Sek Eng and Ms. Lye Pey Pey for their support in processing my orders. I also want to thank all my labmates, Dr. Yang Hong, Dr. Ong Pauline, Dr. Sachdeva Nikhil, Dr. Bera Hriday, Ms. Ng Hui Li, Mr. Li Ka Chun and FYP student Ms. Li Jia Rong and Ms. Ang Xiao Hui for your daily help, and I would not forget your nice collaboration during those safety audits. My dear friends, Mr. Li Jian, Dr. Sun Feng, Dr. Zhang Yaochun, Dr. Wang Likun, Dr. Li Lin, Dr. Wang Zhe, Mr. Li Fang, Ms. Yang Shili, Mr. Liu Yuanjie, Mr. Sun Longwei, and Mr. Tan Kuan Boone, it was so lucky for me to recognize all of you! The time we spent together in playing War Craft Ⅲ or basketball was so precious that I would memorize forever. I am grateful to my dear parents and relatives. I understand that it is difficult for my parents to make a decision that let their son go aboard, and I sincerely appreciate your respect towards my own choice. In addition, I also want to thank my dear girlfriend Ms. Chen Xiao for accompanying me in the past three years. I did not get significant results before our encounter thus I believe it was you who brought me the good fortune. This last paragraph is for those who have ever helped me during the past four years but not mentioned above due to the limited space: Thank you so much! i CONTENTS PAGE ACKNOWLEDGEMENTS i SUMMARY . v ABBREVIATIONS viii LIST OF TABLES . x LIST OF FIGURES xii LIST OF SCHEMES xiv 1. Introduction 1.1 A brief overview of enzyme inhibitors as drugs 1.1.1 Oxidoreductases (EC 1) as targets for developing enzyme inhibitors as drugs 1.1.2 Transferases (EC 2) as targets for developing enzyme inhibitors as drugs 1.1.3 Hydrolase (EC 3) as targets for developing enzyme inhibitors as drugs 1.1.4 Lyases (EC 4) as targets for developing enzyme inhibitors as drugs . 1.2 Thymidine phosphorylase as a target for developing enzyme inhibitors possessing therapeutic values 12 1.2.1 Physiological functions of thymidine phosphorylase 13 1.2.2 Pathological functions of thymidine phosphorylase 14 1.2.2.1 Thymidine phosphorylase in cancers . 14 1.2.2.2 Thymidine phosphorylase in other diseases 20 1.2.3 Thymidine phosphorylase inhibitors and their potential therapeutic values . 22 1.2.3.1 Pyrimidine derivatives as inhibitors of thymidine phosphorylase 23 ii 1.2.3.2 Purine derivatives as inhibitors of thymidine phosphorylase 29 1.2.3.3 Thymidine phosphorylase inhibitors based on other structures 31 1.2.3.4 Therapeutic potential of inhibitors of thymidine phosphorylase 32 1.3 Synthesis of pyrazolo[1,5-a][1,3,5]triazines 34 1.3.1 Synthesis of pyrazolo[1,5-a][1,3,5] triazines from pyrazole scaffold . 35 1.3.2 Synthesis of pyrazolo[1,5-a][1,3,5] triazines from 1,3,5-triazine scaffold 42 1.3.3 Synthesis of pyrazolo[1,5-a][1,3,5] triazines by concurrent formation of both the 1,3,5-triazine and pyrazole rings . 43 1.3.4 Synthesis of pyrazolo[1,5-a][1,3,5] triazines by ring transformation reactions . 44 1.4 Biological activity of pyrazolo[1,5-a][1,3,5]triazines . 47 1.4.1 Enzyme inhibitors containing the pyrazolo[1,5-a][1,3,5]triazine scaffold 47 1.4.2 Other biological activities . 51 1.5 Hypothesis and objectives 54 1.5.1 Hypothesis . 54 1.5.2 Objectives 57 2. Fused bicyclic pyrazolo[1,5-a][1,3,5]triazine derivatives as inhibitors of thymidine phosphorylase . 60 2.1 Chemistry . 62 2.2 Thymidine phosphorylase inhibitory activity 77 2.3 Enzyme inhibition kinetic studies 83 2.4 Antiangiogenic potential studies 85 2.4.1 Cytotoxicity study of selected TP inhibitors against MDA-MB-231 . 86 iii 2.4.2 Inhibition of MMP-9 secretion in MDA-MB-231 by selected TP inhibitors . 89 2.5 Summary 91 3. 5-Chlorouracil-linked-pyrazolo[1,5-a][1,3,5]triazines as inhibitors of thymidine phosphorylase . 94 3.1 Chemistry . 96 3.2 Thymidine phosphorylase inhibitory activity 103 3.3 Enzyme inhibition kinetics studies . 109 3.4 Antiangiogenic potential studies 112 3.4.1 Cytotoxic studies of selected TP inhibitors against MDA-MB-231 . 112 3.4.2 Inhibition of MMP-9 secretion in MDA-MB-231 by selected TP inhibitors . 114 3.5 Summary 118 4. Conclusion and Future work 120 4.1 Conclusion 121 4.2 Future work 129 5. Materials and methods . 133 5.1 Chemistry . 134 5.1.1 Preparation and characterization of intermediates 135 5.1.2 Preparation and characterization of target compounds . 152 5.2 Biological tests . 178 5.2.1 Evaluation of inhibitory activity against thymidine phosphorylase 178 5.2.2 Thymidine phosphorylase inhibition kinetic studies 179 5.2.3 MTT assay 180 5.2.4 Gelatine zymography 181 Bibliography 183 iv SUMMARY Thymidine phosphorylase (TP) is an enzyme that promotes tumour growth and metastasis thus is an attractive druggable target. Currently, the most potent TP inhibitors are pyrimidine derivatives; although some purine based inhibitors have also been reported but their potency against TP is still weak. The goal of this project was to develop new TP inhibitors that are analogues of purine. It was hypothesized that the pyrazolo[1,5-a][1,3,5]triazine scaffold equipped with a homophthalimide moiety would exhibit TP inhibitory activity through proper structural modifications. In addition, it was also hypothesized that compounds consisting of both pyrimidine moiety and purine related moiety could inhibit TP through dual site interaction. In particular, to test the first hypothesis, a total of 59 1,3-dihydro-pyrazolo[1,5-a][1,3,5]triazin-2,4-diones as well as their isosteric 2- thioxo analogues were synthesized and subjected to an in vitro enzyme bioassay. All target compounds were obtained in good yields (32%-94%) via a synthetic approach that required annulation of the 1,3,5-triazine ring onto substituted 3-amino pyrazoles. Results of the subsequent enzyme test showed that although 1,3-dihydro-pyrazolo[1,5-a][1,3,5]triazin-2,4-diones were not active against TP, most of their isosteric 2- thioxo analogues exhibited TP inhibitory activity with IC50 values ranging from 87.3µM to 40nM. The best compound 17r showed an IC50 value of 40nM which is around 800 times more v potent than the lead compound 7DX. Therefore, the first hypothesis was proven to be partially true. Further enzyme inhibitory kinetic studies revealed that 17r was a non-competitive inhibitor, suggesting that it might bind to an allosteric site. To test the second hypothesis, 31 compounds consisting of both pyrimidine moiety and purine moiety designed as 3H-2-(5-chlorouracil-6-methylthio)-pyrazolo[1,5-a][1,3,5]triazin-4-ones were synthesized and evaluated by the in vitro enzyme assay. A multiple-step convergent synthetic scheme was devised to generate the target compounds in good yields (40%-96%). The intermediate 5-chloro-6-chloromethyluracil was synthesized by a 4-step reaction and then coupled with 1,3-dihydro-pyrazolo[1,5-a][1,3,5]triazin-2-thioxo-4-ones to yield the target compounds. Subsequent enzyme tests showed that this type of compounds was active against TP with IC50 values ranging from 67.8µM to 0.36µM, and the second hypothesis in this study was proven to be true. The best compound in this series, 24r, was subjected to enzyme inhibitory kinetic studies. Results revealed that 24r demonstrated a mixed-type of enzyme inhibition kinetics, thus suggesting that it might potentially bind at two different sites on the enzyme. In addition, a total of 26 compounds with IC50 values less than 10µM were selected from the two series of compounds synthesized to explore their vi potential antiangiogenic properties. They were subjected to a gelatin zymography assay that evaluated their potential suppressive effect on the secretion of the angiogenic factor MMP-9 in cancer cells. Based on the results obtained, compounds among them did suppress the secretion of MMP-9 thus might possess some therapeutic value in antiangiogenesis. (Words-458) vii ABBREVIATIONS 2DDR 2-Deoxy-D –ribose 2DDR-1P 2-Deoxy-D –ribose-1-phosphate 2DLR 2-Deoxy-L-ribose 6A5BP 6-Amino-5-bromo-3H-pyrimidin-4-one 6A5BU 6-Amino-5-bromouracil 6A5CU 6-Amino-5-chlorouracil 6AT 6-Aminothymine 7DX 7-Deazaxanthine BNIMU 5-Bromo-2-nitro-imidazolylmethyluracil BPMU 5-Bromo-6-(pyrrolidinylmethyl)uracil CAM Chorio-allantoic membrane CB Cannabinoid CDK2 Cyclin-dependent kinase CIIMU 5-Chloro-6-[(2-iminoimidazolidinyl)methyl]uracil hydrobromide CIPMP 5-Chloro-6-[1-(2-iminopyrrolidinyl)methyl]-3H-pyrimidin-4-one hydrochloride CNIMU 5-Chloro-2-nitro-imidazolylmethyluracil CNS Central nervous system CRF Corticotropin-releasing factor DISC Death-inducing signaling complex dNTPs Deoxyribonucleoside triphosphates ECM Extracellular matrix EPC Endothelial progenitor cells FAK Focal adhesion kinase viii HCMM Hydrazine carboxamide 2-[(1-methyl-2,5-dioxo-4-pentyl -4-imidazolidinyl)methylene] HUVEC Humbilical vein endothelial cells MIC Minimal inhibitory concentration MIMC 3- [(3-Methoxy-4-methylphenyl) imino] methyl-4H-chromen -4-one MMP Matrix metalloproteinase MNEC Maximal non-effective concentration MNGIE Mitochondrial neurogastrointestinal encephalopathy MPIC 3-(2-Methylphenyl) isocoumarin MW Molecular weight PDE Phosphodiesterases PD-ECGF Protein platelet-derived endothelial cell growth factor PMA Phorbol 12-myristate 13-acetate RA Reumatoid arthritis SAR Structure activity relationship SCO2 Synthesis of cytochrome c oxidase TFT Trifluorothymidine TP Thymidine phosphorylase TPI 5-Chloro-6-[1-(2-iminopyrrolidinyl)methyl]uracil hydrochloride TPIPA 3-(2,4,5-Trioxo-3-phenylethyl-imidazolodin-1-yl)propionamide TS Thymidylate synthase VEGF Vascular endothelial growth factor XO Xanthine oxidase ix HTLV-III/LAV replication, to patients with AIDS or AIDS-related complex. 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Lingyi Sun, Hriday Bera, and Wai Keung Chui, “Derivatives of pyrazolo[1,5-a][1,3,5]triazines phosphorylase”, 22nd as International inhibitors Symposium of on thymidine Medicinal Chemistry, 2012, Berlin, German. 2. Lingyi Sun, Hriday Bera, and Wai Keung Chui, “Developing the 1,3dihydro-pyrazolo[1,5-a][1,3,5]triazin-2-thioxo-4-one Novel Thymidine Phosphorylase Inhibitors”, scaffold 2011 into American Association of Pharmaceutical Scientists Annual Meeting and Exposition, 2011, Washington, USA. [...]... formation of focal adhesions as well as the phosphorylation of tyrosine397 of focal adhesion kinase (FAK), which was a nonreceptor protein-tyrosine kinase and played an important role in the attachment and migration of endothelial cells.64 Thymidine phosphorylase promotes tumor metastasis It has been found that high TP expression was associated with metastasis, and TP was able to increase the metastatic potential. .. Hydrolase (EC 3) as targets for developing enzyme inhibitors as drugs Hydrolases are a type of enzymes which catalyze the hydrolysis of their corresponding substrates A few examples of drugs which are enzyme inhibitors of hydrolases are provided below Acetylcholinesterase which hydrolyzes the neurotransmitter acetylcholine is an established target in the treatment of a range of central nervous diseases 8 For... considered as an efficient strategy for the drug development in some cases Many different types of enzymes involved in the regulation of metabolism have been targeted for the development of inhibitors to be used as drugs Some representatives, sorted by enzyme categories, are described below 1.1.1 Oxidoreductases (EC 1) as targets for developing enzyme inhibitors as drugs Oxidoreductase is an enzyme which... independent of its enzymatic activity.39, 49, 57 Finally, the antiapoptotic effect of TP was supported by numerous clinical studies It was found that expression of TP was associated with the decrease in apoptotic cells in colon,77 gastric,78 esophageal,79, 80 ovarian81 as well as oral squamous cell82 carcinomas 1.2.2.2 Thymidine phosphorylase in other diseases Many studies had suggested that TP was involved... recognized by the enzymes, leading to either blockage of the enzymes or the generation of non-functional products One such example is theantifolate-methotrexate It is structurally similar to dihydrofolate, and hence it can inhibit the enzyme dihydrofolate reductase Since many disease processes are found to be associated with the metabolite imbalance, designing enzyme inhibitors as antimetabolites has been considered... increase the structure diversity of its inhibitors 11 1.2 Thymidine phosphorylase as a target for developing enzyme inhibitors possessing therapeutic values Thymidine phosphorylase (TP) occurs widely in many normal tissues and cells Within the cell, TP is found in both the cytoplasm and the nucleus.19 TP catalyses the reverse phosphorolysis of pyrimidine nucleosides (Figure 1) The active site of TP... Triapine 1.1.2 Transferases (EC 2) as targets for developing enzyme inhibitors as drugs The function of transferase is to catalyze the transfer of a functional group from one donor molecule to an acceptor molecule Several examples of using transferases as targets for the drug design are listed below Catechol-O-methyl transferase is an enzyme that degrades dopamine, and it serves as a target for drugs... Synthesis of 2-(5-chloro-1,3-dihydropyrimidin-2,4dioxo-6-ylmethylthio)pyrazolo[1,5-a][1,3,5]triazin-4 (3H)-ones 100 xv 1 Introduction 1 1.1 A brief overview of enzyme inhibitors as drugs Enzyme inhibitors refer to a group of molecules which block the catalytic activity of enzymes According to interacting mechanisms with their targets, enzyme inhibitors can be categorized into either reversible inhibitors. .. several major challenges in the design of enzyme inhibitors as drugs Selectivity is one challenge that refers to how specific inhibitors are able to act against different isoforms of the enzyme which share the same substrates Low selectivity may lead to unwanted interactions with other isoforms, causing some potential side effects In particular, these isoenzymes are often distributed unequally in different... advantage over competitive inhibitors from this aspect The approach to design an enzyme inhibitor is to mimic the structure of the substrate, therefore most enzyme inhibitors are competitive inhibitors 2 Many enzyme inhibitors are antimetabolites, and they act by interfering with the essential metabolic pathways which may lead to disruption of normal cellular functions As antimetabolites often appear structurally . 2) as targets for developing enzyme inhibitors as drugs 6 1.1.3 Hydrolase (EC 3) as targets for developing enzyme inhibitors as drugs 8 1.1.4 Lyases (EC 4) as targets for developing enzyme inhibitors. potential therapeutic values 22 1.2.3.1 Pyrimidine derivatives as inhibitors of thymidine phosphorylase 23 iii 1.2.3.2 Purine derivatives as inhibitors of thymidine phosphorylase 29 1.2.3.3. xii LIST OF SCHEMES xiv 1. Introduction 1 1.1 A brief overview of enzyme inhibitors as drugs 2 1.1.1 Oxidoreductases (EC 1) as targets for developing enzyme inhibitors as drugs 3 1.1.2 Transferases