I:- Design, synthesis and study of nucleotide prodrugs. II:- Synthesis of 5-fluoro-6-[(2-aminoimidazol-1-yl)methyl]uracil (AIFU) and study of its mechanism of action as a thymidine phosphorylase inhibitor. III:- Design and synthesis of novel nucleotide analogs as alternative substrates for studying the mechanism of the thymidylate synthase-catalyzed reaction.

摘要

CHAPTER I: Design, synthesis and study of nucleotide prodrugs. In the past, several phosphate masking strategies have been developed with the objective of delivering nucleoside monophosphates into cells for improving the therapeutic efficacy of anticancer and antiviral nucleosides. Most of the first generation prodrugs of nucleotides (pronucleotides) were designed to deliver the monophosphorylated nucleoside, where they can be further converted into their active forms. In the previous strategies emphasis was on delivery of nucleoside monophosphates, but little or no attempt was made to design phosphate masking groups of no toxicity and/or undesirable activity. In order to overcome the toxicity problem in this project three different strategies were rationally designed and prototype pronucleotides with masking groups of no potential toxicity were synthesized and evaluated. These strategies are referred to as cProTide, bisSAL and aryloxy phosphoramidates (ProTide).Novel cyclic pronucleotides (cProTides) of 5-fluoro-2'-deoxyuridine (FdUrd) were rationally designed, synthesized and studied as potential anticancer agents. New stable amino acid ester-containing phosphoramidating reagents are described, which were used to synthesize prototype cProTides in a single step, under mild conditions and in moderate yields. Diastereomers generated in the reaction were separated by chromatography and analyzed spectroscopically. Plausible hypothetical mechanisms for the intracellular unmasking of these phosphate prodrugs are outlined, and unique structural features of the cProTides are discussed. Methoxy alanine cyclic phosphoramidates of FdUrd were found to be active in human ovarian cancer cell line A2780 and thymidine kinase mutant cell line LM-TK-. In the LM-TK- cell line, a five-fold difference in the cytotoxicity of the diastereomers was observed.Prototype bisSAL derivatives of FdUrd were rationally designed, synthesized and studied. They were synthesized to overcome the diastereomer problem and were obtained in one step from bis(alkylsalicylate)phosphochloridate reagents in mild to moderate yields. BisSAL derivatives were found to be active in the A2780 cell line but were inactive in the LM-TK- cell line. ProTide technology developed by McGuigan et al. was improved upon by replacing phenyl group, which generates phenol of known toxicity with the non-toxic methyl salicylate. ProTides containing alanine methyl ester with methyl salicylate as well as phenyl proTides of FdUrd were synthesized in moderate yields. The methyl salicylate proTide was found to be approximately 2-folds more active than the phenyl analog.In summary, two novel pronucleotides technologies were developed and one existing pronucleotide technology was improved. cProTide and methylsalicylate proTide of FdUrd were found to be cytotoxic in cell culture studies studies. Further studies are required to determine the mechanism of intracellular activation of cProTides.CHAPTER II: Synthesis of 5-fluoro-6-[(2-aminoimidazol-1-yl)methyl]uracil (AIFU) and study of its mechanism of action as a thymidine phosphorylase inhibitor. Thymidine phosphorylase (TP), also known as platelet-derived endothelial cell growth factor (PD-ECGF), has been implicated in tumor angiogenesis, and is considered a promising target for anticancer drug development. A variety of 6-subsituted-5-fluorouracil derivatives were designed by the Kalman group as transition state analogs and their inhibitory activities against E. coli and recombinant human TP were determined. 5-Fluoro-6-[(2-aminoimidazol-1-yl)methyl]uracil (AIFU) was synthesized and found to be the most potent inhibitor among all 6-substituted 5-fluorouracil derivatives with Ki-values of 11 nM (ecTP) and 17 nM (hTP).Molecular modeling studies were performed to show the tentative position of phosphate inside the E. coli TP active site. Enzyme kinetic studies were conducted, which established that the inhibitor must bind to the enzyme-phosphate or enzyme-arsenate binary complex. This explains previous findings that a basic side chain at the 6-position of the pyrimidine ring is required for potent inhibitory activity.CHAPTER III: Design and synthesis of novel nucleotide analogs as alternative substrates for studying the mechanism of the thymidylate synthase-catalyzed reaction. Thymidylate synthase (TS) is a highly conserved enzyme involved in the de novo synthesis of 2'-deoxythymidine monophosphate (thymidylate, dTMP) and has been an important target for various anticancer drugs such as 5-fluorouracil. Genomic studies have revealed that about 30% of the bacteria possess a distinct flavin-dependent TS enzyme (FDTS). Since many pathogenic bacteria contain this enzyme, minor subtleties found in both the enzymes can be useful for selectively targeting flavin-dependent TS for antibacterial drug design. The hydride transfer step of the TS-catalyzed reaction is poorly understood. Study of the covalently-bound exocyclic methylene intermediate 4 (and 9) could provide an insight into this process due to the lack of the crystal structure of methylene bound intermediate with enzyme. In order to generate intermediate 4 (and 9) inside the enzyme active site, nucleotide analogs were designed and synthesized as alternative substrates for TS. The rational design of these analogs was aided by molecular modeling studies.