The synthesis is described of a series of C2-methyl-N10-alkylquinazoline-based antifolates in which the p-aminobenzoate ring is replaced by the heterocycles thiophene, thiazole, thiadiazole, pyridine, and pyrimidine. These were generally elaborated by the reaction of (bromomethyl)quinazoline 18 or its N3-[(pivaloyloxy)methyl]-protected derivative 36 with suitable heterocyclic amines although each heterocyclic system required its own particular synthetic approach. The compounds were tested as inhibitors of partially purified L1210 thymidylate synthase (TS). They were also examined for their inhibition of the growth of L1210 cells in culture. The thiophene system 7 and its related thiazole 8 gave analogues that were considerably more potent than the parent benzene series 2 as inhibitors of L1210 cell growth although in general these heterocycles were somewhat poorer inhibitors of the isolated TS enzyme. The enhanced cytotoxicities of the thiophene and thiazole analogues result, at least in part, from their efficient transport into the cells via the reduced folate carrier mechanism and very good substrate activity for folylpolyglutamate synthetase. The replacement of the C2-methyl group by C2-(fluoromethyl) and C2-(hydroxymethyl) substituents in the thiophene and thiazole series gave derivatives that were only slightly less potent inhibitors of the TS enzyme but which were considerably less cytotoxic.
Several C2-methylquinazoline-based antifolates have been prepared in which the C9,N10 bridge has been replaced by the reversed N9,C10 unit. This series was extensively studied by incorporating further substituents at N9 and C10 as well as by modifications to the p-aminobenzoate ring. The C2-methylquinazoline analogues 29, 30, and 31 containing the methyleneoxa, methylenethia, and thia bridge units were also synthesized. In general these isosteric replacements of the bridge unit in the parent C2-methyl-N10-propargylquinazoline antifolate 2 were much less potent as inhibitors of isolated thymidylate synthase (TS) but several were at least as potent as inhibitors of L1210 cell growth in culture. The fusion of the p-aminobenzoate ring into the bicyclic systems 75 and 76 also reduced activity against TS but again gave highly cytotoxic compounds. The cytotoxicities were largely prevented by thymidine, confirming that TS is the major locus.
The synthesis of a series of new C2-methyl-N10-alkylquinazoline-based thymidylate synthase (TS) inhibitors containing difluroinated p-aminobenzoate rings is described. Derivatives of the N10-propargyl and N10-methylquinazoline antifolates were prepared with 2',3'-, 2',5'-, and 2',6'-difluoro substitution. The synthesis of the 2',5'-difluoro analogues involved oxidation of the difluoronitrotoluene to 2,5-difluoro-4-nitrobenzoic acid followed by glutamation, reduction, and alkylation (propargyl bromide or MeI) to the diethyl N-(4-(alkylamino)-2,5-difluorobenzoyl)-L-glutamates. For the synthesis of the 2',3'- and 2',6'-difluoro compounds a new route was devised starting from methyl 4-((tert-butoxycarbonyl)amino)-2,6-difluorobenzoate and its 2,3-substituted counterpart. Treatment with NaH and then an alkyl halide introduced the N10-substituent. The methyl ester was hydrolyzed and the resulting acid was condensed with diethyl L-glutamate. The secondary amine was liberated using CF3CO2H and coupled with 6-(bromo-methyl)-3,4-dihydro-2-methyl-4-oxoquinazoline to yield the antifolate diesters. Final deprotection with mild alkali completed the synthesis in each case. The target compounds were tested as inhibitors of partially purified L1210 TS and also examined for their inhibition of the growth of L1210 cells in culture. Compared to their nonfluorinated parent compounds all the difluoro analogues were poorer inhibitors of TS. The greatest loss of enzyme activity was seen in the N10-propargyl analogues which contained one of the fluorine atoms ortho to the amine substituent. This loss was less apparent in the N10-methyl derivatives. Despite this lower inhibition of TS the majority of new compounds have equivalent cytotoxicity to their nonfluorinated predecessors.
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