CLASSICAL AND NONCLASSICAL FURO[2,3-D]PYRIMIDINES AS NOVEL ANTIFOLATES - SYNTHESIS AND BIOLOGICAL-ACTIVITIES

Classical antifolate analogues containing a novel furo2,3-d pyrimidi ne ring system which include N-4-N-(2,4-diaminofuro2,3-dpyrimidin -5-yl)m aminobenzoyl-L-glutamic acid (1) and its N-9 methyl analogue 2 were synthesized as potential dual inhibitors of thymidylate syntha se (TS) and dihydrofolate reductase (DHFR) and as antitumor agents. Fo ur nonclassical antifolates, ,4-diamino-5-(anilinomethyl)furo2,3-dpy rimidines 3-6 with 3,4,5-trimethoxy, 3,4,5-trichloro, 3,4-dichloro, an d 2,5-dimethoxy substituents, respectively, in the phenyl ring, were a lso synthesized as potential inhibitors of DHFRs including those from Pneumocystis carinii and Toxoplasma gondii, which are organisms respon sible for opportunistic infections in AIDS patients. The classical and nonclassical analogues were obtained via nucleophilic displacements o f the hey intermediate 2,4-diamino-5- (chloromethyl)furo 2,3-dpyrimi dine with the appropriate (p-aminobenzoyl)-L-glutamate or substituted aniline. The key intermediate was in turn synthesized from 2,4-diamino -6-hydroxypyrimidine and 1,3-dichloroacetone. The final compounds were tested in vitro against rat liver, (recombinant) human, P. carinii, T . gondii, and Lactobacillus casei DHFRs. The classical analogues showe d moderate to good DHFR inhibitory activity (IC50 10(-6)-10(-8) M) wit h the N-CH3 analogue 2 about twice as potent as 1. The nonclassical an alogues were inactive with IC(50)s > 3 x 10(-5) M. The classical analo gues were also evaluated as inhibitors of TS (L. casei, (recombinant) human and human CCRF-CEM), glycinamide ribonucleotide formyltransferas e, and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase and were found to be inactive against these enzymes. The classical an alogues (particularly 2) were significantly cytotoxic toward a variety of tumor cell lines in culture. The nonclassical analogues were margi nally active. Both classical compounds were good substrates for human folylpolyglutamate synthetase. Further evaluation of the cytotoxicity of 1 and 2 in CCRF-CEM cells and its sublines, having defined mechanis ms of methotrexate (MTX) resistance, demonstrated that the analogues u tilize the reduced folate/MTX-transport system and primarily inhibit D HFR and that poly-gamma-glutamylation was crucial to their mechanism o f action. Protection studies in the FaDu squamous cell carcinoma cell line indicated that inhibition was completely reversed by leucovorin o r the combination of thymidine plus hypoxanthine. Furthermore, for com pounds 1 and 2, in contrast to MTX, the FaDu cells were better protect ed by thymidine alone than hypoxanthine alone, suggesting a predominan tly antithymidylate effect.