NOVEL ,4-DIAMINO-5-SUBSTITUTED-PYRROLO[2,3-D]PYRIMIDINES AS CLASSICALAND NONCLASSICAL ANTIFOLATE INHIBITORS OF DIHYDROFOLATE REDUCTASES

Eight novel, nonclassical, antifolate iamino-5-(anilinomethyl)pyrrolo[ 2,3-d]pyrimidines, 1-8, with 3',4',5'-trimethoxyphenyl, 3',4'-dimethox yphenyl, 2',5'-dimethoxyphenyl, 4'-methoxy-phenyl, 2',5'-diethoxypheny l, 3',4'-dichlorophenyl, 1'-naphthyl, and phenyl substituents were syn thesized as potential inhibitors of dihydrofolate reductases (DHFRs). The classical analogue -[(2,4-diaminopyrrolo[2,3-d]pyrimidin-5-yl)meth yl] amino]benzoyl]-L-glutamic acid (9) was also synthesized as an inhi bitor of DHFR and an antitumor agent. The classical and nonclassical a nalogues were obtained via reductive condensations of the key intermed iate 2,4-diamino-5-cyanopyrrolo[2,3-d]pyrimidine (12) with the appropr iate substituted aniline or (p-aminobenzoyl)-L-glutamate followed by r eduction of the intermediate Schiff bases with NaCNBH3. Compounds 1-9 were evaluated in vitro as inhibitors of rat liver (rl), Pneumocystis carinii (pc), and Toxoplasma gondii (tg) DHFRs. The nonclassical analo gues were significantly selective against tgDHFR (vs rat liver DHFR), ranging from 7- to 92-fold. The inhibitory activity was lower in pcDHF R and rlDHFR (IC(50)s > 10(-5) M) than in tgDHFR (IC(50)s 10(-6) M). T he classical analogue had inhibitory activity similar to that of metho trexate (MTX) against the growth of human leukemia CCRF-CEM, A253, and FaDu squamous cell carcinoma (SCC) of the head and neck cell lines. F urther evaluation of 9 against CCRF-CEM and its sublines having define d mechanisms of MTX resistance demonstrated that the analogue utilizes the reduced folate/MTX-transport system and primarily inhibits DHFR a nd poly-gamma-glutamylation plays a role in its mechanism of action. C ompound 9 was found to be 3-fold more efficient than aminopterin as a substrate for human folylpolyglutamate synthetase.