NOVEL 6,5-FUSED RING HETEROCYCLIC ANTIFOLATES - BIOCHEMICAL AND BIOLOGICAL CHARACTERIZATION

Six novel antifolates with 2,4-diaminopyrimidine-fused five-membered r ings containing either pyrrole or cyclopentene rings were characterize d at the cellular and biochemical level. Five of these antifolates wer e more growth inhibitory to the CCRF-CEM human leukemia cell line than methotrexate [MTX; drug concentration effective at inhibiting cell gr owth by 50% relative to untreated control (EC(50)), 12 nM], the antifo late used in the clinic, and two were more potent than 10-ethyl-10-dea zaaminopterin (EC(50), 2.7 nM); similar patterns of response were obta ined in the FaDu and A253 squamous carcinoma cell lines. In addition, the growth inhibitory potency of these antifolates was generally less dependent on exposure time than was MTX. Growth inhibitory effects cou ld be reversed by leucovorin, indicating an antifolate mechanism. Thes e antifolates targeted dihydrofolate reductase (DHFR) based on direct human DHFR inhibition assays [drug concentration inhibiting enzyme act ivity by 50% (IC50), 0.6-28 nM; MTX IC50, 0.8 nM] and the cross-resist ance of MTX-resistant CCRF-CEM cells containing elevated DHFR. Inhibit ion of human thymidylate synthase was generally weak These 6,5-fused r ing heterocyclic antifolates utilized the reduced folate/MTX transport er for uptake, based on the cross-resistance of MTX uptake-impaired CC RF-CEM cells, and were efficient substrates for this uptake system, ba sed on inhibition of [H-3]MTX uptake (IC50, 0.3-5.8 mu M; aminopterin IC50, 2.6 mu M). These analogues were substrates for CCRF-CEM folylpol yglutamate synthetase, with several being among the most active substr ates now known (highest V-rel/K-m 0.73; MTX and 10-ethyl-10-deazaamino pterin, 0.013 and 0.24, respectively). Substrate activity for murine i ntestinal folylpolyglutamate synthetase was also assayed, and a differ ent specificity pattern was observed. These new antifolates are appare ntly not substrates for aldehyde oxidase. Analogues containing the fus ed cyclopentene ring are preferred to those containing the fused pyrro le ring based on growth inhibitory potency, effectiveness against decr eased uptake mutants and apparent affinity for transport, and inhibiti on of DHFR. In addition, fused cyclopentene-containing analogues are e fficiently polyglutamylated. The data indicate that antifolates with 2 ,4-diaminopyrimidine-fused five-membered rings, especially those conta ining the fused cyclopentene ring, are an important new class of antif olates which warrant further exploration at the synthetic and preclini cal levels.