A STRATEGY FOR THE DESIGN OF MEMBRANE-PERMEABLE FOLYLPOLY-GAMMA-GLUTAMATE SYNTHETASE INHIBITORS - BAY-REGION-SUBSTITUTED 2-DESAMINO-2-METHYL-5,8-DIDEAZAFOLATE ANALOGS

Previous attempts to design inhibitors of mammalian folylpolyglutamate synthetase (FPGS) have resulted in three classes of active compounds, all of which have charged moieties in the side chain, but structural alteration of the rest of the folate molecule has not seemed to be an avenue for drug discovery. However, groups in the side chain of folate analogs that bear charge distributions different from that of glutami c acid appear to prevent efficient transport into mammalian cells on t he reduced folate carrier system. We now report that substituents at t he 7-, 2'-, or 3'-position of 2-desamino-2-methyl-4-hydroxyquinazoline antifolates decrease or prevent the catalysis of diglutamate formatio n by FPGS but are compatible with efficient binding to the reduced fol ate carrier system. Thus, 5,8-dideazafolates with a 3'-alkyl group had a lower V-max for FPGS than did the corresponding unsubstituted quina zolines, by a factor of 4-12, but these compounds inhibited the reacti on of control FPGS substrates, indicating that the 3'-groups had much larger effects on catalytic activity than on binding to enzyme. A 7-me thyl substituent affected the V-max of a series of 5,8-dideazafolate c ompounds by a factor of 2-8, but this decrease in the catalytic rate w as also accompanied by an increase in the K-m of the substituted compo unds by a factor of 10-100. The extent of the effect of a 7-methyl sub stituent on V-max appeared to be dependent on the size of the substitu ent at N10. Different substituents at the 2'-position affected the kin etics of the FPGS reaction with one of three patterns, i.e., 1)a 2'-fl uoro substituent both increased V-max and decreased K-m slightly, 2) e ither -OH or -NH2 decreased the V-max without affecting the K-m and 3) 2'-Cl, -CH3, -CF3, or -OCH3 substituents were found to both decrease V-max and increase K-m. Substitutions at the 7-, 2'-, or 3'-position h ad only minor effects on the ability of 2-desamino-2-methyl-4-oxoquina zolines to interfere with the transport of PH]methotrexate into L1210 cells. Hence, these classes of compounds are likely to be efficiently transported by the reduced folate carrier system. We conclude that the region of the folate molecule bounded by the 7-, 6-, 9-, 10-, 3'-, an d 2'-positions, the ''bay region,'' is of major importance both for th e binding of folates and folate analogs to FPGS and for the assumption of a conformation of the enzyme-substrate complex compatible with cat alysis. We also suggest that appropriate substitutions at these positi ons would lead to membrane-permeable, specific FPGS inhibitors potent enough to allow evaluation of this enzyme as a target for cancer chemo therapy.