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 glutamic acid appear to prevent efficient transport into mammalian cells on the reduced folate carrier system. We now report that substituents at the 7-, 2'-, or 3'-position of 2-desamino-2-methyl-4-hydroxyquinazoline antifolates decrease or prevent the catalysis of diglutamate formation by FPGS but are compatible with efficient binding to the reduced folate carrier system. Thus, 5,8-dideazafolates with a 3'-alkyl group had a lower Vmax for FPGS than did the corresponding unsubstituted quinazolines, by a factor of 4-12, but these compounds inhibited the reaction of control FPGS substrates, indicating that the 3'-groups had much larger effects on catalytic activity than on binding to enzyme. A 7-methyl substituent affected the Vmax of a series of 5,8-dideazafolate compounds by a factor of 2-8, but this decrease in the catalytic rate was also accompanied by an increase in the Km of the substituted compounds by a factor of 10-100. The extent of the effect of a 7-methyl substituent on Vmax appeared to be dependent on the size of the substituent at N10. Different substituents at the 2'-position affected the kinetics of the FPGS reaction with one of three patterns, i.e., 1) a 2'-fluoro substituent both increased Vmax and decreased Km slightly, 2) either -OH or -NH2 decreased the Vmax without affecting the Km, and 3) 2'-Cl-, -CH3, -CF3, or -OCH3 substituents were found to both decrease Vmax and increase Km. Substitutions at the 7-, 2'-, or 3'-position had only minor effects on the ability of 2-desamino-2-methyl-4-oxoquinazolines to interfere with the transport of [3H]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'-, and 2'-positions, the "bay region," is of major importance both for the binding of folates and folate analogs to FPGS and for the assumption of a conformation of the enzyme-substrate complex compatible with catalysis.(ABSTRACT TRUNCATED AT 400 WORDS)