| Thymidylate synthase (TS) is an indispensable enzyme for de novo thymidylate biosynthesis in humans, the malaria-causing parasite Plasmodium falciparum, and the tuberculosis-causing bacterium Mycobacterium tuberculosis. Antifolate inhibitors are a promising and attractive route for inhibiting TS. To circumvent resistance mechanisms associated with classical antifolates, lipophilic nonclassical antifolates were designed, synthesized, purified, characterized, and evaluated. The resulting sixty-eight quinazolinone compounds were exploited to probe the active sites of TS enzymes accessible by moieties in the C2, C5, C6, and C8 positions of the quinazolinone pharmacophore. Each library of compounds was evaluated toward human TS, P. falciparum DHFR-TS, M. tuberculosis ThyA and ThyX, as well as the P. falciparum parasite and mammalian cells. Structure-activity relationships toward the enzymes were identified. Compounds exhibiting potency and/or selectivity were discovered for each enzyme and the malaria parasite. Molecular characteristics critical for high level enzyme inhibition included: a C2 amino substituent; a C5 thiol containing a 4-heteroatom, an electron-withdrawing and hydrogen-bonding substituent in the 4-position, or a naphthalene ring; and a C6 methyl group. Compounds bearing a C5 methylene linker exhibited unique activity with most of the library compounds inactive against all screened enzymes and mammalian cells, but remaining highly cytotoxic toward P. falciparum. Achievements made through this project may present opportunities for the unmet medical need in the fields of malaria and tuberculosis. |
