Folates are cofactors crucial for cell proliferation. Antifolates are a class of anticancer drugs that inhibit cell proliferation by inhibiting one-carbon metabolic pathways. Mammals cannot synthesize folates de novo and rely on specialized transporters for folate uptake from the circulation. There are three major folate transporters, the ubiquitously expressed reduced folate carrier (RFC), folate receptors (FRs) α and β, and the proton-coupled folate transporter (PCFT). Clinically approved antifolates such as pemetrexed (PMX) and methotrexate (MTX) have been shown to induce dose-limiting toxicities likely due to their RFC transport into both tumor and normal cells. The design of antifolates that are selectively transported via FRs and/or PFCT could overcome toxicities seen with clinically used antifolates that are primary transported by RFC. We previously reported 5-substituted pyrrolo[2,3-d]pyrimidines AGF127 which lacked RFC transport compared to PMX. Considering AGF127 as the lead, simple isosteric replacement of carbon in the linker with nitrogen afforded AGF367 with different chain lengths, angles, conformation, and extra hydrogen bond donors compared to AGF127. In engineered Chinese hamster ovary cells individually expressing the major human folate transporters, the additional nitrogen in AGF367 improved RFC selectivity vs PMX. However, AGF367 showed a 2-fold reduction in FRα transport. Fluorine’s distinctive properties make it a valuable tool in medicinal chemistry, influencing various aspects of drug design and development. We strategically incorporated fluorine into the benzoyl side chain of AGF367 to afford AGF409. The incorporation of fluorine provides conformational restriction to the L-glutamate chain via a fluorine-hydrogen bond and resulted in additional hydrophobic interactions with the major folate transporters. An additional fluorine in AGF409 improved FRα-mediated drug sensitivity compared to PMX by 20-fold and by 6-fold for FRβ. Towards KB human tumor cells, AGF409 was 7-fold more potent than PMX. Pyrrolo[2,3-d]pyrimidines with a heteroatom side chain provide an avenue for further development of analogs to selectively target tumor cells and limit dose-limiting toxicity.

Charles Dann

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