Discovery of Tumor-Targeted 6-Methyl Substituted Pemetrexed and Related Antifolates with Selective Loss of RFC Transport

Pemetrexed and related 5-substituted pyrrolo[2,3-d]pyrimidine antifolates are substrates for the ubiquitously expressed reduced folate carrier (RFC), and the proton-coupled folate transporter (PCFT) and folate receptors (FRs) which are more tumor-selective. A long-standing goal has been to discover tumor-targeted therapeutics that draw from one-carbon metabolic vulnerabilities of Cancer cells and are selective for transport by FRs and PCFT over RFC. We discovered that a methyl group at the 6-position of the pyrrole ring in the bicyclic scaffold of 5-substituted 2-amino-4-oxo-pyrrolo[2,3-d]pyrimidine antifolates 1-4 (including pemetrexed) abolished transport by RFC with modest impacts on FRs or PCFT. From molecular modeling, loss of RFC transport involves steric repulsion in the scaffold binding site due to the 6-methyl moiety. 6-Methyl substitution preserved antiproliferative activities toward human tumor cells (KB, IGROV3) with selectivity over IOSE 7576 normal ovary cells and inhibition of de novo purine biosynthesis. Thus, adding a 6-methyl moiety to 5-substituted pyrrolo[2,3-d]pyrimidine antifolates affords tumor transport selectivity while preserving antitumor efficacy.