Targeting the proton-coupled folate transporter for selective delivery of 6-substituted pyrrolo[2,3-d]pyrimidine antifolate inhibitors of de novo purine biosynthesis in the chemotherapy of solid tumors

The proton-coupled folate transporter (PCFT) is a folate-proton symporter with an acidic pH optimum, approximating the mi-croenvironments of solid tumors. We tested 6-substituted pyr-rolo[2,3-d]pyrimidine antifolates with one to six carbons in the bridge region for inhibition of proliferation in isogenic Chinese hamster ovary (CHO) and HeLa cells expressing PCFT or reduced folate carrier (RFC). Only analogs with three and four bridge carbons (N-4-[3-2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]-pyrimidin-6-yl)propyl]benzoyl-L-glutamic acid (compound 2) and N-4-[4-2-amino-4-oxo-4,7-dihydro-3H-pyr-rolo[2,3-d]-pyrimidin-6-yl)butyl]benzoyl-L-glutamic acid (com-pound 3), respectively) were inhibitory, with 2 3. Activity toward RFC-expressing cells was negligible. Compound 2 and pemetrexed (Pmx) competed with [3H]methotrexate for PCF