Abstract 4542: The design, synthesis and in vitro evaluation of selective FR α and β and PCFT substrates with potent GARFTase inhibitory activity as antitumor agents

Abstract

Abstract Three folate transporters, the reduced folate carrier (RFC), folate receptors (FR) and the proton-coupled folate transporter (PCFT) account for most of the folate influx activity in mammalian cells. Lack of selectivity for folate transporters is one of the major causes of chemotherapy failure with antifolate drugs. We recently reported the potent glycinamideribonucleotide formyltransferase (GARFTase) inhibitory activity of a series of classical 2-amino-4-oxo-6-substituted pyrrolo[2,3-d]pyrimidines with a thienoyl for benzoyl-substituted side chain with FR and PCFT specificity over RFC. In an attempt to optimize for selective transport by FR and PCFT, and GARFTase inhibitory activity, a furan for benzoyl-substituted side chain analog with a 4-carbon bridge was synthesized. The target compound was tested against KB human tumor cells that express FR alpha, human RFC, and human PCFT using a fluorescence-based cytotoxicity screen, and showed remarkably potent subnanomolar inhibitory activity (IC50 of 0.29 nM). The furan analog was further tested in isogenic Chinese hamster ovary sublines, engineered to express human FR alpha (RT-16), FR beta (D4), hRFC (pC43-10), or hPCFT (R2/hPCFT4). The furan side chain compound again showed a subnanomolar IC50s for both FR alpha and FR beta expressing cells (IC50 of 0.16 nM for RT16, and 0.92 nM for D4). Thus, in comparison with the lead thienoyl side chain compound (IC50 of 2 nM for RT16, and 0.75 nM for D4), the furan analog exhibited similar activities toward FR beta, but a 12.5-fold increased activity toward FR alpha. For PCFT-expressing R2/hPCFT4 cells, the furan side chain compound (IC50 of 439 nM) was about 10-fold less potent than the thienoyl analog (IC50 of 43.4 nM). The furan analog was inactive against human dihydrofolate reductase (hDHFR) or human thymidylate synthase (hTS) (IC50 >10−5 M), suggesting that neither hDHFR nor hTS is a principal target. Metabolite protection from in vitro cytotoxicity in KB cells showed that thymidine (10 μM) did not affect the growth inhibitory effects of the furan analog whereas adenosine (60 μM) and 5-amino-4-imidazolecarboxamide (320 μM) were completely protective, identifying GARFTase as the likely intracellular target. Our results indicate that the side chain heterocyclic ring may control the transport selectivity for both FR and PCFT of the pyrrolo[2,3-d]pyrimidines. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4542.