Abstract 1364: Design, synthesis and evaluation of potent antiproliferative and cytotoxic antifolates with selective FRs and PCFT transport over RFC and potent GARFTase inhibition

Abstract

Abstract Lack of selectivity for folate transporters is one of the major causes of toxicity and chemotherapy failure with clinically used antifolates. 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. We recently reported potent inhibition of cell proliferation by a classical 2-amino-4-oxo-6-substituted pyrrolo[2,3-d]pyrimidine with a thienoyl for benzoyl-substituted side chain and a 4-carbon bridge (AG71). AG71 was selective for transport by FRs and PCFT over RFC and inhibited glycinamideribonucleotide formyl transferase (GARFTase) in de novo purine biosynthesis. In SCID mice with KB tumors, AG71 was highly active against both early and advanced stage tumors. A series of five thienoyl regioisomers of AG71 with [1,3] (AG117 and AG118) and [1,2] (AG114, AG115, AG116) thienoyl substitutions by the 4-carbon bridge and the L-glutamate were synthesized. Analogs were evaluated for anti-proliferative activities in engineered Chinese hamster ovary (CHO) cells expressing RFC, FR, or PCFT to determine the optimum structures for selective transport by FRs and/or PCFT. Additional studies were performed in KB and IGROV1 tumor cells, using inhibition of cell proliferation or colony formation as metrics. Like AG71, none of the novel thienyol regioisomers were substrates for RFC. AG117 and AG118 were potently active toward FR- and PCFT-expressing CHO cells and toward KB and IGROV1 tumor cells, equivalent or exceeding levels for AG71. In colony-forming assays in KB cells, AG117 and AG118 were cytotoxic. AG114, AG115, and AG116 were substantially less active or completely inert. AG117 and AG118, like AG71, were high affinity ligands for FRα by competitive ligand binding with 3H-folic acid, and were potent substrates for PCFT by direct competition 3H-methotrexate transport. By protection assays, AG71 and AG117 preserved potent anti-proliferative activity even in the presence of 50 nM leucovorin; adenosine and 5-imidazole-4-carboxamide were completely protective, establishing the de novo purine biosynthetic pathway and GARFTase as the principal intracellular targets. GARFTase inhibition was confirmed by in situ GARFTase metabolic assays with 14C-glycine as a radiotracer. Our results identify 6-subsituted pyrrolo[2,3-d]pyrimidine thienoyl regioisomers of AG71 as potent anti-proliferative and cytotoxic antifolates, reflecting selective transport by PCFT and FR over RFC and inhibition of GARFTase. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1364. doi:10.1158/1538-7445.AM2011-1364