Abstract 3983: Tumor-targeting with novel dual-targeted 6-substituted thieno[2,3-d]pyrimidine antifolates via cellular uptake by folate receptor α, and inhibition of de novo purine nucleotide biosynthesis

Epithelial ovarian cancer (EOC) is the deadliest gynecologic cancer, with limited progress in targeted therapy or in combating drug resistance. Reflecting this, there is an urgent need to develop novel inhibitors with minimal toxicity. EOC is associated with increasing expression of folate receptor alpha (FRα) with increasing stage and grade of disease. Of particular interest are targeted therapeutics toward FRα which spare transport by the ubiquitously expressed reduced folate carrier (RFC). FRα is a viable target for tumor selectivity with limited expression in normal tissues, where it is only expressed on apical membranes. We previously described 6-substituted thieno[2,3-d]pyrimidine (6TP) benzoyl antifolates with bridge lengths of 2-8 carbons as potent and selective inhibitors of tumor cells including EOCs that express FRs. The most potent compound (AGF50) included a 4-carbon bridge and showed FRα selectivity and inhibition of de novo purine biosynthesis. We synthesized AGF50 analogs with various aryl side-chain modifications (e.g., thiophene, furan, pyridine) including 29fluorine substitutions. Toward isogenic Chinese hamster ovary cell lines individually expressing FRα, RFC and the proton-coupled folate transporter (PCFT), the 6TP analogs were completely selective toward FRα over RFC and PCFT, manifested as inhibition of cell proliferation with nmolar IC50s. Activity of this series extended to KB human tumor cells and human EOC cell lines (e.g., IGROV1). Drug effects were abolished with excess folic acid, confirming FRα uptake. Adenosine completely protected cells from the inhibitory effects of 6TPs in vitro, suggesting inhibition of one-carbon transfers mediated by glycinamide ribonucleotide (GAR) formyltransferase (GARFTase) and/or 5-aminoimidazole-4-carboxamide (AICA) ribonucleotide formyltransferase (AICARFTase), leading to synthesis of purine nucleotides. Protection from 6TPs by AICA was incomplete (in contrast to a “pure” GARFTase inhibitor AGF94), suggesting inhibition of AICARFTase. Whereas AGF94 potently inhibited GARFTase (>98%) in KB cells, reflected in [14C]glycine incorporation into [14C]formyl GAR, inhibition by the 6TPs was incomplete (75-90%) up to 1 μM drug. AICARFTase inhibition was confirmed by measuring accumulation of AICA ribonucleotide (ZMP), although this varied for different 6TP analogs. Our studies establish an important structure-activity relationship for novel FRα-targeted 6TPs with aromatic side-chain modifications and imply the therapeutic potential of targeting AICARFTase for EOC. Citation Format: Adrianne Wallace-Povirk, Nian Tong, Carrie O9Connor, Zhanjun Hou, Aleem Gangjee, Larry Matherly, Xilin Zhou. Tumor-targeting with novel dual-targeted 6-substituted thieno[2,3-d]pyrimidine antifolates via cellular uptake by folate receptor α, and inhibition of de novo purine nucleotide biosynthesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3983.