Abstract 4836: Targeted antifolates with thienoyl regioisomers in the side chain result in improved selectivity and potency against KB human tumor cells
Authors: Gangjee Aleem , Tong Nian , Povrik Adrianne Wallace , Hou Zhanjun , Matherly Larry H. 
Published: July 2016

Abstract
Folates function as cofactors in one-carbon transfer reactions including de novo biosynthesis of nucleotides and play a key in DNA and RNA synthesis, epigenetic processes, cellular proliferation and survival. Cellular folate uptake is mediated by three distinct transporters. Among these, the reduced folate carrier (RFC) is the predominant folate transporter and is critical for antitumor efficacy of currently used classical antifolates such as methotrexate (MTX), pemetrexed (PMX), and raltitrexed (RTX). RFC is ubiquitously expressed in tumor cells and in normal tissues, resulting in dose-limiting toxicity with classical antifolates. We previously reported a series of 6-substituted pyrrolo[2,3-d]pyrimidine antifolates that are selectively transported by folate receptors (FR) and/or by the proton-coupled folate transporter (PCFT) over RFC and inhibit FR/PCFT-expressing human tumor cells (KB and IGROV1) at sub-nanomolar IC50 values. These antifolates are comparatively more potent and reduce the toxicity of clinically available antifolates which have non-selective uptake. As an extension of the SAR, we focused on a novel isosteric series of 6-substituted thieno[2,3-d]pyrimidine analogs, with a 3- or 4- carbon bridge between the thieno[2,3-d]pyrimidine and the thienoyl regioisomeric side chain. This simple isosteric replacement of pyrrole with thiophene in the scaffold, combined with varying the positions of attachment of 3- and 4-carbon bridge and the L-glutamic acid on the thiophene ring, provides compounds with different chain length, conformations and extra hydrogen bond donors and/or acceptors compared to the previously reported compounds. All the analogs (AGF 102, AGF131, AGF 271, AGF 275, AGF 276 and AGF 282) are potently inhibitory toward Chinese hamster ovary (CHO) cells (RT16) expressing human FRá (IC50s of 3.1, 0.33, 0.45, 1.1, 0.83 and 5.6 nM, respectively). All the analogs were inactive in the CHO cell line expressing RFC indicating absolute selectivity for FRá. AGF271, AGF275, and AGF282 showed modest (IC50s from 95-460 nM) with PCFT-expressing CHO cells (R2/PCFT4). AGF102, AGF 131 and AGF 271 AGF 275 AGF 276 AGF 282 were all potently inhibitory toward KB tumor cells (IC50 of 2.10, 1.70, 1.48, 1.81, 5.74 and 7.61nM, respectively). Collectively, our results suggest that AGF102, AGF 131, AGF 271, AGF 275, AGF 276 and AGF 282 are targeted antifolates with activity profiles that suggest that further preclinical studies and optimization are warranted.
Citation Format: Aleem Gangjee, Nian Tong, Adrianne Wallace Povrik, Zhanjun Hou, Larry H. Matherly. Targeted antifolates with thienoyl regioisomers in the side chain result in improved selectivity and potency against KB human tumor cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4836.