Abstract 3824: Design, synthesis and biological evaluation of 4-atom side chain pemetrexed (PMX) homolog AG127 as a potent inhibitor of tumors expressing folate receptors via 5-aminoimidazole-4-carboxamide ribonucleotide formyl transferase (AICARFTase) inhibition
Authors: Wang Yiqiang , Ravindra Manasa Punaha , Ryan Shermaine Mitchell , Kisliuk Roy L. , Matherly Larry H. , Gangjee Aleem 
Published: August 2012

Abstract
Two major hurdles in cancer therapy are the inability of chemotherapeutic agents to selectively target tumor cells over normal tissues, and the development of tumor cell resistance. The multi-enzyme-targeted anticancer drug pemetrexed (PMX) suffers from dose-limiting toxicity, in part due to its membrane transport by the reduced folate carrier (RFC) which is ubiquitously expressed in normal and tumor cells. We recently described a novel series of 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolates. A highly active compound of this series (AG71) included a 4-carbon bridge and was selectively transported into cells by folate receptors (FRs) and the proton-coupled folate transporter (PCFT) but not RFC. AG71 inhibited de novo purine biosynthesis at the level of glycinamide ribonucleotide formyltransferase (GARFTase). AG71 potentially inhibited proliferation of FR- and PCFT-expressing Chinese hamster ovary cells and of both KB and IGROV1 tumors in vitro; in SCID mice with subcutaneous KB tumors, AG71 was highly active against both early and advanced stage tumors. Based on the 5-substituted pyrrolo[2,3-d]pyrimidine scaffold of PMX and our results with the 6-substituted compound AG71, we synthesized 5-substituted pyrrolo[2,3-d]pyrimidine phenyl analogs with 1- to 6-carbon atoms in the bridge (AG124-AG129). Analogs were tested for anti-proliferative effects on KB human tumor cells and on Chinese hamster ovary (CHO) sublines engineered to individually express RFC (PC43-10), PCFT (R2/PCFT4), and FRα (RT-16). The 4-carbon atom chain analog (AG127) showed IC50 value of 1.9 nM, only slightly less than for AG71 (0.55 nM), in inhibiting proliferation of KB tumor cells in culture. In the CHO sublines, both AG127 and AG71 inhibited growth of RT-16 cells (IC50 = 3.1 nM and 1.8 nM respectively). AG71 was inactive toward PC43-10 cells and was inhibitory toward R2/PCFT4 cells (IC50 = 43.4 nM). Compared to RT-16 cells, AG127 showed reduced inhibitory activity toward PC43-10 cells (IC50 = 54 nM) and R2/PCFT4 cells (IC50 = 733 nM). PMX is a multi-targeted agent with its principle inhibitory effects on thymidylate synthase and secondary inhibitory effects on AICARFTase and GARFTase in de novo purine nucleotide biosynthetic. Inhibition of KB cell proliferation by AG127 was abolished by excess adenosine, but not thymidine or AICA, suggesting that AICARFTase was the principle intracellular target. Collectively, our results show that the side chain homologated analog of PMX, AG127, exhibits nanomolar inhibition of proliferation of KB tumor cells in culture. The unique and specific AICARFTase inhibition of AG127 makes it a promising option for cancer chemotherapy.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3824. doi:1538-7445.AM2012-3824