Abstract 4481: Tumor-targeting with novel 6-substituted thienoyl[2,3-d]pyrimidine antifolates via cellular uptake by folate receptor α, and inhibition of de novo purine nucleotide biosynthesis
Authors: Matherly Larry H. , Zhang Xin , Wallace Adrianne , Hou Zhanjun , George Christina , Zhou Xilin , Gangjee Aleem 
Published: August 2015

Abstract
Recent interest has focused on folate receptor (FR) α in delivering folate-based therapeutics to solid tumors, as FRα is expressed in human tumors, notably ovarian and non-small cell lung cancers. While FRα is present in normal epithelial tissues, this occurs at apical membranes where it is inaccessible to the circulation. Selectivity of FR-targeted agents would be enhanced if these were poor substrates for the ubiquitous reduced folate carrier (RFC). We previously described 6-substituted thieno[2,3-d]pyrimidine benzoyl antifolates with varying bridge lengths (n = 2-8 carbons) as potent and selective inhibitors of tumor cells that express FRs. The most potent compound (AGF50) included a 4-carbon bridge and showed FRα selectivity and inhibition of de novo purine biosynthesis. To investigate the role of the side-chain aromatic ring in transport and pathway inhibition, we synthesized AGF50 analogs with a variety of aryl side-chain modifications (AGF102, AGF103, AGF55, AGF65, and AGF132). The various 6-substituted thienoyl[2,3-d]pyrimidine antifolates were tested in proliferation assays with isogenic Chinese hamster ovary (CHO) sublines expressing RFC, proton-coupled folate transporter (PCFT), or FRα, and with KB tumor cells. The analogs were inert toward RFC- and PCFT-expressing cells. Toward FRα-expressing CHO and KB cells, inhibitory potencies were in the order, AGF102 = AGF132 = AGF103 &gt; AGF50 &gt; AGF64 &gt;&gt; AGF55, with IC50s ranging from ∼ 1 nM to 182 nM. Drug effects were abolished with excess folic acid, confirming FR uptake. Inhibition of cell proliferation was reflected in binding affinities to FRα, measured by competition with [3H]folic acid. While PCFT-targeted activity was nominal, all drugs still bound to PCFT, as reflected in inhibition of [3H]methotrexate uptake at pH 5.5 and 6.8. By protection experiments with thymidine or adenosine, de novo purine biosynthesis was confirmed as the principal targeted pathway for the active analogs. While protection by 5-aminoimidazole-4-carboxamide (AICA) was complete with AGF64 (suggesting glycinamide ribonucleotide formyltransferase (GARFTase) as the primary target), for AGF50, AGF102, AGF103 and AGF132, protection was incomplete, implying a dual targeting of GARFTase and AICA ribonucleotide formyltransferase (AICARFTase). Our studies establish an important structure-activity relationship for FRα-targeted antifolates, involving modifications of the side-chain aromatic group that are well tolerated for FRα binding and transport. 6-Substituted thienoyl[2,3-d]pyrimidine analogs were all inhibitors of de novo purine biosynthesis, although relative targeting of GARFTase versus AICARFTase varied with the nature of the aromatic side-chain.
Citation Format: Larry H. Matherly, Xin Zhang, Adrianne Wallace, Zhanjun Hou, Christina George, Xilin Zhou, Aleem Gangjee. Tumor-targeting with novel 6-substituted thienoyl[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 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4481. doi:10.1158/1538-7445.AM2015-4481