Abstract 2798: Folate receptor-targeted cancer chemotherapy with novel 6-substituted thieno[2,3-d]pyrimidine compounds with nitrogen and fluorine containing aromatic side chains as de novo purine biosynthesis inhibitors
Authors: Tong Nian , Gangjee Aleem , Povrik Adrianne W. , Connor Carrie O. , Hou Jianjun , Matherly Larry 
Published: August 2018

Targeted cancer chemotherapy inhibits the growth of cancer cells by interfering with critical pathways required for tumor progression. Three major transporters are involved in membrane transport of folates into mammalian cells and include the reduced folate carrier (RFC), folate receptors (FRs) α and β, and proton-coupled folate transporter (PCFT). The clinically used agents methotrexate, pemetrexed, raltitrexed and pralatrexate are all transported by the ubiquitously expressed RFC into both tumor cells and normal tissues, and hence are non-selective. The lack of tumor selectivity results in dose-limiting toxicity. We reported 6-substituted thieno[2,3-d]pyrimidine (6TP) compounds with a phenyl side-chain and bridge lengths of 2-8 carbons as potent inhibitors that are selectively transported by FRs over RFC and inhibit proliferation of FR-expressing KB tumor cells with low nanomolar IC50s. The most potent compound (AGF50), a thieno[2,3-d]pyrimidine with a 4-carbon bridge and a phenyl side chain, showed FR selectivity over RFC and potent inhibition of de novo purine biosynthesis at least in part due to inhibition of glycinamide ribonucleotide formyltransferase (GARFTase). In this report, fluorine was strategically placed on the aryl side chain which could induce conformational restriction due to possible intramolecular fluorine-hydrogen bonding interactions. We found that fluorine substitution on the phenyl side-chain, ortho (o-) to L-glutamate (AGF304) improved potency and selectivity for FRα (4.5-fold) and FRβ (20-fold) compared to AGF50, as reflected in inhibition of proliferation of Chinese hamster ovary cells expressing human FRs. The pyridine nitrogen can also form H-bonds with NH of the L- glutamate amide, which could improve the potency and selectivity of 6TPs with pyridine side-chains. Analogs with systematic pyridine side-chain modifications, as well as ortho fluorine substitution on the pyridine ring, were designed, modeled, and synthesized. The 2',5'-pyridine analog AGF132 was ~9-fold more active toward FRα-expressing cells but approximately equipotent toward FRβ. The fluorinated 2',5'-pyridine analog AGF305 was ~5-fold less potent for FRα and was &gt;50-fold more potent for FRβ compared to AGF132. AGF132, AGF304 and AGF305 inhibited growth of KB human tumor cells approximately equivalent to AGF50. Whereas adenosine completely protected cells from growth inhibition by the 6TPs, protection with 5-aminoimidazole-4-carboxamide (AICA) was incomplete, suggesting inhibition at AICA ribonucleotide formyltransferase (AICARFTase), along with GARFTase. Our 6TPs with aryl side-chain modifications are slated for further preclinical studies to identify potential clinical candidates that target the de novo purine biosynthesis pathway.

Citation Format: Nian Tong, Aleem Gangjee, Adrianne W. Povrik, Carrie O. Connor, Jianjun Hou, Larry Matherly. Folate receptor-targeted cancer chemotherapy with novel 6-substituted thieno[2,3-d]pyrimidine compounds with nitrogen and fluorine containing aromatic side chains as de novo purine biosynthesis inhibitors [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 2798.