Multitargeted 6-Substituted Thieno[2,3- d ]pyrimidines as Folate Receptor-Selective Anticancer Agents that Inhibit Cytosolic and Mitochondrial One-Carbon Metabolism.

Multitargeted agents with tumor selectivity result in reduced drug resistance and dose-limiting toxicities. We report 6-substituted thieno[2,3- d ]pyrimidine compounds ( 3 - 9 ) with pyridine ( 3 , 4 ), fluorine-substituted pyridine ( 5 ), phenyl ( 6 , 7 ), and thiophene side chains ( 8 , 9 ), for comparison with unsubstituted phenyl ( 1 , 2 ) and thiophene side chain ( 10 , 11 ) containing thieno[2,3- d ]pyrimidine compounds. Compounds 3 - 9 inhibited proliferation of Chinese hamster ovary cells (CHO) expressing folate receptors (FRs) α or β but not the reduced folate carrier (RFC); modest inhibition of CHO cells expressing the proton-coupled folate transporter (PCFT) by 4 , 5 , 6 , and 9 was observed. Replacement of the side-chain 1',4'-phenyl ring with 2',5'-pyridyl, or 2',5'-pyridyl with a fluorine insertion ortho to l-glutamate resulted in increased potency toward FR-expressing CHO cells. Toward KB tumor cells, 4 - 9 were highly active (IC 50 's from 2.11 to 7.19 nM). By metabolite rescue in KB cells and in vitro enzyme assays, de novo purine biosynthesis was identified as a targeted pathway (at 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase (AICARFTase) and glycinamide ribonucleotide formyltransferase (GARFTase)). Compound 9 was 17- to 882-fold more potent than previously reported compounds 2 , 10 , and 11 against GARFTase. By targeted metabolomics and metabolite rescue, 1 , 2 , and 6 also inhibited mitochondrial serine hydroxymethyl transferase 2 (SHMT2); enzyme assays confirmed inhibition of SHMT2. X-ray crystallographic structures were obtained for 4 , 5 , 9 , and 10 with human GARFTase. This series affords an exciting new structural platform for potent multitargeted antitumor agents with FR transport selectivity.