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

Abstract

Multitargeted agents with tumor selectivity result in reduced drug resistance and dose-limiting toxicities. We report 6substituted 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 Lglutamate 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.

Design, Synthesis, and Biological Evaluation of Trisubstituted Piperazine Derivatives as Noncovalent Severe Acute Respiratory Syndrome Coronavirus 2 Main Protease Inhibitors with Improved Antiviral Activity and Favorable Druggability

“…The thiophene α-position is prone to be metabolized, and occupation of the site by substituents such as methyl groups could improve the pharmacokinetic parameters . Additionally, the presence of the methylene group, which is connected to both the aromatic ring and NH of the amide, poses challenges in terms of pharmacokinetics.…”

“…The thiophene αposition is prone to be metabolized, and occupation of the site by substituents such as methyl groups could improve the pharmacokinetic parameters. 27 Additionally, the presence of the methylene group, which is connected to both the aromatic ring and NH of the amide, poses challenges in terms of pharmacokinetics. Therefore, we targeted the (thiophen-2ylmethyl)carbamoyl group and made some preliminary modifications, such as introducing halogen atoms to the thiophene α-position, substitution of the amide NH with methyl, and preparing thiophenesulfonamides to replace the thienylmethylene moiety.…”

A journey into the regulatory secrets of the de novo purine nucleotide biosynthesis

“…This molecule, designated as « multi-targeted antifolate », acts as a broad inhibitor of not only TrifGART, but also ATIC and two other folate dependent enzymes, thymidylate synthase and dihydrofolate dehydrogenase ( Shih et al, 1997 ). Recently, replacing the pyrrolo [2,3-d]pyrimidine core by a thieno [2,3-d]pyrimidine one led to the discovery of novel multitargeted inhibitors ( Tong et al, 2023 ). The advantage of this series is their ability to be transported through folate receptors which confer a more selective delivery route to tumors compared to normal cells.…”