pemetrexed and purine

We previously reported the selective transport of classical 2-amino-4-oxo-6-substituted pyrrolo[2,3-d]pyrimidines with a thienoyl-for-benzoyl-substituted side chain and a three- (3a) and four-carbon (3b) bridge. Compound 3a was more potent than 3b against tumor cells. While 3b was completely selective for transport by folate receptors (FRs) and the proton-coupled folate transporter (PCFT) over the reduced folate carrier (RFC), 3a was not. To determine if decreasing the distance between the bicyclic scaffold and l-glutamate in 3b would preserve transport selectivity and potency against human tumor cells, 3b regioisomers with [1,3] (7 and 8) and [1,2] (4, 5, and 6) substitutions on the thienoyl ring and with acetylenic insertions in the four-atom bridge were synthesized and evaluated. Compounds 7 and 8 were potent nanomolar inhibitors of KB and IGROV1 human tumor cells with complete selectivity for FRα and PCFT over RFC.

Topics: Alkynes; Animals; Antineoplastic Agents; Cell Line, Tumor; CHO Cells; Cricetinae; Cricetulus; Drug Screening Assays, Antitumor; Folate Receptor 1; Humans; Proton-Coupled Folate Transporter; Purines; Pyrimidines; Pyrroles; Reduced Folate Carrier Protein; Stereoisomerism; Structure-Activity Relationship; Thiophenes

A series of seven 2-amino-4-oxo-6-substituted thieno[2,3-d]pyrimidines with bridge length variations (from 2 to 8 carbon atoms) were synthesized as selective folate receptor (FR) alpha and beta substrates and as antitumor agents. The syntheses were accomplished from appropriate allylalcohols and 4-iodobenzoate to afford the aldehydes, which were converted to the appropriate 2-amino-4-carbethoxy-5-substituted thiophenes 23-29. Cyclization with chloroformamidine afforded the thieno[2,3-d]pyrimidines 30-36, which were hydrolyzed and coupled with diethyl-L-glutamate, followed by saponification, to give the target compounds 2-8. Compounds 3-6 were potent growth inhibitors (IC(50) 4.7-334 nM) of human tumor cells (KB and IGROV1) that express FRs. In addition, compounds 3-6 inhibited the growth of Chinese hamster ovary (CHO) cells that expressed FRs but not the reduced folate carrier (RFC) or proton-coupled folate transporter (PCFT). However, the compounds were inactive toward CHO cells that lacked FRs but contained either the RFC or PCFT. By nucleoside and 5-amino-4-imidazole carboxamide (AICA) protection studies, along with in vitro and in situ enzyme activity assays, the mechanism of antitumor activity was identified as the dual inhibition of glycinamide ribonucleotide formyltransferase and, likely, AICA ribonucleotide formyltransferase. The dual inhibitory activity of the active thieno[2,3-d]pyrimidine antifolates and the FR specificity represent unique mechanistic features for these compounds distinct from all other known antifolates. The potent inhibitory effects of compounds 3-6 toward cells expressing FRs but not PCFT provide direct evidence that cellular uptake of this series of compounds by FRs does not depend on the presence of PCFT and argues that direct coupling between these transporters is not obligatory.

Topics: Animals; Anion Transport Proteins; Antineoplastic Agents; Biological Transport; Carrier Proteins; Cell Proliferation; CHO Cells; Cricetinae; Cricetulus; Folate Receptors, GPI-Anchored; Folic Acid Antagonists; Gene Expression Regulation; Humans; Hydrogen Bonding; Membrane Transport Proteins; Nucleosides; Phosphoribosylaminoimidazolecarboxamide Formyltransferase; Protons; Purines; Pyrimidines; Receptors, Cell Surface; Reduced Folate Carrier Protein; Substrate Specificity; Transfection