Synthesis and Biological Activity of a Novel Series of 6-Substituted Thieno[2,3-d]pyrimidine Antifolate Inhibitors of Purine Biosynthesis with Selectivity for High Affinity Folate Receptors over the Reduced Folate Carrier and Proton-Coupled Folate Transporter for Cellular Entry^†

Abstract

A series of seven 2-amino-4-oxo-6-substituted thieno [2,3-d]pyrimidines, with bridge length variations (from 2-8 carbon atoms) were synthesized as selective folate receptor (FR) α and β 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 chlorformamidine 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 to 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.

Targeting the Proton-Coupled Folate Transporter for Selective Delivery of 6-Substituted Pyrrolo[2,3-d]Pyrimidine Antifolate Inhibitors of De Novo Purine Biosynthesis in the Chemotherapy of Solid Tumors

“…As part of our larger drug discovery endeavor to establish pharmacophores for all the major folate transporters (Deng et al, 2008(Deng et al, , 2009 and to develop transporter-specific drugs, we previously generated novel sublines derived from the RFC-, FR-, and PCFT-null MtxRIIOua R 2-4 CHO cells (hereafter, simply R2) that ectopically express hRFC (designated PC43-10) (Wong et al, 1995) and human FRs (Deng et al, 2008).…”

“…This reasoning was the impetus to develop drugs that are selectively transported by FRs over RFC (Gibbs et al, 2005;Hilgenbrink and Low, 2005;Salazar and Ratnam, 2007;Deng et al, 2008Deng et al, , 2009Wang et al, 2010). Such agents can target tumors (e.g., ovarian adenocarcinomas) that express high levels of FRs (Elnakat and Ratnam, 2004).…”

“…For agents such as Pmx that are transported by both RFC and PCFT, loss of tumor selectivity could be due to RFC transport in normal tissues. Although RFC-selective agents without PCFT transport were described (PT523, GW1843U89) (Zhao and Goldman, 2007;Deng et al, 2009), until our recent report of pyrrolo [2,3-d]pyrimidine thienoyl antifolate substrates for PCFT , no analogous PCFT-selective cytotoxic agents without RFC transport had been reported.…”

Structures of human folate receptors reveal biological trafficking states and diversity in folate and antifolate recognition

“…Similarly, AMT and MTX bind in the same mode and are useful for studies of additional diaminopteridine analogs. A number of additional folate metabolites or analogs should be pursued for structural study to increase the diversity of available model complexes, including reduced folates and leucovorin (19), as well as the newly developed ONX801 (13) and 6-substituted thieno-pyrimidines, analogs that show varying degrees of specificity for the folate receptors over the reduced folate carrier (51,52).…”

“…Monoclonal antibodies against hFRα and hFRβ could promote clearance of FRpositive cells by the immune system, folate-conjugates aim to deliver cytotoxic cargo or imaging agents to FR-positive cells, and FR-targeted antifolates would potentially eliminate cytotoxic side effects of current antifolates delivered to normal cells via RFC (12,16,45,(48)(49)(50)(51)(52). Because these therapies have enormous potential for the treatment of cancer and inflammatory disease, we performed a detailed molecular study of hFRs to understand biological trafficking and principles of ligand binding and release of the folate receptors.…”