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

Abstract

Abstract2-Amino-4-oxo-6-substituted pyrrolo [2,3-d]pyrimidines with a thienoyl side chain and 4-6 carbon bridge lengths (compounds 1-3) were synthesized as substrates for folate receptors (FRs) and the proton-coupled folate transporter (PCFT). Conversion of acetylene carboxylic acids to α-bromomethylketones and condensation with 2,4-diamino-6-hydroxypyrimidine afforded the 6-substituted pyrrolo [2,3-d]pyrimidines. Sonogashira coupling with (S)-2-[(5-bromo-thiophene-2-carbonyl)-amino]-pentanedioic acid diethyl ester, followed by hydrogenation and saponification, afforded 1-3. Compounds 1 and 2 potently inhibited KB and IGROV1 human tumor cells that express FRα, reduced folate carrier (RFC), and PCFT. The analogs were selective for FR-and PCFT over RFC. Glycinamide ribonucleotide formyltransferase was the principal cellular target. In SCID mice with KB tumors, 1 was highly active against both early (3.5 log kill, 1/5 cures) and advanced (3.7 log kill, 4/5 complete remissions) stage tumors. Our results demonstrate potent in vitro and in vivo antitumor activity for 1 due to selective transport by FRs and PCFT over RFC.

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

“…Finally, the present results, combined with earlier studies of 6-substituted pyrrolo-and thieno [2,3-d]pyrimidine antifolates (Deng et al, 2009;Wang et al, 2010), shed light on the impact of both aromatic ring systems and the length of the bridge domain on transport by PCFT versus other folate transporters. Thus, antifolates with thieno[2,3-d]pyrimidine and benzoyl rings (designated A and B rings, respectively) (labeled "I" in Fig.…”

“…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).…”

Functional Loss of the Reduced Folate Carrier Enhances the Antitumor Activities of Novel Antifolates with Selective Uptake by the Proton-Coupled Folate Transporter

“…In this study, we expanded on previous reports (Zhao and Goldman, 2007;Goldman et al, 2010;Kugel Desmoulin et al, 2010Wang et al, 2010Wang et al, , 2011) that hPCFT may be We demonstrated that functional loss of hRFC in R5 cells caused a contraction of total cellular THF cofactor pools derived from 5-CHO-THF, which enhanced both C1 and C2 antitumor activities compared with those in WT cells. Of importance, the reduction in total cellular folate pools in R5 cells was exacerbated in the presence of C1 and C2, through direct competition at hPCFT, which further restricted cellular uptake of exogenous 5-CHO-THF.…”

Substrate- and pH-Specific Antifolate Transport Mediated by Organic Anion-Transporting Polypeptide 2B1 (OATP2B1-SLCO2B1)

“…Although the radiolabeled form of raltitrexed was not available, its transport and higher affinity for the carrier relative to pemetrexed were confirmed by the much greater growth inhibition produced by this agent in OATP2B1-transfected cells. New generations of antifolates might have even higher affinities for this transporter (Wang et al, 2010). However, whether transport mediated by OATP2B1 could contribute to the pharmacological activity of antifolates in vivo would depend upon several factors: (i) the level of expression in solid tumors, (ii) the pH in the tumor microenvironment, (iii) and the relative levels of expression and activities of the much higher-affinity and potent folate transporters RFC and PCFT.…”