Therapeutic Targeting of a Novel 6-Substituted Pyrrolo [2,3-d]pyrimidine Thienoyl Antifolate to Human Solid Tumors Based on Selective Uptake by the Proton-Coupled Folate Transporter

ABSTRACT The proton-coupled folate transporter (PCFT) is a proton-folate symporter with an acidic pH optimum. By real-time reverse transcription-polymerase chain reaction, PCFT was expressed in the majority of 53 human tumor cell lines, with the highest levels in Caco-2 (colorectal adenocarcinoma), SKOV3 (ovarian), and HepG2 (hepatoma) cells. A novel 6-substituted pyrrolo [2,3-d]pyrimidine thienoyl antifolate (compound 1) was used to establish whether PCFT can deliver cytotoxic drug under pH conditions that mimic the tumor microenvironment. Both 1 and pemetrexed (Pmx) inhibited proliferation of R1-11-PCFT4 HeLa cells engineered to express PCFT without the reduced folate carrier (RFC) and of HepG2 cells expressing both PCFT and RFC. Unlike Pmx, 1 did not inhibit proliferation of R1-11-RFC6 HeLa cells, which express RFC without PCFT. Treatment of R1-11-PCFT4 cells at pH 6.8 with 1 or Pmx inhibited colony formation with dose and time dependence. Transport of [ 3 H]compound 1 into R1-11-PCFT4 and HepG2 cells was optimal at pH 5.5 but appreciable at pH 6.8. At pH 6.8, [ 3 H]compound 1 was metabolized to 3 H-labeled polyglutamates. Glycinamide ribonucleotide formyltransferase (GARFTase) in R1-11-PCFT4 cells was inhibited by 1 at pH 6.8, as measured by an in situ GARFTase assay, and was accompanied by substantially reduced ATP levels. Compound 1 caused S-phase accumulation and a modest level of apoptosis. An in vivo efficacy trial with severe combined immunodeficient mice implanted with subcutaneous HepG2 tumors showed that compound 1 was active. Our findings suggest exciting new therapeutic possibilities to selectively deliver novel antifolate drugs via transport by PCFT over RFC by exploiting the acidic tumor microenvironment

Synthesis and Biological Activity of 6-Substituted Pyrrolo[2,3-<i>d</i>]pyrimidine Thienoyl Regioisomers as Inhibitors of de Novo Purine Biosynthesis with Selectivity for Cellular Uptake by High Affinity Folate Receptors and the Proton-Coupled Folate Transporter over the Reduced Folate Carrier

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

Therapeutic targeting of a novel 6-substituted pyrrolo [2,3d]pyrimidine thienoyl antifolate to human solid tumors based on selective uptake by the proton-coupled folate transporter

Non-standard abbreviations: AICARFTase, 5-amino-4-imidazolecarboxamide ribonucleotide CHO, Chinese hamster ovary cells dFBS, dialyzed fetal bovine serum DHFR, dihydrofolate reductase DPBS, Dulbecco&apos;s phosphate-buffered saline FITC, fluorescein isothiocyanate FR, folate receptor GAPDH, glyceraldehyde-3-phosphate dehydrogenase GAR, glycinamide ribonucleotide GARFTase, glycinamide ribonucleotide formyltransferase HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HPLC, high performance liquid chromatography hPCFT, human proton-coupled folate transporter hRFC, human reduced folate carrier IC 50 , fifty percent inhibitory concentration LCV, leucovorin Lmx, lometrexol MES, 4-morphilinopropane sulfonic MTAP, methylthioadenosine phosphorylase MTRP, 5-deoxy-5-(methylthio)ribose-1-phosphate Mtx, methotrexate PCFT, proton-coupled folate transporter PG, polyglutamate PI, propidium iodide PIPES, piperazine-N,N′-bis(2-ethanesulfonic acid) Pmx, pemetrexed RFC, reduced folate carrie