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

2-Amino-4-oxo-6-substituted pyrrolo[2,3-d]pyrimidines with a thienoyl side chain and four to six 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 alpha-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 FR alpha, 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.

Figure 1

Structures of novel 6-substituted pyrrolo[2,3-d]pyrimidines with a thienoyl ring in the side chain 1, 2, and 3, and previously reported, benzoyl ring analogs 4 and 5.

Figure 2

Structure of classical antifolates

Figure 3

GARFTase Inhibitors

Scheme 1

a. oxalyl chloride, CH₂Cl₂, reflux, 1h; b. diazomethane, Et₂O, r.t., 1h; c. 48% HBr, 80°C, 2h; d. DMF, r.t., 3d; e. 19, CuI, Pd(0)(PPh₃)₄, Et₃N, DMF, r.t., 12h; f. 5% Pd/C, H₂, 55psi, 2h; g. (i) 1N NaOH, r.t., 6h; (ii) 1N HCl; h. N-methylmorpholine, 2-chloro-4,6-dimethoxy-1,3,5-triazine, L-glutamate diethyl ester hydrochloride, DMF, r.t., 12 h.

Figure 4. Growth inhibition of KB cells by the 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolate 1 in the presence of physiologic concentrations of reduced folate

Cell proliferation inhibition was measured over a range of concentrations of compound 1 in complete folate-free RPMI1640 in the presence of LCV at 2, 25, and 50 nM. Cell densities were measured with CellTiter Blue™ fluorescence dye and a fluorescence plate reader. Results were normalized to cell density in the absence of drug. Results shown are representative data of experiments performed in duplicate.

Figure 5. Colony-forming inhibition assay

KB cells were inoculated into 60 mm dishes with a density of 200 cells per dish, in the presence or absence of a range of concentrations of methotrexate (upper panel) or compound 1 (lower panel). Colonies were enumerated after 10-14 days after staining with methylene blue. Data were calculated as percent of controls treated identically but without drugs. Results are presented as mean values from 3 experiments (plus/minus SEM).

Figure 6. Protection of KB and R2/hRFC4 cells from growth inhibition by the 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolate 1 in the presence of nucleosides and 5-amino-4-imidazole (AICA)

Proliferation inhibition was measured for KB (upper panel) and R2/hPCFT4 (lower panel) cells over a range of concentrations of compound 1, as shown, in complete folate-free RPMI1640 with 2 nM (KB) or 25 nM (R2/hPCFT4) leucovorin, in the presence or absence of adenosine (60 μM) or thymidine (10 μM), or AICA (320 nM). Cell densities were measured with CellTiter Blue™ fluorescence dye and a fluorescence plate reader. Results were normalized to cell density in the absence of drug. Results shown are representative data of experiments performed in triplicate.

Figure 7. Transport parameters for FR, hRFC, and hPCFT cellular uptake

Panel A: Data are shown for the effects of the unlabeled ligands with FRα-expressing RT16 CHO cells and FRβ-expressing D4 CHO cells. Relative binding affinities for assorted folate/antifolate substrates were determined over a range of ligand concentrations and were calculated as the inverse molar ratios of unlabeled ligands required to inhibit [³H]folic acid binding by 50%. By definition, the relative affinity of folic acid is 1. Panel B: PC43-10 cells ectopically expressing hRFC but no FR or PCFT were assayed for [³H]MTX (0.5 μM) uptake at pH 7.2 in the presence of the pyrrolo[2,3-d]pyrimidine antifolates 1-3, or the (anti)folates PMX, RTX, LMTX, or LCV (each at 10 μM). Panel C: R2/hPCFT4 cells ectopically expressing hPCFT but not FR or hRFC were assayed for [³H]MTX (0.5 μM) uptake from pH 5.5 to pH 7.2 in the presence of the pyrrolo[2,3-d]pyrimidine thienoyl antifolates 1-3, or PMX (each at 10 μM). For panels B and C, results are expressed as a percent of control, incubated in parallel but without addition of inhibitor. Details for the transport and binding assays are provided in the Experimental Section. Results are presented as mean values plus/minus SEM from 3 experiments.

Figure 8. In vivo efficacy trial with compound 1

Female ICR SCID mice (12 weeks old; 21 g average body weight) were maintained on a folate-deficient diet ad libitum for 27 days prior to subcutaneous tumor implant to decrease serum folate to a concentration approximating that in human serum. Human KB tumors were implanted bilaterally and mice were non-selectively randomized into 5 mice/group. Compound 1 [dissolved in 5% ethanol (v/v), 1% Tween-80 (v/v), 0.5% NaHCO₃] was administered on a Q4dx4 schedule intravenously (0.2 ml/injection) on days 4, 8, 12, and 16 for early stage disease or on days 29, 33, 37, and 41 for advanced stage disease beginning when the KB tumor burdens were 600-689 mg. Mice were observed and weighed daily; tumors were measured twice per week. For the experiment shown, activity was significant for both early (3.5 log kill, 1/5 cures; T-C = 47 days) and late (3.7 log kill, 4/5 complete remissions; T-C = 49 days) stage tumors.