Synthesis, biological, and antitumor activity of a highly potent 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolate inhibitor with proton-coupled folate transporter and folate receptor selectivity over the reduced folate carrier that inhibits β-glycinamide ribonucleotide formyltransferase

Abstract

2-Amino-4-oxo-6-substituted pyrrolo[2,3-d]pyrimidine antifolates with a thienoyl side chain (compounds 1-3, respectively) were synthesized for comparison with compound 4, the previous lead compound of this series. Conversion of hydroxyl acetylen-thiophene carboxylic esters to thiophenyl-α-bromomethylketones and condensation with 2,4-diamino-6-hydroxypyrimidine afforded the 6-substituted pyrrolo[2,3-d]pyrimidine compounds of type 18 and 19. Coupling with l-glutamate diethyl ester, followed by saponification, afforded 1-3. Compound 3 selectively inhibited the proliferation of cells expressing folate receptors (FRs) α or β, or the proton-coupled folate transporter (PCFT), including KB and IGROV1 human tumor cells, much more potently than 4. Compound 3 was more inhibitory than 4 toward β-glycinamide ribonucleotide formyltransferase (GARFTase). Both 3 and 4 depleted cellular ATP pools. In SCID mice with IGROV1 tumors, 3 was more efficacious than 4. Collectively, our results show potent antitumor activity for 3 in vitro and in vivo, associated with its selective membrane transport by FRs and PCFT over RFC and inhibition of GARFTase, clearly establishing the 3-atom bridge as superior to the 1-, 2-, and 4-atom bridge lengths for the activity of this series.

Figure 1

Structures for classical antifolates and novel pyrrolo- and thieno[2,3-d]pyrimidine antifolates. Compounds 4–8 were previously described^–. MTX is a dihydrofolate reductase inhibitor, whereas PMX and RTX are primarily thymidylate synthase inhibitors. Compound 9 is a novel antifolate and dihydrofolate reductase inhibitor that is an active and highly selective substrate for RFC over PCFT^,.

Figure 2

Structures for previous generation GARFTase inhibitors. Lometrexol was originally synthesized by Taylor et al. and the first antifolate inhibitor of GARFTase^,. Compounds 10a and 10b are second generation GARFTase inhibitors as described in the text^–.

Figure 3. Colony formation assay

R2/hPCFT4 cells were inoculated into 60 mm dishes (200 cells per dish), in the presence or absence of a range of concentrations of compound 3, compound 4, LMTX, or PMX. Colonies were enumerated and results are presented as percent of control treated identically but without drugs, as mean values from 3 experiments (plus/minus SEM). IC₅₀s for 3, 4, PMX, and LMTX were 1.4 nM, 27.2 nM, 4.9 nM, and 29.7 nM, respectively.

Figure 4. Transport assays for RFC and PCFT cellular uptake

Panel A: PC43-10 cells ectopically expressing hRFC but no FRs or hPCFT were assayed for [³H]MTX (0.5 uM) uptake at pH 7.2 in the presence of compounds 2–4, or the established RFC substrates, 9, PMX, RTX, LMTX, or LCV (each at 10 µM). Results are compared to those for transporter-null R2 (VC) cells (labeled “vector”). Panel B: Substrate-induced currents (nA) were recorded in individual Xenopus oocytes injected with wild type hPCFT cRNA and voltage clamped to a holding potential of −90 mV. Oocytes were perfused with ND90 solution at pH 5.5 with PMX, compound 3, or compound 4 (all at 5 µM). The design of these experiments was identical to those previously reported^,. Panel C: R2/hPCFT4 cells, ectopically expressing hPCFT, were assayed for cellular uptake of [³H]MTX (0.5 µM) from pH 5.5 to pH 7.2 in the presence of PMX, 3, 4, or 9 (each at 10 µM). For panels A and C, the results are expressed as percent of control (absence of inhibitors) and as mean values plus/minus SEM from 3 experiments. Details for all the transport and binding assays are in the Experimental Section.

Figure 5. FRα and β binding assays for assorted antifolates

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 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 (FA) is 1. Results are presented as mean values plus/minus SEM from 3 experiments. Experimental details are provided in the Experimental Section.

Figure 6. Protection of R2/hPCFT4 cells from growth inhibition by the 6-substituted pyrrolo[2,3-d] pyrimidine thienoyl antifolate 3 (upper) and PMX (lower) in the presence of nucleosides and 5-amino-4-imidazolecarboxamide (AICA)

Proliferation inhibition was measured for R2/hPCFT4 cells over a range of concentrations of compound 3, in the presence or absence of adenosine (60 µM), thymidine (10 µM), and/or AICA (320 µM), as described in the Experimental Section. Results were normalized to cell density in the absence of drug. Results shown are representative data from experiments performed in triplicate.

Figure 7. Intracellular ATP levels and in situ GARFTase inhibition in R2/hPCFT4 cells treated with compound 3, compound 4, LMTX, and PMX

Panel A: R2/hPCFT4 cells were treated with 1 µM of 3, 4, PMX, or LMTX, or solvent (0.5% DMSO for 3 and 4, H₂O for PMX and LMTX) for 24 h. Cells were washed and nucleotides were extracted and analyzed by HPLC. Results are shown for percentage control ATP after the drug treatments. Panel B: GARFTase activity and inhibition were evaluated in situ with R2/hPCFT4 cells. Accumulation of [¹⁴C]formyl GAR from [¹⁴C]glycine was measured in R2/hPCFT4 cells treated with the antifolates. The production of [¹⁴C]formyl GAR was calculated as a percent of vehicle control over a range of antifolate concentrations. Results are presented as mean values ± standard errors from 3 experiments. Methodologic details are described in the Experimental Section. Results with PMX and LMTX were from Kugel Desmoulin et al. IC₅₀s were as follows: 0.69 nM, 3; 1.96 nM, 4; 31.5 nM LMTX; and 7.3 nM, PMX.

Figure 8. In vivo efficacy trial with compounds 3 and 4 with IGROV1 xenografts

Human IGROV1 tumors were implanted bilaterally into female ICR SCID mice maintained on a folate-deficient diet and mice were non-selectively randomized into 4–7 mice/group. Compounds 3 (40 mg/mg) and 4 (180 mg/kg and 112.5 mg/kg) [dissolved in 5% ethanol (v/v), 1% Tween-80 (v/v), 0.5% NaHCO₃] were administered on a Q4dx5 schedule intravenously (0.2 ml/injection) on days 3, 7, 11, 15, and 19. Mice were observed and weighed daily; tumors were measured twice per week. For the experiment shown, antitumor activity was significant for compound 3 (14% T/C, 2.7 gross log kill) and exceeded that for compound 4 at either dose (32% and 44% T/C, 2.3 and 1.2 gross log kill, respectively at 180 mg/kg and 112.5 mg/kg, respectively). The results are summarized in Table 1S in the Supplement.

Scheme 1^a

^a Reagents and conditions: (a) CuI, PdCl₂, PPh₃, Et₃N, CH₃CN, microwave,100 °C, 10 min; (b) 10% Pd/C, H₂, 55psi, MeOH, 4 h; (c) H₂SO₄, CrO₃, 0 °C~RT; (d) i. oxalyl chloride, CH₂Cl₂, reflux, 1 h; ii. diazomethane, Et₂O, RT, 1h; iii. HBr, 70–80 °C, 2 h; (e) DMF, RT, 3 days; (f) i. 1N NaOH, RT, 12 h; ii. 1N HCl; (g) N-methylmorpholine, 2-chloro-4,6-dimethoxy-1,3,5-triazine, L-glutamate diethyl ester hydrochloride, DMF, RT, 12 h.