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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
J Med Chem. Author manuscript; available in PMC 2012 October 27.
Published in final edited form as:
PMCID: PMC3209708
NIHMSID: NIHMS328244

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 13. Compound 3 selectively inhibited proliferation of cells expressing folate receptors (FRs) α or β, or the proton-coupled folate transporter (PCFT), including human tumor cells KB and IGROV1 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.

INTRODUCTION

In the era of targeted therapeutics for cancer, antifolates continue to occupy a unique niche in the treatment of a variety of malignancies including acute lymphoblastic leukemia, lymphomas, osteosarcoma, and malignant pleural mesothelioma1. Clinically relevant antifolates (Figure 1) such as methotrexate (MTX), pemetrexed (PMX), and raltitrexed (RTX) are all substrates for the ubiquitously expressed reduced folate carrier (RFC) which is the major folate transport system in tissues and tumors2. RFC levels and activities are important determinants of drug efficacy and loss of RFC is a common mode of antifolate drug resistance2,3. However, RFC is also expressed in normal tissues such that it is likely to be a determinant of dose-limiting drug toxicity, although other factors such as systemic folate levels (both circulating and intracellular forms), capacities to synthesize antifolyl polyglutamates, and rates of cell proliferation all come into play2.

Figure 1
Structures for classical antifolates and novel pyrrolo- and thieno[2,3-d]pyrimidine antifolates. Compounds 48 were previously described1214. MTX is a dihydrofolate reductase inhibitor, whereas PMX and RTX are primarily thymidylate synthase ...

Along with RFC, other folate membrane transporters are recognized as important for physiologic functions in relation to in vivo folate homeostasis and likely contribute to assorted clinical manifestations of folate deficiency. For instance, folate receptor (FR) α is expressed in epithelial tissues such as the renal tubules and cerebral microvasculature where it is important in folate reabsorption and folate transport across the blood-brain-barrier, respectively4. A folate-proton symporter, the proton-coupled folate transporter (PCFT), is expressed in the upper small intestine (e.g., jejunum) where it represents the primary mode of dietary folate absorption and its loss is causal in the autosomal recessive folate-deficient condition termed hereditary folate malabsorption57.

Interestingly, these non-RFC transport mechanisms have also attracted attention for their potentials in chemotherapy drug targeting, particularly for cancer, since FRs are expressed in a subset of malignancies such as ovarian and endometrial cancers4,8, and PCFT is present at high levels in assorted solid tumors (including lung cancers, ovarian cancer, and hepatomas)6,9. Further, PCFT is maximally active at pHs approximating those attained in the solid tumor microenvironment6,7,10. Examples of folate-based therapeutics for cancer with selective transport by FRs include a folate-conjugated microtubule inhibitor licensed by Bristol-Myers Squibb (http://www.cancer.gov/drugdictionary/?CdrID=575735), and N-[(4-{(6RS)-2-hydroxymethyl-4-oxo-4,6,7,8-tetrahydro-3H-cyclopenta[g]quinazolin-6-yl](prop-2-yn-1-yl)amino}phenyl)carbonyl]-L-(-glutamyl-D-glutamic acid (ONX0801)11, a small molecule antifolate licensed by Onyx Pharmaceuticals which is a potent inhibitor of thymidylate synthase. Both of these compounds are in clinical trials.

We reported9, 1215 the 6-substituted pyrrolo- and thieno[2,3-d]pyrimidine benzoyl L-glutamate antifolate scaffolds 7 and 8 (Figure 1) to identify analogs with selective membrane transport by PCFT and/or FRs but not by RFC. For both series, the lengths of the bridge region were systematically tested. The most active agents had 3- or 4-carbon bridge lengths and were potent inhibitors of β-glycinamide ribonucleotide (GAR) formyltransferase (GARFTase) which catalyzes the third step in de novo purine biosynthesis in which GAR is converted to formyl GAR with 10-formyl tetrahydrofolate as the one-carbon donor. Drug potencies declined with increasing (>4 carbons) or decreasing (<3 carbons) bridge lengths and were further impacted by isosteric replacement of the benzoyl ring of the pyrrolo[2,3-d]pyrimidine series with a thienoyl ring (compounds 46)9,1215. The 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolate with a 4-carbon bridge (compound 4 in Figure 1) was the most active analog toward both FR- and PCFT-expressing tumors both in vitro and in vivo14. However, analogs of this series with less than 4 carbons in the bridge region were not previously synthesized and tested.

A number of other antifolates have been previously reported to inhibit GARFTase as their principal cellular targets. Lometrexol [(6R)5,10-dideazatetrahydrofolate; LMTX] (Figure 2) originated from a collaboration between academic laboratories and Eli Lilly Corporation in the mid 1980s and shortly thereafter was described as an inhibitor of de novo purine biosynthesis at GARFTase1720. For 2nd generation GARFTase inhibitors, replacement of the 1,4-phenyl ring by a 2,5-thienyl ring increased potency over LMTX (e.g., 10a (LY309887)20,21 and 10b (AG2034)22). LMTX, 10a and 10b were all entered into clinical trials2124; the toxicities experienced were likely due, at least in part, to their transport by RFC and polyglutamylation by folypolyglutamate synthetase in susceptible normal tissues. These early studies provided further impetus to developing agents with FR and PCFT transport selectivity over RFC.

Figure 2
Structures for previous generation GARFTase inhibitors. Lometrexol was originally synthesized by Taylor et al.17 and the first antifolate inhibitor of GARFTase18,19. Compounds 10a and 10b are second generation GARFTase inhibitors as described in the text ...

In this report, we describe the synthesis and biologic properties of compounds 13, analogs of compound 4 with 1 to 3 bridge carbons, respectively, linking a 6-substituted pyrrolo[2,3-d]pyrimidine moiety with a thienoyl ring, for direct comparison with compound 4. Our results demonstrate the dramatically increased potency of compound 3 toward both PCFT- and FR-expressing cells, associated with its greater inhibition of intracellular GARFTase. In an efficacy trial toward severe combined immunodeficient (SCID) mice bearing early stage IGROV1 ovarian carcinoma, compound 3 was substantially more active than compound 4.

CHEMISTRY

The synthesis of target compounds 13 is shown in Scheme 1. A palladium-catalyzed Sonogashira coupling of 5-bromo-thiophene-2-carboxylic acid methyl ester, 12, with prop-2-yn-1-ol, 11a, or but-3-yn-1-ol, 11b, afforded unsaturated alcohols 13a and 13b, respectively, which were catalytically hydrogenated to give the saturated alcohols 14a and 14b in quantitative yield. Subsequent oxidation of 14a and 14b using Jones reagent afforded the carboxylic acids 15b and 15c. Compound 15a is commercially available. The carboxylic acids 15ac were then converted to the acid chlorides and immediately reacted with diazomethane followed by 48% HBr to give the desired α-bromomethylketones 16ac. Condensation of 2,6-diamino-3H-pyrimidin-4-one, 17, with 16ac at room temperature for 3 days afforded the 2-amino-4-oxo-6-substitutedpyrrolo[2,3-d]pyrimidines 18ac. Hydrolysis of 18ac afforded the corresponding free acids 19ac. Subsequent coupling with L-glutamate diethyl ester using 2-chloro-4,6-dimethoxy-1,3,5-triazine as the activating agent afforded the diesters 20ac. Final saponification of the diesters gave the desired compounds 13.

Scheme 1a
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, ...

BIOLOGICAL EVALUATION AND DISCUSSION

6-Substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolates as potent inhibitors of tumor cell proliferation

Our previous studies of 6-substituted pyrrolo- and thieno[2,3-d]pyrimidine benzoyl analogs showed that the intra-molecular distance between the bicyclic scaffold and the L-glutamate was critical to inhibitory potencies, and that an isosteric thienoyl-for-benzoyl substitution in the side chain conferred dramatically enhanced antiproliferative activities for analogs with 4- (compound 4) and 5- (compound 5) carbon bridge lengths (Figure 1) compared to the corresponding benzoyl analogs9,1215. In the present study, we expanded the 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl series of antifolates to include analogs with truncated bridge lengths of 1–3 carbons (compounds 13) (Figure 1).

Compounds 13 were tested for their capacities to inhibit proliferation of a series of isogenic Chinese hamster ovary (CHO) cell lines individually expressing each of the major human (anti)folate uptake systems including human RFC (hRFC) (PC43-10), FRα (RT16), FRβ (D4), and human PCFT (hPCFT) (R2/hPCFT4)12,13,15. Additional drug screening was done with KB (nasopharyngeal) and IGROV1 (ovarian) human tumor cell lines that express RFC, PCFT and FRα. For these experiments, cells were continuously exposed to a wide range of drug concentrations in standard RPMI1640/10% dialyzed fetal bovine serum (dFBS) (for PC43-10), or in folate-free RPMI/10% dFBS, supplemented with 2 nM (RT16, D4, KB, IGROV1) or 25 nM leucovorin [(6R,S)5-formyl tetrahydrofolate (LCV)] (R2/hPCFT4). Relative cell numbers were assessed after 96 h. Negative controls for RFC- and PCFT-expressing CHO sublines included the RFC-, FR-, and PCFT-null MTXRIIOuaR2–4 CHO parental cell line used for transfections (hereafter, designated R2) or vector control R2 cells [R2(VC)], both cultured under the same conditions. For FR-expressing cells, FR-mediated drug uptake was confirmed by treating a parallel culture with the antifolates, along with excess (200 nM) folic acid to block FRs. Results with compounds 13 were compared to those for compound 4 and for classical antifolates including MTX, PMX, LMTX, RTX, and compound 9 (Figures 1 and and2).2). These results are summarized in Table 1.

Table 1
IC50s (in nM) for 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolates 1–4, and classical antifolates in hRFC-, hPCFT-, and FR-expressing cell lines

The known classical antifolates were all growth inhibitory toward the RFC-, PCFT-, and FR-expressing CHO sublines. Compound 3 was more active than the classic agents and likewise more so than compounds 1 and 2, or compound 4, the 4-carbon bridge analog and the previous lead compound of this series14. This was most impressively reflected in the ~11-fold decreased IC50 (~3 nM) toward PCFT-expressing R2/hPCFT4 cells and the ~6-fold decreased IC50 toward FRα-expressing RT16 cells with 3 compared to 4. Activity substantially declined for compounds 1 and 2 with 1- and 2-carbon bridge lengths, respectively. While 1 and 2 were inert toward RFC-expressing PC43-10 cells and RFC-, PCFT-, and FR-null R2 cells up to 1000 nM drug, compound 3 showed evidence of a non-FR, non-PCFT cellular uptake process at higher drug concentrations (seemingly in part mediated by RFC and also by a non-RFC uptake mechanism), although substantial FR- and PCFT-selectivity over RFC (as reflected in relative IC50s) was nonetheless preserved (Table 1). Compounds 3 and 4 showed slightly (~2-fold) increased activities (decreased IC50s) toward FRβ-expressing D4 cells over those for FRα-expressing RT16 cells, although this was statistically significant (i.e., p<0.05) only for compound 4. For RT16 and D4 sublines with both compounds 3 and 4, growth inhibition was abolished with excess (200 nM) folic acid. Analogous results were observed with KB and IGROV1 human tumor cells such that the IC50s for compound 3 were an impressive 0.26 nM and 0.55 nM, respectively, or ~2-fold lower than the IC50s for compound 4.

The results of the cell proliferation assays for R2/hPCFT4 cells were recapitulated in the results of colony-forming assays over 10 days in the continuous presence of 3 (0.5–10 nM), 4 (10–100 nM), PMX (1–20 nM), or LMTX (10–100 nM) (Figure 3). IC50s in colony-forming assays closely approximated those in the cell proliferation assays (Table 1) with R2/hPCFT4 cells and were 1.4 nM, 27.2 nM, 4.94 nM, and 29.7 nM, respectively.

Figure 3
Colony formation assay

Collectively, these results establish significantly higher potencies for compound 3 over 4 as an inhibitor of proliferation for cells expressing FRs and/or PCFT with substantial selectivity for transport by FRs and PCFT over RFC.

Membrane transport characteristics for compound 3

Compound 3 showed demonstrably higher anti-proliferative activities toward FR- and hPCFT-expressing cells (Table 1) compared to compound 4 and the classical antifolate drugs, whereas hRFC-directed activity was substantially decreased (compound 3) or was altogether undetectable up to 1000 nM drug (compounds 1, 2, and 4). To further examine biochemical measures of drug transport by hRFC, FR, and hPCFT, functional assays were performed for compounds 2, 3, and 4, the most active compounds of the series.

As competitive inhibitors of transport of [3H]MTX (0.5 µM) by hRFC in PC43-10 cells (2 min influx, 37° C), compounds 24 (at 10 µM) were all poorly inhibitory, with 3 showing the greatest effect (~20% inhibition relative to control) (Figure 4, panel A). The inhibitory effects of compounds 24 toward hRFC were substantially less than the inhibitions detected with 10 µM of 9, PMX, RTX, LMTX, or LCV, all established hRFC substrates.

Figure 4
Transport assays for RFC and PCFT cellular uptake

With FRs and the most active analogs (compounds 24), we used competitive binding assays to measure the abilities of unlabeled ligands to compete for FR binding with [3H]folic acid. For these assays, RT16 (FRα) and D4 (FRβ) cells were briefly treated with acid-buffered saline to dissociate FR-bound folate, then with [3H]folic acid (at neutral pH) in the presence of unlabeled competitors including compounds 2, 3, and 4, classic antifolates (LMTX, PMX, MTX), or unlabeled folic acid (50 nM). Cells were washed (neutral pH), after which FR-bound [3H]folic acid was measured with results expressed as pmol [3H]folic acid per mg of cellular protein. Relative inhibitor affinities were calculated as the inverse molar ratios of the concentrations of unlabeled (anti)folates required to decrease bound [3H]folic acid by 50%. By this assay, binding of 2 was undetectable and analogous to that for MTX and PMX, both poor substrates for FRs in spite of detectable low levels of growth inhibition toward FR-expressing cells. There was no relationship between binding affinities for FRα or FRβ (Figure 5) and inhibition of proliferation of RT16 or D4 CHO cells, respectively, by compounds 3 and 4 (Table 1).

Figure 5
FRα and β binding assays for assorted antifolates

To confirm membrane transport of compound 3 by PCFT, we used electrophysiology experiments with Xenopus oocytes microinjected with hPCFT cRNA and clamped at −90 mV using a bath pH of 5.5. As for established PCFT substrates (e.g., PMX)15,16, compounds 3 and 4 at 5 µM induced currents, whereas current (Figure 4, panel B) was not induced in oocytes microinjected with H2O in lieu of hPCFT cRNA (not shown). There was no obvious difference in the current induced by compound 3 and that by compound 4 or PMX (all at 5 µM).

As an additional measure of transport by hPCFT, we compared compound 3 to 4 and to PMX or 9 (at 10 µM) as competitors of hPCFT-mediated transport of [3H]MTX (0.5 µM) in R2/hPCFT4 cells from pH 5.5 to pH 7.2. Whereas 9 did not inhibit [3H]MTX transport by hPCFT at any pH, compounds 3, 4 and PMX were all potent and essentially equivalent inhibitors, with the relative extents of inhibition dramatically increasing from pH 6.8 to pH 5.5 (Figure 4, panel C).

To calculate Ki values as measures of relative binding affinities for hPCFT, we performed Dixon analyses at pH 5.5 and pH 6.8 with [3H]MTX and variable concentrations of unlabeled substrates, including compounds 3, 4, and PMX. By this analysis, Kis for compounds 3 and 4 were nearly identical at both pHs 5.5 and 6.8, and only slightly higher (~35% and 18%, respectively) than the Kis for PMX at pH 5.5 and pH 6.8 (Table 2).

Table 2
Kinetic constants for hPCFT

Collectively, these results demonstrate that transport characteristics for compounds 3 and 4 for FRs and hPCFT are nominally different, and for hPCFT only slightly different from those for PMX. However, unlike PMX, transport of 3 and 4 by hRFC appears to be minimal. Clearly, transport differences between 3 and 4 do not account for the substantial differences in inhibitions of cell proliferation or colony formation.

Identification of GARFTase as the primary intracellular target for compound 3

The dramatic increase in growth inhibitory potency of compound 3 over 4 or PMX toward hPCFT- or FR-expressing cell lines including KB and IGROV1 human tumor cells does not appear to reflect differences in FR or hPCFT transport parameters for these drugs. We previously reported that compound 4 was a potent inhibitor of GARFTase, the 3rd step in the de novo purine nucleotide biosynthetic pathway, and that reduced cell proliferation was directly attributed to GARFTase inhibition in cultured cells14. To identify the targeted pathway for compound 3 (de novo purine nucleotide versus thymidylate biosynthesis), we tested exogenous nucleosides for their capacities to circumvent growth inhibition by this analog toward R2/hPCFT4 cells. Results were compared to those for PMX, reported to act as an inhibitor of both purine nucleotide [primarily at 5-amino-4-imidazolecarboxamide ribonucleotide (AICAR) formyltransferase (AICARFTase) and less so at GARFTase] and of thymidylate synthase26,27, and of LMTX, an established inhibitor of GARFTase1820.

Whereas thymidine (10 µM) also protected R2/hPCFT4 cells from the growth inhibitory effects of low concentrations (<50 nM) of PMX, at higher PMX concentrations, protection was incomplete with thymidine either in the presence or absence of 5-amino-4-imidazolecarboxamide (AICA) (320 µM) (Figure 6, lower panel) which is metabolized to AICAR (circumvents drug effects at GARFTase). Adenosine (60 µM) could not circumvent effects of PMX. However, growth inhibition by PMX was completely reversed by adenosine combined with thymidine. Analogous results were previously described for nucleoside protection from the inhibitory effects of PMX for CCRF-CEM cells.27 In contrast, for both LMTX (results not shown) and compound 3 (Figure 6, upper panel), growth inhibition was abolished by either adenosine or AICA alone. These results establish de novo purine nucleotide biosynthesis as the targeted pathway for compound 3 and GARFTase as the principal intracellular enzyme target, analogous to compound 414 and LMTX18.

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)

To confirm the inhibition of purine nucleotide biosynthesis by compounds 3 and 4, we measured ATP pools by HPLC in R2/hPCFT4 cells treated with 1 µM of these antifolates for 24 h at pH 6.8. Results were compared to those of PMX and LMTX (both at 1 µM) (Figure 7A). Whereas PMX treatment only minimally impacted ATP pools (~17%), for both 3 and 4, ATP pools were profoundly depleted (~90%) and exceeded the decrease resulting from treatment with LMTX (~76%).

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

To directly measure GARFTase activity in R2/hPCFT4 cells by growth inhibitory antifolates (3, 4, PMX, LMTX), we used an in situ GARFTase activity assay which measures incorporation of [14C]glycine into [14C]formyl GAR in the presence of azaserine12,18. For these experiments, the protocol was modified15 such that the medium pH was ~6.9 during 15 h of antifolate treatment, after which cells were washed and resuspended in fresh medium (pH ~7.2) with azaserine, L-glutamine and [14C]glycine for an additional 8 h. Cells were treated with trichloroacetic acid; the acid soluble metabolites were ether-extracted and fractionated by anion exchange chromatography so that [14C]formyl GAR could be quantitated.

The results showed that in R2/hPCFT4 cells for which PCFT is the only mode of transport, GARFTase was inhibited in the order 3 > 4 > PMX > LMTX (Figure 7B). For compounds 3 and 4, IC50s for in situ inhibition of GARFTase were 0.69 nM and 1.96 nM, respectively. Thus, the IC50s for GARFTase inhibition in R2/PCFT4 cells generally parallel those for inhibition of cell proliferation (Table 1). However, the IC50s for GARFTase inhibition were substantially lower.

These results establish that compound 3 is a potent inhibitor of GARFTase in cells and that the increased anti-proliferative effects of 3 over 4 are likely due primarily to its increased inhibition of intracellular GARFTase rather than to differences in membrane transport. For both compounds 3 and 4, GARFTase inhibition results in a profound inhibition of de novo purine nucleotide biosynthesis, as reflected in a dramatic decrease in total cellular ATP pools exceeding that for LMTX. The failure of PMX to significantly deplete ATP pools reflects its poor inhibition of GARFTase, as previously reported27.

In vivo antitumor efficacy with compound 3

An in vivo drug efficacy trial was designed with 10 week old female ICR SCID mice (Taconic) implanted with subcutaneous human IGROV1 ovarian tumors which express hRFC, hPCFT, and FRα. Mice were maintained ad libitum on a folate-deficient diet in order to decrease levels of serum folates (~5-fold) to concentrations approximating those reported in humans on a folate-fortified diet14. Mice maintained on a standard folate-replete diet were included as controls. Mice were pooled and implanted subcutaneously with IGROV1 tumor fragments, then non-selectively randomized into control and treatment groups. A Q4dx5 schedule was used, with intravenous administration of compounds 3 (40 and 25 mg/kg/injection) and 4 (180 mg/kg/injection and 112.5 mg/kg/injection) on days 3, 7, 11, 15, and 19 post-implantation. Mice were observed and weighed daily; tumors were measured twice per week. Figure 8 shows results for compound 4 at both 112.5 and 180 mg/kg/injection and for compound 3 at 40 mg/kg/injection. Complete results for the in vivo experiment shown in Figure 8 are summarized in Table 1S in the Supplement.

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

For mice maintained on the folate-deficient diet, antitumor activities for compound 3 (40 and 25 mg/kg/injection) were striking with T/C values of 14% and 16%, respectively, and gross log kill values of 2.7 and 3.3, respectively. The treatment regimen with compound 3 was very well tolerated and the only adverse dose-limiting symptom observed was reversible body weight loss. Recovery was excellent with good host recovery time (≤ 10 days) when maintained on a folate-deficient diet. Weight loss ranged from 6.3% (25 mg/kg/inj, nadir day 13, full recovery day 27 or 8 days post last treatment) to 9.4% (40 mg/kg/inj; nadir day 21; full recovery 10 days post last treatment). By comparison, compound 4 (180 mg/kg/inj) gave a 32% T/C and 2.3 gross log kill against the IGROV1 tumor model with one lethality, resulting from GI epithelial damage (based on necropsy). At 112.5 mg/kg/injection, compound 4 produced a 44% T/C with no lethalities. For compound 4 at 180 mg/kg/injection, a weight loss nadir of 8.8% was reached on day 21 with 99% recovery by day 31.

Whereas antitumor activity for 4 was completely ablated in mice maintained on the standard folate replete diet, for compound 3 with a standard diet, a T/C of 67% and gross log kill of 0.8 were measured with minimal toxicity (2.7–5% reversible body weight loss). The latter was sufficient to be considered as “minimally active” (by log kill standards), even in the presence of substantially elevated serum folate.

The results of the in vivo efficacy trial with the IGROV1 tumor demonstrate potent antitumor activities with compound 3, exceeding that for compound 4 and reflecting cellular uptake by FRα and hPCFT over hRFC, and potent inhibition of intracellular GARFTase.

Conclusions

In this report, we expanded upon our previous report on a series of 2-amino-4-oxo-6-substituted-pyrrolo[2,3-d]pyrimidines with a side chain thienoyl ring with 4–6 bridge carbons as selective transport substrates for FRs and PCFT over RFC14. We systemically examined the effects of analogs with 1–3 bridge carbons compared to compound 4, the 4-carbon bridge analog and the previous lead compound for this series14, on proliferation of CHO cells with individual expressions of FRs, hPCFT, or hRFC, and on IGROV1 and KB human tumor cells expressing all three transport systems, in order to assess the effects of systematically varying the distance between the bicyclic scaffold and the L-glutamate moiety.

Our results establish that the 3-carbon bridge analog, compound 3, is substantially more active in vitro toward CHO cells that exclusively express FRs and/or hPCFT (~6- and 11-fold, respectively) than 4, characterized by a 4-carbon bridge. For FR-expressing CHO cells, 1 (one carbon bridge) showed low level activity (394-fold less than compound 3 with FRα-expressing RT16 cells) and 3 was 38- (FRβ) and 125- (FRα) fold more active than 2 (two carbon bridge). Against hPCFT-expressing (R2/hPCFT4) cells, 3 was 40- and 87-fold more potent than 2 and 1, respectively. Thus, increasing the bridge lengths from 1- to 3-carbon atoms sequentially increases potencies toward both FR- and hPCFT-expressing cells, and potencies further decrease with increasing the chain length to 4 carbons. Accordingly, the 3-atom carbon bridge appears to be optimal for this series. Analogous results with compounds 14 were obtained in vitro with human KB and IGROV1 tumors with activity in order 3 > 4 >> 2 > 1.

Interestingly, between 3 and 4, there were no significant differences in transport properties for FRs or hPCFT that could explain differences in their anti-proliferative effects. While low level RFC and/or non-RFC uptake components were implied for compound 3, these would occur only at substantially higher drug concentrations than those that targeted FR- and hPCFT-expressing cells and would thus be unlikely to appreciably contribute to the inhibitory effects of this compound on cell proliferation or clonogenicity. Differences in inhibitory potencies between 3 and 4 toward intracellular GARFTase predominated. Although we were not able to detect differences in inhibitory potencies of monoglutamyl 3 and 4 toward isolated recombinant murine GARFTase (data not shown), the relevance of these findings to the intracellular mileau where antifolyl polyglutamates predominate3 and are likely far more potent inhibitors of intracellular GARFTase19 is uncertain. Against intracellular GARFTase, both 3 and 4 were inhibitory at concentrations substantially less than those required for inhibition of cell proliferation or clonogencity, suggesting that sustained inhibition of GARFTase and de novo purine biosynthesis are essential for cell killing in this CHO cell line model15. GARFTase inhibition was accompanied by substantial suppression of de novo purine nucleotide biosynthesis, as reflected in depletion of cellular ATP pools.

We extended our promising in vitro findings with compound 3 in vivo using a challenging tumor model (IGROV1) with substantially lower levels of FRα than those in the KB tumor previously reported for 414. With subcutaneous IGROV1 tumors in SCID mice, compound 3 was more efficacious than compound 4, as reflected in T/C and gross log kill and at much lower doses. Further, at the highest drug dose (180 mg/kg), compound 4 was lethal, resulting in death of 1 of 7 mice in this trial. Clearly, compound 3 would permit a greater margin of safety than compound 4, as less than maximum tolerated dosing can be administered with reduced toxicity accompanying substantially increased antitumor activity. This property would also be beneficial with potentially additive or synergistic drug combination regimens. Interestingly, compound 3 also preserved some level of activity even in the presence of elevated serum folate, unlike 4 which was inactive under these conditions.

Collectively, our results establish that the pyrrolo[2,3-d]pyrimidine thienoyl scaffold is particularly amenable to selective FR and PCFT transport compared to RFC, and to potent GARFTase inhibition. Anti-proliferative activity is substantially increased for this series with compound 3, characterized by a 3-carbon bridge and a reduced intra-molecular distance between the bicyclic scaffold and the L-glutamate compared to compound 4. Our in vitro data for compounds 3 and 4 strongly imply that their relative inhibitory potencies toward intracellular GARFTase are the major determinants of relative drug activities for this series of analogs, likely secondary to their metabolism to polyglutamates. While it cannot be established with complete certainty from the available data that in vivo tumor selectivity for the active analogs derives from their selective cellular uptake by FRα and PCFT over RFC in tumors versus normal tissues, based on our in vitro results this is not unreasonable.

Although early preclinical studies with LMTX showed its substantial efficacy against a number of transplanted tumors accompanying its membrane transport by all the major systems including RFC, PCFT, and FRs20, when administered to patients in phase I, toxicity was not insignificant23. This is likely due at least in part to its excellent RFC substrate activity and its polyglutamylation in susceptible normal tissues. A similar explanation may account for the toxicity encountered with the 2nd generation GARFTase inhibitors (10a and 10b) in patients21,22,24.

Based on this promising lead analog, future studies with compound 3 will further examine major biochemical determinants of its in vitro and in vivo activities, including its membrane transport properties and its metabolism to polyglutamates. Additional studies will expand in vivo testing to include a broader cohort of clinically relevant human tumor models expressing FRs and PCFT, including late stage tumors. These results will be included in future reports.

EXPERIMENTAL PROCEDURES

All evaporations were carried out in vacuum with a rotary evaporator. Analytical samples were dried in vacuo (0.2 mmHg) in a CHEM-DRY drying apparatus over P2O5 at 60 °C. Melting points were determined on a MEL-TEMP II melting point apparatus with FLUKE 51 K/J electronic thermometer and are uncorrected. Nuclear magnetic resonance spectra for proton (1H NMR) were recorded on Bruker Avance II 400 (400 MHz) and 500 (500 MHz) spectrometer. The chemical shift values are expressed in ppm (parts per million) relative to tetramethylsilane as an internal standard: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad singlet. Thin-layer chromatography (TLC) was performed on Whatman Sil G/UV254 silica gel plates with a fluorescent indicator, and the spots were visualized under 254 and 366 nm illumination. Proportions of solvents used for TLC are by volume. Column chromatography was performed on a 230–400 mesh silica gel (Fisher, Somerville, NJ) column. The amount (weight) of silica gel for column chromatography was in the range of 50–100 times the amount (weight) of the crude compounds being separated. Columns were dry-packed unless specified otherwise. Elemental analyses were performed by Atlantic Microlab, Inc., Norcross, GA. Element compositions are within ±0.4% of the calculated values. Fractional moles of water or organic solvents frequently found in some analytical samples of antifolates could not be prevented despite 24–48 h of drying in vacuo and were confirmed where possible by their presence in the 1H NMR spectra. High-resolution mass spectrometry (HRMS) was performed on a Waters Q-TOF (API-US) by Department of Chemistry, University of Pittsburgh, Pittsburgh, PA. Elemental analysis was used to determine the purity of the final compounds 1–3. All solvents and chemicals were purchased from Aldrich Chemical Co. and Fisher Scientific and were used as received. Purity of the final compounds 1–3, 7–9, 10a, and 10b were >95% and were determined by elemental (C, H, N, S) analysis.

General procedure for the synthesis of compounds 13a–b

To a 20 mL vial for microwave reaction, were added a mixture of palladium chloride (71 mg, 0.40 mmol), triphenylphosphine (131 mg, 0.40 mmol), triethylamine (10.1 g, 100 mmol), 5-bromo-thiophene-2-carboxylic acid methyl ester, 12 (2.21 g, 10 mmol) and anhydrous acetonitrile (10 mL). To the stirred mixture, were added copper(I) iodide (304 mg, 1.60 mmol), and prop-2-yn-1-ol, 11a, or but-3-yn-1-ol, 11b (1.05 equ.), and the vial was sealed and put into the microwave reactor at 100 °C for 10 min. Silica gel (5 g) was added and the solvent was evaporated under reduced pressure. The resulting plug was loaded on to a silica gel column (3.5 × 12 cm) and eluted with hexane followed by 20% EtOAc in hexane. The desired fraction (TLC) was collected and the solvent was evaporated under reduced pressure to afford 13a–b.

5-(3-Hydroxy-prop-1-ynyl)-thiophene-2-carboxylic acid methyl ester (13a)

Compound 13a was prepared using the general method described for the preparation of 13a–b, from prop-2-yn-1-ol, 11a (588 mg, 10.5 mmol) to give 1.45 g (74%) of 13a as a yellow oil. TLC Rf 0.33 (hexane/EtOAc 1:1); 1H NMR (DMSO-d6): δ 3.82 (s, 3H, COOCH3), 4.33–4.34 (d, J = 6.0 Hz, 2H, CH2), 5.45–5.48 (t, J = 6.0 Hz, 1H, OH, exch), 7.34–7.35 (d, J = 4.0 Hz, 1H, Ar), 7.72–7.73 (d, J = 4.0 Hz, 1H, Ar).

5-(4-Hydroxy-but-1-ynyl)-thiophene-2-carboxylic acid methyl ester (13b)

Compound 13b was prepared using the general method described for the preparation of 13a–b, from prop-2-yn-1-ol, 11b (588 mg, 8.4 mmol) to give 1.17 g (70%) of 13b as yellow oil. TLC Rf 0.33 (hexane/EtOAc 1:1); 1H NMR (DMSO-d6): δ 2.59–2.63 (t, J = 6.4 Hz, 2H, CH2), 3.56–3.59 (t, J = 6.4 Hz, 2H, CH2), 3.81 (s, 3H, COOCH3), 4.95–4.98 (t, J = 5.6 Hz, 1H, OH, exch), 7.27–7.28 (d, J = 4.0 Hz, 1H, Ar), 7.69–7.70 (d, J = 4.0 Hz, 1H, Ar).

General procedure for the synthesis of compounds 14a–b

To a Parr flask was added 13a–b, 10% palladium on activated carbon (50% w/w), and MeOH (100 mL). Hydrogenation was carried out at 55 psi of H2 for 4 h. The reaction mixture was filtered through Celite, washed with MeOH (100 mL) and concentrated under reduced pressure to give 14a–b.

5-(3-Hydroxy-propyl)-thiophene-2-carboxylic acid methyl ester (14a)

Compound 14a was prepared using the general method described for the preparation of 14a–b, from 13a (1.45 g, 7.4 mmol) to give 1.4 g (95%) of 14a as a yellow oil. TLC Rf 0.34 (hexane/EtOAc 1:1); 1H NMR (DMSO-d6): δ 1.86–1.93 (m, 2H, CH2), 2.68–2.71 (t, J = 6.8 Hz, 2H, CH2), 3.06–3.09 (t, J = 6.8 Hz, 2H, CH2), 3.76 (s, 3H, COOCH3), 4.02–4.05 (t, J = 6.0 Hz, 1H, OH, exch), 6.97–6.98 (d, J = 4.0 Hz, 1H, Ar), 7.61–7.63 (d, J = 4.0 Hz, 1H, Ar).

5-(4-Hydroxy-butyl)-thiophene-2-carboxylic acid methyl ester (14b)

Compound 14b was prepared using the general method described for the preparation of 14a–b, from 13b (1.17 g, 5.6 mmol) to give 1.14 g (95%) of 14b as a yellow oil. TLC Rf 0.34 (hexane/EtOAc 1:1); 1H NMR (DMSO-d6): δ 1.41–1.48 (m, 2H, CH2), 1.61–1.68 (m, 2H, CH2), 2.81–2.85 (t, J = 7.2 Hz, 2H, CH2), 3.37–3.42 (m, 2H, CH2), 3.77 (s, 3H, COOCH3), 4.40–4.43 (t, J = 5.2 Hz, 1H, OH, exch), 6.94–6.95 (d, J = 3.6 Hz, 1H, Ar), 7.63–7.64 (d, J = 3.6 Hz, 1H, Ar).

General procedure for the synthesis of compounds 15b–c

To a solution of 14a–b in acetone (15 mL) was added dropwise to a cold solution (ice bath) of CrO3 (6 equ.) in sulfuric acid (30 mL) and water (90 mL). After the addition, the resulting solution was stirred in an ice bath for an additional 2 h and the solution was allowed to warm to room temperature overnight. The solution was extracted with 5 × 30 mL of ethyl ether and dried over Na2SO4. After evaporation of the solvent under reduced pressure, the resulting residue was flash chromatographed through silica gel column (3.5 × 15 cm) using hexane/EtOAc (2:1) as eluent. The desired fraction (TLC) was collected and the solvent was evaporated under reduced pressure to afford 15b–c.

5-(2-Carboxy-ethyl)-thiophene-2-carboxylic acid methyl ester (15b)

Compound 15b was prepared using the general method described for the preparation of 15b–c, from 14a (1.4 g, 7.0 mmol) to give 642 mg (43%) of 15b as a colorless oil. TLC Rf 0.55 (hexane/EtOAc 1:1); 1H NMR (DMSO-d6): δ 2.59–2.62 (t, J = 7.2 Hz, 2H, CH2), 3.03–3.07 (t, J = 7.2 Hz, 2H, CH2), 3.78 (s, 3H, COOCH3), 6.97–6.98 (d, J = 3.6 Hz, 1H, Ar), 7.62–7.63 (d, J = 3.6 Hz, 1H, Ar), 12.32 (br, 1H, COOH, exch).

5-(3-Carboxy-propyl)-thiophene-2-carboxylic acid methyl ester (15c)

Compound 15c was prepared using the general method described for the preparation of 15b–c, from 14b (1.14 g, 5.3 mmol) to give 570 mg (47%) of 15c as a colorless oil. TLC Rf 0.58 (hexane/EtOAc 1:1); 1H NMR (DMSO-d6): δ 1.79–1.87 (m, 2H, CH2), 2.24–2.27 (t, J = 7.2 Hz, 2H, CH2), 2.82–2.86 (t, J = 7.2 Hz, 2H, CH2), 3.77 (s, 3H, COOCH3), 6.95–6.96 (d, J = 3.6 Hz, 1H, Ar), 7.63–7.64 (d, J = 3.6 Hz, 1H, Ar), 12.17 (br, 1H, COOH, exch). HRMS calcd for C10H12O4S 228.0456, found 228.0458.

General procedure for the synthesis of compounds 16b–c

To 15b–c in a 100 mL flask was added oxalyl chloride (6 equ.) and anhydrous CH2Cl2 (20 mL). The resulting solution was refluxed for 1 h and then cooled to room temperature. After evaporating the solvent under reduced pressure, the residue was dissolved in 20 mL of Et2O. The resulting solution was added dropwise to an ice-cooled diazomethane (generated in situ from 10 g of diazald by using Aldrich Mini Diazald Apparatus) in an ice bath over 10 min. The resulting mixture was allowed to stand for 30 min and then stirred for an additional 1 h. To this solution was added 48% HBr (20 mL). The resulting mixture was refluxed for 1.5 h. After cooling to room temperature, the organic layer was separated and the aqueous layer was extracted with Et2O (2 × 50 mL). The combined organic layer and Et2O extract was washed with two portions of 10% Na2CO3 solution and dried over Na2SO4. Evaporation of the solvent under reduced pressure afforded 16b–c.

5-(4-Bromo-3-oxo-butyl)-thiophene-2-carboxylic acid methyl ester (16b)

Compound 16b was prepared using the general method described for the preparation of 16b–c, from 15b (642 mg, 3.0 mmol) to give 803 mg (92%) of 16b as a colorless oil. TLC Rf 0.68 (hexane/EtOAc 1:1); 1H NMR (DMSO-d6): δ 3.00–3.06 (m, 4H, CH2CH2), 3.78 (s, 3H, COOCH3), 4.37 (s, 2H, CH2Br), 6.96–6.97 (d, J = 3.6 Hz, 1H, Ar), 7.62–7.63 (d, J = 3.6 Hz, 1H, Ar).

5-(5-Bromo-4-oxo-pentyl)-thiophene-2-carboxylic acid methyl ester (16c)

Compound 16c was prepared using the general method described for the preparation of 16b–c, from 15c (570 mg, 2.5 mmol) to give 730 mg (96%) of 16c as a colorless oil. TLC Rf 0.71 (hexane/EtOAc 1:1); 1H NMR (CDCl3-d): δ 1.99–2.07 (m, 2H, CH2), 2.71–2.75 (t, J = 7.2 Hz, 2H, CH2), 2.87–2.91 (t, J = 7.2 Hz, 2H, CH2), 3.87 (s, 3H, COOCH3), 3.88 (s, 2H, CH2Br), 6.81–6.82 (d, J = 3.6 Hz, 1H, Ar), 7.64–7.65 (d, J = 3.6 Hz, 1H, Ar). HRMS calcd for C11H13BrO3S 303.9769, found 303.9759.

Methyl 5-[(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)methyl]thiophene-2-carboxylate (18a)

To commercially available 5-carboxymethyl-thiophene-2-carboxylic acid methyl ester 15a (1 g, 5.0 mmol) in a 100 mL flask was added oxalyl chloride (3.8 g, 30 mmol) and anhydrous CH2Cl2 (20 mL). The resulting solution was refluxed for 1 h and then cooled to room temperature. After evaporating the solvent under reduced pressure, the residue was dissolved in 20 mL of Et2O. The resulting solution was added dropwise to an ice-cooled diazomethane (generated in situ from 10 g of diazald by using Aldrich Mini Diazald Apparatus) in an ice bath over 10 min. The resulting mixture was allowed to stand for 30 min and then stirred for an additional 1 h. To this solution was added 48% HBr (20 mL). The resulting mixture was refluxed for 1.5 h. After cooling to room temperature, the organic layer was separated and the aqueous layer was extracted with Et2O (2 × 50 mL). The combined organic layer and Et2O extract was washed with two portions of 10% Na2CO3 solution and dried over Na2SO4. To this residue in anhydrous DMF (15 mL) was added 2,6-diamino-3H-pyrimidin-4-one, 17 (630 mg, 5 mmol). The resulting mixture was stirred under N2 at room temperature for 3 days. Silica gel (1.6 g) was then added and the solvent was evaporated under reduced pressure. The resulting plug was loaded on to a silica gel column (1.5 × 12 cm) and eluted with CHCl3 followed by 3% MeOH in CHCl3 and then 5% MeOH in CHCl3. The desired fraction (TLC) was collected and the solvent was evaporated under reduced pressure to afford 180 mg (12%) of 18a as a light yellow powder. mp 200–201 °C; TLC Rf 0.18 (CHCl3/MeOH 10:1); 1H NMR (DMSO-d6): δ 3.78 (s, 3H, COOCH3), 4.10 (s, 2H, CH2), 6.01 (s, 1H, C5-CH), 6.05 (s, 2H, 2-NH2, exch), 7.00–7.01 (d, J = 4.0 Hz, 1H, Ar), 7.65–7.66 (d, J = 4.0 Hz, 1H, Ar), 10.21 (s, 1H, 3-NH, exch), 11.04 (s, 1H, 7-NH, exch).

General procedure for the synthesis of compounds 18b–c

To a suspension of 2,6-diamino-3H-pyrimidin-4-one, 17 (1 equ.) in anhydrous DMF (15 mL) was added 16b–c. The resulting mixture was stirred under N2 at room terperature for 3 days. Silica gel (1.6 g) was then added and the solvent was evaporated under reduced pressure. The resulting plug was loaded on to a silica gel column (1.5 × 12 cm) and eluted with CHCl3 followed by 3% MeOH in CHCl3 and then 5% MeOH in CHCl3. The desired fraction (TLC) was collected and the solvent was evaporated under reduced pressure to afford 18b–c.

Methyl 5-[(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)ethyl]thiophene-2-carboxylate (18b)

Compound 18b was prepared using the general method described for the preparation of 18b–c, from 16b (803 mg, 2.8 mmol) to give 320 mg (36%) of 18b as a light yellow powder. mp 200–201 °C; TLC Rf 0.18 (CHCl3/MeOH 10:1); 1H NMR (DMSO-d6): δ 2.84–2.87 (t, J = 7.2 Hz, 2H, CH2), 3.15–3.17 (t, J = 7.2 Hz, 2H, CH2), 3.77 (s, 3H, COOCH3), 5.91 (s, 1H, C5-CH), 6.09 (s, 2H, 2-NH2, exch), 6.95–6.96 (d, J = 3.6 Hz, 1H, Ar), 7.61–7.62 (d, J = 3.6 Hz, 1H, Ar), 10.24 (s, 1H, 3-NH, exch), 10.94 (s, 1H, 7-NH, exch).

Methyl 5-[(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)propyl]thiophene-2-carboxylate (18c)

Compound 18c was prepared using the general method described for the preparation of 18b–c, from 16c (730 mg, 2.4 mmol) to give 300 mg (38%) of 18c as white powder. mp 175–176 °C; TLC Rf 0.17 (CHCl3/MeOH 10:1); 1H NMR (DMSO-d6): δ 1.89–1.97 (m, 2H, CH2), 2.49–2.54 (t, J = 7.2 Hz, 2H, CH2), 2.82–2.85 (t, J = 7.2 Hz, 2H, CH2), 3.78 (s, 3H, COOCH3), 5.89 (s, 1H, C5-CH), 5.96 (s, 2H, 2-NH2, exch), 6.97–6.98 (d, J = 3.6 Hz, 1H, Ar), 7.64–7.65 (d, J = 3.6 Hz, 1H, Ar), 10.13 (s, 1H, 3-NH, exch), 10.82 (s, 1H, 7-NH, exch).

General procedure for the synthesis of compounds 19a–c

To a solution of 18a–c in 10 mL of MeOH was added 1 N NaOH (10 mL) and the mixture was stirred under N2 at room temperature for 16 h. TLC showed the disappearance of the starting material and one major spot at the origin. The reaction mixture was evaporated to dryness under reduced pressure. The residue was dissolved in water (10 mL), the resulting solution was cooled in an ice bath, and the pH was adjusted to 3–4 with dropwise addition of 1 N HCl. The resulting suspension was frozen in a dry ice-acetone bath, thawed to 4–5 °C in the refrigerator, and filtered. The residue was washed with a small amount of cold water and dried in vacuum using P2O5 to afford 19a–c.

5-[(2-Amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)methyl)thiophene-2-carboxylic acid (19a)

Compound 19a was prepared using the general method described for the preparation of 19a–c, from 18a (180 mg, 0.6 mmol) to give 163 mg (95%) of 19a as a white powder. mp 200–201 °C; 1H NMR (DMSO-d6): δ 4.08 (s, 2H, CH2), 6.00 (s, 1H, C5-CH), 6.06 (s, 2H, 2-NH2, exch), 6.96–6.97 (d, J = 4.0 Hz, 1H, Ar), 7.56–7.57 (d, J = 4.0 Hz, 1H, Ar), 10.22 (s, 1H, 3-NH, exch), 11.03 (s, 1H, 7-NH, exch), 12.97 (br, 1H, COOH, exch).

5-[(2-Amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)ethyl)thiophene-2-carboxylic acid (19b)

Compound 19b was prepared using the general method described for the preparation of 19a–c, from 18b (320 mg, 1.0 mmol) to give 274 mg (90%) of 19b as a white powder. mp 200–201 °C; 1H NMR (DMSO-d6): δ 2.82–2.86 (t, J = 7.2 Hz, 2H, CH2), 3.12–3.16 (t, J = 7.2 Hz, 2H, CH2), 5.90 (s, 1H, C5-CH), 6.01 (s, 2H, 2-NH2, exch), 6.89–6.90 (d, J = 3.6 Hz, 1H, Ar), 7.50–7.51 (d, J = 3.6 Hz, 1H, Ar), 10.18 (s, 1H, 3-NH, exch), 10.89 (s, 1H, 7-NH, exch), 12.90 (br, 1H, COOH, exch).

5-[(2-Amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)propyl)thiophene-2-carboxylic acid (19c)

Compound 19c was prepared using the general method described for the preparation of 19a–c, from 18c (300 mg, 0.9 mmol) to give 254 mg (89%) of 19c as a white powder. mp 175–176 °C; 1H NMR (DMSO-d6): δ 1.89–1.96 (m, 2H, CH2), 2.49–2.55 (t, J = 7.2 Hz, 2H, CH2), 2.80–2.84 (t, J = 7.2 Hz, 2H, CH2), 5.88 (s, 1H, C5-CH), 5.98 (s, 2H, 2-NH2, exch), 6.92–6.93 (d, J = 3.6 Hz, 1H, Ar), 7.55–7.56 (d, J = 3.6 Hz, 1H, Ar), 10.14 (s, 1H, 3-NH, exch), 10.83 (s, 1H, 7-NH, exch) 12.86 (br, 1H, COOH, exch).

Diethyl N-({5-[(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)methyl]thiophen-2-yl}carbonyl)-L-glutamate (20a)

To a solution of 19a (118 mg, 0.4 mmol) in anhydrous DMF (10 mL) was added N-methylmorpholine (73 mg, 0.72 mmol) and 2-chloro-4,6-dimethoxy-1,3,5-triazine (127 mg, 0.72 mmol). The resulting mixture was stirred at room temperature for 2 h. To this mixture was added N-methylmorpholine (73 mg, 0.72 mmol) and L-glutamate diethyl ester hydrochloride (144 mg, 0.6 mmol). The reaction mixture was stirred for an additional 4 h at room temperature. Silica gel (400 mg) was then added and the solvent was evaporated under reduced pressure. The resulting plug was loaded on to a silica gel column (1.5 × 15 cm) and with 5% CHCl3 in MeOH as the eluent. Fractions that showed the desired spot (TLC) were pooled and the solvent evaporated to dryness to afford 120 mg (63%) of 20a as a yellow powder. mp 78–79 °C; 1H NMR (DMSO-d6): δ 1.14–1.20 (m, 6H, COOCH2CH3), 1.93–2.11 (m, 2H, β-CH2), 2.40–2.43 (t, J = 7.2 Hz, 2H, γ-CH2), 4.02–4.13 (m, 4H, COOCH2CH3), 4.31 (s, 2H, CH2), 4.34–4.40 (m, 1H, α-CH), 6.07 (s, 1H, C5-CH), 6.94–6.95 (d, J = 4.0 Hz, 1H, Ar), 7.71–7.72 (d, J = 4.0 Hz, 1H, Ar), 8.66–8.68 (d, J = 7.2 Hz, 1H, CONH, exch), 11.18 (s, 1H, 3-NH, exch), 11.43 (s, 1H, 7-NH, exch).

Diethyl N-({5-[(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)propyl]thiophen-2-yl}carbonyl)-L-glutamate (20c)

To a solution of 19c (254 mg, 0.8 mmol) in anhydrous DMF (10 mL) was added N-methylmorpholine (145 mg, 1.44 mmol) and 2-chloro-4,6-dimethoxy-1,3,5- triazine (253 g, 1.44 mmol). The resulting mixture was stirred at room temperature for 2 h. To this mixture were added N-methylmorpholine (145 mg, 1.44 mmol) and L-glutamate diethyl ester hydrochloride (290 mg, 1.2 mmol). The reaction mixture was stirred for an additional 4 h at room temperature and then evaporated to dryness under reduced pressure. The residue was dissolved in the minimum amount of CHCl3/MeOH (4:1) and chromatographed on a silica gel column (2 × 15 cm) and with 5% CHCl3 in MeOH as the eluent. Fractions that showed the desired spot (TLC) were pooled and the solvent evaporated to dryness to afford 252 mg (63%) of 20c as yellow powder. mp 85–86 °C; TLC Rf 0.13 (CHCl3/MeOH 10:1); 1H NMR (DMSO-d6): δ 1.14–1.21 (m, 6H, COOCH2CH3), 1.81–2.05 (m, 4H, β-CH2, CH2), 2.32–2.39 (t, J = 7.6 Hz, 2H, γ-CH2), 2.49–2.52 (t, J = 7.2 Hz, 2H, CH2), 2.78–2.81 (t, J = 7.2 Hz, 2H, CH2), 4.02–4.07 (m, 4H, COOCH2CH3), 4.30–4.35 (m, 1H, α-CH), 5.88 (s, 1H, C5-CH), 5.94 (s, 2H, 2-NH2, exch), 6.89–6.90 (d, J = 3.6 Hz, 1H, Ar), 7.68–7.69 (d, J = 3.6 Hz, 1H, Ar), 8.61–8.63 (d, J = 8 Hz, 1H, CONH, exch), 10.71 (s, 1H, 3-NH, exch), 11.19 (s, 1H, 7-NH, exch).

N-({5-[(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)methyl]thiophen-2-yl}carbonyl)-L-glutamic acid (1)

To a solution of 20a (120 mg, 0.25 mmol) in MeOH (10 mL) was added 1 N NaOH (4 mL). The mixture was then stirred under N2 at room temperature for 16 h. TLC showed the disappearance of the starting material (Rf 0.15) and one major spot at the origin (CHCl3/MeOH 10:1). The reaction mixture was then evaporated to dryness under reduced pressure. The residue was dissolved in water (10 mL), the resulting solution was cooled in an ice bath, and the pH was adjusted to 3–4 with dropwise addition of 1 N HCl. The resulting suspension was frozen in a dry ice-acetone bath, thawed to 4–5 °C in the refrigerator, and filtered. The residue was washed with a small amount of cold water and dried in vacuum using P2O5 to afford 73 mg (69%) of 1 as a light yellow powder. mp 167–168 °C; 1H NMR (DMSOd6): δ 1.85–2.11 (m, 2H, β-CH2), 2.31–2.35 (t, J = 6.8 Hz, 2H, γ-CH2), 4.06 (s, 2H, CH2), 4.30–4.36 (m, 1H, α-CH), 6.00 (s, 1H, C5-CH), 6.07 (s, 2H, 2-NH2, exch), 6.92–6.93 (d, J = 4.0 Hz, 1H, Ar), 7.69–7.70 (d, J = 4.0 Hz, 1H, Ar), 8.52–8.54 (d, J = 8 Hz, 1H, CONH, exch), 10.22 (s, 1H, 3-NH, exch), 11.03 (s, 1H, 7-NH, exch), 12.46 (br, 2H, COOH, exch). Anal. (C17H17N5O6S) C, H, N, S.

N-({5-[(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)ethyl]thiophen-2-yl}carbonyl)-L-glutamic acid (2)

To a solution of 19b (274 mg, 0.9 mmol) in anhydrous DMF (10 mL) was added N-methylmorpholine (164 mg, 1.62 mmol) and 2-chloro-4,6-dimethoxy-1,3,5-triazine (285 mg, 1.62 mmol). The resulting mixture was stirred at room temperature for 2 h. To this mixture was added N-methylmorpholine (164 mg, 1.62 mmol) and L-glutamate diethyl ester hydrochloride (324 mg, 1.35 mmol). The reaction mixture was stirred for an additional 4 h at room temperature. Silica gel (600 mg) was then added and the solvent was evaporated under reduced pressure. The resulting plug was loaded on to a silica gel column (1.5 × 15 cm) and with 5% CHCl3 in MeOH as the eluent. Fractions that showed the desired spot (TLC) were pooled and the solvent evaporated to dryness to afford a residue. To this residue was added MeOH (10 mL) and 1 N NaOH (10 mL) and the mixture was stirred under N2 at room temperature for 16 h. TLC showed the disappearance of the starting material (Rf 0.15) and one major spot at the origin (CHCl3/MeOH 10:1). The reaction mixture was then evaporated to dryness under reduced pressure. The residue was dissolved in water (10 mL), the resulting solution was cooled in an ice bath, and the pH was adjusted to 3–4 with dropwise addition of 1 N HCl. The resulting suspension was frozen in a dry ice-acetone bath, thawed to 4–5 °C in the refrigerator, and filtered. The residue was washed with a small amount of cold water and dried in vacuum using P2O5 to afford 262 mg (67%) of 2 as a light yellow powder. mp 184–185 °C; 1H NMR (DMSO-d6): δ 1.84–2.10 (m, 2H, β-CH2), 2.30–2.34 (t, J = 7.4 Hz, 2H, γ-CH2), 2.81–2.85 (t, J = 7.4 Hz, 2H, CH2), 3.10–3.13 (t, J = 7.4 Hz, 2H, CH2), 4.29–4.35 (m, 1H, α-CH), 5.91 (s, 1H, C5-CH), 6.03 (s, 2H, 2-NH2, exch), 6.87–6.88 (d, J = 3.6 Hz, 1H, Ar), 7.66–7.67 (d, J = 3.6 Hz, 1H, Ar), 8.49–8.51 (d, J = 8.0 Hz, 1H, CONH, exch), 10.18 (s, 1H, 3-NH, exch), 10.90 (s, 1H, 7-NH, exch), 12.41 (br, 2H, COOH, exch). Anal. (C18H19N5O6S) C, H, N, S.

N-({5-[(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)propyl]thiophen-2-yl}carbonyl)-L-glutamic acid (3)

To a solution of 20c (252 mg, 0.5 mmol) in MeOH (10 mL) was added 1 N NaOH (10 mL) and the mixture was stirred under N2 at room temperature for 16 h. TLC showed the disappearance of the starting material (Rf 0.13) and one major spot at the origin (CHCl3/MeOH 10:1). The reaction mixture was evaporated to dryness under reduced pressure. The residue was dissolved in water (10 mL), the resulting solution was cooled in an ice bath, and the pH was adjusted to 3–4 with dropwise addition of 1 N HCl. The resulting suspension was frozen in a dry ice-acetone bath, thawed to 4–5 °C in the refrigerator, and filtered. The residue was washed with a small amount of cold water and dried in vacuum using P2O5 to afford 212 mg (95%) of 3 as white powder. mp 168–169 °C; 1H NMR (DMSO-d6): δ 1.88–2.10 (m, 4H, β-CH2, CH2), 2.31–2.34 (t, J = 7.6 Hz, 2H, γ-CH2), 2.49–2.54 (t, J = 7.2 Hz, 2H, CH2), 2.78–2.81 (t, J = 7.2 Hz, 2H, CH2), 4.30–4.35 (m, 1H, α-CH), 5.88 (s, 1H, C5-CH), 5.97 (s, 2H, 2-NH2, exch), 6.89–6.90 (d, J = 3.6 Hz, 1H, Ar), 7.68–7.69 (d, J = 3.6 Hz, 1H, Ar), 8.50–8.52 (d, J = 8 Hz, 1H, CONH, exch), 10.13 (s, 1H, 3-NH, exch), 10.82 (s, 1H, 7-NH, exch) 12.42 (br, 2H, COOH, exch). Anal. (C19H21N5O6S) C, H, N, S.

Reagents for biological studies

[3’, 5’, 7-3H]MTX (20 Ci/mmol), [3’, 5’, 7, 9-3H] folic acid (25 Ci/mmol), and [14C(U)]-glycine (87mCi/mmol) were purchased from Moravek Biochemicals (Brea, CA). Unlabeled folic acid was purchased from the Sigma Chemical Co. (St. Louis, MO). LCV [(6R,S) 5-formyl tetrahydrofolate] was provided by the Drug Development Branch, National Cancer Institute, Bethesda, MD. The sources of the classical antifolate drugs were as follows: MTX, Drug Development Branch, National Cancer Institute (Bethesda, MD); RTX [N-(5-[N-(3,4-dihydro-2-methyl-4-oxyquinazolin-6-ylmethyl)-N-methyl-amino]-2-thienoyl)-L-glutamic acid], AstraZeneca Pharmaceuticals (Maccesfield, Cheshire, England); LMTX (5,10-dideaza-5,6,7,8-tetrahydrofolate) and PMX [N-{4-[2-(2-amino-3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl}-L-glutamic acid] (Alimta), Eli Lilly and Co. (Indianapolis, IN); and PT523 [Nα-(4-amino-4-deoxypteroyl)-Nδ-hemiphthaloyl-L-ornithine], Dr. Andre Rososky (Boston, MA). Other chemicals were obtained from commercial sources in the highest available purity.

Cell lines and assays of antitumor drug activities

The engineered CHO sublines including RFC- and FRα-null MTXRIIOuaR2–4 (R2), and hRFC- (pC43-10), hPCFT-(R2/PCFT4), or FRα- (RT16) expressing CHO sublines were previously described1215. The pcDNA3.1 vector transfected control CHO cells (R2/VC) were previously described14. The CHO cells were cultured in α-minimal essential medium (MEM) supplemented with 10% bovine calf serum (Invitrogen, Carlsbad, CA), penicillin-streptomycin solution and L-glutamine at 37°C with 5% CO2. All the R2 transfected cells [PC43-10, RT16, R2/hPCFT4, R2(VC)] were routinely cultured in α-MEM plus 1.5 mg/ml G4181416. Prior to the cell proliferation assays (see below), RT16 cells were cultured for 3 days in complete folate-free RPMI 1640 (without added folate) with dFBS plus 1.5 mg/ml G418. KB human cervical cancer cells were purchased from the American Type Culture Collection (Manassas, VA), whereas IGROV1 ovarian carcinoma cells were a gift of Dr. Manohar Ratnam (University of Toledo). Both IGROV1 and KB cells were routinely cultured in folate-free RPMI 1640 medium, supplemented with 10% fetal bovine serum, penicillin-streptomycin solution, and 2 mM L-glutamine at 37°C with 5% CO2.

For growth inhibition studies, cells (CHO, KB, or IGROV1) were plated in 96 well dishes (~2500–5000 cells/well, total volume of 200 µl medium) with various antifolate concentrations. The medium was standard RPMI 1640 with 10% dialyzed serum and antibiotics for experiments with R2 and PC43-10 cells. For experiments with RT16, KB, and IGROV1 cells, the cells were cultured in folate-free RPMI medium with 10% dFBS (Invitrogen) and antibiotics; the medium was supplemented with 2 nM LCV. The requirement for FR-mediated drug uptake in these assays was established in parallel incubations including 200 nM folic acid, along with the cytotoxic antifolate. For the growth inhibition studies with R2/hPCFT4 cells, cells were cultured in folate-free RPMI 1640 (pH 7.2) containing 25 nM LCV, supplemented with 10% dFBS and antibiotics. Cells were routinely incubated for up to 96 h, during which the medium pH decreased to ~6.8–6.9. Metabolically active cells (reflecting cell viability) were assayed with CellTiter-blue Cell Viability Assay (Promega, Madison, WI), with fluorescence measured (590 nm emission, 560 nm excitation) using a fluorescence plate reader. Raw data were exported from Softmax Pro software to an Excel spreadsheet for determinations of IC50s, corresponding to the drug concentrations that result in 50% loss of cell growth.

For some of the in vitro growth inhibition studies, inhibitory effects of the antifolate drugs on de novo thymidylate synthase and de novo purine nucleotide biosynthesis (GARFTase and AICARFTase) were tested by co-incubations with thymidine (10 µM) and adenosine (60 µM), respectively. For de novo purine biosynthesis, additional protection experiments used AICA (320 µM) to distinguish inhibitory effects at GARFTase from those at AICARFTase1214.

For assays of colony formation in the presence of the antifolate drugs, R2/hPCFT4 cells were harvested, diluted and 200 cells were plated into 60 mm dishes in folate-free RPMI1640 medium supplemented with 25 nM LCV, 10% dFBS, penicillin-streptomycin, and 2 mM L-glutamine, in the presence of antifolate drugs. The dishes were incubated at 37°C with 5% CO2 for 10–14 days. At the end of the incubations, the dishes were rinsed with Dulbecco’s phosphate-buffered saline (DPBS), 5% trichloroacetic acid, and borate buffer (10 mM, pH 8.8), followed by 30 min incubation in 1% methylene blue in borate buffer. The dishes were rinsed with borate buffer and colonies were counted for calculating percent colony-forming efficiency normalized to control.

FR binding assay

[3H]Folic acid binding was used to evaluate binding affinities12. Briefly, RT16 and D4 cells (~1.6×106) were rinsed twice with DPBS followed by two washes with an acidic buffer (10 mM sodium acetate, 150 mM NaCl, pH 3.5) to remove FR-bound folates. Cells were washed twice with ice-cold HEPES-buffered saline (20 mM HEPES, 140 mM NaCl, 5 mM KCl, 2 mM MgCl2, 5 mM glucose, pH7.4) (HBS), then incubated in HBS with [3H]folic acid (50 nM, specific activity 0.5 Ci/mmol) in the presence and absence of unlabeled folic acid or antifolate (over a range of concentrations) for 15 min at 0°C. The dishes were rinsed (3x) with ice-cold HBS, after which the cells were solubilized (0.5 N NaOH) and aliquots of the alkaline homogenates were measured for radioactivity and protein contents. Protein concentrations were measured with Folin-phenol reagent28. Bound [3H]folic acid was calculated as pmol/mg protein and relative binding affinities were calculated as the inverse molar ratios of unlabeled ligands required to inhibit [3H]folic acid binding by 50%. By definition, the relative affinity for folic acid is 1.

Transport assays

For membrane transport assays, R2/hPCFT4, PC43-10, and R2(VC) CHO cells were grown as monolayers and used to seed spinner flasks. For experiments to determine the inhibitions of transport by antifolate substrates, cells were collected and washed with DPBS and resuspended in 2 ml of physiologic Hank’s balanced salts solution (HBSS) for PC43-10 cells, and in HBS, adjusted to pH 7.2 or 6.8, or in 4-morpholinepropanesulfonic acid (MES)-buffered saline (20 mM MES, 140 mM NaCl, 5 mM KCl, 2 mM MgCl2, and 5 mM glucose), adjusted to pH 6.5, 6.0 or 5.5 for R2/hPCFT4 cells. Uptakes of [3H]MTX (0.5 µM) were measured over 2 minutes at 37 °C, in the presence and absence of unlabeled antifolates (10 µM). Uptakes of [3H]MTX were quenched with ice-cold DPBS. Cells were washed with ice-cold DPBS (3x) and solubilized with 0.5 N NaOH. Levels of intracellular radioactivity were expressed as pmol/mg protein, calculated from direct measurements of radioactivity and protein contents of cell homogenates. Protein concentrations were measured with Folin-phenol reagent28. Percent MTX transport inhibition was calculated by comparing the levels of [3H]MTX uptake in the presence and absence of the inhibitors. Kinetic constants (Kt, Vmax) and Kis were calculated from Lineweaver-Burke and Dixon plots, respectively. These methods are analogous to those previously published1315.

Electrophysiology experiments

Xenopus oocytes were used to determine the currents associated with transport of the antifolate substrates. Briefly, hPCFT cRNA (50 nl of 0.5 µg/µl, i.e., 25 ng) or water (50 nl) was injected into stage V/VI oocytes and electrophysiological measurements were recorded 3–5 days later15,25. Oocytes were voltage clamped to −90 mV to maximize folate-induced currents, a technique that was previously used in studies on the divalent metal transporter, DMT129,30 and PCFT15,16. Oocyte solutions were adjusted to pH 5.5 using MES (pH 5.5). During these experiments, oocytes were continuously superfused with solution (with and without substrates as indicated) at 5 ml/min.

In situ GARFTase enzyme inhibition assay

To measure intracellular GARFTase activity in R2/hPCFT4 cells, we measured incorporation of [14C(U)]glycine into [14C]formyl β-glycinamide ribonucleotide (GAR)15. Briefly, R2/hPCFT4 cells were seeded in 5 ml of folate-free RPMI 1640 plus 25 nM LCV, 10% dFBS, 2 mM L-glutamine and penicillin-streptomycin in T25 flasks at a density of 2×105 cells per flask. After 48 h, antifolate inhibitor or DMSO (control) was added to the culture medium. The cells were incubated for another 15 h (the pH was ~6.9 at this time). Cells were washed (2X) with DPBS and resuspended in 5 mL folate-free, L-glutamine-free RPMI 1640 plus penicillin-streptomycin, 10% dFBS, 0.46 g/L NaHCO3 and 1.21 g/L NaCl medium, with or without 0.5–100 nM antifolate and azaserine (4 µM final concentration), and incubated for 30 min. L-glutamine (2 mM final concentration) and [14C]glycine (final specific activity, 0.1 mCi/L) were added; cells were incubated at 37° C for 8 h, after which time the cells were trypsinized and washed twice with ice-cold DPBS. Cell pellets were treated with 2 mL of 5% trichloroacetic acid at 0° C. Cell debris was removed by centrifugation, samples were solubilized in 0.5 N NaOH and assayed for protein contents28. The supernatants were extracted twice with 2 mL of ice-cold ether to remove the trichloroacetic acid. The aqueous layer was passed through a 1 cm column of AG1×8 (chloride form, 100–200 mesh) (BioRad), washed with 10 mL of 0.5 N formic acid, followed by 10 mL of 4 N formic acid, and eluted with 8 mL of 1 N HCl solution. The eluants were collected as 1 mL fractions and determined for radioactivity. The accumulation of [14C]formyl GAR was calculated as pmol per mg protein over a range of inhibitor concentrations. IC50s were calculated as the concentrations of inhibitors that resulted in a 50% decrease in [14C]formyl GAR synthesis.

Measurement of intracellular ATP levels

For analysis of ATP levels following antifolate treatments, R2/hPCFT4 cells were seeded in 10 ml of folate-free RPMI 1640, 10% dFBS, 2 mM L-glutamine and penicillin-streptomycin plus 25 nM leucovorin (pH 7.2), then replaced after 24 h with the same media buffered to pH 6.8 with 25 mM PIPES/25 mM HEPES. Compounds 3, 4, PMX, or LMTX (each at 1 µM), or vehicle (0.5% DMSO for 3 and 4, H2O for PMX and LMTX) (controls) was added to the culture medium. Cells were incubated for an additional 24 h, after which they were trypsinized and washed (2x) with ice-cold DPBS. Nucleotides were extracted and ATP levels were quantitated by HPLC exactly as previously described15.

In vivo efficacy study in IGROV1 cells

The methods for protocol design, drug treatment, toxicity evaluation, data analysis, quantification of tumor cell kill, tumor model systems, and the biological significance of the drug treatment results with transplantable tumors have been described previously14,3135. Cultured IGROV1 human ovarian tumor cells were grown in folate-deficient media and were implanted subcutaneously (5 × 106 cells/flank) to establish a solid tumor xenograft model in female ICR SCID mice (obtained from Taconic Laboratories). For the efficacy study, mice were 10 weeks old on day 0 (tumor implant) with an average body weight of 25.9 g. Mice were supplied food and water ad libitum. Study mice were maintained on either a folate- deficient diet from Harlan-Teklad (TD.00434) or a folate-replete diet from Lab Diet (5021; autoclavable mouse breeder diet) starting 24 days before subcutaneous tumor implant to ensure serum folate levels would approximate those of humans. Folate serum levels were determined prior to tumor implant and post study via L. casei bioassay36. The animals were pooled and implanted bilaterally subcutaneously with 30 to 60 mg tumor fragments by a 12 gauge trocar, and again pooled before unselective distribution to the various treatment and control groups. Chemotherapy was begun 3 days after tumor implantation, when the number of cells was relatively small (107 to 108 cells; before the established limit of palpation). Tumors were measured with a caliper two or three times weekly (depending on the doubling time of the tumor). Mice were sacrificed when the cumulative tumor burden reached 1500 mg. Tumor weights were estimated from two-dimensional measurements [i.e., tumor mass (in mg) = (a × b2)/2, where a and b are the tumor length and width in mm, respectively]. For calculation of endpoints, both tumors on each mouse were added together, and the total mass per mouse was used. The following quantitative end-points were used to assess anti-tumor activity: (i)T/C [where T is the median time in days required for the treatment group tumors to reach a predetermined size (e.g., 1000 mg), and C is the median time in days for the control group tumors to reach the same size; tumor-free survivors are excluded from these calculations]; and (ii) tumor cell kill [log10 cell kill total (gross) = (T − C)/(3.32)(Td), where T − C is the tumor growth delay, and Td is the tumor volume doubling time in days, estimated from the best fit straight line from a log-linear growth plot of control group tumors in exponential growth (100 to 800 mg range)].

Supplementary Material

1_si_001

Acknowledgement

This work was supported, in part, by grants from the National Institutes of Health, National Cancer Institute, CA125153 (AG), CA152316 (LHM and AG), and CA53535 (LHM), a grant from the Mesothelioma Applied Research Foundation, a pilot grant from the Barbara Ann Karmanos Cancer Institute, and support from the Duquesne University Adrian Van Kaam Chair in Scholarly Excellence (AG). Ms. Kugel Desmoulin was supported by a Doctoral Research Award from the Canadian Institutes of Health Research (CIHR).

Abbreviations

AICA
5-amino-4-imidazolecarboxamide
AICAR
5-amino-4-imidazolecarboxamide ribonucleotide
AICARFTase
5-amino-4-imidazolecarboxamide ribonucleotide formyltransferase
CHO
Chinese hamster ovary
dFBS
dialyzed fetal bovine serum
DPBS
Dulbecco’s phosphate-buffered saline
FGAR
formyl glycinamide ribonucleotide
FR
folate receptor
GAR
glycinamide ribonucleotide
GARFTase
glycinamide ribonucleotide formyltransferase
HBSS
Hank’s balanced salts solution
HBS
HEPES-buffered saline
hPCFT
human PCFT
hRFC
human RFC
LCV
leucovorin
LMTX
Lometrexol
MTX
methotrexate
PMX
Pemetrexed
PCFT
proton-coupled folate transporter
RTX
Raltitrexed
RFC
reduced folate carrier
SCID
severe combined immunodeficient

Footnotes

Supporting Information. Elemental analysis and high-resolution mass spectra (HRMS) (ESI). This material is available free of charge via the Internet at http://pubs.acs.org.

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