James R. Piper

A new analogue of 10-deazaaminopterin with markedly enhanced curative effects against human tumor xenografts in mice

Purpose: These studies sought to evaluate the biochemical and cellular pharmacokinetic properties, cytotoxicity and antitumor efficacy of a new analogue of 10-deaza-aminopterin (PDX) against human tumors. Methods: Studies were conducted with a group of human tumor cell lines in culture examining PDX and other folate analogues as permeants for mediated membrane transport, as inhibitors of dihdrofolate reductase and as substrates for folylpolyglutamate synthetase. These same analogues were examined for their cytotoxicity following a 3-h pulse exposure, in experiments providing a value for IC50. Other studies with these analogues were conducted in nude mice bearing subcutaneously implanted human tumors. Treatment of the mice was initiated 4 days after implantation of the tumor using a schedule of administration of one dose per day for 5 days. The tumors were measured 6 days after cessation of therapy and compared to controls for assessment of response. Results: In the CCRF-CEM cell system, PDX was 2- to 3-fold less effective as an inhibitor of dihydrofolate reductase than aminopterin (AMT), methotrexate (MTX) or edatrexate (EDX) but much more effective as a permeant for one-carbon, reduced folate transport inward (PDX >AMT ≃ EDX >MTX) and substrate for folylpolyglutamate synthetase (PDX >AMT >EDX >MTX). As predicted by these results, PDX was 15- to 40-fold more cytotoxic than MTX and 3- to 4-fold more cytotoxic than the highly potent EDX following a 3-h pulse exposure in culture of CCRF-CEM cells and cells from a panel of three human breast and two human nonsmall-cell (NSC) lung cancers. The same relative differences were shown for the therapeutic efficacy of these three analogues at equitoxic doses in studies with the human MX-1 and LX-1 tumors and the human A549 NSC lung tumor xenografted in nude mice. On a schedule of qd × 5 given 3–4 days posttransplant, MTX was minimally active (modest tumor growth delay) against all three tumors. EDX was highly active (25–35% complete regressions and 5–10% cures) against the MX-1 and LX-1 tumors but very modestly active (no regressions) against the A549 tumor. In contrast, PDX was even more active (75–85% complete regressions and 25–30% cures) than EDX against the MX-1 and LX-1 tumors and highly active (30% complete regressions and 20% cures) against the A549 tumor. Conclusions: These studies showed significantly enhanced antitumor properties of PDX compared with MTX and EDX. Based upon these results, clinical trials of PDX in patients with metastatic breast and NSC lung cancer appear to be warranted.

Atomic structures of human dihydrofolate reductase complexed with NADPH and two lipophilic antifolates at 1.09 a and 1.05 a resolution.

The crystal structures of two human dihydrofolate reductase (hDHFR) ternary complexes, each with bound NADPH cofactor and a lipophilic antifolate inhibitor, have been determined at atomic resolution. The potent inhibitors 6-([5-quinolylamino]methyl)-2,4-diamino-5-methylpyrido[2,3-d]pyrimidine (SRI-9439) and (Z)-6-(2-[2,5-dimethoxyphenyl]ethen-1-yl)-2,4-diamino-5-methylpyrido[2,3-d]pyrimidine (SRI-9662) were developed at Southern Research Institute against Toxoplasma gondii DHFR-thymidylate synthase. The 5-deazapteridine ring of each inhibitor adopts an unusual puckered conformation that enables the formation of identical contacts in the active site. Conversely, the quinoline and dimethoxybenzene moieties exhibit distinct binding characteristics that account for the differences in inhibitory activity. In both structures, a salt-bridge is formed between Arg70 in the active site and Glu44 from a symmetry-related molecule in the crystal lattice that mimics the binding of methotrexate to DHFR.

Human thymidylate synthetase—III: Effects of methotrexate and folate analogs

Abstract The structure-activity relationship of human thymidylate synthetase (EC 2.1.1.45) was studied with two groups of folate analogs: (1) methotrexate (MTX) analogs modified at the glutamate residue and N 10 ; and (2) tetrahydrofolate (H 4 PteGlu) analogs modified at N 5 and N 10 . With respect to MTX analogs, it was found that: (1) substitution of the glutamate side chain by α-aminoadipic acid. α-aminopimelic acid or β-aminoglutaric acid slightly affects its K i ; (2) a free α-carboxyl group on the amino acid side chain of MTX, or any free carboxyl group in that vicinity plays an important role in the inhibitory potency of MTX analogs to the enzyme; (3)esterification or amidation of the α-carboxyl group of MTX decreases the inhibitory potency; and (4) free aspartyl or glutamyl conjugation through a peptide linkage to the γ-carboxyl group of the glutamate side chain decreases its K i to the enzyme by 5- and 8-fold respectively. Tetrahydrofolate analogs formed by inserting an ethylene, iminyl or a carbonyl bridge between the nitrogen at N 5 and N 10 or by substitution at the N 5 position were found to be poor inhibitors under our assay conditions.

Liposome-mediated delivery of pteridine antifolates to cells in vitro: potency of methotrexate, and its α and γ substituents

Abstract We have examined the growth-inhibitory potency of several pteridines encapsulated in negatively charged liposomes, including methotrexate, methotrexate-γ-methylamide, methothrexate-γ-dimethylamide, methotrexate-α-aspartate, and a lipophilic methotrexate-phosphatidylethanolamine conjugate. The potency of encapsulated methotrexate is greater than the potency of the free drug for CV1-P cells, but not for other cell lines. The potency of methotrexate-γ-methylamide and mehtotrexate-γ-dimethylamide is only minimally improved by encapsulation. The potency of methotrexate-α-aspartate is increased by encapsulation. In addition, the lipophilic methotrexate derivative has demonstrable potency when incorporated in liposomes. We have also examined the potency of several pteridines under conditions where cells are exposed to the drug for periods shorter than the entire growth assay. Reduction of the exposure time decreases the potency of both encapsulated and free drugs. However, the difference in potency between the encapsulated and free drug is increased, because the potency of the encapsulated drug is affected less. Consequently, encapsulated methotrexate-γ-aspartate is 300-fold more potent than free drug, if CV1-P cells are exposed to drug for 4 h. Moreover, encapsulated methotrexate is more potent than free methotrexate for growth inhibition of L929 fibroblasts, if the term of exposure is less than 8 h. Potency is least affected by reduction of exposure length for the lipophilic methotrexate derivative.

Growth inhibitory, transport and biochemical properties of the γ-glutamyl and γ-aspartyl peptides of methotrexate inL1210 leukemia cells in vitro

Abstract Both methotrexate-γ-glutamate and methotrexate-γ-aspartate are equivalent to metho-trexate as inhibitors of L1210 cell dihydrofolate reductase. However, the initial influx of both peptides into L1210 cells during transport studies is substantially lower than that of methotrexate. The apparent K m tor influx of methotrexate-γ-glutamate and methotrexate-γ-aspartate is 15-fold and 100-fold greater than methotrexate respectively. Efflux measurements, which were possible only for methotrexate-γ-glutamate, showed a similar rate for this peptide and methotrexate. The intracelluiar accumulation and subsequent metabolism to methotrexate of methotrexate-γ-glutamate, but not of methotrexate-γ-aspartate, were confirmed by bioautographic analysis of cell extracts. After correction for extracellular cleavage of both peptides mediated by enzymes in calf serum supplementing the culture medium, the relative growth (L1210 cell)-inhibitory potency for the three agents was 1:18:210 for methotrexate, methotrexate-γ-glutamate and methotrexate-γ-aspartate respectively. Both the relative inhibitory potency and the difference in absolute inhibitory concentration among the three agents were predictable solely from the data on the influx of each measured during transport studies. Methotrexate-γ-aspartate is apparently more resistant to enzymic cleavage than is methotrexate-γ-glutamate.

Folylpolyglutamate synthetase inhibition and cytotoxic effects of methotrexate analogs containing 2,ω-diaminoalkanoic acids

The properties of a series of methotrexate analogs containing 2,omega-diaminoalkanoic acids have been investigated. The compounds were potent inhibitors of dihydrofolate reductase but, unlike methotrexate, they were also inhibitors of mammalian folylpolyglutamate synthetases. The potency of synthetase and reductase inhibition increased with increasing length of the 2,omega-diaminoalkanoate moiety. The most cytotoxic compound and the most potent inhibitor of both dihydrofolate reductase (I50 = 2.5 to 4 nM) and folylpolyglutamate synthetase (Ki ca. 4 microM) contained 2,5-diaminopentanoic acid (ornithine). These compounds were 70- to 100-fold less cytotoxic than methotrexate to human leukemia cell lines; however, they retained their potency against sublines resistant to methotrexate via defective transport. Their dual loci of enzyme inhibition and their efficacy against methotrexate transport-defective cell lines indicate that these compounds may be an important new class of antifol.

In Vitro Activities of Several Diaminomethylpyridopyrimidines against Mycobacterium avium Complex

ABSTRACT Three recently synthesized dihydrofolate reductase (DHFR) inhibitors designated SoRI 8890, 8895, and 8897 were evaluated for their in vitro activities against 25 isolates of Mycobacterium avium complex. The MICs at which 50 and 90% of isolates were inhibited were 1 and 2, 4 and 8, and 4 and 8 μg/ml for SoRI 8890, 8895, and 8897, respectively. Although the addition of dapsone at 0.5 μg/ml did not significantly enhance the in vitro activities of these compounds, their activities alone were comparable to, if not better than, results seen with other DHFR inhibitors, such as pyrimethamine or WR99210.

Lipophilic Antifolates as Candidates against Opportunistic Infections

The problems in treating immunocompromised patients infected by Pneumocystis carinii and Toxoplasma gondii have prompted intense efforts to develop improved therapy against these pathogens.1,2 Lipophilic antifolates trimethoprim and pyrimethamine (structures shown under Table 1) are used in combination with sulfa drugs in current treatment regimens which inhibit the ability of the microorganism to synthesize reduced folates. In such treatment, the antifolate inhibits dihydrofolate reductase (DHFR), and the sulfa drug inhibits utilization by the microorganism of 4-aminobenzoic acid in its vital biosynthesis of dihydropteroic acid.2–4 Adverse reactions that frequently occur with these regimens often necessitate discontinuation of the therapy. New agents or combinations of agents of greater therapeutic effectiveness in terms of lower toxicity and shorter treatment periods are needed.

Synthesis and antifolate evaluation of the aminopterin analogue with a bicyclo[2.2.2]octane ring in place of the benzene ring

N-[4-[[2,4-diamino-6-pteridinyl)methyl]amino]bicyclo[2.2.2]octane-1-carbonyl]-L-glutamic acid (1) was synthesized and tested for antifolate activity. N-(4-Aminobicyclo[2.2.2]octane-1-carbonyl-L-glutamic acid dimethyl ester (6), the side chain precursor to subject compound 1, was synthesized readily via reported bicyclo[2.2.2]octane-1,4-dicarboxylic acid monoethyl ester (2). The side chain precursor 6 was alkylated by 6-(bromomethyl)-2,4-pteridinediamine (7). Subsequent ester hydrolysis then afforded 1. Antifolate and antitumor evaluation of 1 verses L1210 dihydrofolate reductase (DHFR) and three tumor cell lines (L1210, S180, and HL60) showed it to be ineffective. Although compound 1 was very similar to aminopterin structurally, the bicyclo[2.2.2]octane ring system in place of the phenyl ring in the p-aminobenzoate moiety effectively negates the stoichiometric binding displayed by many classical DHFR inhibitors bearing appropriate aromatic ring systems in the side chain.

Structural specificity of inhibition of human folylpolyglutamate synthetase by ornithinecontaining folate analogs

A series of folate analogs containing ornithine instead of glutamate was synthesized and tested for inhibition of folylpolyglutamate synthetase (FPGS) and other folate-dependent enzymes of human leukemia cell lines. Reduced derivatives of 2-amino-4-oxo-10-methyl-pteroyl-ornithine had dramatically increased inhibitory potency against FPGS compared to the oxidized parent. The amino-pterin analog (2,4-diamino-pteroylornithine) was a potent inhibitor of both dihydrofolate reductase and FPGS. It was a much more potent linear competitive inhibitor of human FPGS than the corresponding methotrexate derivative previously described (Ki = 0.15-0.26 and 3 microM respectively). A quinazoline folate analog, 2-amino-4-oxo-5,8-dideazapteroyl-ornithine, was a relatively poor inhibitor of isolated dihydrofolate reductase and thymidylate synthase; however, it is the most potent human FPGS inhibitor identified to date (Ki = 100-150 nM). Because of the lack of appreciable interaction with other folate-dependent enzymes, structures incorporating the 2-amino-4-oxo-5,8-dideazapteroate nucleus may thus lead to selective inhibition of FPGS. Substitution of ornithine for glutamate caused a profound decrease in cytotoxic potency for these analogs; this was apparently the result of poor transport. Together with earlier studies, these data indicate that the potency of FPGS inhibition by an analog containing ornithine closely parallels the relative substrate activity of its glutamate-containing counterpart. The substitution of ornithine apparently does not perturb the pterin specificity of FPGS. The close parallel between substrate and inhibitor specificity may thus allow the use of currently available structure-activity studies on FPGS to design more potent and more selective inhibitors of FPGS.