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 approximately equal to 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 x 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.

Stabilized analogs of thymopentin. 1. 4,5-Ketomethylene pseudopeptides.

The pentapeptide, thymopentin (Arg1-Lys2-Asp3-Val4-Tyr5) is known for its activity as an immunomodulating drug, but with limited half-life in plasma. In this first paper of a series of three studies, the synthesis of analogs stabilized at the peptide bond between the C-terminal amino acids via insertion of a ketomethylene moiety is described. N-Blocked pseudopeptides containing Val(k)Phe, Ala(k)Phe, and Val(k)Val units were prepared and attached to chloromethyl Merrifield resin via the carboxy terminal. Removal of the N-BOC group by trifluoroacetic acid was followed by sequential coupling with N-BOC dipeptides of aspartic acid to yield resin-bound N-BOC pseudotetrapeptides. Removal of N-BOC and coupling with N-BOC-r-N-tosylarginine followed by total cleavage of blocking groups and resin by HF afforded the target pseudopentapeptides. The analogs were found to compete favorably with thymopentin for binding to CEM cells, but binding was reduced by about 20-30% on average. All analogs showed significant enhancement of half-life versus thymopentin in mouse serum, but most showed only modest improvement in human serum. Insertion of proline or norleucine at position 2 in the chain caused a substantial increase in half-life (3-4-fold), while N-methylnorleucine conferred complete stability in the analogs.

Stabilized analogs of thymopentin. 2. 1,2- and 3,4-ketomethylene pseudopeptides.

In this second paper in a series of three studies of stable analogs of thymopentin (Arg1-Lys2-Asp3-Val4-Tyr5), the synthesis of analogs stabilized at peptide bonds 1,2 and 3,4 via insertion of ketomethylene units is described. A tris(carbobenzyloxy)arginyl(k)norleucine pseudopeptide was synthesized and coupled to Asp-Val-Phe-resin units followed by HF cleavage to prepare Arg(k)Nle-Asp-Val-Phe analogs. Preparation of N-BOC Asp(k)Val and N-BOC Asp(k)Ala units followed by coupling to Phe- or Tyr-resin units provided resin-bound pseudotripeptide substrates for attachment of various arginyl dipeptides. Cleavage from the resin afforded 3,4-ketomethylene-stabilized pseudopeptide analogs of thymopentin. The Arg-Lys-Asp(k)Val-Phe and Arg-Lys-Asp(k)Val-Tyr analogs were more strongly bound to CEM cells than thymopentin itself. There was significant enhancement of stability in serum for the analogs, especially those containing Arg(k)Nle or Arg-NMeLys moieties at the 1,2-peptide bond.

Stabilized analogs of thymopentin. 3. Evaluation of ketomethylene pseudopeptides for antiarthritic properties.

This study analyzed the role of ketomethylene pseudopeptides of thymopentin as potential agents for the treatment of arthritis. The analogs were tested in vivo using assessment of inflammation and antibody production in the mouse type II collagen arthritis model and the rat adjuvant arthritis model. The compounds were also tested for immune-potentiating activity in vitro using induction of the lymphocyte marker, Thy-1.2, in mouse spleen cells and stimulation of T-cell proliferation. The results show that certain of the compounds exhibit disease-remitting properties for arthritis as evidenced by reduction of paw swelling in the mouse and rat models and decreased incidence of disease in the mouse model. The active compounds were dose specific and represented a range in efficacy. In spite of effects on arthritis, type II collagen antibody levels were not altered in the mouse model. Selected compounds also exhibited immune potentiating properties as evidenced by induction of Thy-1.2 expression and stimulation of T-cell proliferation. The absence of effect of the compounds on type II collagen antibody production suggests that the antiarthritic activity of the effective compounds results from alteration of cell-mediated immunity.

Analogues of methotrexate in rheumatoid arthritis. 1. Effects of 10-deazaaminopterin analogues on type II collagen-induced arthritis in mice.

Carbonation of the dianions (LDA) of 5-methylthiophene-2-carboxylic, 2-methylpyridine-5-carboxylic, and 3-methylpyridine-6-carboxylic acids provided the respective carboxy heteroarylacetic acids. The crude diacids were directly esterified in MeOH-HCl to afford the diesters. Alkylation of the sodio anions with ethyl iodide yielded the appropriate alpha-ethyl diesters. The anions of the various diester substrates were then alkylated by 2,4-diamino-6-(bromomethyl)-pteridine followed by ester saponification at room temperature to afford the respective 2,4-diamino-4-deoxy-10-carboxy-10-deazapteroic acids. The 10-carboxyl group was readily decarboxylated by heating in DMSO at temperatures of 110-135 degrees C to give the diamino 10-deaza heteropteroic acid intermediates. Coupling with diethyl L-glutamate followed by ester hydrolysis afforded the target aminopterins. The analogues were evaluated for antiinflammatory effect in the mouse type II collagen model. The thiophene analogue of 10-ethyl-10-deazaaminopterin was found to be an effective inhibitor in terms of reduced visual evidence of inflammation and swelling as determined by caliper measurement.

Analogues of methotrexate in rheumatoid arthritis. 2. Effects of 5-deazaaminopterin, 5,10-dideazaaminopterin, and analogues on type II collagen-induced arthritis in mice.

Twenty-six compounds derived from the 5-deaza- and 5,10-dideazaaminopterin series of aminopterin analogues were evaluated for antiarthritic activity in the mouse type II collagen model. New compounds in the 5-deaza series were prepared by alkylation of an appropriate N-substituted (4-aminobenzoyl)-L-glutamic acid dialkyl ester or N-(5-amino-2-thenoyl)-L-glutamate diester with a 2,4-diamino-5-alkyl-6-(bromomethyl)-5-deazapteridine. The resultant 5-deazaaminopterin diesters were saponified to provide the target 5-deaza analogues. 5,10-Dideazaaminopterins were synthesized by similar alkylation of the carbanions of appropriate 4-carboxyphenylacetic, (5-carboxy-2-thienyl)acetic, or (5-carboxy-2-pyridyl)acetic acid dimethyl esters. The diesters of the 2,4-diamino-4-deoxy-10-carboxy-5,10-dideazapteroic acid types so obtained were saponified and then readily decarboxylated by heating in Me2SO solution to provide the 2,4-diamino-5,10-dideazapteroic acid-type intermediates. Peptide coupling with diethyl L-glutamate followed by ester hydrolysis at room temperature afforded the new 5,10-dideazaaminopterin analogues. 5-Deazaaminopterins bearing an alkyl substituent at the 5-position were generally quite effective as antiinflammatory agents. Thus 5-propyl-5-deazaaminopterin, 5-methyl-10-propargyl-5-deazaaminopterin, 5-methyl-10-allyl-5-deazaaminopterin, 5-ethyl-5-deazamethotrexate, and 2,5-disubstituted thiophene analogue of 5-methyl-5-deazaaminopterin showed potencies greater than methotrexate by intraperitoneal or oral administration and were active over a considerably broader dose range. Useful activity in the 5,10-dideaza series was only observed for 5,10-dideazaaminopterin and its 10-methyl analogue. Alkyl substitution at C-5 or C-10 was generally detrimental to antiinflammatory activity in this series.

Synthesis and antitumor activity of 10-propargyl-10-deazaaminopterin.

Successive alkylation of dimethyl homoterephthalate with propargyl bromide and 2,4-diamino-6-(bromomethyl)pteridine followed by ester saponification at room temperature afforded 2,4-diamino-4-deoxy-10-carboxy-10-propargyl-10-deazapteroic acid. The 10-COOH was readily decarboxylated by heating in DMSO at a temperature of only 120 degrees C to yield the diamino-10-propargyl-10-deazapteroic acid intermediate. Coupling with diethyl L-glutamate and ester hydrolysis gave the title compound. The 10-propargyl analogue was about 5 times more potent than MTX as an inhibitor of growth in L1210 cells, but was only one-third as potent as an inhibitor of DHFR from L1210. The analogue was transported inward very effectively in L1210 cells showing a 10-fold advantage over MTX. At a dose of 36 mg/kg the 10-propargyl compound caused shrinkage of the E0771 solid murine mammary tumor to only 1% of untreated controls.

Synthesis and antifolate properties of 5,10-ethano-5,10-dideazaaminopterin.

2-Carbomethoxy-4-(p-carbomethoxyphenyl)cyclohexanone was prepared in a four-step process and thermally condensed with 2,4,6-triaminopyrimidine to afford methyl 2,4-diamino-4-deoxy-7-hydroxy-5,10-ethano-5,10-dideazapteroate+ ++. Reduction of the 7-oxo function with borane gave the 7,8-dihydro pterin which was subsequently oxidized to the fully aromatic pteroate ester with dicyanodichlorobenzoquinone. Saponification of the benzoate ester, coupling with diethyl glutamate and final ester hydrolysis afforded the title compound. This novel deazaaminopterin analogue was approximately as potent as methotrexate in vitro in terms of DHFR and L1210 cell growth inhibition. There are indications of diastereomeric differences in the enzyme inhibition measurements. A significant transport advantage over MTX for influx into L1210 cells was observed. The compound was active against the E 0771 murine mammary solid tumor, but further investigation with individual diastereomers is required to define the ED50.

Synthesis and antifolate properties of 10-alkyl-5,10-dideaza analogues of methotrexate and tetrahydrofolic acid.

Synthesis of the 10-methyl and 10-ethyl analogues of 5,10-dideazatetrahydrofolic acid (DDTHF), a potent inhibitor of glycinamide ribotide (GAR) formyltransferase, is reported. Key intermediates in the process were 10-methyl- and 10-ethyl-4-amino-4-deoxy-5,10-dideazapteroic acid. Condensation of the piperidine enamines of branched 4-(p-carbomethoxyphenyl)butyraldehydes with (acetoxymethylene)malononitrile afforded 1,1-dicyano-4-piperidinobutadiene 5a,b. Subsequent reaction with alcoholic ammonium hydroxide yielded the appropriately substituted 2-amino-3-cyanopyridines 6a,b. Ring closure with guanidine gave 10-methyl- and 10-ethyl-4-amino-4-deoxy-5,10-dideazapteroic acids (7a,b). Coupling with diethyl glutamate followed by ester hydrolysis afforded 10-alkyl-5,10-dideazaminopterin analogues 9a,b. Hydrolysis of the 4-amino group of 7a,b yielded the 10-alkylpteroic acids, which were coupled with diethyl glutamate, hydrogenated over PtO2, and saponified to afford 10-alkyl-5,10-dideazatetrahydrofolic acids 13a,b. Aminopterin analogues 9a,b were effective inhibitors of DHFR derived from L1210, but were less potent than methotrexate for inhibition of growth of L1210 in culture. The 10-ethyl (13b) analogue of 5,10-DDTHF was about twice as potent an inhibitor of L1210 cell growth as 5,10-DDTHF, but was only 1/7 as potent for inhibition of GAR formyltransferase. 10-Methyl analogue 13a was similar in potency to 5,10-DDTHF. All of the compounds showed moderately improved transport into L1210 cells relative to methotrexate.

Synthesis and antifolate properties of 9-alkyl-10-deazaminopterins.

Reformatski condensation of benzyl 2-bromopropionate with 4-carbomethoxybenzaldehyde, followed by dehydration afforded benzyl 2-methyl-p-carbomethoxycinnamate (4a). Hydrogenation over a Pd catalyst gave the hydrocinnamic acid 5a. Conversion to the chloromethyl (6a) and azidomethyl ketone (7a) was followed by hydrogenation to the aminomethyl ketone (8a). Direct N-alkylation by 2,4-diamino-5-nitro-6-chloropyrimidine followed by reductive ring closure in Zn-HOAc and subsequent saponification of the benzoate ester yielded 4-amino-4-deoxy-9-methyl-10-deazapteroic acid (11a). Coupling with diethyl L-glutamate and saponification afforded 9-methyl-10-deazaminopterin (13a). The 9-ethyl analogue (13b) was similarly prepared from benzyl 2-bromobutyrate. The 9-methyl analogue (13a) was 21 times more potent than MTX as an inhibitor of cell growth in L1210 cells. The reason for this enhanced cytotoxicity in L1210 is unclear, since enzyme inhibition and transport parameters were similar to those of MTX. In human Manca leukemia cells growth inhibition was not dramatic and paralleled MTX.

Intracavitary therapy of murine ovarian cancer with cis-diamminedichloroplatinum(II) and 10-ethyl-10-deazaaminopterin incorporating systemic leucovorin protection.

Administration i.p. of 10-ethyl-10-deazaaminopterin (10EDAM) with cis-diamminedichloroplatinum(II) (cis-Pt) had significant antitumor activity against the murine ovarian tumor. This tumor is a teratoma originating in the ovary with pathogenesis and metastatic properties similar to those of human ovarian cancer. Drug was given on a schedule of once every 3 days for 3 doses 1 or 2 days after i.p. implant of 10(7) tumor cells. Despite the 2-fold attenuation of dosage required, antitumor activity of the combination (increased life span, 161%) was approximately twice that obtained with maximum tolerated doses of either agent alone and tumor-free, long-term survivors were obtained. Incorporation of s.c. calcium leucovorin administration 16 h after each dose of 10EDAM and cis-Pt allowed a 4-fold increase in dosage of 10-EDAM without an increase in toxicity, increased median survival by an additional 120%, and quadrupled the number of tumor-free, long-term survivors to 40% of treated animals. By comparison, methotrexate was only modestly active against this tumor model either as a single agent, with cis-Pt, or with delayed s.c. calcium leucovorin administration. These results appear to suggest that 10EDAM with cis-Pt may have considerable potential for intracavitary therapy of human cancer, including ovarian carcinoma, particularly when incorporating delayed systemic calcium leucovorin administration.

Synthesis and biological evaluation of 8-deazahomofolic acid and its tetrahydro derivative.

The syntheses of 8-deazahomofolic acid and its tetrahydro derivative, potential inhibitors of thymidylate synthase (TS) and other folate related enzymes, are described. Wittig condensation of 2-acetamido-6-formyl-4-pyrimidinol with the triphenylphosphine ylide 3 derived from N-acetyl-4-(p-carbethoxyanilino)-1-chloro-2-butanone, hydrogenation of the enone intermediate 5, introduction of a 5-amino group via diazonium coupling, and reductive ring closure yielded ethyl N11-acetyl-8-deazahomopteroate (8). Alkaline hydrolysis gave 8-deazahomopteroic acid, which was blocked as the 11-trifluoroacetyl derivative, coupled with diethyl L-glutamate, and the blocking groups saponified to afford 8-deazahomofolic acid (12). Hydrogenation of the glutamate diester intermediate and subsequent saponification yielded the tetrahydro-8-deazahomofolate (14). Growth inhibition of Streptococcus faecium, Lactobacillus casei, and L1210 cells in culture by the target compounds was modest. They were also weak inhibitors of thymidylate synthase, dihydrofolate reductase, glycinamide-ribonucleotide transformylase, and aminoimidazolecarboxamide ribonucleotide transformylase. In contrast, 8-deazafolate showed moderate inhibition of aminoimidazolecarboxamide ribonucleotide transformylase, suggesting that inhibition of this enzyme may be related to its cytotoxic action. Tetrahydro-8-deazahomofolate showed low substrate activity with thymidylate synthase.

Combination chemotherapy with a new folate analog: activity of 10-ethyl-10-deaza-aminopterin compared to methotrexate with 5-fluorouracil and alkylating agents against advanced metastatic disease in murine tumor models.

A new folate analog, 10-ethyl-10-deaza-aminopterin (10EDAM), was compared to methotrexate (MTX) in combination with 5-fluorouracil (5-FU) and/or alkylating agents against three models of advanced metastatic (E0771 mammary adenocarcinoma and T241 fibrosarcoma) or disseminated (L1210 leukemia) disease in BD2F1 mice. Of all the agents examined on a schedule of every 3 days X 3, 10EDAM and cyclophosphamide (CPA) were the most active overall against the tumor models employed. Against the E0771 tumor, antitumor potency was in the descending order of CPA, 10EDAM, cisplatin (Cis Pt), melphalan (L-PAM), MTX, and 5-FU. Similar ranking in relative potency was also derived with the T241 tumor. Against both of these models, MTX and 5-FU were only minimally active. Against the L1210 leukemia, 10EDAM was the most active agent followed in descending order by MTX, CPA, 5-FU, L-PAM, and Cis Pt. All two-drug combinations required attenuation to one-half the LD10 dosage for each. The results show that 10EDAM with CPA was the most effective combination employed in all three tumor models. Therapeutic activity was far greater than that obtained with each agent alone, with a substantial number of long-term survivors. 10EDAM was also highly active in combination with L-PAM and Cis Pt against the E0771 tumor and with Cis Pt against the L1210 leukemia. Some increase in therapeutic activity beyond that obtained with each agent alone, but to a substantially lesser extent, was documented for MTX and each of the three alkylating agents against the L1210 tumor. MTX with 5-FU was not an effective combination, but against the E0771 tumor gave therapeutic effects beyond that obtained with each agent alone when 5-FU was given 7 hours after MTX. Similar effects were seen with 10EDAM and 5-FU on these schedules, but 10EDAM alone was more effective than MTX and 5-FU given together against this tumor. A combination of either antifolate with 5-FU and CPA was ineffective against all three tumor models, in part because of the further attenuation of dosage required in this mouse strain and the minimal overall activity of 5-FU in these models. Results documented with 10EDAM and CPA and, perhaps, other alkylating agents are interpreted as indicative of therapeutic synergism between these agents. These results appear to suggest substantial potential for 10EDAM in clinically employed combination therapy currently including MTX.

Toxicity, elimination, and metabolism of 10-ethyl-10-deazaaminopterin in rats and dogs.

10-Ethyl-10-deazaaminopterin (10-EdAM) is an antifolate compound with greater therapeutic activity than methotrexate against transplanted tumors in mice. When given weekly for 3 weeks, the 10% lethal dose in rats was 125 mg/kg (i.p.) and in dogs it was 2.5 mg/kg (i.v.). The major histopathological findings in intoxicated animals were damage to the mucosa of the gastrointestinal tract in rats and dogs and hypocellularity of the marrow in rats. The elimination of 50 mg/kg of 10-EdAM from the plasma of rats was triexponential with a terminal phase t1/2 of 18.5 h but a mean residence time of 0.7 h. The primary route of elimination in rats was biliary secretion of parent compound and eventual excretion of the parent compound and the deglutamate metabolite in the feces; the 7-hydroxy metabolite was also present in plasma, bile, and feces. Biliary elimination was independent of dose over a 5-fold range. The elimination of 10-EdAM from the plasma of dogs was also triexponential with a mean terminal phase t1/2 of 9.1 h and a mean residence time of 2.5 h; nonrenal clearance was the primary route of elimination. The pharmacokinetic parameters were independent of dose over the range of 0.25 to 5.0 mg/kg. High tissue concentrations of 10-EdAM were observed initially in liver, kidney, and small intestine of rats, while concentrations in bone marrow were low. Some polyglutamate formation was observed in these tissues as early as 0.5 h after drug administration but declined over 72 h.

10-Ethyl-10-deaza-aminopterin: structural design and biochemical, pharmacologic, and antitumor properties.

The new folate analog 10-ethyl-10-deaza-aminopterin (10EdAM) was equivalent to methotrexate (MTX) as an inhibitor of dihydrofolate reductase, but was more effectively transported and polyglutamylated in most tumor cells. Also, the transport and polyglutamylation of 10EdAM in tumor cells vis-a-vis normal proliferative tissue is substantially increased compared to MTX, favoring much greater accumulation of 10EdAM as cytotoxic polyglutamates in some of these tumor cells. 10EdAM was superior to MTX against 4 of 6 murine ascites tumors (L1210, S180, Ehrlich and Tapper) and far superior against 4 of 6 solid murine tumors (S180, Tapper, E0771 mammary AC, T241 fibrosarcoma). 10EdAM produced 10% to 30% complete regressions against S180, E0771 and T241 tumors. Both agents showed similar activity against P288 and 1498c leukemias and the Lewis lung tumor, but were inactive against B16 melanoma. Marked superiority of 10EdAM compared to MTX was also shown against the following human tumor xenografts: MX-1 (mammary carcinoma), LX-1 (small cell lung carcinoma) and CX-1 (colon carcinoma). 10EdAM produced 30% to 40% complete regressions against the MX-1 tumor.

Synthesis and antifolate properties of 5,10-methylenetetrahydro-8,10-dideazaminopterin.

The synthesis of the 5,10-methylene analogue of 5,6,7,8-tetrahydro-8,10-dideazaminopterin, a potential dual inhibitor of dihydrofolate reductase (DHFR) and thymidylate synthase (TS) enzymes, is described. The dimethyl ester of 10-carboxy-4-amino-4-deoxy-8,10-dideazapteroic acid was converted to the tetrahydro derivative by hydrogenation. Thermally induced cyclization of the 10-carbomethoxy and the 5-NH groups afforded the 5,10-carbonyl analogue. Reduction of the lactam with borane readily yielded the key 5,10-methylene-4-amino-4-deoxy-8,10-dideazatetrahydropteroic acid methyl ester. Saponification of the benzoate ester and coupling with L-glutamate concluded the synthesis. The title compound was a modest inhibitor of growth in folate-dependent bacteria. Streptococcus faecium and Lactobacillus casei, but inhibition of DHFR or TS derived from L. casei was poor. The compound was also a weak inhibitor of DHFR derived from L1210 murine leukemia and was a weak inhibitor of L1210 growth in culture.

Inhibitory action of 10-deazaaminopterins and their polyglutamates on human thymidylate synthase.

The action of 10-deazaaminopterin, its 10-alkyl derivatives, and their polyglutamates against thymidylate synthase (TMPS) from human acute myeloblastic leukemia was examined. Comparison of aminopterin with methotrexate showed that the methylation of the N10-position (methotrexate) increased the inhibitory effect of aminopterin on TMPS. In contrast, alkylation of the 10-position of 10-deazaaminopterin decreased inhibition of TMPS, and the 50% inhibitory concentration values were progressively higher, in the order 10,10-dimethyl-, 10-methyl-, and 10-ethyl-derivatives. The addition of gamma-glutamyl moieties to both 10-deazaaminopterin, and one of its alkylated analogs, 10-ethyl-10-deazaaminopterin, enhanced inhibition. The maximum inhibition was achieved with the addition of three glutamyl moieties to 10-deazaaminopterin and two glutamyl moieties to 10-ethyl-10-deazaaminopterin, respectively. Thus, 10-deazaaminopterin-tetraglutamate was 138-fold and 10-ethyl-10-deazaaminopterin-triglutamate was greater than 51-fold more active than their respective parental compound. The compounds 10-deazaaminopterin and its polyglutamates, 10-methyl- and 10,10-dimethyl-analogs, inhibited TMPS in a noncompetitive fashion with respect to 5,10-methylene-tetrahydropteroylglutamate. Ki values for the monoglutamates were 220 microM, 310 microM, and 225 microM, respectively. In contrast, 10-ethyl-10-deazaaminopterin and its polyglutamates inhibited TMPS in a competitive fashion with a Ki value of 410 microM for the monoglutamate. With 5,10-methylene-tetrahydropteroylpentaglutamate as a substrate, 10-deazaaminopterin and its polyglutamates behaved as mixed type inhibitors, and 10-ethyl-10-deazaaminopterin, monoglutamate and diglutamate, behaved as noncompetitive inhibitors, whereas its pentaglutamate behaved as a mixed-type inhibitor. These results suggest that the addition of gamma-glutamyl moieties to the substrate also caused the change in the mode of inhibitory action of these compounds. These findings also show that both replacement of the N10-position of the 4-aminopteroyl structure with a methylene group and its alkylation caused interesting and unexpected changes in the structure-activity relationships and the mode of action for these 4-aminopteroyl antifolates as inhibitors of TMPS, which may be therapeutically relevant.

Synthesis and biological activity of resolved C-10 diastereomers of 10-methyl- and 10-ethyl-10-deazaminopterin.

Synthesis and evaluation of the antitumor drugs 10-methyl- and 10-ethyl-10-deazaminopterin (15a,b) were previously reported for the diastereomeric mixtures, lacking resolution at the C-10 position. In order to assess biological properties of the individual diastereomers, the C-10 isomers of 4-amino-4-deoxy-10-methyl- and 10-ethyl-10-deazapteroic acids (13a,b) were prepared by total synthesis. Coupling with L-glutamate afforded the appropriate diastereomers of the title compounds. Biochemical, transport, and cell growth inhibitory properties in L1210 cells and folate-dependent bacteria were measured. Differences were generally less than 2-fold between diastereomeric pairs, but a factor of 3 was noted for d,L-15b vs. l,L-15b in inhibition of DHFR from L1210 cells and in cytotoxicity toward L1210 cells. An in vivo comparison of the isomers of 15b with racemic compound against L1210 in mice did not show a significant efficacy difference (ILS) among the compounds. However, d,L-15b showed an acute toxicity about 2.5 times that of l,L-15b.