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.

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.

Synthesis and antifolate properties of 10-alkyl-8,10-dideazaminopterins.

The synthesis of 10-alkyl analogues of the potent antitumor agent 8,10-dideazaminopterin is described. Alkylation of appropriate alpha-alkyl homoterephthalate esters with 2,4-diamino-6-(bromomethyl)-8-deazapteridine afforded 10-alkyl-10-carboxy-4-amino-4-deoxy-8,10-dideazapteroic acid diesters. Ester cleavage and decarboxylation at C-10 were accomplished by heating with sodium cyanide in Me2SO at 170-180 degrees C to afford the 2,4-diamino-10-alkyl-8,10-dideazapteroic acids. The acids were coupled with diethyl glutamate, followed by saponification, to give the 10-alkyl-8,10-dideazaminopterins. The compounds were potent inhibitors of growth in folate-dependent bacteria, Streptococcus faecium and Lactobacillus casei. The 10-methyl and 10-ethyl analogues gave the highest percent increases in life span for mice infected with L1210 leukemia with ILS values of +203 and +235%, respectively.

Synthesis and biological activity of an amino analogue of a tripeptide inhibitor of angiotensin-converting enzyme.

An amino analogue of N-benzoyl-phenylalanyl-glycyl-proline, a tripeptide inhibitor of angiotensin-converting enzyme, was synthesized. The analogue (III) has the phenylalanyl-glycine amide linkage of N-benzoyl-phenylalanyl-glycyl-proline reduced to a methylene amine. Compound III was tested as an inhibitor of porcine plasma angiotensin-converting enzyme and has an I50 of 620 microM compared with an I50 of 9.6 microM for its parent tripeptide. These results are explained in terms of a proposed model of the converting-enzyme active site.