RESUMEN
Two-dimensional gel electrophoresis of in vitro phosphorylated proteins coprecipitated by CD2 monoclonal antibody (mAb) from Brij58 lysates of resting human T lymphocytes and natural killer (NK) cells resulted in the identification of a novel 29/30-kD disulfide-linked dimer (pp29/30). Comparative two-dimensional analysis of CD2, CD3, CD4, CD5, and CD8 immunoprecipitates revealed that pp29/30 associates with these signaling receptor complexes but not with CD18, CD27, and CD29 in human T lymphocytes. Analysis of CD2 immunoprecipitates prepared from T cell antigen receptor/CD3-modulated T lymphocytes indicated that pp29/30 preferentially associates and comodulates with the human T cell antigen receptor (TCR). Since tyrosine phosphorylated pp29/30 selectively interacts with the Src homology type 2 domains (SHZ) of the protein tyrosine kinases p56lck and p59fyn but not ZAP70 the present data suggest that pp29/30 represents a novel signaling receptor associated phosphoprotein likely involved in the activation of human T lymphocytes and NK cells.
Asunto(s)
Antígenos de Diferenciación de Linfocitos T/análisis , Células Asesinas Naturales/química , Fosfoproteínas/sangre , Receptores Inmunológicos/análisis , Linfocitos T/química , Antígenos de Diferenciación de Linfocitos T/inmunología , Antígenos CD2 , Electroforesis en Gel Bidimensional , Humanos , Células Asesinas Naturales/fisiología , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito , Peso Molecular , Fosfoproteínas/inmunología , Fosfoproteínas/fisiología , Fosforilación , Pruebas de Precipitina , Proteínas Proto-Oncogénicas/análisis , Proteínas Proto-Oncogénicas c-fyn , Complejo Receptor-CD3 del Antígeno de Linfocito T/fisiología , Receptores Inmunológicos/inmunología , Linfocitos T/fisiologíaRESUMEN
Trimetrexate (TMTX), 5,10-dideazatetrahydrofolate (DDATHF), and 10-propargyl-5,8-dideazafolate (PDDF, CB3717) are antifolates whose primary intracellular targets are dihydrofolate reductase, glycinamide ribonucleotide formyltransferase, and thymidylate synthase, respectively. Varying the medium folic acid (PteGlu) concentration over the range of 0.5 to 100 microM increasingly blocks the growth inhibitory effects of the individual antifolates in Manca human lymphoma cells, but increasingly enhances the synergistic interaction of both TMTX + DDATHF and TMTX+ PDDF combinations. Drug interactions were quantitated using the universal response surface approach, which consists of fitting a concentration-effect surface to experimental data with weighted nonlinear regression, enabling the estimation of the synergism parameter, alpha. Estimates for alpha are larger (more intense synergism) for the TMTX + DDATHF combination (alpha = 4.68 +/- 0.66 at 2 microM PteGlu; alpha = 53.6 +/- 3.7 at 40 microM PteGlu) than for the TMTX + PDDF combination (alpha = 0.690 +/- 0.25 at 2 microM PteGlu; alpha = 7.20 +/- 1.8 at 40 microM PteGlu). However, the relative increase brought about by increasing the PteGlu concentration from 2 microM to 40 microM is similar in each instance, 11- and 10-fold, respectively. These experiments suggest that the enhanced cytotoxic interaction brought about by increased PteGlu concentration with the TMTX + DDATHF combination and the TMTX + PDDF combination may share a common mechanism. The dramatic intensity of the synergism between DDATHF and TMTX at 40 microM PteGlu, along with the concept of modulation of antifolate synergism by folates, suggests future in vivo and/or clinical applications of combinations of these (or similar) compounds.
Asunto(s)
Antagonistas del Ácido Fólico/farmacología , Ácido Fólico/análogos & derivados , Ácido Fólico/farmacología , Quinazolinas/farmacología , Tetrahidrofolatos/farmacología , Trimetrexato/farmacología , División Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Ácido Fólico/administración & dosificación , Humanos , Linfoma/patología , Células Tumorales CultivadasRESUMEN
Polyglutamyl derivatives of methotrexate (MTX) and 10-deazaaminopterin (10-DAM) containing a total of one through six glutamate residues (Glu residues) were tested as inhibitors of dihydrofolate reductase (DHFR) derived from sheep, chicken, and beef liver. The ability of dihydropteroylpentaglutamate to antagonize the inhibitory activity of these analogues was also studied. The most striking effects were seen with sheep liver DHFR, where polyglutamylation of MTX causes stepwise decreases in the concentration required for 50% inhibition (IC50) with each additional Glu residue until MTX with a total of six Glu residues has an IC50 value 1/3 that of MTX. With 10-DAM the pattern is more complex. The IC50 values increase with addition of Glu residues until a maximum is reached with 10-DAM having a total of three Glu residues which has a value twice that of 10-DAM. 10-DAM with a total of four Glu residues and 10-DAM with a total of five Glu residues have progressively lower IC50 values, the latter being equipotent with 10-DAM. With dihydropteroylpentaglutamate as substrate instead of dihydrofolate, the IC50 values are increased 2- to 5-fold for both MTX and 10-DAM derivatives. The results obtained with chicken liver and beef liver DHFR are generally similar to those described for the sheep liver enzyme, but the effects of polyglutamylation are less pronounced. The addition of 0.2 M KCl to the assay system reduces the differences in inhibitory potency of the polyglutamyl derivatives with all three enzymes tested. We conclude that polyglutamylation can alter the interaction of folate analogues and dihydrofolate with DHFR.
Asunto(s)
Aminopterina/análogos & derivados , Antagonistas del Ácido Fólico , Ácido Fólico/análogos & derivados , Metotrexato/análogos & derivados , Péptidos/farmacología , Ácido Poliglutámico/farmacología , Aminopterina/farmacología , Animales , Bovinos , Pollos , Ácido Fólico/farmacología , Metotrexato/farmacología , Ácido Poliglutámico/análogos & derivados , Cloruro de Potasio/farmacología , Ovinos , Relación Estructura-ActividadRESUMEN
In order to determine the biochemical basis for the cytotoxicity of homofolates, poly-gamma-glutamyl derivatives of homofolate (HPteGlu) and tetrahydrohomofolate (H4HPteGlu) were synthesized and tested as inhibitors of glycinamide ribonucleotide formyltransferase (GARFT), aminoimidazolecarboxamide ribonucleotide formyltransferase (AICARFT), thymidylate synthase, and serine hydroxymethyltransferase (SHMT) in extracts of Manca human lymphoma and L1210 murine leukemia cells. The most striking inhibitions are that of GARFT by (6R,S)-H4HPteGlu4-6 with IC50 values from 1.3 to 0.3 microM. Both diastereomers, (6R)-H4HPteGlu6 and (6S)-H4HPteGlu6, inhibit GARFT activity. In Manca cell extracts, the (6S) form is more potent than the (6R) form whereas in the murine system the reverse is true. The (6R,S)-H4HPteGlu polyglutamates are weak inhibitors of human AICARFT (IC50, 6-10 microM). Polyglutamates of HPteGlu, however, are more inhibitory to AICARFT, with HPteGlu4-6 having IC50 values close to 2 microM. Polyglutamates of HPteGlu and of H4HPteGlu are weaker inhibitors of thymidylate synthase (IC50, 8 microM for HPteGlu5-6 and greater than 20 microM for H4HPteGlu1-5). Polyglutamates of HPteGlu and of H4HPteGlu are poor inhibitors of SHMT (IC50, greater than 20 microM). Manca cell growth is inhibited 50% by HPteGlu and (6R,S)-5-methyl-H4HPteGlu at 6 and 8 microM, respectively. Both of these effects are reversed by 0.1 mM inosine. Trimetrexate at a subinhibitory concentration, 10 nM, antagonizes growth inhibition by HPteGlu, raising the IC50 from 6 to 64 microM, but enhances inhibition by (6R,S)-5-methyl-H4HPteGlu, lowering the IC50 from 8 to 5 microM. Our results support the view that homofolates become toxic after conversion to H4HPteGlu polyglutamates which block GARFT, a step in purine biosynthesis.
Asunto(s)
Aciltransferasas/antagonistas & inhibidores , Ácido Fólico/análogos & derivados , Transferasas de Hidroximetilo y Formilo , Leucemia L1210/enzimología , Linfoma/enzimología , Ácidos Pteroilpoliglutámicos/farmacología , Animales , Ácido Fólico/metabolismo , Ácido Fólico/farmacología , Humanos , Cinética , Leucemia L1210/patología , Linfoma/patología , Ratones , Conformación Molecular , Fosforribosilaminoimidazolcarboxamida-Formiltransferasa , Fosforribosilglicinamida-Formiltransferasa , Quinazolinas/farmacología , Relación Estructura-Actividad , TrimetrexatoRESUMEN
The phosphonate analogue (6) of 2'-deoxy-5-fluorouridylic acid has been prepared via a Pfitzner--Moffatt oxidation and Witting reaction. This compound was found to inhibit thymidylate synthetase from three sources and to be cytotoxic to H.Ep.-2 cells in culture.
Asunto(s)
Nucleótidos de Desoxiuracil/síntesis química , Fluorodesoxiuridilato/síntesis química , Antineoplásicos/síntesis química , Fluorodesoxiuridilato/análogos & derivados , Timidilato Sintasa/antagonistas & inhibidoresRESUMEN
The synthesis of 5,10-methylene-5-deazatetrahydrofolic acid (2), a stable, rigid analogue of 5,10-methylenetetrahydrofolate (1), is reported as a potential inhibitor of thymidylate synthase. The target compound was obtained by a Fisher-indole type cyclization of the hydrazone 16 from 2-amino-6-hydrazino-4-oxopyrimidine (10) and diethyl N-[4-(3-formyl-1-pyrrolyl)benzoyl]-L-glutamate (15) followed by catalytic reduction of the product 17. Similarly, modification of the Fisher-indole type cyclization of the appropriate hydrazone precursors 11 and 12 afforded the nonclassical analogues 3-amino-7,8,9-trimethyl-2H-pyrrolo[3',4':4,5]pyrido[2,3-d]pyrimidin-1- one (4) and 3-amino-8-benzyl-7,9-dimethyl-2H-pyrrolo[3',4':4,5]pyrido [2,3-d]pyrimidin-1-one (5), respectively. The target compound 2, its aromatic precursor 18, and the nonclassical analogue 4 were evaluated as inhibitors of the growth of Manca human lymphoma cells and also as inhibitors of human dihydrofolate reductase, human thymidylate synthase, glycinamide ribonucleotide formyltransferase, and aminoimidazole carboxamide ribonucleotide formyltransferase. Compound 18 showed weak inhibition of lymphoma cell growth (IC50 = 42 microM) and of AICAR formylTF (IC50 = 17 microM). Compounds 2 and 4 did not inhibit lymphoma cell growth or thymidylate synthase. The inactivity of 2 was attributed to its lack of flexibility leading to its inability to bind to thymidylate synthase.
Asunto(s)
Antineoplásicos/síntesis química , Ácido Fólico/análogos & derivados , Tetrahidrofolatos/síntesis química , Antineoplásicos/farmacología , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Ácido Fólico/síntesis química , Ácido Fólico/farmacología , Humanos , Pirroles/síntesis química , Tetrahidrofolatos/farmacología , Timidilato Sintasa/antagonistas & inhibidores , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
The 10-thia analogs of pteroic acid, folic acid, their esters, and their 4-amino analogs were synthesized through a reaction sequence involving, as a key step, the condensation of 2-amino-3-cyano-5-chloromethylpyrazine with appropriately substituted thiols. The abilities of the products to inhibit the growth of methotrexate (MTX)-sensitive and MTX-resistant microorganisms were investigated as were their abilities to inhibit dihydrofolic acid reductase and thymidylic acid synthetase. Several compounds had high activity.
Asunto(s)
Ácido Fólico/análogos & derivados , Pterinas/síntesis química , Enterococcus faecalis/efectos de los fármacos , Ácido Fólico/síntesis química , Ácido Fólico/farmacología , Antagonistas del Ácido Fólico , Lacticaseibacillus casei/efectos de los fármacos , Lacticaseibacillus casei/enzimología , Espectroscopía de Resonancia Magnética , Pterinas/farmacología , Timidilato Sintasa/antagonistas & inhibidoresRESUMEN
The synthesis of 1',2',3',4',5',6'-hexahydrohomofolic acid (3), a close analogue of homofolic acid (2), has been carried out by replacement of the benzene ring of 2 with a cyclohexane ring. The synthetic methods employed here were based on the Boon-Leigh strategy to obtain products with unambiguous structures. Based on a number of chemical and spectral observations, a tentative cis stereochemistry was assigned to the 1,4-substituents of the cyclohexane ring of both the homopteroate analogue 13 and the target compound 3. We investigated hexahydrohomopteroic acid (13), hexahydrohomofolic acid (3), and their 7,8-dihydro and d,l-5,6,7,8-tetrahydro derivatives for antifolate activity employing several biological test systems. The dihydro and tetrahydro derivatives of both 13 and 3 were active against Streptococcus faecium, whereas they were inactive against Lactobacillus casei. These compounds were neither substrates nor inhibitors of L. casei dihydrofolate reductase or thymidylate synthase.
Asunto(s)
Antagonistas del Ácido Fólico/síntesis química , Ácido Fólico/análogos & derivados , Bioensayo , Ácido Fólico/síntesis química , Ácido Fólico/farmacología , Antagonistas del Ácido Fólico/farmacología , Indicadores y Reactivos , Relación Estructura-Actividad , Tetrahidrofolato Deshidrogenasa/metabolismo , Timidilato Sintasa/metabolismoRESUMEN
Folic acid analogues containing an additional nitrogen atom between the phenyl ring and the carbonyl group of the side chain were synthesized. None of the compounds showed significant inhibitory activity against human lymphoblastic leukemia cells (CCRF-CEM) in culture or against Lactobacillus casei (ATCC 7469) growth. Against L1210 leukemia in mice, the aza homologue of folic acid, 4, and the aspartic acid analogue, 14, showed no increase in life span over control animals. These compounds were more toxic in vivo than the corresponding methotrexate analogues. Compound 4 supported the growth of Streptococcus faecium (ATCC 8043), and its tetrahydro derivative supported the growth of Pediococcus cerevisiae (ATCC 8081). These results strongly suggest that 4 can substitute for folate derivatives as cofactors for serine transhydroxymethylase, thymidylate synthetase, and dihydrofolate reductase.
Asunto(s)
Ácido Fólico/análogos & derivados , Animales , Antineoplásicos/síntesis química , Ácido Fólico/síntesis química , Ácido Fólico/uso terapéutico , Humanos , Técnicas In Vitro , Lacticaseibacillus casei/efectos de los fármacos , Lacticaseibacillus casei/enzimología , Lacticaseibacillus casei/crecimiento & desarrollo , Leucemia L1210/tratamiento farmacológico , Leucemia Linfoide/tratamiento farmacológico , Masculino , Ratones , Pediococcus/efectos de los fármacos , Pediococcus/crecimiento & desarrollo , Streptococcus/efectos de los fármacos , Streptococcus/crecimiento & desarrollo , Timidilato Sintasa/antagonistas & inhibidoresRESUMEN
The diastereoisomers of 5,10-methylene 5,6,7,8-tetrahydropteroyl-D-glutamate were resolved and tested as substrates and inhibitors of Lactobacillus casei thymidylate synthetase. No activity was observed. The compounds were neither growth factors nor inhibitors for Lactobacillus casei, Streptococcus faecium, or Pediococcus cerevisiae. 7,8-Dihydropteroyl-D-glutamate is 50% as active as 7,8-dihydropteroyl-L-glutamate (dihydrofolate) as a substrate for L. casei dihydrofolate reductase.
Asunto(s)
Tetrahidrofolatos , Lacticaseibacillus casei/efectos de los fármacos , Lacticaseibacillus casei/enzimología , Conformación Molecular , Pediococcus/efectos de los fármacos , Estereoisomerismo , Streptococcus/efectos de los fármacos , Relación Estructura-Actividad , Tetrahidrofolatos/metabolismo , Tetrahidrofolatos/farmacología , Timidilato Sintasa/antagonistas & inhibidores , Timidilato Sintasa/metabolismoRESUMEN
The synthesis of 11-thiohomoaminopterin (1), which is a close analogue of 11-thiohomofolic acid (2), has been carried out by modification of the Boon-Leigh procedure. Treatment of 1-chloro-4-[p-(carbomethoxy)thiopenoxy]-2-butanone (5) with sodium azide gave 1-azido-4-[p-(carbomethoxy)thiophenoxy]-2-butanone (6). After protection of the carbonyl group of 6, the product 7 was catalytically hydrogenated to 1-amino-4-[p-(carbomethoxy)thiophenoxy]-2-butanone ketal (3). Reaction of 32 with 6-chloro-2,4-diaminmo-5-nitropyrimidine gave the desired pyrimidine intermediate, which was elaborated to 4-amino-4-deoxy-11-thiohomopteroic acid (20) by standard procedures. Alternately, 1-azido-4-[p-(carbomethoxy)thiophenoxy]-2-butanone ketal (7) was hydrolyzed to the corresponding acid (8) and coupled with diethyl L-glutamate to obtain diethyl N-[p-(1-azido-2-oxo-4-thiobutanoyl)benzoyl]-L-glutamate ketal (10), which was used for the large-scale preparation of 11-thiohomoaminopterin (1). Although 11-thiohomoaminopterin showed antifolate activity against two folate-requiring microorganisms and inhibited Lactobacillus casei dihydrogolate reductase, it did not exhibit any antitumor activity against L-1210 lymphoid leukemia in mice at a maximum dose of 48 mg/kg.
Asunto(s)
Antagonistas del Ácido Fólico/síntesis química , Ácido Fólico/análogos & derivados , Animales , Antineoplásicos/síntesis química , Ácido Fólico/síntesis química , Ácido Fólico/farmacología , Lacticaseibacillus casei/efectos de los fármacos , Lacticaseibacillus casei/crecimiento & desarrollo , Leucemia L1210/tratamiento farmacológico , Ratones , Streptococcus/efectos de los fármacos , Streptococcus/crecimiento & desarrollo , Timidilato Sintasa/antagonistas & inhibidoresRESUMEN
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.
Asunto(s)
Aminopterina/análogos & derivados , Antagonistas del Ácido Fólico/farmacología , Aminopterina/síntesis química , Aminopterina/farmacología , Animales , División Celular/efectos de los fármacos , Fenómenos Químicos , Química , Lacticaseibacillus casei/efectos de los fármacos , Lacticaseibacillus casei/crecimiento & desarrollo , Leucemia L1210/enzimología , Leucemia L1210/patología , Metotrexato/farmacología , Ratones , Streptococcus/efectos de los fármacos , Streptococcus/crecimiento & desarrollo , Timidilato Sintasa/antagonistas & inhibidoresRESUMEN
N10-Propargylfolic acid (2), which is the closest pteridine analogue of the thymidylate synthase inhibitor N10-propargyl-5,8-dideazafolic acid (PDDF), was synthesized starting from diethyl [p-(N-propargylamino)benzoyl]-L-glutamate (5) and N-(3-bromo-2-oxopropyl)phthalimide (8). The 7,8-dihydro derivative of propargylfolic acid served as a synthetic substrate of Lactobacillus casei dihydrofolate reductase. Propargylfolic acid and its reduced derivatives were weak inhibitors of L. casei thymidylate synthase compared to PDDF. All derivatives of propargylfolate were active against the growth of Streptococcus faecium, but with the exception of 7,8-dihydropropargylfolic acid, all were inactive against L. casei. Although less potent than PDDF, marked inhibition of thymidylate synthase by 2 was observed in permeabilized L1210 cells.
Asunto(s)
Ácido Fólico/análogos & derivados , Ácido Fólico/síntesis química , Animales , Ácido Fólico/farmacología , Indicadores y Reactivos , Lacticaseibacillus casei/enzimología , Leucemia L1210/enzimología , Ratones , Pruebas de Sensibilidad Microbiana , Streptococcus/efectos de los fármacos , Relación Estructura-Actividad , Especificidad por Sustrato , Tetrahidrofolato Deshidrogenasa/metabolismo , Timidilato Sintasa/antagonistas & inhibidoresRESUMEN
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.
Asunto(s)
Aminopterina/análogos & derivados , Antagonistas del Ácido Fólico , Aminopterina/síntesis química , Aminopterina/farmacología , Aminopterina/uso terapéutico , Animales , Fenómenos Químicos , Química , Humanos , Leucemia/enzimología , Leucemia L1210/tratamiento farmacológico , Leucemia L1210/enzimología , Metotrexato/farmacología , Estructura Molecular , Células Tumorales CultivadasRESUMEN
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.
Asunto(s)
Aciltransferasas/antagonistas & inhibidores , Antagonistas del Ácido Fólico/síntesis química , Transferasas de Hidroximetilo y Formilo , Metotrexato/análogos & derivados , Tetrahidrofolatos , Animales , Antimetabolitos Antineoplásicos/síntesis química , Antimetabolitos Antineoplásicos/farmacología , División Celular/efectos de los fármacos , Leucemia L1210/tratamiento farmacológico , Metotrexato/síntesis química , Ratones , Fosforribosilaminoimidazolcarboxamida-Formiltransferasa , Fosforribosilglicinamida-Formiltransferasa , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
The chemical synthesis of a series of poly-gamma-glutamyl metabolites of the experimental anticancer drugs 10-deazaaminopterin (10-DAAM) and 10-ethyl-10-deazaaminopterin (10-EDAAM) has been carried out by the solid-phase procedure. The synthetic products were identical with the poly-gamma-glutamyl metabolites of radiolabeled 10-DAAM and 10-EDAAM produced by normal mouse tissues with regard to elution volume from [(diethylamino)ethyl]cellulose columns and susceptibility to hydrolysis by human plasma folylpolyglutamate hydrolase. Poly-gamma-glutamyl metabolites with a glutamate chain length of up to four glutamate residues were detected in the tissues. The antifolate activity was evaluated with methotrexate (MTX) sensitive and MTX-resistant strains of Lactobacillus casei and Streptococcus faecium. In general, inhibitory potency decreases with increasing Glu chain length. However there are two exceptions. Addition of one Glu residue to 10-DAAM enhances its potency for MTX-resistant L. casei and addition of one Glu residue to 10-EDAAM enhances its potency for the MTX-sensitive L. casei. As shown earlier for MTX polyglutamates, polyglutamylation greatly enhances the inhibitory potency of 10-DAAM and 10-EDAAM for L. casei thymidylate synthase. MTX polyglutamates are 15-30 times more inhibitory than the corresponding 10-DAAM derivatives and 30-60 times more inhibitory than the corresponding 10-EDAAM derivatives. Polyglutamylation of 10-DAAM had little influence on its ability to inhibit L. casei dihydrofolate reductase; however, with 10-EDAAM, addition of one or two Glu residues enhanced its inhibitory potency 2.3-fold.
Asunto(s)
Aminopterina/análogos & derivados , Antibacterianos/síntesis química , Antagonistas del Ácido Fólico/farmacología , Péptidos/síntesis química , Ácido Poliglutámico/síntesis química , Aminopterina/farmacología , Animales , Femenino , Humanos , Lacticaseibacillus casei/efectos de los fármacos , Metotrexato/farmacología , Ratones , Ratones Endogámicos , Pruebas de Sensibilidad Microbiana , Ácido Poliglutámico/análogos & derivados , Ácido Poliglutámico/farmacología , Streptococcus/efectos de los fármacos , Relación Estructura-Actividad , Especificidad por Sustrato , gamma-Glutamil Hidrolasa/sangreRESUMEN
Representative examples of folate and antifolate poly-gamma-glutamyl metabolites were synthesized via the [(9-fluorenylmethoxy)oxy]carbonyl (Fmoc) chemistry using the KH polyamide resin. Polyglutamate yields were consistently better in all cases compared to the previous Merrifield method, and the crude products were obtained in greater than 85% purity. The symmetrical anhydride (7) derived from alpha-tert-butyl N-Fmoc-L-glutamate (6) was used for the initial coupling of the first glutamate residue to the KH resin and also for subsequent chain elongation. The alpha-tert-butyl protective groups were not labile under the conditions used for the cleavage of the finished peptide from the resin. A series of poly-gamma-glutamyl metabolites of methotrexate (MTX) with a chain length ranging from two to five glutamyl residues were synthesized and coupled with poly(L-lysine) having an average molecular weight of 27,000 and 52,000. Each conjugate was tested for its ability to inhibit the growth of wild type (H35) and MTX transport resistant (H35R) strains of hepatoma cells in culture, the latter having a 100-fold reduced sensitivity to MTX. 4-Amino-4-deoxy-N10-methylpteroylglutamyl-gamma-glutamylpoly (L-lysine) conjugate [MTX(G2)-poly-L-Lys-52000] and MTX(G4)-poly-L-Lys-52000 were among the most active (I50 = 8.0 and 10 nM against H35 cells) MTX-polylysines synthesized to date, and they were somewhat more inhibitory to the transport resistant cells. MTX(G5)-poly-L-Lys-52000 was approximately 1000 times more effective than MTX(G5)-poly-D-Lys-52000 in inhibiting the growth of H35R hepatoma cells in culture, indicating that internal cleavage of the gamma-glutamate chain of the conjugate with subsequent release of MTX or shorter chain polyglutamates of MTX is unlikely to be an important determinant of MTX-polyglutamate polylysine cytotoxicity. The results indicate that MTX-polyglutamate poly(L-lysine) conjugates are taken up by the cells independently of MTX and probably via endocytosis.
Asunto(s)
Antimetabolitos Antineoplásicos/síntesis química , Antagonistas del Ácido Fólico/síntesis química , Metotrexato/análogos & derivados , Antimetabolitos Antineoplásicos/farmacología , Carcinoma Hepatocelular , Fenómenos Químicos , Química , Química Física , Resistencia a Medicamentos , Espacio Extracelular/metabolismo , Antagonistas del Ácido Fólico/farmacología , Humanos , Técnicas In Vitro , Neoplasias Hepáticas , Ácido Poliglutámico , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
The synthesis of 5,11-methylene-5-deazatetrahydrohomofolate (5), a stable, semirigid mimic of 5,10-methylenetetrahydrofolate (4) is reported as a potential inhibitor of thymidylate synthases (TS). The key intermediate 3-amino-1-oxo-tetrahydropyrimido[4,5-c] [2,6]naphthyridine (6) was obtained by the regiospecific cyclocondensation of 2,4,6-triaminopyrimidine with ethyl 1-benzyl-3-oxo-4-piperidinecarboxylate followed by halogenation (of the resulting lactam 9) and catalytic hydrogenolysis. Selective reduction of 6 followed by arylation with tert-butyl p-fluorobenzoate, saponification, and coupling with diethyl L-glutamate followed by saponification afforded the target compound 5. The title compound was tested as an inhibitor of the growth of Manca human lymphoma cells and also as an inhibitor of TS from Manca cells and Lactobacillus casei and was found to be inactive. In addition, compound 5 also failed to inhibit glycinamide ribonucleotide formyltransferase from L. casei and from Manca cells.
Asunto(s)
Tetrahidrofolatos/síntesis química , Timidilato Sintasa/antagonistas & inhibidores , Línea Celular , Fenómenos Químicos , Química , Humanos , Linfoma/tratamiento farmacológico , Estereoisomerismo , Tetrahidrofolatos/farmacologíaRESUMEN
Two analogues of dihydrofolic acid possessing a 7,8-dihydro-8-oxapterin ring system have been synthesized and evaluated for their antifolate activities. These compounds, N-[(2-amino-4-hydroxy-7,8-dihydro-8-oxa-6-pteridinyl)benzoyl]-L-glutamic acid (3) and N-[[(2-amino-4-hydroxy-7,8-dihydro-8-oxa-6-pteridinyl) methyl]benzoyl]-L-glutamic acid (4), were synthesized by reacting the appropriately substituted alpha-halo ketones with 2,5-diamino-4,6-dihydroxypyrimidine (2). Elaboration of p-carbomethoxybenzaldehyde (5) to p-carbomethoxyphenacyl bromide (7) was accomplished by its oxidation with Jones reagent and the successive treatment of the oxidation product with SOCl2, CH2N2, and HBr. Commercially available p-vinylbenzoic acid (11) was converted to its glutamate conjugate 12 and was further converted to the bromo ketone, diethyl N-[p-(1-bromo-2-oxopropyl)benzoyl]-L-glutamate (17), by a series of reactions involving epoxidation, oxirane ring opening with HBr, Jones oxidation, Zn/HOAc reduction, and successive treatment of the reduction product 16 with SOCl2, CH2N2, and HBr. These bromo ketones, 7 and 17, upon reaction with pyrimidine 2, gave the diethyl esters of the target compounds, which were hydrolyzed to 3 and 4 with NaOH. Compound 4 underwent an interesting acid-catalyzed isomerization where the double bond of 4 was shifted from the 5,6-position to the 6,9-position to give the isomer 19. Both compounds 3 and 4 were inactive against Lactobacillus casei (ATCC 7469) and did not serve as synthetic substrates of L. casei dihydrofolate reductase. Compound 4 showed activity against Streptococcus faecium (ATCC 8043), but 3 was inactive against this organism.
Asunto(s)
Ácido Fólico/análogos & derivados , Enterococcus faecalis/efectos de los fármacos , Ácido Fólico/síntesis química , Ácido Fólico/farmacología , Lacticaseibacillus casei/efectos de los fármacos , Espectroscopía de Resonancia Magnética , Metotrexato/farmacologíaRESUMEN
A close analogue of the antileukemic agent 5,8-dideaza-N10 propargylfolic acid (2) was synthesized by replacing the propargyl moiety of 2 with a cyanomethyl group. This compound, N10-(cyanomethyl)-5,8-dideazafolic acid (3), was evaluated for its antifolate and antitumor activities in several biological test systems. Alkylation of diethyl N-(4-aminobenzoyl)-L-glutamate with bromoacetonitrile gave diethyl N-[4-[(cyanomethyl)amino]benzoyl]-L-glutamate (7). Reaction of 7 with 2 amino-6-(bromomethyl)-4-hydroxyquinazoline (9) in dimethylacetamide gave the corresponding diethyl ester 11, which was hydrolyzed to the target compound 3. The known antileukemic agent 2 was also synthesized for comparative studies by employing a modified procedure, which resulted in a better yield of this product. Both compounds 2 and 3 were evaluated for their antifolate activities by using two folate-requiring microorganisms, Streptococcus faecium and Lactobacillus casei. They were further evaluated as inhibitors of thymidylate synthase and dihydrofolate reductase derived from the above organisms, as well as for their antitumor activity by using selected tumor cells in culture. Compound 2 was found to be as equally potent as methotrexate (MTX) against S. faecium, and it was an excellent inhibitor of L. casei thymidylate synthase. The cyanomethyl analogue 3 was less active than 2 in all the test systems, except the inhibition of dihydrofolate reductase.