RESUMO
In an effort to optimize the structural requirements for combined cytostatic and cytotoxic effects in single agents, a series of 5-(arylthio)-9H-pyrimido[4,5-b]indole-2,4-diamines 3-7 were synthesized and evaluated as inhibitors of receptor tyrosine kinases (RTKs) as well as thymidylate synthase (TS). The synthesis of these compounds involved the nucleophilic displacement of the common intermediate 5-bromo/5-chloro-9H-pyrimido[4,5-b]indole-2,4-diamine with appropriate aryl thiols. A novel four step synthetic scheme to the common intermediate was developed which is more efficient relative to the previously reported six-step sequence. Biological evaluation of these compounds indicated dual activity in RTKs and human TS (hTS). In the VEGFR-2 assay, compound 5 was equipotent to the standard compound semaxanib and was better than standard TS inhibitor pemetrexed, in the hTS assay. Compounds 3, 6 and 7 were nanomolar inhibitors of hTS and were several fold better than pemetrexed.
Assuntos
Antineoplásicos/farmacologia , Compostos Heterocíclicos com 3 Anéis/farmacologia , Indóis/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Timidilato Sintase/antagonistas & inibidores , Inibidores da Angiogênese/síntese química , Inibidores da Angiogênese/farmacologia , Animais , Antineoplásicos/síntese química , Linhagem Celular Tumoral , Cisplatino/farmacologia , Antagonistas do Ácido Fólico/síntese química , Antagonistas do Ácido Fólico/farmacologia , Compostos Heterocíclicos com 3 Anéis/síntese química , Humanos , Indóis/síntese química , Camundongos , Pemetrexede/farmacologia , Inibidores de Proteínas Quinases/síntese química , Pirimidinas/síntese química , Pirróis/farmacologia , Receptor beta de Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidores , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
We designed, synthesized and evaluated 13 novel tricyclic indeno[2,1-d]pyrimidines as RTK inhibitors. These analogues were synthesized via a Dieckmann condensation of 1,2-phenylenediacetonitrile followed by cyclocondensation with guanidine carbonate to afford the 2-amino-3,9-dihydro-indeno[2,1-d]pyrimidin-4-one. Sulfonation of the 4-position followed by displacement with appropriately substituted anilines afforded the target compounds. These compounds were potent inhibitors of platelet-derived growth factor receptor ß (PDGFRß) and inhibited angiogenesis in the chicken embryo chorioallantonic membrane (CAM) assay compared to standards. In addition, compound 7 had a two digit nanomolar GI(50) against nine tumor cell lines, a submicromolar GI(50) against 29 of other tumor cell lines in the preclinical NCI 60 tumor cell line panel. Compound 7 also demonstrated significant in vivo inhibition of tumor growth and angiogenesis in a B16-F10 syngeneic mouse melanoma model.
Assuntos
Inibidores da Angiogênese/química , Antineoplásicos/química , Indenos/química , Pirimidinas/química , Inibidores da Angiogênese/uso terapêutico , Inibidores da Angiogênese/toxicidade , Animais , Antineoplásicos/uso terapêutico , Antineoplásicos/toxicidade , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Embrião de Galinha , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Indenos/uso terapêutico , Indenos/toxicidade , Melanoma Experimental/tratamento farmacológico , Camundongos , Pirimidinas/uso terapêutico , Pirimidinas/toxicidade , Receptor beta de Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidores , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismoRESUMO
Classical antifolates (4-7) with a tricyclic benzo[4,5]thieno[2,3-d]pyrimidine scaffold and a flexible and rigid benzoylglutamate were synthesized as dual thymidylate synthase (TS) and dihydrofolate reductase (DHFR) inhibitors. Oxidative aromatization of ethyl 2-amino-4-methyl-4,5,6,7-tetrahydro-1-benzothiophene-3-carboxylate (±)-9 to ethyl 2-amino-4-methyl-1-benzothiophene-3-carboxylate 10 with 10% Pd/C was a key synthetic step. Compounds with 2-CH3 substituents inhibited human (h) TS (IC50 =0.26-0.8 µM), but not hDHFR. Substitution of the 2-CH3 with a 2-NH2 increases hTS inhibition by more than 10-fold and also affords excellent hDHFR inhibition (IC50 = 0.09-0.1 µM). This study shows that the tricyclic benzo[4,5]thieno[2,3-d]pyrimidine scaffold is highly conducive to single hTS or dual hTS-hDHFR inhibition depending on the 2-position substituents. The X-ray crystal structures of 6 and 7 with hDHFR reveal, for the first time, that tricyclics 6 and 7 bind with the benzo[4,5]thieno[2,3-d]pyrimidine ring in the folate binding mode with the thieno S mimicking the 4-amino of methotrexate.
Assuntos
Inibidores Enzimáticos/síntese química , Pirimidinas/química , Tetra-Hidrofolato Desidrogenase/química , Timidilato Sintase/antagonistas & inibidores , Cristalografia por Raios X , Desenho de Fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Antagonistas do Ácido Fólico/química , Antagonistas do Ácido Fólico/farmacologia , Humanos , Conformação Molecular , Pirimidinas/síntese química , Pirimidinas/farmacologia , Tetra-Hidrofolato Desidrogenase/metabolismo , Timidilato Sintase/metabolismoRESUMO
A novel classical antifolate N-{4-[(2,4-diamino-5-methyl-furo[2,3-d]pyrimidin-6-yl)thio]-benzoyl}-l-glutamic acid 5 and 11 nonclassical antifolates 6-16 were designed, synthesized, and evaluated as inhibitors of dihydrofolate reductase (DHFR) and thymidylate synthase (TS). The nonclassical compounds 6-16 were synthesized from 20 via oxidative addition of substituted thiophenols using iodine. Peptide coupling of the intermediate acid 21 followed by saponification gave the classical analog 5. Compound 5 is the first example, to our knowledge, of a 2,4-diamino furo[2,3-d]pyrimidine classical antifolate that has inhibitory activity against both human DHFR and human TS. The classical analog 5 was a nanomolar inhibitor and remarkably selective inhibitor of Pneumocystis carinii DHFR and Mycobacterium avium DHFR at 263-fold and 2107-fold, respectively, compared to mammalian DHFR. The nonclassical analogs 6-16 were moderately potent against pathogen DHFR or TS. This study shows that the furo[2,3-d]pyrimidine scaffold is conducive to dual human DHFR-TS inhibitory activity and to high potency and selectivity for pathogen DHFR.
Assuntos
Inibidores Enzimáticos/farmacologia , Antagonistas do Ácido Fólico/farmacologia , Ácido Glutâmico/análogos & derivados , Pirimidinas/farmacologia , Tetra-Hidrofolato Desidrogenase/metabolismo , Timidilato Sintase/antagonistas & inibidores , Animais , Cristalografia por Raios X , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Escherichia coli/enzimologia , Antagonistas do Ácido Fólico/síntese química , Antagonistas do Ácido Fólico/química , Ácido Glutâmico/síntese química , Ácido Glutâmico/química , Ácido Glutâmico/farmacologia , Humanos , Fígado/enzimologia , Modelos Moleculares , Estrutura Molecular , Mycobacterium avium/enzimologia , Oxirredução , Pneumocystis carinii/enzimologia , Pirimidinas/síntese química , Pirimidinas/química , Ratos , Estereoisomerismo , Relação Estrutura-Atividade , Toxoplasma/enzimologiaRESUMO
We designed and synthesized a classical antifolate N-{4-[(2-amino-6-methyl-4-oxo-3,4-dihydro-5 H-pyrrolo[3,2- d]pyrimidin-5-yl)methyl]benzoyl}- l-glutamic acid 4 and 11 nonclassical analogues 5- 15 as potential dual thymidylate synthase (TS) and dihydrofolate reductase (DHFR) inhibitors. The key intermediate in the synthesis was N-(4-chloro-6-methyl-5 H-pyrrolo[3,2- d]pyrimidin-2-yl)-2,2-dimethylpropanamide, 29, to which various 5-benzyl substituents were attached. For the classical analogue 4, the ester obtained from the N-benzylation reaction was deprotected and coupled with diethyl l-glutamate followed by saponification. Compound 4 was a potent dual inhibitor of human TS (IC 50 = 46 nM, about 206-fold more potent than pemetrexed) and DHFR (IC 50 = 120 nM, about 55-fold more potent than pemetrexed). The nonclassical analogues were marginal inhibitors of human TS, but four analogues showed potent T. gondii DHFR inhibition along with >100-fold selectivity compared to human DHFR.
Assuntos
Antagonistas do Ácido Fólico/síntese química , Pirimidinas/síntese química , Pirróis/síntese química , Tetra-Hidrofolato Desidrogenase/química , Timidilato Sintase/antagonistas & inibidores , Timidilato Sintase/química , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Desenho de Fármacos , Escherichia coli/enzimologia , Antagonistas do Ácido Fólico/química , Humanos , Modelos Moleculares , Pirimidinas/química , Pirróis/química , Relação Estrutura-Atividade , Toxoplasma/enzimologiaRESUMO
The classical antifolate N-{4-[(2,4-diamino-5-ethyl-7H-pyrrolo[2,3-d]pyrimidin-6-yl)sulfanyl]benzoyl}-l-glutamic acid (2) and 15 nonclassical analogues (3-17) were synthesized as potential dihydrofolate reductase (DHFR) inhibitors and as antitumor agents. 5-Ethyl-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamine (20) served as the key intermediate to which various aryl thiols and a heteroaryl thiol were appended at the 6-position via an oxidative addition reaction. The classical analogue 2 was synthesized by coupling the benzoic acid derivative 18 with diethyl l-glutamate followed by saponification. The classical compound 2 was an excellent inhibitor of human DHFR (IC50 = 66 nM) as well as a two digit nanomolar (<100 nM) inhibitor of the growth of several tumor cells in culture. Some of the nonclassical analogues were potent and selective inhibitors of DHFR from two pathogens (Toxoplasma gondii and Mycobacterium avium) that cause opportunistic infections in patients with compromised immune systems.
Assuntos
Antineoplásicos/síntese química , Antagonistas do Ácido Fólico/síntese química , Pirimidinas/síntese química , Pirróis/síntese química , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Desenho de Fármacos , Resistencia a Medicamentos Antineoplásicos , Ensaios de Seleção de Medicamentos Antitumorais , Escherichia coli/enzimologia , Antagonistas do Ácido Fólico/química , Antagonistas do Ácido Fólico/farmacologia , Humanos , Modelos Moleculares , Mycobacterium avium/enzimologia , Pneumocystis carinii/enzimologia , Pirimidinas/química , Pirimidinas/farmacologia , Pirróis/química , Pirróis/farmacologia , Ratos , Relação Estrutura-Atividade , Tetra-Hidrofolato Desidrogenase/química , Toxoplasma/enzimologiaRESUMO
Two boron-containing, ortho-icosahedral carborane lipophilic antifolates were synthesized, and the crystal structures of their ternary complexes with human dihydrofolate reductase (DHFR) and dihydronicotinamide adenine dinucleotide phosphate were determined. The compounds were screened for activity against DHFR from six sources (human, rat liver, Pneumocystis carinii, Toxoplasma gondii, Mycobacterium avium, and Lactobacillus casei) and showed good to modest activity against these enzymes. The compounds were also tested for antibacterial activity against L. casei, M. tuberculosis H37Ra, and three M. avium strains and for cytotoxic activity against seven different human tumor cell lines. Antibacterial and cytotoxic activity was modest, with one sample, the closo-carborane 4, showing about 10-fold greater activity. The less toxic nido-carborane 2 was also tested as a candidate for boron neutron capture therapy, but showed poor tumor retention and low selectivity ratios for boron distribution in tumor tissue versus normal tissue.
Assuntos
Boro/química , Antagonistas do Ácido Fólico/síntese química , Antagonistas do Ácido Fólico/farmacologia , Animais , Terapia por Captura de Nêutron de Boro , Linhagem Celular Tumoral , Cristalografia por Raios X , Antagonistas do Ácido Fólico/química , Humanos , Testes de Sensibilidade Microbiana , Modelos Moleculares , Estrutura Molecular , Ratos , Tetra-Hidrofolato Desidrogenase/efeitos dos fármacosRESUMO
We designed and synthesized a classical analogue N-[4-[(2-amino-6-ethyl-3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d]pyrimidin-5-yl)thio]benzoyl]-L-glutamic acid (4) and thirteen nonclassical analogues 5-17 as potential dual thymidylate synthase (TS) and dihydrofolate reductase (DHFR) inhibitors and as antitumor agents. The key intermediate in their synthesis was 2-amino-6-ethyl-3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d]pyrimidine, 22, to which various aryl thiols were conveniently attached at the 5-position via an oxidative addition reaction using iodine. For the classical analogue 4, the ester obtained from the reaction was deprotected and coupled with diethyl L-glutamate followed by saponification. Compound 4 was a potent dual inhibitor of human TS (IC(50) = 90 nM) and human DHFR (IC(50) = 420 nM). Compound 4 was not a substrate for human FPGS. Metabolite protection studies established TS as its principal target. Most of the nonclassical analogues were only inhibitors of human TS with IC(50) values of 0.23-26 microM.
Assuntos
Antineoplásicos/síntese química , Antagonistas do Ácido Fólico/síntese química , Glutamatos/síntese química , Pirimidinas/síntese química , Pirimidinonas/síntese química , Pirróis/síntese química , Tetra-Hidrofolato Desidrogenase/química , Timidilato Sintase/antagonistas & inibidores , Timidilato Sintase/química , Antineoplásicos/química , Antineoplásicos/farmacologia , Sítios de Ligação , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Escherichia coli/enzimologia , Antagonistas do Ácido Fólico/química , Antagonistas do Ácido Fólico/farmacologia , Glutamatos/química , Glutamatos/farmacologia , Humanos , Modelos Moleculares , Pirimidinas/química , Pirimidinas/farmacologia , Pirimidinonas/química , Pirimidinonas/farmacologia , Pirróis/química , Pirróis/farmacologia , Relação Estrutura-Atividade , TermodinâmicaRESUMO
We report, for the first time, the biological activities of four-carbon-atom bridged classical antifolates on dihydrofolate reductase (DHFR), thymidylate synthase (TS), and folylpolyglutamate synthetase (FPGS) as well as antitumor activity. Extension of the bridge homologation studies of classical two-carbon bridged antifolates, a 5-substituted 2,4-diaminofuro[2,3-d]pyrimidine (1) and a 6-subsituted 2-amino-4-oxopyrrolo[2,3-d]pyrimidine (2), afforded two four-carbon bridged antifolates, analogues 5 and 6, with enhanced FPGS substrate activity and inhibitory activity against tumor cells in culture (EC(50) < or = 10(-7) M) compared with the two-carbon bridged analogues. These results support our original hypothesis that the distance and orientation of the side chain p-aminobenzoyl-L-glutamate moiety with respect to the pyrimidine ring are a crucial determinant of biological activity. In addition, this study demonstrates that, for classical antifolates that are substrates for FPGS, poor inhibitory activity against isolated target enzymes is not necessarily a predictor of a lack of antitumor activity.
Assuntos
Antineoplásicos/síntese química , Antagonistas do Ácido Fólico/síntese química , Furanos/síntese química , Ácido Glutâmico/análogos & derivados , Pirimidinas/síntese química , Pirimidinonas/síntese química , Pirróis/síntese química , Antineoplásicos/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Antagonistas do Ácido Fólico/química , Antagonistas do Ácido Fólico/farmacologia , Furanos/química , Furanos/farmacologia , Ácido Glutâmico/síntese química , Ácido Glutâmico/química , Ácido Glutâmico/farmacologia , Humanos , Peptídeo Sintases/antagonistas & inibidores , Peptídeo Sintases/química , Pirimidinas/química , Pirimidinas/farmacologia , Pirimidinonas/química , Pirimidinonas/farmacologia , Pirróis/química , Pirróis/farmacologia , Relação Estrutura-Atividade , Tetra-Hidrofolato Desidrogenase/química , Tetra-Hidrofolato Desidrogenase/metabolismo , Timidilato Sintase/antagonistas & inibidores , Timidilato Sintase/químicaRESUMO
Two novel classical antifolates N-{4-[(2,4-diamino-5-methyl-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)thio]benzoyl}-L-glutamic acid 3 and N-{4-[(2-amino-4-oxo-5-methyl-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)thio]benzoyl}-L-glutamic acid 4 were designed, synthesized, and evaluated as antitumor agents. Compounds 3 and 4 were obtained from 2,4-diamino-5-methylpyrrolo[2,3-d]pyrimidine 7 and 2-amino-4-oxo-5-methylpyrrolo[2,3-d]pyrimidine 12, respectively, in a concise three-step sequence. Compound 3 is the first example, to our knowledge, of a 2,4-diamino classical antifolate that has potent inhibitory activity against both human dihydrofolate reductase (DHFR) and human thymidylate synthase (TS). Compound 4 was a dual DHFR-TS inhibitor against the bifunctional enzyme derived from Toxoplasma gondii (tg). Further evaluation of the mechanism of action of 3 implicated DHFR as its primary intracellular target. Both 3 and 4 were folylpolyglutamate synthetase (FPGS) substrates. Compound 3 also inhibited the growth of several human tumor cell lines in culture with GI50 < 10(-8) M. This study shows that the pyrrolo[2,3-d]pyrimidine scaffold is conducive to dual DHFR-TS and tumor inhibitory activity, and the potency is determined by the 4-position substituent.
Assuntos
Antineoplásicos/síntese química , Antagonistas do Ácido Fólico/síntese química , Glutamatos/síntese química , Pirimidinas/síntese química , Tetra-Hidrofolato Desidrogenase/metabolismo , Timidilato Sintase/antagonistas & inibidores , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Escherichia coli/enzimologia , Antagonistas do Ácido Fólico/química , Antagonistas do Ácido Fólico/farmacologia , Glutamatos/química , Glutamatos/farmacologia , Humanos , Modelos Moleculares , Pirimidinas/química , Pirimidinas/farmacologia , Relação Estrutura-Atividade , Timidilato Sintase/química , Timidilato Sintase/metabolismo , Toxoplasma/enzimologiaRESUMO
N-[4-[1-Ethyl-2-(2,4-diaminofuro[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-L-glutamic acid 3 was designed and synthesized to investigate the effect of homologation of a C9-methyl to an ethyl on dihydrofolate reductase (DHFR) inhibition and on antitumor activity. Compound 3 was obtained via a concise seven step synthesis starting from palladium-catalyzed carbonylation of 4-propionylphenol, followed by a Wittig reaction with 2,4-diamino-5-(chloromethyl)furo[2,3-d]pyrimidine (6), catalytic hydrogenation, hydrolysis, and standard peptide coupling with diethyl L-glutamate. The biological results indicated that extending the C9-methyl group to an ethyl on the C8-C9 bridge region (analogue 3) doubled the inhibitory potency against recombinant human (rh) DHFR (IC(50) = 0.21 microM) as compared to the C9-methyl analogue 1 and was 4-fold more potent than the C9-H analogue 2. As compared to 1, compound 3 demonstrated increased growth inhibitory potency against several human tumor cell lines in culture with GI(50) values < 1.0 x 10(-8) M. Compound 3 was also a weak inhibitor of rh thymidylate synthase. Compounds 1 and 3 were efficient substrates of human folylpolyglutamate synthetase (FPGS). Further evaluation of the cytotoxicity of 3 in methotrexate-resistant CCRF-CEM cell sublines and metabolite protection studies implicated DHFR as the primary intracelluar target. Thus, alkylation of the C9 position in the C8-C9 bridge of the classical 5-substituted 2,4-diaminofuro[2,3-d]pyrimidine is highly conducive to DHFR and tumor inhibitory activity as well as FPGS substrate efficiency.
Assuntos
Antineoplásicos/síntese química , Antagonistas do Ácido Fólico/síntese química , Ácido Glutâmico/análogos & derivados , Ácido Glutâmico/síntese química , Pirimidinas/síntese química , Antineoplásicos/química , Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Antagonistas do Ácido Fólico/química , Antagonistas do Ácido Fólico/farmacologia , Ácido Glutâmico/química , Ácido Glutâmico/farmacologia , Humanos , Espectroscopia de Ressonância Magnética , Pirimidinas/química , Pirimidinas/farmacologia , Relação Estrutura-Atividade , Timidilato Sintase/antagonistas & inibidores , Células Tumorais CultivadasRESUMO
Seven novel 2,4-diamino-5-deaza-6,7,8,9-tetrahydropyrido[3,4-g]pteridine derivatives 3-9 with different benzyl and a benzoyl substitution at the N7 position were designed and synthesized, as classical and nonclassical, partially restricted, linear tricyclic 5-deaza antifolates. The purpose was to investigate the effect of conformational restriction of the C6-C9 (tau(1)) and C9-N10 (tau(2)) bonds via an ethyl bridge from the N10 to the C7 position of 5-deaza methotrexate (MTX) on the inhibitory potency against dihydrofolate reductase (DHFR) from different sources and on antitumor activity. The synthetic methodology for most of the target compounds was a concise five-step total synthesis to construct the tricyclic nucleus, 2,4-diamino-5-deaza-7H-6,7,8,9-tetrahydropyrido[3,4-g]pteridine (23), followed by regioselective alkylation of the N7 nitrogen. Biological results indicated that this partial conformational modification for the classical analogue N-[4-[(2,4-diamino-5-deaza-6,7,8,9-tetrahydropyrido[3,4-g]pteridin-7-yl)methyl]benzoyl]-L-glutamic acid 3 was detrimental to DHFR inhibitory activity as well as to antitumor activity compared to MTX or 5-deaza MTX. However, the classical analogue 3 was a better substrate for folypolyglutamate synthetase (FPGS) than MTX. These results show that a classical 5-deaza folate partially restricted via a bridge between the N10 and C7 positions retains FPGS substrate activity and that the antitumor activity of classical tricyclic analogues such as 3 would be influenced by FPGS levels in tumor systems. Interestingly, the nonclassical analogues 4-9 showed moderate to good selectivity against DHFR from pathogenic microbes compared to recombinant human DHFR. These results support the idea that removal of the 5-methyl group of piritrexim along with restriction of tau(1) and tau(2) can translate into selectivity for DHFR from pathogens.
Assuntos
Antagonistas do Ácido Fólico/síntese química , Compostos Heterocíclicos com 3 Anéis/síntese química , Metotrexato/análogos & derivados , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/farmacologia , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Resistencia a Medicamentos Antineoplásicos , Escherichia coli/química , Antagonistas do Ácido Fólico/química , Antagonistas do Ácido Fólico/farmacologia , Compostos Heterocíclicos com 3 Anéis/química , Compostos Heterocíclicos com 3 Anéis/farmacologia , Humanos , Metotrexato/síntese química , Metotrexato/química , Metotrexato/farmacologia , Conformação Molecular , Peptídeo Sintases/metabolismo , Pneumocystis/química , Proteínas Recombinantes/química , Especificidade da Espécie , Relação Estrutura-Atividade , Especificidade por Substrato , Tetra-Hidrofolato Desidrogenase/química , Toxoplasma/química , Células Tumorais CultivadasRESUMO
In an attempt to circumvent resistance to and toxicity of clinically used folate-based thymidylate synthase (TS) inhibitors that require folylpoly-gamma-glutamate synthetase (FPGS) for their antitumor activity, we designed and synthesized two classical 6-5 ring-fused analogues, N-[4-[(2-amino-6-methyl-3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d]pyrimidin-5-yl)thio]-2'-fluorobenzoyl]-l-glutamic acid (4) and N-[4-[(2-amino-6-methyl-3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d]pyrimidin-5-yl)thio]-2'-chlorobenzoyl]-l-glutamic acid (5), as TS inhibitors and antitumor agents. The key intermediates in the synthesis of these classical analogues were the mercaptans 10 and 11, which were obtained from the corresponding nitro compounds 6 and 7 respectively, by reduction of the nitro groups followed by diazotization of the amines. The syntheses of analogues 4 and 5 were achieved via the oxidative addition of the sodium salt of ethyl 2-halo-substituted-4-mercaptobenzoate (16 or 17) to 2-amino-6-methyl-3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d]pyrimidine (18) in the presence of iodine. The esters obtained from the reaction were deprotected and coupled with diethyl-l-glutamate followed by saponification. Compounds 4 and 5 were both more potent inhibitors of human TS (IC(50) values of 54 and 51 nM, respectively) than were PDDF and the clinically used ZD1694 and LY231514. Compounds 4 and 5 were not substrates for human FPGS up to 250 muM. In addition, 4 and 5 were growth inhibitory against CCRF-CEM cells as well as a number of other tumor cell lines in culture, and protection studies established TS as the principal target of these analogues.
Assuntos
Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Antagonistas do Ácido Fólico/farmacologia , Timidilato Sintase/antagonistas & inibidores , Linhagem Celular Tumoral , Humanos , Pirimidinas/farmacologia , Relação Estrutura-AtividadeRESUMO
Two novel analogues, N-[2-amino-4-ethyl[(pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-l-glutamic acid (2) and N-[2-amino-4-ethyl-6-methyl[(pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-l-glutamic acid (4), were designed and synthesized as potent dual inhibitors of thymidylate synthase (TS) and dihydrofolate reductase (DHFR) and as antitumor agents. Compound 2 had inhibitory potency against human DHFR similar to N-[4-[2-(amino-3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-L-glutamic acid (LY231514) and 1, whereas 4 was inactive against human DHFR. Both 2 and 4 were more potent than LY231514 against E. coliTS. Against human TS, 2 was 7-fold less potent than LY231514 and 4 showed similar inhibitory activity as LY231514. In contrast to 2, which was an efficient substrate of human folypolyglutamate synthetase (FPGS), 4 was a poor substrate of FPGS. Compound 2 showed GI50 values in the nanomolar range against more than 18 human tumor cell lines in the standard NCI preclinical in vitro screen.
Assuntos
Antineoplásicos/síntese química , Inibidores Enzimáticos/síntese química , Ácido Glutâmico/síntese química , Pirimidinas/síntese química , Tetra-Hidrofolato Desidrogenase/metabolismo , Timidilato Sintase/antagonistas & inibidores , Antineoplásicos/química , Antineoplásicos/farmacologia , Divisão Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Escherichia coli/química , Antagonistas do Ácido Fólico/síntese química , Antagonistas do Ácido Fólico/química , Antagonistas do Ácido Fólico/farmacologia , Ácido Glutâmico/análogos & derivados , Ácido Glutâmico/química , Ácido Glutâmico/farmacologia , Humanos , Lacticaseibacillus casei/química , Peptídeo Sintases/metabolismo , Pirimidinas/química , Pirimidinas/farmacologia , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
Bridge homologation of the previously reported classical two-carbon-bridged antifolates, a 5-substituted 2,4-diaminofuro[2,3-d]pyrimidine (1) [which is a 6-regioisomer of LY231514 (Alimta)] and a 6-subsituted 2-amino-4-oxopyrrolo[2,3-d]pyrimidine, afforded the three-carbon-bridged antifolates analogues 4 and 5, with enhanced inhibitory activity against tumor cells in culture (EC(50) values in the 10(-8)-10(-7) M range or less). These two analogues were synthesized via a 10-step synthetic sequence starting from methyl 4-bromobenzoate (14), which was elaborated to the alpha-chloromethyl ketone (8) followed by condensation with 2,6-diamino-pyrimidin-4-one (7) to afford the substituted furo[2,3-d]pyrimidine 9 and the pyrrolo[2,3-d]pyrimidine 10. Subsequent coupling of each regioisomer with diethyl-l-glutamate followed by saponification afforded 4 and 5. The biological results indicate that elongation of the C8-C9 bridge of the classical 5-substituted 2,4-diaminofuro[2,3-d]pyrimidine and 6-substituted 2-amino-4-oxopyrrolo[2,3-d]pyrimidine are highly conducive to antitumor activity in vitro, despite a lack of increase in inhibitory activity against the target enzymes. This supports our original hypothesis that truncation of the B-ring of a highly potent 6-6 ring system to a 6-5 ring system can be compensated by bridge homologation to restore the overall length of the molecule.
Assuntos
Antimetabólitos Antineoplásicos/síntese química , Antagonistas do Ácido Fólico/síntese química , Pirimidinas/síntese química , Antimetabólitos Antineoplásicos/farmacologia , Antagonistas do Ácido Fólico/farmacologia , Humanos , Peptídeo Sintases/metabolismo , Pirimidinas/farmacologia , Relação Estrutura-Atividade , Tetra-Hidrofolato Desidrogenase/efeitos dos fármacos , Timidilato Sintase/antagonistas & inibidoresRESUMO
Opportunistic infections caused by Pneumocystis jirovecii (P. jirovecii, pj), Toxoplasma gondii (T. gondii, tg), and Mycobacterium avium (M. avium, ma) are the principal causes of morbidity and mortality in patients with acquired immunodeficiency syndrome (AIDS). The absence of any animal models for human Pneumocystis jirovecii pneumonia and the lack of crystal structures of pjDHFR and tgDHFR make the design of inhibitors challenging. A novel series of pyrido[2,3-d]pyrimidines as selective and potent DHFR inhibitors against these opportunistic infections are presented. Buchwald-Hartwig coupling reaction of substituted anilines with pivaloyl protected 2,4-diamino-6-bromo-pyrido[2,3-d]pyrimidine was successfully explored to synthesize these analogues. Compound 26 was the most selective inhibitor with excellent potency against pjDHFR. Molecular modeling studies with a pjDHFR homology model explained the potency and selectivity of 26. Structural data are also reported for 26 with pcDHFR and 16 and 22 with variants of pcDHFR.
Assuntos
Antagonistas do Ácido Fólico/síntese química , Modelos Moleculares , Piridinas/síntese química , Pirimidinas/síntese química , Aminação , Compostos de Anilina/química , Cristalografia por Raios X , Desenho de Fármacos , Antagonistas do Ácido Fólico/química , Humanos , Mycobacterium avium/enzimologia , Pneumocystis carinii/enzimologia , Piridinas/química , Pirimidinas/química , Proteínas Recombinantes/química , Especificidade da Espécie , Relação Estrutura-Atividade , Tetra-Hidrofolato Desidrogenase/químicaRESUMO
Combinations of antiangiogenic agents (AAs) with cytotoxic agents have shown significant promise in cancer treatment, and several such clinical trials are currently underway. We have designed, synthesized, and evaluated two compounds that each inhibit vascular endothelial growth factor receptor-2 (VEGFR-2) and platelet-derived growth factor receptor-beta (PDGFR-beta) for antiangiogenic effects and also inhibit human thymidylate synthase (hTS) for cytotoxic effects in single agents. The synthesis of these compounds involved the nucleophilic displacement of the common intermediate 5-chloro-9H-pyrimido[4,5-b]indole-2,4-diamine with appropriate benzenethiols. The inhibitory potency of both these single agents against VEGFR-2, PDGFR-beta, and hTS is better than or close to standards. In a COLO-205 xenograft mouse model, one of the analogs significantly decreased tumor growth (tumor growth inhibition (TGI) = 76% at 35 mg/kg), liver metastases, and tumor blood vessels compared with a standard drug and with control and thus demonstrated potent tumor growth inhibition, inhibition of metastasis, and antiangiogenic effects in vivo. These compounds afford combination chemotherapeutic potential in single agents.
Assuntos
Antineoplásicos/síntese química , Indóis/síntese química , Pirimidinas/síntese química , Receptor beta de Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidores , Timidilato Sintase/antagonistas & inibidores , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Inibidores da Angiogênese/síntese química , Inibidores da Angiogênese/química , Inibidores da Angiogênese/farmacologia , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Embrião de Galinha , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Indóis/química , Indóis/farmacologia , Masculino , Camundongos , Camundongos Nus , Modelos Moleculares , Transplante de Neoplasias , Pirimidinas/química , Pirimidinas/farmacologia , Relação Estrutura-Atividade , Compostos de Sulfidrila/síntese química , Compostos de Sulfidrila/química , Compostos de Sulfidrila/farmacologia , Transplante HeterólogoRESUMO
N-{4-[(2-Amino-6-ethyl-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidin-5-yl)thio]benzoyl}-L-glutamic acid 2 and 13 nonclassical analogues 2a-2m were synthesized as potential dual thymidylate synthase (TS) and dihydrofolate reductase (DHFR) inhibitors and as antitumor agents. The key intermediate in the synthesis was 2-amino-6-ethyl-5-iodothieno[2,3-d]pyrimidin-4(3H)-one, 7, to which various arylthiols were attached at the 5-position. Coupling 8 with L-glutamic acid diethyl ester and saponification afforded 2. X-ray crystal structures of 2 and 1 (the 6-methyl analogue of 2), DHFR, and NADPH showed for the first time that the thieno[2,3-d]pyrimidine ring binds in a "folate" mode. Compound 2 was an excellent dual inhibitor of human TS (IC50 = 54 nM) and human DHFR (IC50 = 19 nM) and afforded nanomolar GI50 values against tumor cells in culture. The 6-ethyl substitution in 2 increases both the potency (by 2-3 orders of magnitude) as well as the spectrum of tumor inhibition in vitro compared to the 6-methyl analogue 1. Some of the nonclassical analogues were potent and selective inhibitors of DHFR from Toxoplasma gondii.
Assuntos
Antineoplásicos/síntese química , Inibidores Enzimáticos/síntese química , Antagonistas do Ácido Fólico/síntese química , Pirimidinas/síntese química , Tetra-Hidrofolato Desidrogenase/efeitos dos fármacos , Timidilato Sintase/antagonistas & inibidores , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Cristalografia por Raios X , Desenho de Fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Antagonistas do Ácido Fólico/química , Antagonistas do Ácido Fólico/farmacologia , Humanos , Pirimidinas/química , Pirimidinas/farmacologia , Relação Estrutura-Atividade , Toxoplasma/enzimologiaRESUMO
Novel classical antifolates (3 and 4) and 17 nonclassical antifolates (11-27) were synthesized as antitumor and/or antiopportunistic infection agents. Intermediates for the synthesis of 3, 4, and 11-27 were 2,4-diamino-5-alkylsubstituted-7H-pyrrolo[2,3-d]pyrimidines, 31 and 38, prepared by a ring transformation/ring annulation sequence of 2-amino-3-cyano-4-alkyl furans to which various aryl thiols were attached at the 6-position via an oxidative addition reaction using I2. The condensation of alpha-hydroxy ketones with malonodinitrile afforded the furans. For the classical analogues 3 and 4, the ester precursors were deprotected, coupled with diethyl-L-glutamate, and saponified. Compounds 3 (IC50 = 60 nM) and 4 (IC50 = 90 nM) were potent inhibitors of human DHFR. Compound 3 inhibited tumor cells in culture with GI50
Assuntos
Anti-Infecciosos/síntese química , Antineoplásicos/síntese química , Antagonistas do Ácido Fólico/síntese química , Pirimidinas/síntese química , Pirimidinas/farmacologia , Pirróis/síntese química , Pirróis/farmacologia , Tetra-Hidrofolato Desidrogenase/metabolismo , Alquilação , Anti-Infecciosos/química , Anti-Infecciosos/classificação , Anti-Infecciosos/farmacologia , Antineoplásicos/química , Antineoplásicos/classificação , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Antagonistas do Ácido Fólico/química , Antagonistas do Ácido Fólico/classificação , Antagonistas do Ácido Fólico/farmacologia , Humanos , Modelos Moleculares , Estrutura Molecular , Pirimidinas/química , Pirróis/química , Relação Estrutura-Atividade , Tetra-Hidrofolato Desidrogenase/químicaRESUMO
N-{4-[(2-Amino-6-methyl-4-oxo-3,4-dihydrothieno[2,3- d]pyrimidin-5-yl)sulfanyl]benzoyl}-L-glutamic acid (4) and nine nonclassical analogues 5-13 were synthesized as potential dual thymidylate synthase (TS) and dihydrofolate reductase (DHFR) inhibitors. The key intermediate in the synthesis was 2-amino-6-methylthieno[2,3-d]pyrimidin-4(3 H)-one (16), which was converted to the 5-bromo-substituted compound 17 followed by an Ullmann reaction to afford 5-13. The classical analogue 4 was synthesized by coupling the benzoic acid derivative 19 with diethyl L-glutamate and saponification. Compound 4 is the most potent dual inhibitor of human TS (IC 50 = 40 nM) and human DHFR (IC 50 = 20 nM) known to date. The nonclassical analogues 5- 13 were moderately potent against human TS with IC 50 values ranging from 0.11 to 4.6 microM. The 4-nitrophenyl analogue 7 was the most potent compound in the nonclassical series, demonstrating potent dual inhibitory activities against human TS and DHFR. This study indicated that the 5-substituted 2-amino-4-oxo-6-methylthieno[2,3-d]pyrimidine scaffold is highly conducive to dual human TS-DHFR inhibitory activity.