RESUMO
UDP-N-acetylmuramic acid (UDP-MurNAc) is a substrate of MurC, an important enzyme in the intracellular pathway of bacterial peptidoglycan biosynthesis. Various approaches towards preparation of UDP-MurNAc have been published but these synthetic preparations were shown to include many problematic steps. An optimization study with the focus on muramyl phosphate and UMP-morpholidate coupling was performed, resulting in a synthetic procedure enabling robust and easily reproducible production on a multi-gram scale.
Assuntos
Uridina Difosfato Ácido N-Acetilmurâmico/síntese química , Fenômenos Químicos , Química , Cromatografia Líquida de Alta Pressão , Indicadores e Reagentes , Espectroscopia de Ressonância Magnética , FosforilaçãoRESUMO
Design, synthesis and biochemical evaluation of a series of novel non-covalent thrombin inhibitors with a 1-amidinopiperidine moiety are presented. Replacement of the planar benzamidine group in azaphenylalanine derivatives with 1-amidinopiperidine resulted in lower activity but higher selectivity for this type of compounds. The binding conformation of inhibitors in the active site of thrombin was revealed by molecular modelling studies.
Assuntos
Piperidinas/síntese química , Piperidinas/farmacologia , Trombina/antagonistas & inibidores , Coagulação Sanguínea/efeitos dos fármacos , Testes de Coagulação Sanguínea , Desenho de Fármacos , Humanos , Técnicas In Vitro , Indicadores e Reagentes , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Conformação Molecular , Tempo de Tromboplastina Parcial , Tempo de Protrombina , Espectrofotometria Infravermelho , Tempo de Trombina , Inibidores da Tripsina/síntese química , Inibidores da Tripsina/química , Inibidores da Tripsina/farmacologiaRESUMO
Thrombin is the key serine proteinase of the coagulation cascade and therefore a suitable target for inhibition of blood coagulation. A number of pharmacologically active secondary metabolites from mushrooms have already been isolated, thus providing the rationale for screening for new thrombin inhibitors in mushrooms. In this study, inhibitory activities of mushroom extracts on thrombin and trypsin were measured using the chromogenic substrates H-D-phenylalanine-L-pipecolyl-L-arginine-paranitroaniline dihydrochloride (S-2238) for thrombin and N-benzoyl-D,L-Arg-p-nitroanilide (BAPNA) for trypsin. The inhibitory activities of extracts from 95 Basidiomycete species have been determined. The majority of samples inhibited trypsin and thrombin with various potencies; however, some extracts showed no activity against one or both of the enzymes. An aqueous extract of Gleophyllum odoratum exhibited high inhibitory activity on both thrombin and trypsin (72 and 60%, respectively), while extracts of Clitocybe gibba, Amanita virosa, Cantharellus lutescens, Suillus tridentinus, Hypoloma fasciculare and Lactarius badiosanguineus considerably inhibited thrombin (49, 48, 36, 34, 32 and 31%, respectively) and showed no inhibitory activity on trypsin. The results at this point are promising for further research with the objective of finding an effective and safe thrombin inhibitor.
Assuntos
Agaricales/química , Basidiomycota/química , Trombina/antagonistas & inibidores , Benzoilarginina Nitroanilida/metabolismo , Compostos Cromogênicos/metabolismo , Dipeptídeos/metabolismo , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Especificidade da Espécie , Trombina/metabolismo , Tripsina/metabolismo , Inibidores da Tripsina/isolamento & purificaçãoRESUMO
Soluble polyvinylpyrrolidones are very useful and versatile pharmaceutical auxiliaries. The different types of povidone are characterised by their viscosity measured in water, expressed as a K-value. We have developed a rapid, accurate, reliable, and non-destructive near infrared (NIR) spectroscopy method for the determination of PVP type and consequently identification thereof. We have implemented chemometrics onto NIR spectra collected in diffuse reflectance mode using fibre optics to build a qualitative model that enables us to obtain useful analytical information. A principal component analysis and a modelling technique soft independent modelling of class analogy (SIMCA) were applied. An approach to validate the method was developed.
Assuntos
Técnicas de Química Analítica/métodos , Excipientes Farmacêuticos/análise , Povidona/análise , Povidona/análogos & derivados , Reprodutibilidade dos Testes , Espectroscopia de Luz Próxima ao InfravermelhoRESUMO
Differential scanning calorimetry (DSC), Fourier transform infra-red spectroscopy (FT-IR), HPLC and TLC were used to investigate the interactions between the mucolytic drug acetylcysteine and a number of commonly used tablet and capsule excipients. Acetylcysteine was found to be compatible with microcrystalline cellulose (Avicel PH 101), sodium carboxymethylcellulose, amorphous silicon dioxide (Aerosil), PVP, cross-linked PVP (Polyplasdone XL), corn starch, saccharose and magnesium stearate. Acetylcysteine thermal stability (onset degradation temperature) was decreased in mixtures with corn starch, magnesium stearate, saccharose and lactose. Interactions of acetylcysteine with lactose, PEG 4000 and 6000, glycine, adipic acid and saccharin sodium were found using DSC and studied in detail with FT-IR, HPLC and TLC. The results suggest that acetylcysteine in mixtures with PEG 4000, glycine or saccharin sodium is degraded during storage at conditions of high temperature and humidity.
Assuntos
Acetilcisteína/química , Excipientes/química , Cromatografia Líquida de Alta Pressão , Cromatografia em Camada Fina , Interações Medicamentosas , ComprimidosRESUMO
Design, synthesis and biological evaluation of a series of novel non-covalent azaphenylalanine thrombin inhibitors are presented. Replacement of the basic benzamidine moiety in azaphenylalanine derivatives by benzylamine (P1 part of a molecule) and the introduction of a N-cyclopentyl-N-methylamine moiety in the P2 part of a molecule resulted in the thrombin inhibitor LK-733 with greatly increased selectivity against trypsin and an in vitro Ki of 31 nM.
Assuntos
Hidrazinas/síntese química , Hidrazinas/farmacologia , Fenilalanina/análogos & derivados , Trombina/antagonistas & inibidores , Sítios de Ligação , Inibidores do Fator Xa , Indicadores e Reagentes , Cinética , Espectroscopia de Ressonância Magnética , Fenilalanina/síntese química , Fenilalanina/farmacologia , Inibidores de Serina Proteinase , Inibidores da Tripsina/síntese química , Inibidores da Tripsina/farmacologiaRESUMO
Two new adamantyl-desmuramyldipeptides LK 415 and LK 517 with 1-adamantylcarboxamido moiety as a replacement for muramyldipeptide's N-acetylglucosamine fragment were synthesized. Their efficacy to modulate the production of cytokines was measured in vitro in ionomycin and phorbol-12-myristate-13-acetate (PMA) activated cultures of human peripheral blood mononuclear cells (PBMC), co-incubated with the substances tested. The results were compared with the activity of muramyldipeptide (MDP). All three substances are strong up-regulators of IL-12 synthesis and hence of the IFN gamma synthesis as well. While MDP and LK 415 are relatively ineffective in modulation of IL-2, IL-4 and IL-10 production in vitro, the synthesis of all three cytokines is considerably up-regulated when peripheral blood mononuclear cells are co-incubated with LK 517. It seems likely that the introduction of the diethyl phosphonate moiety into LK 517 is of great importance for the augmented T-cell cytokine production.
Assuntos
Acetilmuramil-Alanil-Isoglutamina/farmacologia , Adamantano/síntese química , Adamantano/farmacologia , Adjuvantes Imunológicos/síntese química , Adjuvantes Imunológicos/farmacologia , Citocinas/biossíntese , Dipeptídeos/síntese química , Dipeptídeos/farmacologia , Acetilmuramil-Alanil-Isoglutamina/análogos & derivados , Adamantano/análogos & derivados , Células Cultivadas , Humanos , Interferon gama/biossíntese , Interleucinas/biossíntese , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Fator de Necrose Tumoral alfa/biossínteseRESUMO
A series of N-(5-phthalimidopentanoyl)-, N-[2-(2-ethoxy)acetyl]-, and N-(7-oxooctanoyl)-phosphono and phosphinoalanine derivatives has been synthesized and evaluated for inhibition of the D-glutamic acid-adding enzyme (MurD) of peptidoglycan biosynthesis.
Assuntos
Alanina/síntese química , Inibidores Enzimáticos/síntese química , Acetilmuramil-Alanil-Isoglutamina/química , Alanina/análogos & derivados , Alanina/farmacologia , Inibidores Enzimáticos/farmacologia , Indicadores e ReagentesRESUMO
New inhibitors of serine proteases with azaphenylalanine scaffold were synthesized and their activity was evaluated in vitro. We studied the effect of different substituents in the part of a molecule that binds in the distal pocket of the thrombin active site. Modifications generally led to decreased activity, however two derivatives are promising lead compounds as new thrombin and dual thrombin-factor Xa inhibitors.
Assuntos
Fenilalanina/análogos & derivados , Fenilalanina/síntese química , Fenilalanina/farmacologia , Inibidores de Serina Proteinase/síntese química , Inibidores de Serina Proteinase/farmacologia , Tiadiazóis/síntese química , Tiadiazóis/farmacologia , Cromatografia em Camada Fina , Indicadores e Reagentes , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Espectrofotometria Infravermelho , Relação Estrutura-Atividade , Trombina/antagonistas & inibidoresRESUMO
In this paper the synthesis and antithrombotic activity of a series of novel thrombin inhibitors with azaphenylalanine scaffold are described. By systematic structural modifications for this series we have identified optimal groups for achieving nanomolar potency, that led to potent inhibitors of thrombin with Ki values up to 11 nM.
Assuntos
Anticoagulantes/síntese química , Trombina/antagonistas & inibidores , Anticoagulantes/farmacocinética , Anticoagulantes/farmacologia , Coagulação Sanguínea , Estabilidade de Medicamentos , Humanos , Técnicas In Vitro , Tempo de Tromboplastina Parcial , Tempo de Protrombina , Espectrofotometria Ultravioleta , Relação Estrutura-Atividade , Tempo de TrombinaRESUMO
The effects of muramyl dipeptide (MDP) synthetic analogue LK415 on the immune response of chickens immunized with a live vaccine against infectious bursal disease (IBD) were studied in two independent trials, using levamisole hydrochloride as comparative immunostimulant. Groups of five-week-old commercial chickens (Isa Brown) were immunized orally with 10 doses of the vaccine strain of IBDV (Winterfield strain). The chickens were then given four injections of the MDP analogue LK415 in a dosage of either 0.25 mg/kg body weight (b.w.) or 2.5 mg/kg b.w. or levamisole at a daily dose of 15 mg/kg b.w. for four consecutive days, starting from the day of immunization. Histological examinations of bursal tissue collected on days 2, 4 and 7 postimmunization (p.i.) showed a lower degree of destruction of bursal follicles and earlier renewal of bursal tissue in LK415-treated chickens compared to levamisole-treated and untreated immunized groups. Compared to the other groups, the LK415-treated chickens showed a significantly higher antibody response to IBDV on days 14 and 28 p.i. (P < 0.01) as measured by commercial ELISA. The present study indicates some potent immunostimulatory effects of the MDP analogue LK415 on the chicken immune system.
Assuntos
Acetilmuramil-Alanil-Isoglutamina/análogos & derivados , Galinhas/imunologia , Imunização/veterinária , Vírus da Doença Infecciosa da Bursa/imunologia , Acetilmuramil-Alanil-Isoglutamina/imunologia , Acetilmuramil-Alanil-Isoglutamina/farmacologia , Adjuvantes Imunológicos/administração & dosagem , Animais , Formação de Anticorpos , Infecções por Birnaviridae/prevenção & controle , Relação Dose-Resposta a Droga , Levamisol/administração & dosagemAssuntos
Acetilmuramil-Alanil-Isoglutamina/análogos & derivados , Acetilmuramil-Alanil-Isoglutamina/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Indicadores e Reagentes , Células Tumorais CultivadasRESUMO
Some synthetic analogues of the immunomodulatory agent muramyl dipeptide (MDP), i.e. phthalimido- (LK-511, LK-413, LK-512, LK-423, LK-508), adamantyl- (LK-415, LK-517), 7-oxoalkyl-(LK-409) desmuramylpeptides were assessed for the tumour necrosis factor (TNF) inducing activity and the ability to modulate TNF production in in vitro phorbol 12-myristate 13-acetate (PMA) & ionomycin stimulated cultures of human peripheral blood mononuclear cells. A kinetic study over a 40-hour period indicated that desmuramyldipeptides were weak TNF inducers compared to romurtide, PMA & ionomycin or lipopolysaccharide. By contrast, they showed the potential to up- or down-regulate the production of TNF evoked by PMA & ionomycin, which was strongly dependent on the time of the stimulation. After 4h of stimulation, the TNF secretion was augmented by LK-508, LK-409 and LK-511, after 18 h by LK-409 and LK-423, and after 40 h by LK-423, LK-511, LK-415 and LK-512. However, LK-517 and LK-512 inhibited the secretion of TNF after the 18-h period.
Assuntos
Acetilmuramil-Alanil-Isoglutamina/análogos & derivados , Adjuvantes Imunológicos/farmacologia , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Fator de Necrose Tumoral alfa/biossíntese , Combinação de Medicamentos , Humanos , Ionomicina/farmacologia , Ionóforos/farmacologia , Acetato de Tetradecanoilforbol/farmacologia , Fatores de TempoRESUMO
Microspheres containing the mucoadhesive polymer chitosan hydrochloride, with matrix polymer Eudragit RS, pipemidic acid as a model drug and agglomeration preventing agent magnesium stearate were prepared by the solvent evaporation method. The amount of magnesium stearate was varied and the following methods were used for microsphere evaluation: sieve analysis, drug content and dissolution determination, scanning electron microscopy, x-ray diffractometry, DSC and FTIR spectroscopy. The results showed that average particle size decreased with increasing amount of magnesium stearate used for microsphere preparation. This is probably a consequence of stabilization of the emulsion droplets with magnesium stearate. Higher pipemidic acid content in the microspheres was observed in larger particle size fractions and when higher amounts of magnesium stearate were used. It was also found that these two parameters significantly influenced the dissolution rate. The important reason for the differences in drug content in microspheres of different particle sizes is the diffusion of pipemidic acid from the acetone droplets in liquid paraffin during the preparation procedure. The physical state of pipemidic acid changed from crystalline to mostly amorphous with its incorporation in microspheres, as shown by x-ray diffractometry and differential scanning calorimetry. No differences were observed in the physical state of pipemidic acid and in microsphere shape and surface between different size fractions of microspheres, prepared with different amounts of magnesium stearate. Additionally, no correlation between the physical state of the drug in different microspheres and their biopharmaceutical properties was found.
Assuntos
Sistemas de Liberação de Medicamentos , Microesferas , Ácidos Esteáricos , Resinas Acrílicas , Adesividade , Varredura Diferencial de Calorimetria , Cápsulas , Quitina/análogos & derivados , Quitosana , Composição de Medicamentos , Humanos , Técnicas In Vitro , Microscopia Eletrônica de Varredura , Mucosa , Tamanho da Partícula , Ácido Pipemídico/administração & dosagem , Difração de Raios XRESUMO
The preparation of immunologically active phthalimido desmuramylpeptide analogues 2e-h, 4c-d, and 7b is described. The N-acetylmuramic acid in the muramyl dipeptide has been replaced by a phthaloylated acyclic moiety such as N-phthaloylated amino acids 1a-c, 2-(2-phthalimidoethoxy)acetic acid 3, or by the carbocyclic rac. trans-2-(2'-phthalimidocyclohexyloxy)acetic acid 6.
Assuntos
Adjuvantes Imunológicos/síntese química , Peptídeos/síntese química , Ftalimidas/síntese química , Adjuvantes Imunológicos/farmacologia , Animais , Ciclofosfamida/antagonistas & inibidores , Ciclofosfamida/farmacologia , Imunossupressores/antagonistas & inibidores , Imunossupressores/farmacologia , Camundongos , Peptídeos/farmacologia , Ftalimidas/farmacologiaRESUMO
A new phthalimido compound, N-[2-(2-phthalimidoethoxy)acetyl]-L-alanyl-D-glutamic acid (CAS 142489-47-2, LK 423), was examined for its possible activity to modulate levels and species of cytokines in mice carrying a specific inflamed organ. Colonic inflammation was induced in mice by giving 5% dextran sulfate sodium (DSS) solution as drinking water. The capacity of spleen cells obtained from the DSS-inflamed mice to produce interleukin-10 (IL-10) in response to mitogen was significantly reduced when compared with the capacity of spleen cells from intact mice. Treatment of the mice administered DSS by subcutaneous multiple injections with a low dose of LK423 resulted in delaying the progression to full-blown inflammation in the colon. The mitogen-stimulated spleen cells obtained from the LK423-treated mice yielded significantly greater amounts of IL-10 and IL-6 than the untreated DSS group, and the peritoneal cells from the LK423-treated mice produced significantly lower levels of tumor necrosis factor alpha (TNF alpha). Based on this prophylactic effect of LK423 in the murine colitis model, its therapeutic effect was examined in rats in which colitis had been induced by feeding 3% DSS for 12 days. Intracolonic administration of LK423 to these rats for 7 days resulted in diminishing the ulcerative area in the colon. The immunological characteristics of this new compound are discussed from the point of view of its possible application as a therapeutic agent for inflammatory bowel diseases (IBD) and other inflammatory diseases.
Assuntos
Adjuvantes Imunológicos/farmacologia , Colite/tratamento farmacológico , Sulfato de Dextrana , Dipeptídeos/farmacologia , Interleucina-10/biossíntese , Ftalimidas/farmacologia , Animais , Células Cultivadas , Colite/induzido quimicamente , Colite/patologia , Colite Ulcerativa/induzido quimicamente , Colite Ulcerativa/tratamento farmacológico , Colite Ulcerativa/patologia , Colo/patologia , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Sangue Oculto , Cavidade Peritoneal/citologia , Ratos , Ratos Endogâmicos , Baço/efeitos dos fármacos , Baço/metabolismo , Baço/patologia , Fator de Necrose Tumoral alfa/biossínteseRESUMO
A new phthalimido compound, N-[2-(2-phthalimidoethoxy)acetyl]-L-alanyl-D-glutamic acid (CAS 142489-47-2, LK423), was examined along with other N-acyl-desmuramyldipeptide compounds for possible immunomodulating activities in cyclophosphamide (Cy)-treated mice. Reverse transcriptase-polymerase chain reaction (RT-PCR) and Northern blot assays demonstrated that multiple subcutaneous injections of LK423 (1 to 50 micrograms/days, for 4 days) into these mice resulted in upregulating interleukin-10 (IL-10) gene expression in the spleen. In contrast to IL-10, expression of interferon-gamma (IFN-gamma) gene was reduced by treating with this compound. The culture supernatant of spleen cells obtained from the mice that had received LK423 injections was found to contain a larger amount of IL-10 protein than in the culture supernatant of the spleen obtained from the mice that received no LK423 injections when tested by enzyme-linked immunosorbent assay (ELISA). Conversely to IL-10, the concentration of IFN-gamma was lower in the culture supernatant from the LK423-treated group than that in the control group. In contrast to this compound, other N-acyl-desmuramyldipeptide derivatives carrying a L-alanyl-D-isoglutamine moiety, and other immunological stimulants showed an activity to augment production of IFN-gamma and reduce the gene expression of IL-10. The immunological activities of this new phthalimido desmuramyldipeptide compound, LK423, are discussed from the point of view of its therapeutic application.
Assuntos
Adjuvantes Imunológicos/farmacologia , Dipeptídeos/farmacologia , Interleucina-10/biossíntese , Ftalimidas/farmacologia , Animais , Northern Blotting , Células Cultivadas , Ciclofosfamida/toxicidade , Ensaio de Imunoadsorção Enzimática , Feminino , Regulação da Expressão Gênica , Imunossupressores/toxicidade , Interferon gama/biossíntese , Interferon gama/genética , Interleucina-10/genética , Camundongos , RNA Mensageiro/biossíntese , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Baço/citologia , Baço/efeitos dos fármacos , Baço/metabolismoRESUMO
Theoretical and structural studies followed by the directed synthesis and in vitro biological tests lead us to novel noncovalent thrombin pseudopeptide inhibitors. We have incorporated an azapeptide scaffold into the central part of the classical tripeptide D-Phe-Pro-Arg inhibitor structure thus eliminating one stereogenic center from the molecule. A series of compounds has been designed to optimize the occupancy of the S2 pocket of thrombin. Increased hydrophobicity at P2 provides an enhanced fit into this active site S2 pocket. In the present paper, we also report on the structure of these inhibitors in solution and conformational analysis of inhibitors in the active site in order to asses the consequences of the replacement of the central alpha-CH by a nitrogen functionality. In vitro biological testing of the designed inhibitors shows that elimination of R, S stereoisomerism and restriction of conformational freedom influences the binding of inhibitors in a favorable fashion.