RESUMO
Small-molecule hepatitis C virus (HCV) NS3 protease inhibitors such as boceprevir (SCH 503034) have been shown to have antiviral activity when they are used as monotherapy and in combination with pegylated alpha interferon and ribavirin in clinical trials. Improvements in inhibitor potency and pharmacokinetic properties offer opportunities to increase drug exposure and to further increase the sustained virological response. Exploration of the structure-activity relationships of ketoamide inhibitors related to boceprevir has led to the discovery of SCH 900518, a novel ketoamide protease inhibitor which forms a reversible covalent bond with the active-site serine. It has an overall inhibition constant (K*(i)) of 7 nM and a dissociation half-life of 1 to 2 h. SCH 900518 inhibited replicon RNA at a 90% effective concentration (EC(90)) of 40 nM. In biochemical assays, SCH 900518 was active against proteases of genotypes 1 to 3. A 2-week treatment with 5x EC(90) of the inhibitor reduced the replicon RNA level by 3 log units. Selection of replicon cells with SCH 900518 resulted in the outgrowth of several resistant mutants (with the T54A/S and A156S/T/V mutations). Cross-resistance studies demonstrated that the majority of mutations for resistance to boceprevir and telaprevir caused similar fold losses of activity against all three inhibitors; however, SCH 900518 retained more activity against these mutants due to its higher intrinsic potency. Combination treatment with alpha interferon enhanced the inhibition of replicon RNA and suppressed the emergence of resistant replicon colonies, supporting the use of SCH 900518-pegylated alpha interferon combination therapy in the clinic. In summary, the results of the preclinical characterization of the antiviral activity of SCH 900518 support its evaluation in clinical studies.
Assuntos
Antivirais/farmacologia , Dipeptídeos/farmacologia , Hepacivirus/efeitos dos fármacos , Hepacivirus/enzimologia , Inibidores de Proteases/farmacologia , Sulfonas/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Antivirais/administração & dosagem , Antivirais/química , Ciclopropanos , Dipeptídeos/administração & dosagem , Dipeptídeos/química , Descoberta de Drogas , Avaliação Pré-Clínica de Medicamentos , Farmacorresistência Viral/genética , Genótipo , Hepacivirus/genética , Humanos , Técnicas In Vitro , Interferon alfa-2 , Interferon-alfa/administração & dosagem , Cinética , Leucina/análogos & derivados , Mutação , Prolina/análogos & derivados , Prolina/farmacologia , Inibidores de Proteases/administração & dosagem , Inibidores de Proteases/química , Proteínas Recombinantes , Replicon/efeitos dos fármacos , Sulfonas/administração & dosagem , Sulfonas/química , UreiaRESUMO
Substitutions on the P(1) cyclobutyl side chain of SCH 503034 were studied by introduction of hydroxyl and fluoro substituents. Additionally, effects of fluoro substitution on other P1 moieties were evaluated.
Assuntos
Prolina/análogos & derivados , Inibidores de Serina Proteinase/síntese química , Inibidores de Serina Proteinase/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/química , Área Sob a Curva , Química Farmacêutica/métodos , Desenho de Fármacos , Flúor/química , Hepatite C/tratamento farmacológico , Humanos , Concentração Inibidora 50 , Cinética , Modelos Químicos , Conformação Molecular , Prolina/síntese química , Prolina/farmacologia , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
We have been developing a series of nonpeptidic, small molecule farnesyl protein transferase inhibitors that share a common tricyclic nucleus and compete with peptide/protein substrates for binding to farnesyl protein transferase. Here, we report on pharmacological and in vivo studies with SCH 66336, a lead compound in this structural class. SCH 66336 potently inhibits Ha-Ras processing in whole cells and blocks the transformed growth properties of fibroblasts and human tumor cell lines expressing activated Ki-Ras proteins. The anchorage-independent growth of many human tumor lines that lack an activated ras oncogene is also blocked by treatment with SCH 66336. In mouse, rat, and monkey systems, SCH 66336 has excellent oral bioavailability and pharmacokinetic properties. In the nude mouse, SCH 66336 demonstrated potent oral activity in a wide array of human tumor xenograft models including tumors of colon, lung, pancreas, prostate, and urinary bladder origin. Enhanced in vivo efficacy was observed when SCH 66336 was combined with various cytotoxic agents (cyclophosphamide, 5-fluorouracil, and vincristine). In a Ha-Ras transgenic mouse model, prophylactic treatment with SCH 66336 delayed tumor onset, reduced the average number of tumors/mouse, and reduced the average tumor weight/animal. In a therapeutic mode in which gavage treatment was initiated after the transgenic mice had developed palpable tumors, significant tumor regression was induced by SCH 66336 in a dose-dependent fashion. This was associated with increased apoptosis and decreased DNA synthesis in tumors of animals treated with SCH 66336. Enhanced efficacy was also observed in this model when SCH 66336 was combined with cyclophosphamide. SCH 66336 is presently being evaluated in Phase I clinical trials.
Assuntos
Alquil e Aril Transferases/antagonistas & inibidores , Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Genes ras/fisiologia , Neoplasias Experimentais/tratamento farmacológico , Piperidinas/farmacologia , Piridinas/farmacologia , Células 3T3 , Administração Oral , Animais , Bromodesoxiuridina/metabolismo , Divisão Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Humanos , Macaca fascicularis , Masculino , Camundongos , Transplante de Neoplasias , Ratos , Transplante HeterólogoRESUMO
We previously reported compound 1 as a potent farnesyl protein transferase (FPT) inhibitor that exhibited reasonable pharmacokinetic stability and showed moderate in vivo activity against a variety of tumor cell lines. The analogous C-11 single compound, pyridylacetamide 2, was found to be more potent than 1 in FPT inhibition. Further studies showed that modification of the ethano bridge of the tricyclic ring system by conversion into a double bond with concomitant introduction of a single bond at C-11 piperidine resulted in compound 3 that had superior FPT activity and pharmacokinetic stability. Compound 4, a 5-bromo-substituted analogue of 3, showed improved FPT activity, had good cellular activity, and demonstrated a remarkably improved pharmacokinetic profile with AUC of 84.9 and t1/2 of 82 min. Compound4 inhibited the growth of solid tumor in DLD-1 model by 70% at 50 mpk and 52% at 10 mpk.
Assuntos
Alquil e Aril Transferases/antagonistas & inibidores , Antineoplásicos , Óxidos N-Cíclicos , Inibidores Enzimáticos , Piridinas , Administração Oral , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/síntese química , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Disponibilidade Biológica , Óxidos N-Cíclicos/administração & dosagem , Óxidos N-Cíclicos/síntese química , Óxidos N-Cíclicos/farmacocinética , Óxidos N-Cíclicos/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacocinética , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Camundongos , Camundongos Nus , Transplante de Neoplasias , Proteína Oncogênica p21(ras)/antagonistas & inibidores , Piridinas/administração & dosagem , Piridinas/síntese química , Piridinas/farmacocinética , Piridinas/farmacologia , Células Tumorais CultivadasRESUMO
Crystallographic and thermodynamic studies of farnesyl protein transferase (FPT) complexed with novel tricyclic inhibitors provide insights into the observed SAR for this unique class of nonpeptidic FPT inhibitors. The crystallographic structures reveal a binding pattern conserved across the mono-, di-, and trihalogen series. In the complexes, the tricycle spans the FPT active site cavity and interacts with both protein atoms and the isoprenoid portion of bound farnesyl diphosphate. An amide carbonyl, common to the tricyclic compounds described here, participates in a water-mediated hydrogen bond to the protein backbone. Ten high-resolution crystal structures of inhibitors complexed with FPT are reported. Included are crystallographic data for FPT complexed with SCH 66336, a compound currently undergoing clinical trials as an anticancer agent (SCH 66336, 4-[2-[4-(3,10-dibromo-8-chloro-6,11-dihydro-5H-benzo[5, 6]cyclohepta[1, 2-b]pyridin-11-yl)-1-piperidinyl]-2-oxoethyl]-1-piperidinecarbo xamide ). Thermodynamic binding parameters show favorable enthalpies of complex formation and small net entropic contributions as observed for 4-[2-[4-(3,10-dibromo-8-chloro-6,11-dihydro-11H-benzo[5, 6]cyclohepta[1, 2-b]pyridin-11-ylidene)-1-piperidinyl]-2-oxoethyl]pyridine N-oxide where DeltaH degrees bind = -12.5 kcal/mol and TDeltaS degrees bind = -1.5 kcal/mol.
Assuntos
Alquil e Aril Transferases/antagonistas & inibidores , Antineoplásicos/química , Óxidos N-Cíclicos/química , Inibidores Enzimáticos/química , Compostos Heterocíclicos com 3 Anéis/química , Piperidinas/química , Prenilação de Proteína , Piridinas/química , Sítios de Ligação , Calorimetria , Cristalografia por Raios X , Ligação de Hidrogênio , Modelos Moleculares , TermodinâmicaRESUMO
Novel tricyclic Ras farnesyl-protein transferase (FPT) inhibitors are described. A comprehensive structure-activity relationship (SAR) study of compounds arising from substitution at the 3-position of the tricyclic pyridine ring system has been explored. In the case of halogens, the chloro, bromo, and iodo analogues 19, 22, and 28 were found to be equipotent. However, the fluoro analogue 17 was an order of magnitude less active. Whereas a small alkyl substituent such as a methyl group resulted in a very potent FPT inhibitor (SCH 56580), introduction of bulky substituents such as tert-butyl, compound 33, or a phenyl group, compound 29, resulted in inactive FPT inhibitors. Polar groups at the 3-position such as amino 5, alkylamino 6, and hydroxyl 12 were less active. Whereas compound SCH 44342 did not show appreciable in vivo antitumor activity, the 3-bromo-substituted pyridyl N-oxide amide analogue 38 was a potent FPT inhibitor that reduced tumor growth by 81% when administered q.i.d. at 50 mpk and 52% at 10 mpk. These compounds are nonpeptidic and do not contain sulfhydryl groups. They selectively inhibit FPT and not geranylgeranyl-protein transferase-1 (GGPT-1). They also inhibit H-Ras processing in COS monkey kidney cells and soft agar growth of Ras-transformed cells.
Assuntos
Alquil e Aril Transferases/antagonistas & inibidores , Antineoplásicos/síntese química , Inibidores Enzimáticos/síntese química , Piperidinas/síntese química , Piridinas/síntese química , Células 3T3 , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Células COS , Desenho de Fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Espectroscopia de Ressonância Magnética , Camundongos , Camundongos Nus , Estrutura Molecular , Neoplasias Experimentais/tratamento farmacológico , Piperidinas/química , Piperidinas/farmacologia , Prenilação de Proteína , Piridinas/farmacologia , Relação Estrutura-Atividade , Transfecção/genética , Proteínas ras/metabolismoRESUMO
We have previously shown that appropriate modification of the benzocycloheptapyridine tricyclic ring system can provide potent farnesyl protein transferase (FPT) inhibitors with good cellular activity. Our laboratories have also established that incorporation of either pyridinylacetyl N-oxide or 4-N-carboxamidopiperidinylacetyl moieties results in pharmacokinetically stable inhibitors that are orally efficacious in nude mice. We now demonstrate that further elaboration of the tricyclic ring system by introducing a bromine atom at the 7- or the 10-position of the 3-bromo-8-chlorotricyclic ring system provides compounds that have superior potency and selectivity in FPT inhibition. These compounds have good serum levels and half-lives when given orally to rodents and primates. In vitro and in vivo evaluation of a panel of these inhibitors has led to identification of 15 (SCH 66336) as a highly potent (IC50 = 1.9 nM) antitumor agent that is currently undergoing human clinical trials.
Assuntos
Alquil e Aril Transferases/antagonistas & inibidores , Antineoplásicos/síntese química , Inibidores Enzimáticos/síntese química , Piperidinas/síntese química , Prenilação de Proteína/efeitos dos fármacos , Piridinas/síntese química , Animais , Antineoplásicos/química , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Células COS , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacocinética , Inibidores Enzimáticos/farmacologia , Humanos , Macaca fascicularis , Camundongos , Camundongos Nus , Piperidinas/química , Piperidinas/farmacocinética , Piperidinas/farmacologia , Piridinas/química , Piridinas/farmacocinética , Piridinas/farmacologia , Estereoisomerismo , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
Farnesyl protein transferase (FPT) is a promising target for the development of cancer chemotherapeutics because it is responsible for the farnesylation of oncogenic p21 Ras proteins which are found in nearly 30% of all human cancers and necessary for cellular development and growth. The recent discovery and progression to phase II clinical trials of trihalobenzocycloheptapyridine Sch-66336 as a potent inhibitor of FPT with oral, in vivo efficacy in mice have spawned extensive structure-activity relationship studies (SAR) of this class of compounds. Of the many trihalobenzocycloheptapyridine analogues prepared, we have identified several which inhibit FPT and cellular proliferation at single-digit nanomolar concentrations and which have good pharmacokinetic properties in mice.
Assuntos
Alquil e Aril Transferases/antagonistas & inibidores , Inibidores Enzimáticos/síntese química , Piperidinas/síntese química , Piridinas/síntese química , Sulfonamidas/síntese química , Compostos de Sulfonilureia/síntese química , Administração Oral , Animais , Disponibilidade Biológica , Células COS , Divisão Celular/efeitos dos fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacocinética , Haplorrinos , Camundongos , Camundongos Nus , Piperidinas/química , Piperidinas/farmacocinética , Prenilação de Proteína , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Piridinas/química , Piridinas/farmacocinética , Relação Estrutura-Atividade , Sulfonamidas/química , Sulfonamidas/farmacocinética , Compostos de Sulfonilureia/química , Compostos de Sulfonilureia/farmacocinéticaRESUMO
[formula: see text] Synthesis of C-11 methyl-substituted benzocycloheptylpyridine tricyclic compounds has been achieved via two different methods. Methylation of C-11 has been effected by treatment of amine 4 with BuLi followed by Mel quenching. In a similar procedure, introduction of a C-11 substituent with concomitant rearrangement of the exocyclic double bond has been carried out. Potent farnesyl protein transferase inhibitors have been synthesized using the above methodologies.
Assuntos
Alquil e Aril Transferases/antagonistas & inibidores , Inibidores Enzimáticos/síntese química , Piridinas/síntese química , Inibidores Enzimáticos/farmacologia , Piridinas/farmacologiaRESUMO
5-Hydroxyl-1-neopentylpyrrole-2-carbaldehyde, 2-(2-hydroxyacetyl)-1-neopentylpyrrole, in decreasing order or abundance, have been isolated and the structures characterized. These compounds were obtained from the reaction of a mixture of D-glucose and neopentylamine under physiological conditions of pH and temperature. In addition, 4H-dihydropyran-4-one, a known intermediate product of the Maillard reaction, was detected. The neopentylamine adducts were already detectable after one week of incubation, but rapid acid and alkaline degradation explains the lack of detection in body proteins.
Assuntos
Glucose , Propilaminas , Pirróis/análise , Fenômenos Químicos , Química , Cromatografia Gasosa-Espectrometria de Massas , Concentração de Íons de Hidrogênio , Indicadores e Reagentes , Espectrofotometria , TemperaturaRESUMO
Cleavage of the hepatitis C virus (HCV) polyprotein by the viral NS3 protease releases functional viral proteins essential for viral replication. Recent studies by Foy and coworkers strongly suggest that NS3-mediated cleavage of host factors may abrogate cellular response to alpha interferon (IFN-alpha) (E. Foy, K. Li, R. Sumpter, Jr., Y.-M. Loo, C. L. Johnson, C. Wang, P. M. Fish, M. Yoneyama, T. Fujita, S. M. Lemon, and M. Gale, Jr., Proc. Natl. Acad. Sci. USA 102:2986-2991, 2005, and E. Foy, K. Li, C. Wang, R. Sumpter, Jr., M. Ikeda, S. M. Lemon, and M. Gale, Jr., Science 300:1145-1148, 2003). Blockage of NS3 protease activity therefore is expected to inhibit HCV replication by both direct suppression of viral protein production as well as by restoring host responsiveness to IFN. Using structure-assisted design, a ketoamide inhibitor, SCH 503034, was generated which demonstrated potent (overall inhibition constant, 14 nM) time-dependent inhibition of the NS3 protease in cell-free enzyme assays as well as robust in vitro activity in the HCV replicon system, as monitored by immunofluorescence and real-time PCR analysis. Continuous exposure of replicon-bearing cell lines to six times the 90% effective concentration of SCH 503034 for 15 days resulted in a greater than 4-log reduction in replicon RNA. The combination of SCH 503034 with IFN was more effective in suppressing replicon synthesis than either compound alone, supporting the suggestion of Foy and coworkers that combinations of IFN with protease inhibitors would lead to enhanced therapeutic efficacy.
Assuntos
Antivirais/farmacologia , Hepacivirus/efeitos dos fármacos , Interferon-alfa/farmacologia , Inibidores de Proteases/uso terapêutico , Replicon/efeitos dos fármacos , Proteínas não Estruturais Virais/antagonistas & inibidores , Sítios de Ligação , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Células Clonais , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Hepacivirus/enzimologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/virologia , Humanos , Hidrólise , Neoplasias Hepáticas/patologia , Modelos Moleculares , Conformação Molecular , Inibidores de Proteases/química , Inibidores de Proteases/metabolismo , Ligação Proteica , Estrutura Secundária de ProteínaRESUMO
Modification of the P(2) and P(1) side chains of earlier P(3)-capped alpha-ketoamide inhibitor of HCV NS3 serine protease 1 resulted in the discovery of compound 24 with about 10-fold improvement in potency.
Assuntos
Alanina/química , Inibidores de Serina Proteinase/química , Inibidores de Serina Proteinase/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Difração de Raios XRESUMO
We have discovered that introduction of appropriate amino acid derivatives at P'2 position improved the binding potency of P3-capped alpha-ketoamide inhibitors of HCV NS3 serine protease. X-ray crystal structure of one of the inhibitors (43) bound to the protease revealed the importance of the P'2 moiety.
Assuntos
Hepacivirus/química , Inibidores de Serina Proteinase/química , Proteínas não Estruturais Virais/antagonistas & inibidores , Sítios de Ligação , Cristalografia por Raios X , Estrutura Molecular , Ligação Proteica , Relação Estrutura-Atividade , Proteínas não Estruturais Virais/químicaRESUMO
The chemistry of the Maillard reaction is one of the most challenging to study due to its extreme complexity. When it come to elucidating the structure of Maillard products bound to macromolecules, whether in foodstuffs or biological proteins, the difficulty becomes enormous. Although the structure of a variety of Maillard compounds has been elucidated in model systems at higher temperature, it is presently unknown to what extent they occur in vivo. Past and current approaches to obtain clues on the chemical nature of Maillard products formed under physiological conditions are reviewed.
Assuntos
Cetoses/metabolismo , Reação de Maillard , Aminas/metabolismo , Metabolismo dos Carboidratos , Fenômenos Químicos , Química , Glicoproteínas/metabolismo , Glicosilação , Proteínas/metabolismoRESUMO
2-(2-Furoyl)-4(5)-(2-furanyl)-1H-imidazole (FFI) is a fluorescent molecule which was originally discovered in chloroform extract of ammoniacal solution of acid-hydrolyzed glycated proteins and proposed to represent a protein cross-link. The absence of a lysyl residue side chain and other observations promoted a detailed study of its mechanism of formation. Glycated alpha-t-Butoxycarbonyllysine was incubated for 29 days and periodically assayed for FFI and FFI-like fluorescence. Whereas fluorescence increased over time, FFI recovery was unexpectedly highest on day 0 and lowest on day 29, suggesting that FFI was directly derived from Amadori products. FFI was also recovered from hydrolysates of glycated neopentylamine, furosine, and browned poly-L-lysine but was virtually undetectable in similar solutions basified with NaOH, triethylamine, or pyridine instead of ammonia. Gas chromatography-mass spectrometry analysis of FFI from similar hydrolysates basified in the presence of 15N-enriched NH4Cl revealed for all precursors a parent ion peak at 230 instead of 228 m/e units, suggesting that the two imidazole nitrogen atoms had been incorporated from free ammonia into FFI. Spontaneous FFI synthesis occurred when furosine was reacted with aqueous ammonia at room temperature. These results do not support the proposition that FFI is an advanced glycosylation end product or a protein cross-link. They suggest that FFI is formed from ammonia and furosine which are by-products of acid-hydrolyzed glycated proteins.
Assuntos
Reagentes de Ligações Cruzadas , Imidazóis , Proteínas , Cromatografia Líquida de Alta Pressão , Glicosilação , Indicadores e Reagentes , Cinética , Espectrometria de Massas , Polilisina , EspectrofotometriaRESUMO
Recent work from this laboratory revealed that glucose-derived pyrroles can form with model amines under physiological conditions (Niroge, F. G., Sayre, L. M., and Monnier, V. M. (1987) Carbohydr. Res. 167, 211-220). The major extractable product, 5-hydroxymethyl-1-alkylpyrrole-2-carbaldehyde (named by us pyrraline) was labile to acid hydrolysis. To allow its detection in proteins undergoing advanced glycosylation, an enzyme-linked immunosorbent assay was developed. An immunogen consisting of epsilon-caproyl pyrraline (hapten) was linked onto poly-L-lysine (114:1) and used to raise polyclonal antibodies in the rabbit. High antibody titers were obtained 16 weeks after immunization. The antibody cross-reacted with butyl pyrraline (88%), propyl pyrraline (8%), lysyl pyrraline (2%), and neopentyl pyrraline (1.3%). A time-related increase in pyrraline immunoreactivity was observed in bovine serum albumin incubated with glucose (1000 mM), glycated lysine (50 mM), and 3-deoxyglucosone (50 mM) which reached 25, 300, and 350 pmol/mg, respectively, after 30 days. Mean level of protein pyrraline immunoreactivity were 27.0 +/- 7.2 and 43.3 +/- 11.7 pmol/mg in serum albumin from control and diabetic subjects, respectively (p less than 0.001). The pathobiological relevance of pyrraline may relate to its reported antiproteolytic and mutagenic properties. In addition, glucose-derived pyrroles may play a role in diabetic neuropathy in analogy to pyrroles formed during hexane-induced neuropathy.
Assuntos
Envelhecimento , Glucose , Reação de Maillard , Soroalbumina Bovina , Ensaio de Imunoadsorção Enzimática , Concentração de Íons de Hidrogênio , Técnicas In Vitro , PirróisRESUMO
Quantitative estimation of isotopic enrichment and concentrations of keto analogs of branched-chain amino acids in biological fluid has been used for the study of protein metabolism in animal and human studies. At present, O-trimethylsilyl-quinoxalinol derivative is used widely in the quantification of branched-chain alpha-keto acids. In the present study, N-methyl-quinoxalone derivative was developed and its use in quantification in human studies verified. O-phenylenediamine and alpha-keto acid react in acidic media to yield phenolic and amide tautomers. O-trimethylsilyl-quinoxalinol derivative of the phenolic tautomer is used at present for quantification by chemical ionization/selected ion monitoring. We have prepared N-methyl-quinoxalone derivative using N,N-dimethyl formamide dimethyl acetal. This derivative is characterized by a major amide form and a minor phenolic form. The mass spectrum has a characteristic fragment, which facilitates easy identification and quantitation by electron impact/selected ion monitoring. Because m/z 174 was observed as the base peak for alpha-ketoisocaproate, alpha-keto-beta-methylvalerate and alpha-ketoisovalerate, "single ion monitoring' could be performed for the quantification of isotopic enrichment as well as plasma concentration of these three branched-chain alpha-keto acids. Isotopic enrichment from 0.25 to 7.5 at% excess could be measured easily, with an average coefficient of variation of less than 8%. Plasma concentrations as low as 10 microM l-1 in a 200-microliters aliquot could be measured. Methyl migration was an interesting feature of the mass spectrometric fragmentation pattern of the alpha-keto acids. The mechanism of methyl migration is proposed and discussed. This paper also describes some of the studies involved in the formation of isomeric O- and N-alkyl, -quinoxaline and -quinoxalone using a number of N,N-dimethyl formamide dialkyl acetals.
Assuntos
Líquidos Corporais/análise , Cetoácidos/análise , Alquilação , Cromatografia Gasosa-Espectrometria de Massas/métodos , Humanos , Indicadores e Reagentes , Espectroscopia de Ressonância Magnética/métodos , Quinoxalinas , Espectrofotometria Infravermelho/métodos , Relação Estrutura-AtividadeRESUMO
The hepatitis C virus (HCV) encodes a chymotrypsin-like serine protease responsible for the processing of HCV nonstructural proteins and which is a promising target for antiviral intervention. Its relatively low catalytic efficiency has made standard approaches to continuous assay development only modestly successful. In this report, four continuous spectrophotometric substrates suitable for both high-throughput screening and detailed kinetic analysis are described. One of these substrates, Ac-DTEDVVP(Nva)-O-4-phenylazophenyl ester, is hydrolyzed by HCV protease with a second-order rate constant (kcat/Km) of 80,000 +/- 10,000 M-1 s-1. Together with its negligible rate of nonenzymatic hydrolysis under assay conditions (0.01 h-1), analysis of as little as 2 nM protease can be completed in under 10 min.
Assuntos
Hepacivirus/enzimologia , Serina Endopeptidases/análise , Espectrofotometria/métodos , Sequência de Aminoácidos , Compostos Cromogênicos/síntese química , Compostos Cromogênicos/química , Hepacivirus/efeitos dos fármacos , Humanos , Técnicas In Vitro , Cinética , Oligopeptídeos/síntese química , Oligopeptídeos/química , Serina Endopeptidases/metabolismo , Inibidores de Serina Proteinase/farmacologia , Especificidade por Substrato , Proteínas não Estruturais Virais/análise , Proteínas não Estruturais Virais/metabolismoRESUMO
The Tg.AC mouse carries an activated v-Ha-ras oncogene fused to an embryonic zeta-globin promoter and develops cutaneous papillomas in response to specific chemicals, full thickness wounding, and ultraviolet radiation. Papilloma development in these mice has been suggested to be dependent upon activation of ras transgene expression, thus providing a potential model for studying ras-inhibitory compounds. Farnesyl transferase inhibitors (FTIs) prevent a critical posttranslational modification step necessary for activation of ras proteins. Our studies demonstrated that a tricyclic FTI (SCH 56582) applied directly to the skin of homozygous Tg.AC mice 1 h prior to administration of the tumor promoter TPA decreased tumor multiplicity compared to TPA-only controls. In addition, a reduction of TPA-induced tumor development was seen in similarly treated hemizygous Tg.AC mice either on an FVB/N strain background or 50% C57BL/6. Histological examination of skin from Tg. AC(+/-):FVB/N mice revealed no differences with respect to 12-O-tetradecamoylpharbol-13-acetate (TPA)-mediated hyperplasia. Keratinocytes isolated from treated and control skin were assayed for ras transgene expression by reverse transcription-polymerase chain reaction, and expression was detected in both TPA- and FTI+TPA-treated tissue, although the appearance of transgene positive pre-papillomas was observed only in histological sections taken 21 d after the first treatment. In summary, we have used a regimen of topical application of an FTI (SCH 56582) to suppress TPA-mediated papillomagenesis in v-Ha-ras transgenic Tg.AC mice. These studies demonstrate that TPA-induced epidermal hyperplasia is a ras-independent process, while papilloma development in response to TPA treatment requires the function of activated ras.
Assuntos
Alquil e Aril Transferases/antagonistas & inibidores , Benzazepinas/farmacologia , Inibidores Enzimáticos/farmacologia , Genes ras , Papiloma/prevenção & controle , Neoplasias Cutâneas/prevenção & controle , Pele/patologia , Acetato de Tetradecanoilforbol/toxicidade , Animais , Cruzamentos Genéticos , Farnesiltranstransferase , Feminino , Globinas/genética , Homozigoto , Hiperplasia , Queratinócitos/efeitos dos fármacos , Queratinócitos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Camundongos Transgênicos , Papiloma/induzido quimicamente , Papiloma/genética , Regiões Promotoras Genéticas , Pele/efeitos dos fármacos , Neoplasias Cutâneas/induzido quimicamente , Neoplasias Cutâneas/genética , Fatores de TempoRESUMO
The resolution of secondary amines via enzyme-catalyzed acylation is a relatively rare process. The kinetic resolution of a series of intermediates of SCH66336 (1), by either enzymatic acylation of the pendant piperidine (4, 5) or hydrolysis of the corresponding carbamate 3, was investigated. In the case of 4, the molecule exists as a pair of enantiomers due to atropisomerism about the exocyclic double bond. The enzymatic acylation of (+/-)-4 was optimized in terms of acylating agent, solvent, and moisture content. The use of lipase, Toyobo LIP-300, and trifluoroethyl isobutyrate as acylating agent resulted in isobutyrylation of the (+)-enantiomer, which is easily separated from the unwanted (-)-4. Hydrolysis of the isobutyramide 6c yielded the desired (+)-4 in high enantiomeric excess. (-)-4 may be recovered from the resolution step, racemized, and resubjected to enzymatic acylation to increase material throughput.