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1.
Org Lett ; 21(22): 9198-9202, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31647672

RESUMO

This communication highlights the use of chiral sulfinamides as nitrogen nucleophiles in intermolecular aza-Michael reactions. When chiral sulfinamides are coupled to a chloroethyl group, the corresponding novel annulating reagents can be used to streamline the stereoselective synthesis of complex pyrrolidine-containing molecules. As a result, it has enabled a medicinal chemistry campaign for the synthesis of biologically active RORγt inverse agonists.

2.
J Med Chem ; 62(21): 9931-9946, 2019 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-31638797

RESUMO

RORγt is an important nuclear receptor that regulates the production of several pro-inflammatory cytokines such as IL-17 and IL-22. As a result, RORγt has been identified as a potential target for the treatment of various immunological disorders such as psoriasis, psoriatic arthritis, and inflammatory bowel diseases. Structure and computer-assisted drug design led to the identification of a novel series of tricyclic RORγt inverse agonists with significantly improved in vitro activity in the reporter (Gal4) and human whole blood assays compared to our previous chemotype. Through careful structure activity relationship, several potent and selective RORγt inverse agonists have been identified. Pharmacokinetic studies allowed the identification of the lead molecule 32 with a low peak-to-trough ratio. This molecule showed excellent activity in an IL-2/IL-23-induced mouse pharmacodynamic study and demonstrated biologic-like efficacy in an IL-23-induced preclinical model of psoriasis.

3.
J Pharm Biomed Anal ; 171: 30-34, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-30959317

RESUMO

Conjugation of macromolecular drugs to polyethylene glycol (PEG) improves their therapeutic potential by reducing their rate of degradation, thereby extending the drugs half life. As a substantial component of the drug, it is necessary to measure the pharmacokinetic (PK) characteristics of PEG in vivo. A quantitative NMR-based method was developed and successfully applied to measuring double-branched polyethylene glycol 40 kDa (PEG40) in serum samples, enabling determination of PK parameters of PEG40 in preclinical species. NMR is ideal for measuring such polymers because a single, sharp peak is obtained for all the equivalent methylene protons; this amplifies the signal and makes the method insensitive to polymeric heterogeneity. High field NMR (600 MHz) with proton-observe cryoprobe technology allowed for analysis of samples in 300 nM range. Mice received 50 mg/kg of PEG40 intravenously (IV) and serum samples were collected at regular intervals for up to 72 h after dosing. The serum samples were analyzed for PEG40 using the NMR method and PK parameters were calculated using non-compartmental analysis. The volume of distribution was determined to be 0.17 L/kg for IV dosing, indicating limited distribution to interstitial space. A low clearance and observed half life of 18 h is consistent with previous reports on the PK properties of a variety of different PEG molecules ranging from 3 kDa to 190 kDa using 125I-labeled PEG in mice. The current NMR technique is easy to implement and does not require labeling of the PEG. Additionally, this is the first report, to our knowledge, of NMR spectroscopy application to PK profiling in serum.


Assuntos
Espectroscopia de Ressonância Magnética/métodos , Veículos Farmacêuticos/farmacocinética , Polietilenoglicóis/farmacocinética , Animais , Meia-Vida , Injeções Intravenosas , Limite de Detecção , Masculino , Camundongos , Veículos Farmacêuticos/administração & dosagem , Polietilenoglicóis/administração & dosagem
4.
ACS Med Chem Lett ; 10(3): 300-305, 2019 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-30891130

RESUMO

We encountered a dilemma in the course of studying a series of antagonists of the G-protein coupled receptor CC chemokine receptor-2 (CCR2): compounds with polar C3 side chains exhibited good ion channel selectivity but poor oral bioavailability, whereas compounds with lipophilic C3 side chains exhibited good oral bioavailability in preclinical species but poor ion channel selectivity. Attempts to solve this through the direct modulation of physicochemical properties failed. However, the installation of a protonation-dependent conformational switching mechanism resolved the problem because it enabled a highly selective and relatively polar molecule to access a small population of a conformer with lower polar surface area and higher membrane permeability. Optimization of the overall properties in this series yielded the CCR2 antagonist BMS-741672 (7), which embodied properties suitable for study in human clinical trials.

5.
Org Lett ; 20(2): 337-340, 2018 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-29283585

RESUMO

The purpose of this letter is to document the use of protected chloroethyl and chloropropyl amines as conformationally unrestricted ambiphilic reagents that undergo annulation reactions with Michael acceptors. This reaction is wide in scope and utilizes reagents that are commercially available, inexpensive, and stable. Furthermore, this reaction is easy to execute and proceeds rapidly.

6.
J Lipid Res ; 55(7): 1366-74, 2014 07.
Artigo em Inglês | MEDLINE | ID: mdl-24755647

RESUMO

A method is described that allows noninvasive identification and quantitative assessment of lipid classes present in sebaceous excretions in rodents. The method relies on direct high-field proton NMR analysis of common group lipid protons in deuterated organic solvent extracts of fur. Extracts from as little as 15 mg of fur from rat, mouse, and hamster provided acceptable results on a 600 MHz NMR equipped with a cryogenically cooled proton-observe probe. In rats, sex- and age-related differences in lipid composition are larger than differences in fur collected from various body regions within an individual and much larger than interanimal differences in age- and sex-matched specimens. The utility of this method to noninvasively monitor drug-induced sebaceous gland atrophy in rodents is demonstrated in rats dosed with a stearoyl-CoA desaturase 1 (SCD1) inhibitor. In this model, a 35% reduction in sebum lipids, extracted from fur, was observed. Finally, structural elucidation of cholesta-7,24-dien-3ß-ol ester as the most prominent, previously unidentified sebum sterol ester in male Syrian hamsters is described. The utility of this method for drug and cosmetic safety and efficacy assessment is discussed.


Assuntos
Pelo Animal/metabolismo , Inibidores Enzimáticos/efeitos adversos , Metabolismo dos Lipídeos/efeitos dos fármacos , Doenças das Glândulas Sebáceas/metabolismo , Estearoil-CoA Dessaturase/antagonistas & inibidores , Animais , Inibidores Enzimáticos/farmacologia , Feminino , Masculino , Mesocricetus , Camundongos , Ressonância Magnética Nuclear Biomolecular , Ratos Sprague-Dawley , Doenças das Glândulas Sebáceas/induzido quimicamente , Estearoil-CoA Dessaturase/metabolismo
7.
Toxicol Sci ; 122(2): 587-97, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21561886

RESUMO

Organic anion-transporting polypeptides (Oatp) 1a1 and 1a4 were deleted by homologous recombination, and mice were characterized for Oatp expression in liver and kidney, transport in isolated hepatocytes, in vivo disposition of substrates, and urinary metabolomic profiles. Oatp1a1 and Oatp1a4 proteins were undetected in liver, and both lines were viable and fertile. Hepatic constitutive messenger RNAs (mRNAs) for Oatp1a4, 1b2, or 2b1 were unchanged in Oatp1a1⁻/⁻ mice, whereas renal Oatp1a4 mRNA decreased approximately 50% (both sexes). In Oatp1a4⁻/⁻ mice, no changes in constitutive mRNAs for other Oatps were observed. Uptake of estradiol-17ß-D-glucuronide and estrone-3-sulfate in primary hepatocytes decreased 95 and 75%, respectively, in Oatp1a1⁻/⁻ mice and by 60 and 30%, respectively, in Oatp1a4⁻/⁻ mice. Taurocholate uptake decreased by 20 and 50% in Oatp1a1⁻/⁻ and Oatp1a4⁻/⁻ mice, respectively, whereas digoxin was unaffected. Plasma area under the curve (AUC) for estradiol-17ß-D-glucuronide increased 35 and 55% in male and female Oatp1a1⁻/⁻ mice, respectively, with a concurrent 50% reduction in liver-to-plasma ratios. In contrast, plasma AUC or tissue concentrations of estradiol-17ß-D-glucuronide were unchanged in Oatp1a4⁻/⁻ mice. Plasma AUCs for dibromosulfophthalein increased nearly threefold in male Oatp1a1⁻/⁻ and Oatp1a4⁻/⁻ mice, increased by 40% in female Oatp1a4⁻/⁻ mice, and were unchanged in female Oatp1a1⁻/⁻ mice. In both lines, no changes in serum ALT, bilirubin, and cholesterol were noted. NMR analyses showed no generalized increase in urinary excretion of organic anions. However, urinary excretion of taurine decreased by 30-40% and was accompanied by increased excretion of isethionic acid, a taurine metabolite generated by intestinal bacteria, suggesting some perturbations in intestinal bacteria distribution.


Assuntos
Deleção de Genes , Recombinação Homóloga , Metabolômica , Transportadores de Ânions Orgânicos/metabolismo , Animais , Área Sob a Curva , Transporte Biológico/genética , Western Blotting , Estradiol/análogos & derivados , Estradiol/sangue , Estradiol/farmacocinética , Estrona/análogos & derivados , Estrona/sangue , Estrona/farmacocinética , Feminino , Hepatócitos/efeitos dos fármacos , Ácido Isetiônico/urina , Rim/efeitos dos fármacos , Fígado/efeitos dos fármacos , Masculino , Camundongos , Camundongos Knockout , Transportadores de Ânions Orgânicos/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Taurina/urina , Ácido Taurocólico/farmacocinética
8.
Anal Chem ; 76(14): 4123-7, 2004 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-15253652

RESUMO

In the present experiments, in vivo microdialysis techniques together with nanoprobe NMR spectroscopy were used to evaluate the neurochemical environment of the rat frontal cortex. Metabonomics techniques of data reduction and pattern recognition were used to examine whether collected neurochemicals were sensitive to tetrodotoxin (TTX), a neurotoxin that when infused into discrete brain regions can help distinguish between the neuronal versus glial origin of neurochemicals in cerebrospinal fluid microdialysate. (1)H NMR spectra recorded on samples collected from the rat frontal cortex before and after an intracortical TTX infusion (10 microM for 60 min) were subjected to multivariate statistical analysis. Glutamate, isoleucine, valine, alanine, and alpha- and beta-hydroxybutyrate were found to have decreased concentrations after the addition of TTX, suggesting that their release is likely from cortical neurons. In contrast, lactate, formate, acetate, glucose, creatinine, pyruvate, and other neurochemicals remained unchanged following local application of TTX. The present findings extend our previous work combining the analytical technology of small-volume nanoprobe NMR spectroscopy with in vivo microdialysis in freely moving animals and show that it is possible to apply metabonomics methodology to this important class of biofluid to monitor changes in neurochemical composition of the rat brain.


Assuntos
Química Encefálica , Espectroscopia de Ressonância Magnética/métodos , Microdiálise/instrumentação , Anestésicos Locais/farmacologia , Animais , Masculino , Nanotecnologia , Córtex Pré-Frontal/química , Córtex Pré-Frontal/metabolismo , Ratos , Tetrodotoxina/farmacocinética , Tetrodotoxina/farmacologia
9.
J Neurosci Methods ; 133(1-2): 181-9, 2004 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-14757359

RESUMO

Nuclear magnetic resonance (NMR) spectroscopy was used to study the chemical composition of cerebrospinal fluid (CSF) microdialysate from the rat brain. In vivo microdialysis techniques were used in several brain regions including the frontal cortex, amygdala, striatum, nucleus accumbens and third ventricle and dialysate samples (20microl) were subsequently analyzed by one and two-dimensional 1H NMR experiments using a Varian nanoprobe. Neurochemical resonances were assigned on the basis of published chemical shifts [Lindon et al., Ann. Rep. NMR Spectrosc. 38 (1999) 1-88], correlation experiments and addition of standard compounds. Glucose, lactate, formate, pyruvate, creatinine, gamma-hydroxybutyrate, acetate, glutamate, glycine, tyrosine, isoleucine, leucine, alanine and choline were some of the neurochemicals unambiguously assigned. Additional studies in the frontal cortex showed that amino acids such as glutamate, alanine and isoleucine were sensitive to local tetrodotoxin (TTX) infusion. The NMR spectra were also subjected to multivariate statistical methods to compare the different brain regions examined. To our knowledge, the present experiments are the first to describe the combination of nanoprobe NMR technology with in vivo microdialysis for the analysis of brain neurochemistry in freely-moving rats.


Assuntos
Química Encefálica/fisiologia , Encéfalo/metabolismo , Espectroscopia de Ressonância Magnética/métodos , Microdiálise/métodos , Nanotecnologia/métodos , Animais , Encéfalo/anatomia & histologia , Masculino , Análise Multivariada , Neuroquímica/métodos , Ratos , Ratos Sprague-Dawley , Software/estatística & dados numéricos
10.
Biochemistry ; 41(38): 11425-37, 2002 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-12234185

RESUMO

Virulence of pathogenic bacteria of the genus Yersinia requires the injection of six effector proteins into the cytoplasm of host cells. The amino-terminal domain of one of these effectors, the tyrosine phosphatase YopH, is essential for translocation of YopH, as well as for targeting it to phosphotyrosine-containing substrates of the type pYxxP. We report the high-resolution solution structure of the N-terminal domain (residues 1-129) from the Yersinia pseudotuberculosis YopH (YopH-NT) in complex with N-acetyl-DEpYDDPF-NH(2), a peptide derived from an in vivo protein substrate. In contrast to the domain-swapped dimer observed in a crystal structure of the same protein (Smith, C. L., Khandelwal, P., Keliikuli, K., Zuiderweg, E. R. P., and Saper, M. A. (2001) Mol. Microbiol. 42, 967-979), YopH-NT is monomeric in solution. The peptide binding site is located on a beta-hairpin that becomes the crossover point in the dimer structure. The binding site has several characteristics that are reminiscent of SH2 domains, which also bind to pYxxP sequences.


Assuntos
Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Fosfolipídeos/metabolismo , Proteínas Tirosina Fosfatases/química , Proteínas Tirosina Fosfatases/metabolismo , Yersinia pseudotuberculosis/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Dimerização , Ligações de Hidrogênio , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Conformação Proteica , Estrutura Secundária de Proteína , Soluções
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