RESUMO
The free-state solution behaviors of small molecules profoundly affect their respective properties. It is becoming more obvious that compounds can adopt a three-phase equilibrium when placed in an aqueous solution, among soluble-lone molecule form, self-assembled aggregate form (nano-entities), and solid precipitate form. Recently, correlations have emerged between the existence of self-assemblies into drug nano-entities and unintended side effects. This report describes our pilot study involving a selection of drugs and dyes to explore if there may be a correlation between the existence of drug nano-entities and immune responses. We first implement practical strategies for detecting the drug self-assemblies using a combination of nuclear magnetic resonance (NMR), dynamic light scattering (DLS), transmission electron microscopy (TEM), and confocal microscopy. We then used enzyme-linked immunosorbent assays (ELISA) to monitor the modulation of immune responses on two cellular models, murine macrophage and human neutrophils, upon exposure to the drugs and dyes. The results suggest that exposure to some aggregates correlated with an increase in IL-8 and TNF-α in these model systems. Given this pilot study, further correlations merit pursuing on a larger scale given the importance and potential impact of drug-induced immune-related side effects.
Assuntos
Corantes , Água , Animais , Humanos , Camundongos , Projetos Piloto , Água/química , Espectroscopia de Ressonância Magnética , ImunidadeRESUMO
A classic synthetic issue that remains unresolved is the reaction that involves the control of N- versus O-alkylation of ambident anions. This common chemical transformation is important for medicinal chemists, who require predictable and reliable protocols for the rapid synthesis of inhibitors. The uncertainty of whether the product(s) are N- and/or O-alkylated is common and can be costly if undetermined. Herein, we report an NMR-based strategy that focuses on distinguishing inhibitors and intermediates that are N- or O-alkylated. The NMR strategy involves three independent and complementary methods. However, any combination of two of the methods can be reliable if the third were compromised due to resonance overlap or other issues. The timely nature of these methods (HSQC/HMQC, HMBC. ROESY, and (13)C shift predictions) allows for contemporaneous determination of regioselective alkylation as needed during the optimization of synthetic routes.
Assuntos
Descoberta de Drogas , Inibidores Enzimáticos/química , Espectroscopia de Ressonância Magnética , Nitrogênio/química , Oxigênio/química , Alquilação , Cristalografia por Raios X , Transcriptase Reversa do HIV/antagonistas & inibidores , Humanos , Concentração Inibidora 50 , Estrutura MolecularRESUMO
Replacement of the benzimidazole core of allosteric Thumb Pocket 1 HCV NS5B finger loop inhibitors by more lipophilic indole derivatives provided up to 30-fold potency improvements in cell-based subgenomic replicon assays. Optimization of C-2 substitution on the indole core led to the identification of analogs with EC(50)<100 nM and modulated the pharmacokinetic properties of the inhibitors based on preliminary data from in vitro ADME profiles and in vivo rat PK.
Assuntos
Amidas/química , Benzimidazóis/síntese química , Hepacivirus/efeitos dos fármacos , Indóis/síntese química , Proteínas não Estruturais Virais/antagonistas & inibidores , Regulação Alostérica , Amidas/síntese química , Amidas/farmacologia , Animais , Benzimidazóis/química , Benzimidazóis/farmacologia , Células CACO-2 , Hepacivirus/enzimologia , Hepacivirus/genética , Humanos , Indóis/química , Indóis/farmacologia , Concentração Inibidora 50 , Estrutura Molecular , Ratos , Solubilidade , Relação Estrutura-Atividade , Proteínas não Estruturais Virais/genéticaRESUMO
In this part 2, new indole 5-carboxamide Thumb Pocket 1 inhibitors of HCV NS5B polymerase are described. Structure-activity relationships (SAR) were explored at the central amino acid linker position and the right-hand-side of the molecule in an attempt to identify molecules with a balanced overall profile of potency (EC(50)<100 nM), physicochemical properties and ADME characteristics.
Assuntos
Aminoácidos/química , Benzimidazóis/síntese química , Hepacivirus/efeitos dos fármacos , Hepacivirus/enzimologia , Indóis/síntese química , Proteínas não Estruturais Virais/antagonistas & inibidores , Regulação Alostérica , Sequência de Aminoácidos , Aminoácidos/síntese química , Aminoácidos/farmacologia , Animais , Benzimidazóis/química , Benzimidazóis/farmacologia , Células CACO-2 , Hepacivirus/genética , Humanos , Indóis/química , Indóis/farmacologia , Concentração Inibidora 50 , Dados de Sequência Molecular , Estrutura Molecular , Ratos , Solubilidade , Relação Estrutura-Atividade , Proteínas não Estruturais Virais/genéticaRESUMO
Significant advances have led to receptor induced-fit and conformational selection models for describing bimolecular recognition, but a more comprehensive view must evolve to also include ligand shape and conformational changes. Here, we describe an example where a ligand's "structural hinge" influences potency by inducing an "L-shape" bioactive conformation, and due to its solvent exposure in the complex, reasonable conformation-activity-relationships can be qualitatively attributed. From a ligand design perspective, this feature was exploited by successful linker hopping to an alternate "structural hinge" that led to a new and promising chemical series which matched the ligand bioactive conformation and the pocket bioactive space. Using a combination of X-ray crystallography, NMR and modeling with support from binding-site resistance mutant studies and photoaffinity labeling experiments, we were able to derive inhibitor-polymerase complexes for various chemical series.
Assuntos
Diamida/química , Diamida/farmacologia , Descoberta de Drogas , Hepacivirus , Indóis/química , Conformação Molecular , Proteínas não Estruturais Virais/antagonistas & inibidores , Regulação Alostérica , Diamida/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Concentração Inibidora 50 , Ligantes , Modelos Moleculares , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismoRESUMO
Previously described SAR of benzimidazole-based non-nucleoside finger loop (Thumb Pocket I) inhibitors of HCV NS5B polymerase was expanded. Prospecting studies using parallel synthesis techniques allowed the rapid identification of novel cinnamic acid right-hand sides that provide renewed opportunities for further optimization of these inhibitors. Novel diamide derivatives such as 44 exhibited comparable potency (enzymatic and cell-based HCV replicon) as previously described tryptophan-based inhibitors but physicochemical properties (e.g., aqueous solubility and lipophilicity) have been improved, resulting in molecules with reduced off-target liabilities (CYP inhibition) and increased metabolic stability.
Assuntos
Antivirais/síntese química , Benzimidazóis/química , Diamida/síntese química , Inibidores Enzimáticos/síntese química , Proteínas não Estruturais Virais/antagonistas & inibidores , Regulação Alostérica , Antivirais/química , Antivirais/farmacologia , Cinamatos/química , Diamida/química , Diamida/farmacologia , Descoberta de Drogas , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Microssomos Hepáticos/metabolismo , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Proteínas não Estruturais Virais/metabolismoRESUMO
SAR at the C-2 position of benzimidazole-based Thumb Pocket I inhibitors of HCV NS5B polymerase revealed parallel activity for distinct sub-series that harbor 5-hydroxytryptophan amides, neutral thiazole isosteres or recently disclosed cinnamic acid diamides. The consistent SAR among the three sub-series suggest a common binding mode to the Thumb Pocket I allosteric site. New inhibitors with sub-micromolar cell-based replicon potency and improved 'drug-like' features are disclosed along with preliminary characterization of their ADME-PK profile.
Assuntos
Benzimidazóis/farmacologia , Inibidores Enzimáticos/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Administração Oral , Animais , Benzimidazóis/sangue , Benzimidazóis/química , Inibidores Enzimáticos/sangue , Inibidores Enzimáticos/química , Humanos , Ratos , Relação Estrutura-AtividadeRESUMO
SAR studies at the N(1)-position of allosteric indole-based HCV NS5B inhibitors has led to the discovery of acetamide derivatives with good cellular potency in subgenomic replicons (EC(50) <200 nM). This class of inhibitors displayed improved physicochemical properties and favorable ADME-PK profiles over previously described analogs in this class.
Assuntos
Acetamidas/química , Antivirais/síntese química , Ácidos Carboxílicos/química , Descoberta de Drogas , Hepacivirus/enzimologia , RNA Polimerase Dependente de RNA/antagonistas & inibidores , Proteínas não Estruturais Virais/antagonistas & inibidores , Acetamidas/farmacologia , Regulação Alostérica/efeitos dos fármacos , Regulação Alostérica/fisiologia , Antivirais/farmacologia , Células CACO-2 , Ácidos Carboxílicos/farmacologia , Linhagem Celular , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , RNA Polimerases Dirigidas por DNA/metabolismo , Descoberta de Drogas/métodos , Hepacivirus/efeitos dos fármacos , Humanos , Microssomos Hepáticos/enzimologia , RNA Polimerase Dependente de RNA/metabolismo , Proteínas não Estruturais Virais/metabolismoRESUMO
A high-throughput screen based on a viral replication assay was used to identify inhibitors of the human cytomegalovirus. Using this approach, hit compound 1 was identified as a 4 µM inhibitor of HCMV that was specific and selective over other herpes viruses. Time of addition studies indicated compound 1 exerted its antiviral effect early in the viral life cycle. Mechanism of action studies also revealed that this series inhibited infection of MRC-5 and ARPE19 cells by free virus and via direct cell-to-cell spread from infected to uninfected cells. Preliminary structure-activity relationships demonstrated that the potency of compound 1 could be improved to a low nanomolar level, but metabolic stability was a key optimization parameter for this series. A strategy focused on minimizing metabolic hydrolysis of the N1-amide led to an alternative scaffold in this series with improved metabolic stability and good pharmacokinetic parameters in rat.
RESUMO
A previously disclosed series of non-nucleoside allosteric inhibitors of the NS5B polymerase of the hepatitis C virus (HCV) was optimized to yield novel compounds with improved physicochemical properties and activity in cell-based assays. Replacement of ionizable carboxylic acids with neutral substituents in lead compounds produced inhibitors with cellular permeability and antiviral activity in a cell-based assay of subgenomic HCV RNA replication (replicon EC(50) as low as 1.7 microM). The improvement in potency in this ex vivo model of HCV RNA replication validates, in part, the mechanism by which this class of allosteric benzimidazole derivatives inhibits the polymerase and represents a significant step forward in the discovery of novel HCV therapeutics.
Assuntos
Antivirais/síntese química , Benzimidazóis/síntese química , Inibidores Enzimáticos/síntese química , Hepacivirus/efeitos dos fármacos , Replicon/efeitos dos fármacos , Proteínas não Estruturais Virais/antagonistas & inibidores , Regulação Alostérica , Antivirais/farmacologia , Benzimidazóis/farmacologia , Inibidores Enzimáticos/farmacologia , Hepacivirus/genética , Humanos , Relação Estrutura-AtividadeRESUMO
We have developed a highly convergent and stereoselective synthesis of BILA 2157 BS, a potent and orally active renin inhibitor. The synthesis proceeds in 15 distinct chemical steps (with several integrated, multistep operations) from aminodiol 4. The key step in the synthesis involves the use of an enantiospecific, enzyme-catalyzed hydrolysis of a substituted succinate diester to provide a homochiral succinic acid derivative in 98% enantiomeric excess (>/=2.5 kg scale). Recycling of the unwanted enantiomer is accomplished through base-catalyzed racemization, leading to an efficient deracemization of the starting racemic diester. The entire sequence proceeds without chromatographic purifications and delivers the product with >97% homogeneity. In addition, compared to the previously reported syntheses of BILA 2157 BS, this approach avoids the use of expensive chiral auxiliaries and cryogenics and, thus, should be amenable to the preparation of large quantities of this peptidomimetic inhibitor.
RESUMO
Conformational restrictions of flexible torsion angles were used to guide the identification of new chemotypes of HCV NS5B inhibitors. Sites for rigidification were based on an acquired conformational understanding of compound binding requirements and the roles of substituents in the free and bound states. Chemical bioisosteres of amide bonds were explored to improve cell-based potency. Examples are shown, including the design concept that led to the discovery of the phase III clinical candidate deleobuvir (BI 207127). The structure-based strategies employed have general utility in drug design.
Assuntos
Antivirais/farmacologia , Benzimidazóis/farmacologia , Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Hepacivirus/efeitos dos fármacos , Indóis/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Antivirais/química , Benzimidazóis/química , Cristalografia por Raios X , Inibidores Enzimáticos/química , Hepacivirus/enzimologia , Indóis/química , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Conformação MolecularRESUMO
An anthranilic acid series of allosteric thumb pocket 2 HCV NS5B polymerase inhibitors exhibited hindered rotation along a covalent bond axis, and the existence of atropisomer chirality was confirmed by NMR, HPLC analysis on chiral supports, and computational studies. A thorough understanding of the concerted rotational properties and the influence exerted by substituents involved in this steric phenomenon was attained through biophysical studies on a series of truncated analogues. The racemization half-life of a compound within this series was determined to be 69 min, which was consistent with a class 2 atropisomer (intermediate conformational exchange). It was further found by X-ray crystallography that one enantiomer of a compound bound to the intended HCV NS5B polymerase target whereas the mirror image atropisomer was able to bind to an unrelated HIV matrix target. Analogues were then identified that selectively inhibited the former. These studies highlight that atropisomer chirality can lead to distinct entities with specific properties, and the phenomenon of atropisomerism in drug discovery should be evaluated and appropriately managed.
Assuntos
Antivirais/farmacologia , HIV/efeitos dos fármacos , Hepacivirus/efeitos dos fármacos , Proteínas não Estruturais Virais/antagonistas & inibidores , Regulação Alostérica , Antivirais/química , Antivirais/farmacocinética , Cromatografia Líquida de Alta Pressão , Cristalografia por Raios X , Hepacivirus/enzimologia , Espectroscopia de Ressonância Magnética , EstereoisomerismoRESUMO
The development of interferon-free regimens for the treatment of chronic HCV infection constitutes a preferred option that is expected in the future to provide patients with improved efficacy, better tolerability, and reduced risk for emergence of drug-resistant virus. We have pursued non-nucleoside NS5B polymerase allosteric inhibitors as combination partners with other direct acting antivirals (DAAs) having a complementary mechanism of action. Herein, we describe the discovery of a potent follow-up compound (BI 207524, 27) to the first thumb pocket 1 NS5B inhibitor to demonstrate antiviral activity in genotype 1 HCV infected patients, BILB 1941 (1). Cell-based replicon potency was significantly improved through electronic modulation of the pKa of the carboxylic acid function of the lead molecule. Subsequent ADME-PK optimization lead to 27, a predicted low clearance compound in man. The preclinical profile of inhibitor 27 is discussed, as well as the identification of a genotoxic metabolite that led to the discontinuation of the development of this compound.
Assuntos
Acrilatos/síntese química , Antivirais/síntese química , Antivirais/metabolismo , Indóis/síntese química , Proteínas não Estruturais Virais/antagonistas & inibidores , Acrilatos/metabolismo , Acrilatos/farmacocinética , Animais , Antivirais/farmacologia , Cinamatos/síntese química , Cinamatos/metabolismo , Cinamatos/farmacocinética , Cães , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacocinética , Hepatite C Crônica , Humanos , Indóis/metabolismo , Indóis/farmacocinética , Macaca mulatta , RNA Polimerase Dependente de RNA/antagonistas & inibidores , Ratos , Relação Estrutura-AtividadeRESUMO
The design and preliminary SAR of a new series of 1H-quinazolin-4-one (QAZ) allosteric HCV NS5B thumb pocket 2 (TP-2) inhibitors was recently reported. To support optimization efforts, a molecular dynamics (MD) based modeling workflow was implemented, providing information on QAZ binding interactions with NS5B. This approach predicted a small but critical ligand-binding induced movement of a protein backbone region which increases the pocket size and improves access to the backbone carbonyl groups of Val 494 and Pro 495. This localized backbone shift was consistent with key SAR results and was subsequently confirmed by X-ray crystallography. The MD protocol guided the design of inhibitors, exploiting novel H-bond interactions with the two backbone carbonyl groups, leading to the first thumb pocket 2 NS5B inhibitor with picomolar antiviral potency in genotype (gt) 1a and 1b replicons (EC50 = 120 and 110 pM, respectively) and with EC50 ≤ 80 nM against gt 2-6.
Assuntos
Antivirais/farmacologia , Inibidores Enzimáticos/farmacologia , Hepacivirus/efeitos dos fármacos , Replicon/efeitos dos fármacos , Proteínas não Estruturais Virais/antagonistas & inibidores , Regulação Alostérica , Antivirais/química , Linhagem Celular , Cristalografia por Raios X , Inibidores Enzimáticos/química , Hepacivirus/enzimologia , Hepacivirus/genética , Simulação de Dinâmica Molecular , Relação Estrutura-AtividadeRESUMO
The pharmaceutical industry has recognized that many drug-like molecules can self-aggregate in aqueous media and have physicochemical properties that skew experimental results and decisions. Herein, we introduce the use of a simple NMR strategy for detecting the formation of aggregates using dilution experiments that can be performed on equipment prevalent in most synthetic chemistry departments. We show that (1)H NMR resonances are sensitive to large molecular-size entities and to smaller multimers and mixtures of species. Practical details are provided for sample preparation and for determining the concentrations of single molecule, aggregate entities, and precipitate. The critical concentrations above which aggregation begins can be found and were corroborated by comparisons with light scattering techniques. Disaggregation can also be monitored using detergents. This NMR assay should serve as a practical and readily available tool for medicinal chemists to better characterize how their compounds behave in aqueous media and influence drug design decisions.
Assuntos
Descoberta de Drogas/métodos , Preparações Farmacêuticas/química , Detergentes/química , Espectroscopia de Ressonância Magnética , SolubilidadeRESUMO
Combinations of direct acting antivirals (DAAs) that have the potential to suppress emergence of resistant virus and that can be used in interferon-sparing regimens represent a preferred option for the treatment of chronic HCV infection. We have discovered allosteric (thumb pocket 1) non-nucleoside inhibitors of HCV NS5B polymerase that inhibit replication in replicon systems. Herein, we report the late-stage optimization of indole-based inhibitors, which began with the identification of a metabolic liability common to many previously reported inhibitors in this series. By use of parallel synthesis techniques, a sparse matrix of inhibitors was generated that provided a collection of inhibitors satisfying potency criteria and displaying improved in vitro ADME profiles. "Cassette" screening for oral absorption in rat provided a short list of potential development candidates. Further evaluation led to the discovery of the first thumb pocket 1 NS5B inhibitor (BILB 1941) that demonstrated antiviral activity in patients chronically infected with genotype 1 HCV.
Assuntos
Antivirais/farmacologia , Inibidores Enzimáticos/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Genótipo , Humanos , Modelos Moleculares , Proteínas não Estruturais Virais/químicaAssuntos
Benzimidazóis/química , Hepacivirus/enzimologia , RNA Polimerase Dependente de RNA/antagonistas & inibidores , Proteínas não Estruturais Virais/antagonistas & inibidores , Benzimidazóis/farmacologia , Inibidores Enzimáticos/química , Humanos , Concentração Inibidora 50 , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Ligação Proteica , Relação Estrutura-AtividadeRESUMO
Benzimidazole-based allosteric inhibitors of the hepatitis C virus (HCV) NS5B polymerase were diversified to a variety of topologically related scaffolds. Replacement of the polar benzimidazole core by lipophilic indoles led to inhibitors with improved potency in the cell-based subgenomic HCV replicon system. Transposing the indole scaffold into a previously described series of benzimidazole-tryptophan amides generated the most potent inhibitors of HCV RNA replication in cell culture reported to date in this series (EC(50) approximately 50 nM).
Assuntos
Benzimidazóis/farmacologia , Hepacivirus/efeitos dos fármacos , Indóis/farmacologia , Replicon/efeitos dos fármacos , Proteínas não Estruturais Virais/antagonistas & inibidores , Regulação Alostérica , Linhagem Celular , Humanos , Concentração Inibidora 50 , RNA Polimerase Dependente de RNA/antagonistas & inibidores , Relação Estrutura-AtividadeRESUMO
(1R,2S)-1-Amino-2-vinylcyclopropanecarboxylic acid (vinyl-ACCA) is a key building block in the synthesis of potent inhibitors of the hepatitis C virus NS3 protease such as BILN 2061, which was recently shown to dramatically reduce viral load after administration to patients infected with HCV genotype 1. We have developed a scalable process that delivers derivatives of this unusual amino acid in >99% ee. The strategy was based on the dialkylation of a glycine Schiff base using trans-1,4-dibromo-2-butene as an electrophile to produce racemic vinyl-ACCA, which was subsequently resolved using a readily available, inexpensive esterase enzyme (Alcalase 2.4L). Factors that affect diastereoselection in the initial dialkylation steps were examined and the conditions optimized to deliver the desired diastereomer selectively. Product inhibition, which was encountered during the enzymatic resolution step, initially resulted in prolonged cycle times. Enrichment of racemic vinyl-ACCA through a chemical resolution via diastereomeric salt formation or the use of forcing conditions in the enzymatic reaction both led to improvements in throughput and the development of a viable process. The chemistry described herein was scaled up to produce multikilogram quantities of this building block.