RESUMEN
HCV infections are the leading causes for hepatocellular carcinoma and liver transplantation in the United States. Recent advances in drug discovery have identified direct acting antivirals which have significantly improved cure rates in patients. Current efforts are directed towards identification of novel direct acting antiviral targeting different mechanism of actions which could become part of all oral therapies. We recently disclosed the identification of a novel tricyclic indole derived inhibitors of HCV NS5B polymerase that bound to the enzyme close to the active site. In this manuscript we describe further optimization of potency and pharmacokinetics (PK) of these inhibitors to identify compounds in low nM potency against gt-1b. These analogs also demonstrate excellent PK in rats and monkeys when administered as a dimethyl ethyl amino ester prodrug.
Asunto(s)
Ésteres/farmacocinética , Hepacivirus/efectos de los fármacos , Indoles/farmacocinética , Proteínas no Estructurales Virales/antagonistas & inhibidores , Administración Oral , Animales , Descubrimiento de Drogas , Ésteres/química , Haplorrinos , Hepacivirus/enzimología , Humanos , Indoles/química , Profármacos/farmacología , Ratas , Relación Estructura-ActividadRESUMEN
The discovery of lead compound 2e was described. Its covalent binding to HCV NS5B polymerase enzyme was investigated by X-ray analysis. The results of distribution, metabolism and pharmacokinetics were reported. Compound 2e was demonstrated to be potent (replicon GT-1b EC50 = 0.003 µM), highly selective, and safe in in vitro and in vivo assays.
Asunto(s)
Inhibidores Enzimáticos/química , Hepacivirus/enzimología , Indoles/química , Quinolinas/química , Proteínas no Estructurales Virales/antagonistas & inhibidores , Animales , Cristalografía por Rayos X , Perros , Evaluación Preclínica de Medicamentos , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacocinética , Inhibidores Enzimáticos/farmacología , Haplorrinos , Humanos , Indoles/síntesis química , Indoles/farmacocinética , Indoles/farmacología , Masculino , Simulación de Dinámica Molecular , Unión Proteica , Estructura Terciaria de Proteína , Quinolinas/síntesis química , Quinolinas/farmacocinética , Quinolinas/farmacología , Ratas , Ratas Sprague-Dawley , Proteínas no Estructurales Virales/metabolismoRESUMEN
The characterization of HCV genome has identified various vital functional proteins involved in the life cycle of hepatitis C virus. This has resulted in many novel enzymatic targets that are potential for development of therapeutic agents. The HCV RNA dependent RNA polymerase (HCV NS5B) is one such essential enzyme for HCV replication that has been well characterized and studied by various groups to develop novel therapies for hepatitis C. In this paper, we describe our efforts towards the identification and structure-activity relationship (SAR) of novel tricyclic indole derivatives that bind close to the palm site of the NS5B polymerase. X-ray crystal structure of an inhibitor bound to the polymerase is also described.
Asunto(s)
Antivirales/química , Antivirales/farmacología , Hepacivirus/enzimología , Indoles/química , Indoles/farmacología , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , Sitios de Unión , Cristalografía por Rayos X , Hepacivirus/química , Hepatitis C/tratamiento farmacológico , Hepatitis C/virología , Humanos , Simulación del Acoplamiento Molecular , ARN Polimerasa Dependiente del ARN/química , ARN Polimerasa Dependiente del ARN/metabolismo , Relación Estructura-ActividadRESUMEN
Development of SAR at the C2 position of indole lead 1, a palm site inhibitor of HCV NS5B polymerase (NS5B IC(50)=0.053µM, replicon EC(50)=4.8µM), is described. Initial screening identified an acyl sulfonamide moiety as an isostere for the C2 carboxylic acid group. Further SAR investigation resulted in identification of acyl sufonamide analog 7q (NS5B IC(50)=0.039µM, replicon EC(50)=0.011µM) with >100-fold improved replicon activity.
Asunto(s)
Antivirales/farmacología , Indoles/química , Proteínas no Estructurales Virales/antagonistas & inhibidores , Química Farmacéutica/métodos , Cristalografía por Rayos X/métodos , Diseño de Fármacos , Humanos , Enlace de Hidrógeno , Concentración 50 Inhibidora , Modelos Químicos , Modelos Moleculares , Conformación Molecular , Relación Estructura-Actividad , Sulfonamidas/químicaRESUMEN
The installation of geminal substitution at the C5' position of the carbosugar in our pyrimidine-derived hepatitis C inhibitor series is reported. SAR studies around the C5' position led to the installation of the dimethyl group as the optimal functionality. An improved route was subsequently designed to access these substitutions. Expanded SAR at the C2 amino position led to the utilization of C2 ethers. These compounds exhibited good potency, high selectivity, and excellent plasma exposure and bioavailability in rodent as well as in higher species.
Asunto(s)
Antivirales/síntesis química , Carbohidratos/química , Pirimidinas/química , Animales , Antivirales/química , Antivirales/farmacocinética , Disponibilidad Biológica , Perros , Semivida , Haplorrinos , Hepacivirus/efectos de los fármacos , Hepacivirus/metabolismo , Pirimidinas/síntesis química , Pirimidinas/farmacocinética , Ratas , Relación Estructura-Actividad , Replicación Viral/efectos de los fármacosRESUMEN
Introduction of a nitrogen atom into the benzene ring of a previously identified HCV replication (replicase) benzothiazole inhibitor 1, resulted in the discovery of the more potent pyridothiazole analogues 3. The potency and PK properties of the compounds were attenuated by the introductions of various functionalities at the R(1), R(2) or R(3) positions of the molecule (compound 3). Inhibitors 38 and 44 displayed excellent potency, selectivity (GAPDH/MTS CC(50)), PK parameters in all species studied, and cross genotype activity.
Asunto(s)
Antivirales/química , Antivirales/farmacología , Hepacivirus/efectos de los fármacos , Pirimidinas/química , Pirimidinas/farmacología , Replicación Viral/efectos de los fármacos , Animales , Antivirales/farmacocinética , Perros , Hepatitis C/tratamiento farmacológico , Hepatitis C/virología , Humanos , Pirimidinas/farmacocinética , Ratas , Relación Estructura-Actividad , Tiazoles/química , Tiazoles/farmacocinética , Tiazoles/farmacologíaRESUMEN
Based on a previously identified HCV replication (replicase) inhibitor 1, SAR efforts were conducted around the pyrimidine core to improve the potency and pharmacokinetic profile of the inhibitors. A benzothiazole moiety was found to be the optimal substituent at the pyrimidine 5-position. Due to potential reactivity concern, the 4-chloro residue was replaced by a methyl group with some loss in potency and enhanced rat in vivo profile. Extensive investigations at the C-2 position resulted in identification of compound 16 that demonstrated very good replicon potency, selectivity and rodent plasma/target organ concentration. Inhibitor 16 also demonstrated good plasma levels and oral bioavailability in dogs, while monkey exposure was rather low. Chemistry optimization towards a practical route to install the benzothiazole moiety resulted in an efficient direct C-H arylation protocol.
Asunto(s)
Antivirales/química , Benzotiazoles/química , Hepacivirus/efectos de los fármacos , Pirimidinas/química , Replicación Viral/efectos de los fármacos , Animales , Perros , Haplorrinos , Hepacivirus/fisiología , Metilación , Roedores , Especificidad de la EspecieRESUMEN
SAR development of indole-based palm site inhibitors of HCV NS5B polymerase exemplified by initial indole lead 1 (NS5B IC(50)=0.9 µM, replicon EC(50)>100 µM) is described. Structure-based drug design led to the incorporation of novel heterocyclic moieties at the indole C3-position which formed a bidentate interaction with the protein backbone. SAR development resulted in leads 7q (NS5B IC(50)=0.032 µM, replicon EC(50)=1.4 µM) and 7r (NS5B IC(50)=0.017 µM, replicon EC(50)=0.3 µM) with improved enzyme and replicon activity.
Asunto(s)
Descubrimiento de Drogas , Inhibidores Enzimáticos/farmacología , Compuestos Heterocíclicos/farmacología , Indoles/farmacología , Proteínas no Estructurales Virales/antagonistas & inhibidores , Ácidos Carboxílicos , Dominio Catalítico/efectos de los fármacos , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Compuestos Heterocíclicos/síntesis química , Compuestos Heterocíclicos/química , Indoles/síntesis química , Indoles/química , Modelos Moleculares , Estructura Molecular , Estereoisomerismo , Relación Estructura-Actividad , Proteínas no Estructurales Virales/metabolismoRESUMEN
Blood borne hepatitis C infections are the primary cause for liver cirrhosis and hepatocellular carcinoma. HCV NS3 protease, a pivotal enzyme in the replication cycle of HCV virus has been the primary target for development of new drug candidates. Boceprevir and telaprevir are two novel ketoamide derived inhibitors that are currently undergoing phase-III clinical trials. These inhibitors include ketoamide functionality as serine trap and have an acidic alpha-ketoamide center that undergoes epimerization under physiological conditions. Our initial attempts to arrest this epimerization by introducing quaternary amino acids at P(1) had resulted in significantly diminished activity. In this manuscript we describe alpha quaternized P(1) group that result in potent inhibitors in the enzyme assay and demonstrate cellular activity comparable to boceprevir.
Asunto(s)
Amidas/farmacología , Antivirales/farmacología , Hepacivirus/enzimología , Hepatitis C/tratamiento farmacológico , Proteínas no Estructurales Virales/antagonistas & inhibidores , Proteínas no Estructurales Virales/metabolismo , Amidas/química , Antivirales/química , Humanos , Prolina/análogos & derivados , Prolina/farmacología , Inhibidores de Proteasas/química , Inhibidores de Proteasas/farmacología , Relación Estructura-ActividadRESUMEN
In the search for a second generation HCV protease inhibitor, molecular modeling studies of the X-ray crystal structure of Boceprevir1 bound to the NS3 protein suggest that expansion into the S4 pocket could provide additional hydrophobic Van der Waals interactions. Effective replacement of the P4 tert-butyl with a cyclohexylmethyl ligand led to inhibitor 2 with improved enzyme and replicon activities. Subsequent modeling and SAR studies led to the pyridine 38 and sulfone analogues 52 and 53 with vastly improved PK parameters in monkeys, forming a new foundation for further exploration.
Asunto(s)
Antivirales/química , Prolina/análogos & derivados , Inhibidores de Proteasas/química , Proteínas no Estructurales Virales/antagonistas & inhibidores , Animales , Antivirales/farmacocinética , Antivirales/farmacología , Área Bajo la Curva , Disponibilidad Biológica , Cristalografía por Rayos X , Haplorrinos , Modelos Moleculares , Prolina/química , Prolina/farmacocinética , Prolina/farmacología , Inhibidores de Proteasas/farmacocinética , Inhibidores de Proteasas/farmacología , Ratas , Relación Estructura-ActividadRESUMEN
Hepatitis is a disease characterized by inflammation of the liver, usually producing swelling and, in many cases, permanent damage to liver tissues. Viral hepatitis C (HCV), a small (+)-RNA virus, infects chronically 3% of the world's population. Boceprevir, SCH 503034, (1) our first generation HCV inhibitor, has already established proof-of- concept and is currently in late stage (phase III) clinical trials. In view of the positive data from our first generation compound, further work aimed at optimizing its overall profile was undertaken. Herein, we report that extension of our earlier inhibitor to the P(4) pocket by introducing a new sulfonamide moiety and optimization of the P1/P(1)' capping led to the discovery of a novel series of inhibitors of the HCV NS3 serine protease. Optimization of the P(1) residue significantly improved potency and selectivity. The combination of optimal moieties led to the discovery of compound 47 which, in addition to being a potent inhibitor of HCV subgenomic RNA replication, was also found to have good PK profile in rat, dog and monkey.
Asunto(s)
Amidas/química , Antivirales/química , Inhibidores de Serina Proteinasa/química , Sulfonamidas/química , Urea/análogos & derivados , Proteínas no Estructurales Virales/antagonistas & inhibidores , Animales , Antivirales/síntesis química , Antivirales/farmacocinética , Sitios de Unión , Simulación por Computador , Perros , Evaluación Preclínica de Medicamentos , Proteínas de Escherichia coli , Haplorrinos , Humanos , Proteínas de la Membrana , Modelos Moleculares , Ratas , Inhibidores de Serina Proteinasa/síntesis química , Inhibidores de Serina Proteinasa/farmacocinética , Sulfonamidas/síntesis química , Sulfonamidas/farmacocinética , Urea/síntesis química , Urea/química , Urea/farmacocinética , Proteínas no Estructurales Virales/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
Chronic hepatitis C infection is the primary cause for cirrhosis of the liver and hepatocellular carcinoma leading to liver failure and transplantation. The etiological agent hepatitis C virus produces a single positive strand RNA that is processed further with the help of NS3 serine protease to produce mature virus. Inhibition of this protease can potentially be used to develop drugs for HCV infections. Boceprevir is a ketoamide derived novel inhibitor of HCV NS3 protease that has been progressed to clinical trials and proven to be efficacious in humans. Herein, we report our efforts in identifying an aza-peptide derivative as a potential second generation compound, that lacks electrophilic ketoamide group and are potent in enzyme and replicon assay.
Asunto(s)
Antivirales/química , Compuestos Aza/química , Hepacivirus/enzimología , Péptidos/química , Inhibidores de Serina Proteinasa/química , Proteínas no Estructurales Virales/antagonistas & inhibidores , Antivirales/síntesis química , Antivirales/farmacología , Compuestos Aza/síntesis química , Compuestos Aza/farmacología , Hepatitis C/tratamiento farmacológico , Humanos , Péptidos/síntesis química , Péptidos/farmacología , Inhibidores de Serina Proteinasa/síntesis química , Inhibidores de Serina Proteinasa/farmacología , Proteínas no Estructurales Virales/metabolismoRESUMEN
Chronic hepatitis C infection is the leading causes for cirrhosis of the liver and hepatocellular carcinoma, leading to liver failure and liver transplantation. The etiological agent, HCV virus produces a single positive strand of RNA that is processed with the help of serine protease NS3 to produce mature virus. Inhibition of NS3 protease can be potentially used to develop effective drugs for HCV infections. Numerous efforts are now underway to develop potent inhibitors of HCV protease that contain ketoamides as serine traps. Herein we report the synthesis of a series of potent inhibitors that contain a boronic acid as a serine trap. The activity of these compounds were optimized to 200pM. X-ray structure of compound 17 bound to NS3 protease is also discussed.
Asunto(s)
Antivirales/síntesis química , Ácidos Borónicos/síntesis química , Proteínas no Estructurales Virales/antagonistas & inhibidores , Amidas/síntesis química , Amidas/farmacología , Antivirales/farmacología , Ácidos Borónicos/farmacología , Cristalografía por Rayos X , Estructura Molecular , Inhibidores de Proteasas/síntesis química , Inhibidores de Proteasas/farmacologíaRESUMEN
Hepatitis C Virus (HCV) infection is the major cause of chronic liver disease, leading to cirrhosis and hepatocellular carcinoma, which affects more than 200 million people worldwide. Currently the only therapeutic regimens are subcutaneous interferon-alpha or PEG-interferon alone or in combination with oral ribavirin. Although combination therapy is reasonably successful with the majority of genotypes, its efficacy against the predominant genotype (genotype 1) is moderate at best, with only approximately 50% of the patients showing sustained virological response. We recently disclosed the discovery of Boceprevir, SCH 503034 (1), which is a novel, potent, selective, orally bioavailable NS3 protease inhibitor that has been shown to be efficacious in humans and is currently undergoing clinical trials. As second generation compounds, we have further explored various novel structures with the aim of improving enzyme and cellular binding activities of 1. Herein, we disclose our efforts toward the identification of a novel P(3) sulfonamide-capped inhibitor that demonstrated improved binding and cellular activity compared to 1. X-ray structure of one of these inhibitors bound to the enzyme revealed a hydrogen bond of the P(3) sulfonamide group to Cys-159 which resulted in improved binding and cellular potency.
Asunto(s)
Antivirales/farmacología , Hepacivirus/efectos de los fármacos , Hepacivirus/enzimología , Sulfonamidas/farmacología , Proteínas no Estructurales Virales/antagonistas & inhibidores , Proteínas no Estructurales Virales/metabolismo , Animales , Antivirales/química , Antivirales/farmacocinética , Cristalografía por Rayos X , Descubrimiento de Drogas , Humanos , Modelos Moleculares , Unión Proteica , Ratas , Relación Estructura-Actividad , Sulfonamidas/química , Sulfonamidas/farmacocinética , Replicación Viral/efectos de los fármacosRESUMEN
The structures of both the native holo-HCV NS3/4A protease domain and the protease domain with a serine 139 to alanine (S139A) mutation were solved to high resolution. Subsequently, structures were determined for a series of ketoamide inhibitors in complex with the protease. The changes in the inhibitor potency were correlated with changes in the buried surface area upon binding the inhibitor to the active site. The largest contribution to the binding energy arises from the hydrophobic interactions of the P1 and P2 groups as they bind to the S1 and S2 pockets [the numbering of the subsites is as defined in Berger, A.; Schechter, I. Philos. Trans. R. Soc. London, Ser. B 1970, 257, 249-264]. This correlation of the changes in potency with increased buried surface area contributed directly to the design of a potent tripeptide inhibitor of the HCV NS3/4A protease that is currently in clinical trials.
Asunto(s)
Antivirales/síntesis química , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/química , Hepacivirus/enzimología , Prolina/análogos & derivados , Inhibidores de Serina Proteinasa/química , Proteínas no Estructurales Virales/antagonistas & inhibidores , Proteínas no Estructurales Virales/química , Proteínas Virales/antagonistas & inhibidores , Proteínas Virales/química , Antivirales/química , Sitios de Unión , Cristalografía por Rayos X , Péptidos y Proteínas de Señalización Intracelular , Modelos Moleculares , Prolina/síntesis química , Prolina/química , Conformación Proteica , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
An efficient synthetic approach for the preparation of macrocyclic peptidomimetics for inhibition of HCV NS3 is presented. The macrocyclic core is built using ring-closing metathesis (RCM) of a tripeptidic diene. The presented approach allows the introduction of heteroatoms in strategic places along the macrocyclic ring. The methyl ester moiety in the RCM products was synthetically manipulated to install a keto-amide moiety via a Passerini reaction.
Asunto(s)
Inhibidores Enzimáticos/síntesis química , Hepacivirus/enzimología , Péptidos/química , Pirroles/síntesis química , Catálisis , Ciclización , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Imitación Molecular , Estructura Molecular , Pirroles/química , Pirroles/farmacologíaRESUMEN
This paper describes the development and qualification of a fast, sensitive and specific ultra-performance liquid chromatography/tandem mass spectrometric (UPLC/MS/MS) method for the determination of diastereomers of SCH 503034 in monkey plasma. The analytical method involves direct protein precipitation with a mixture of methanol/acetonitrile (10/90) containing an internal standard, followed by separation of the stereoisomers on an Acquity UPLC C(18) column and detected by selected reaction monitoring (SRM) in positive ionization mode using atmospheric pressure chemical ionization (APCI). The effects of ion-pairing agents on separation and ionization efficiency were investigated. The two diastereomers were well separated (R=1.3) with a runtime of 5 min under an isocratic condition. The method was qualified. The linear concentration range was 1-2500 ng/ml for the both stereoisomers. Inter-assay mean bias and relative standard deviation (R.S.D.) were in the range of -1.2% to 3.6% and 2.8-10%, respectively. Intra-assay mean bias and R.S.D. were in the range of -1.3% to 5.5% and 2.3-7.8%, respectively. Recoveries of the stereoisomers at concentration levels of 2.5, 50 and 1000 ng/ml were 87.2-90.0%, 89.1-90.4% and 92.3-94.3%, respectively. The LLOQ for this assay was 1 ng/ml. No matrix interferences were observed in six different sources of blank monkey plasma.
Asunto(s)
Cromatografía Liquida/métodos , Prolina/análogos & derivados , Espectrometría de Masas en Tándem/métodos , Animales , Macaca fascicularis , Masculino , Prolina/sangre , Prolina/química , Estándares de Referencia , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , EstereoisomerismoRESUMEN
GPR120 (FFAR4) is a fatty acid sensing G protein coupled receptor (GPCR) that has been identified as a target for possible treatment of type 2 diabetes. A selective activator of GPR120 containing a chromane scaffold has been designed, synthesized, and evaluated in vivo. Results of these efforts suggest that chromane propionic acid 18 is a suitable tool molecule for further animal studies. Compound 18 is selective over the closely related target GPR40 (FFAR1), has a clean off-target profile, demonstrates suitable pharmacokinetic properties, and has been evaluated in wild-type/knockout GPR120 mouse oGTT studies.
RESUMEN
Type 2 diabetes mellitus (T2DM) is an ever increasing worldwide epidemic, and the identification of safe and effective insulin sensitizers, absent of weight gain, has been a long-standing goal of diabetes research. G-protein coupled receptor 120 (GPR120) has recently emerged as a potential therapeutic target for treating T2DM. Natural occurring, and more recently, synthetic agonists have been associated with insulin sensitizing, anti-inflammatory, and fat metabolism effects. Herein we describe the design, synthesis, and evaluation of a novel spirocyclic GPR120 agonist series, which culminated in the discovery of potent and selective agonist 14. Furthermore, compound 14 was evaluated in vivo and demonstrated acute glucose lowering in an oral glucose tolerance test (oGTT), as well as improvements in homeostatic measurement assessment of insulin resistance (HOMA-IR; a surrogate marker for insulin sensitization) and an increase in glucose infusion rate (GIR) during a hyperinsulinemic euglycemic clamp in diet-induced obese (DIO) mice.
RESUMEN
The hepatitis C virus (HCV) NS3 protease is essential for viral replication. It has been a target of choice for intensive drug discovery research. On the basis of an active pentapeptide inhibitor, 1, we envisioned that macrocyclization from the P2 proline to P3 capping could enhance binding to the backbone Ala156 residue and the S4 pocket. Thus, a number of P2 proline-based macrocyclic alpha-ketoamide inhibitors were prepared and investigated in an HCV NS3 serine protease continuous assay (K(i*)). The biological activity varied substantially depending on factors such as the ring size, number of amino acid residues, number of methyl substituents, type of heteroatom in the linker, P3 residue, and configuration at the proline C-4 center. The pentapeptide inhibitors were very potent, with the C-terminal acids and amides being the most active ones (24, K(i*) = 8 nM). The tetrapeptides and tripeptides were less potent. Sixteen- and seventeen-membered macrocyclic compounds were equally potent, while fifteen-membered analogues were slightly less active. gem-Dimethyl substituents at the linker improved the potency of all inhibitors (the best compound was 45, K(i*) = 6 nM). The combination of tert-leucine at P3 and dimethyl substituents at the linker in compound 47 realized a selectivity of 307 against human neutrophil elastase. Compound 45 had an IC(50) of 130 nM in a cellular replicon assay, while IC(50) for 24 was 400 nM. Several compounds had excellent subcutaneous AUC and bioavailability in rats. Although tripeptide compound 40 was 97% orally bioavailable, larger pentapeptides generally had low oral bioavailability. The X-ray crystal structure of compounds 24 and 45 bound to the protease demonstrated the close interaction of the macrocycle with the Ala156 methyl group and S4 pocket. The strategy of macrocyclization has been proved to be successful in improving potency (>20-fold greater than that of 1) and in structural depeptization.