Development of BET Inhibitors as Potential Treatments for Cancer: Optimization of Pharmacokinetic Properties.

We describe the synthesis of triazole-containing carboline derivatives and their utility as bromodomain and extra-terminal (BET) inhibitors. A convergent synthetic route permitted the detailed investigation of deuteration and fluorination strategies to reduce clearance while maintaining a favorable profile. This work led to the identification of a potent BET inhibitor, 2-{8-fluoro-3-[4-(H)methyl-1-methyl-1-1,2,3-triazol-5-yl]-5-[()-(oxan-4-yl)(phenyl)methyl]-5-pyrido[3,2-]indol-7-yl}propan-2-ol (), which demonstrated reduced clearance and an improved pharmacokinetic (PK) profile across preclinical species.

Development of BET inhibitors as potential treatments for cancer: A new carboline chemotype.

We describe our efforts to introduce structural diversity to a previously described triazole-containing N1-carboline series of bromodomain and extra-terminal (BET) inhibitors. N9 carbolines were designed to retain favorable binding interactions that the N1-carbolines possess. A convergent synthetic route enabled modifications to reduce clearance, enhance physicochemical properties, and improve the overall in vitro profile.

Development of BET inhibitors as potential treatments for cancer: A search for structural diversity.

We describe our efforts to identify structurally diverse leads in the triazole-containing N1-carboline series of bromodomain and extra-terminal inhibitors. Replacement of the N5 "cap" phenyl moiety with various heteroaryls, coupled with additional modifications to the carboline core, provided analogs with similar potency, improved pharmacokinetic properties, and increased solubility compared to our backup lead, BMS-986225 (2). Rapid SAR exploration was enabled by a convergent, synthetic route.

Pharmacodynamics-based approach for efficacious human dose projection of BMS-986260, a small molecule transforming growth factor beta receptor 1 inhibitor.

Transforming growth factor beta (TGF-β) is a pleiotropic cytokine that has a wide array of biological effects. For decades, tumor biology implicated TGF-β as an attractive therapeutic target due to its immunosuppressive effects. Toward this end, multiple pharmaceutical companies developed a number of drug modalities that specifically target the TGF-β pathway.

Discovery of BMS-986144, a Third-Generation, Pan-Genotype NS3/4A Protease Inhibitor for the Treatment of Hepatitis C Virus Infection.

The discovery of a pan-genotypic hepatitis C virus (HCV) NS3/4A protease inhibitor based on a P1-P3 macrocyclic tripeptide motif is described. The all-carbon tether linking the P1-P3 subsites of is functionalized with alkyl substituents, which are shown to effectively modulate both potency and absorption, distribution, metabolism, and excretion (ADME) properties. The CFBoc-group that caps the P3 amino moiety was discovered to be an essential contributor to metabolic stability, while positioning a methyl group at the C1 position of the P1' cyclopropyl ring enhanced plasma trough values following oral administration to rats.

Structure-based amelioration of PXR transactivation in a novel series of macrocyclic allosteric inhibitors of HIV-1 integrase.

Previous studies have identified a series of imidazo[1,2-a]pyridine (IZP) derivatives as potent allosteric inhibitors of HIV-1 integrase (ALLINIs) and virus infection in cell culture. However, IZPs were also found to be relatively potent activators of the pregnane-X receptor (PXR), raising the specter of induction of CYP-mediated drug disposition pathways. In an attempt to modify PXR activity without affecting anti-HIV-1 activity, rational structure-based design and modeling approaches were used.

Identification and Preclinical Evaluation of the Bicyclic Pyrimidine γ-Secretase Modulator BMS-932481.

A triazine hit identified from a screen of the BMS compound collection was optimized for potency, in vivo activity, and off-target profile to produce the bicyclic pyrimidine γ-secretase modulator BMS-932481. The compound showed robust reductions of Aβ and Aβ in the plasma, brain, and cerebrospinal fluid of mice and rats. Consistent with the γ-secretase modulator mechanism, increases in Aβ and Aβ were observed, with no change in the total amount of Aβ produced.

Improving Metabolic Stability with Deuterium: The Discovery of BMT-052, a Pan-genotypic HCV NS5B Polymerase Inhibitor.

Iterative structure-activity analyses in a class of highly functionalized furo[2,3-]pyridines led to the identification of the second generation pan-genotypic hepatitis C virus NS5B polymerase primer grip inhibitor BMT-052 (), a potential clinical candidate. The key challenge of poor metabolic stability was overcome by strategic incorporation of deuterium at potential metabolic soft spots. The preclinical profile and status of BMT-052 () is described.

Development, optimization and implementation of a centralized metabolic soft spot assay.

Aim: High clearance is a commonly encountered issue in drug discovery. Here we present a centralized metabolic soft spot identification assay with adequate capacity and turnaround time to support the metabolic optimization needs of an entire discovery organization.

Methodology: An integrated quan/qual approach utilizing both an orthogonal sample-pooling methodology and software-assisted structure elucidation was developed to enable the assay.

The discovery of a pan-genotypic, primer grip inhibitor of HCV NS5B polymerase.

The development of a series of novel 7-azabenzofurans exhibiting pan-genotype inhibition of HCV NS5B polymerase binding to the primer grip site is presented. Many challenges, including poor oral bioavailability, high clearance, bioactivation, high human serum shift, and metabolic stability were encountered and overcome through SAR studies. This work culminated in the selection of BMS-986139 () as a preclinical candidate.

Discovery of a Potent Acyclic, Tripeptidic, Acyl Sulfonamide Inhibitor of Hepatitis C Virus NS3 Protease as a Back-up to Asunaprevir with the Potential for Once-Daily Dosing.

The discovery of a back-up to the hepatitis C virus NS3 protease inhibitor asunaprevir (2) is described. The objective of this work was the identification of a drug with antiviral properties and toxicology parameters similar to 2, but with a preclinical pharmacokinetic (PK) profile that was predictive of once-daily dosing. Critical to this discovery process was the employment of an ex vivo cardiovascular (CV) model which served to identify compounds that, like 2, were free of the CV liabilities that resulted in the discontinuation of BMS-605339 (1) from clinical trials.

Comprehensive evaluation of liver microsomal cytochrome P450 3A (CYP3A) inhibition: comparison of cynomolgus monkey and human.

1. Members of the cytochrome P450 3A (CYP3A) subfamily metabolize numerous compounds and serve as the loci of drug-drug interactions (DDIs). Because of high amino acid sequence identity with human CYP3A, the cynomolgus monkey has been proposed as a model species to support DDI risk assessment.

Coupling Laser Diode Thermal Desorption with Acoustic Sample Deposition to Improve Throughput of Mass Spectrometry-Based Screening.

The move toward label-free screening in drug discovery has increased the demand for mass spectrometry (MS)-based analysis. Here we investigated the approach of coupling acoustic sample deposition (ASD) with laser diode thermal desorption (LDTD)-tandem mass spectrometry (MS/MS). We assessed its use in a cytochrome P450 (CYP) inhibition assay, where a decrease in metabolite formation signifies CYP inhibition.

Identification and Preclinical Pharmacology of the γ-Secretase Modulator BMS-869780.

Alzheimer's disease is the most prevalent cause of dementia and is associated with accumulation of amyloid-β peptide (Aβ), particularly the 42-amino acid Aβ1-42, in the brain. Aβ1-42 levels can be decreased by γ-secretase modulators (GSM), which are small molecules that modulate γ-secretase, an enzyme essential for Aβ production. BMS-869780 is a potent GSM that decreased Aβ1-42 and Aβ1-40 and increased Aβ1-37 and Aβ1-38, without inhibiting overall levels of Aβ peptides or other APP processing intermediates.

The discovery of asunaprevir (BMS-650032), an orally efficacious NS3 protease inhibitor for the treatment of hepatitis C virus infection.

The discovery of asunaprevir (BMS-650032, 24) is described. This tripeptidic acylsulfonamide inhibitor of the NS3/4A enzyme is currently in phase III clinical trials for the treatment of hepatitis C virus infection. The discovery of 24 was enabled by employing an isolated rabbit heart model to screen for the cardiovascular (CV) liabilities (changes to HR and SNRT) that were responsible for the discontinuation of an earlier lead from this chemical series, BMS-605339 (1), from clinical trials.

Discovery and early clinical evaluation of BMS-605339, a potent and orally efficacious tripeptidic acylsulfonamide NS3 protease inhibitor for the treatment of hepatitis C virus infection.

The discovery of BMS-605339 (35), a tripeptidic inhibitor of the NS3/4A enzyme, is described. This compound incorporates a cyclopropylacylsulfonamide moiety that was designed to improve the potency of carboxylic acid prototypes through the introduction of favorable nonbonding interactions within the S1' site of the protease. The identification of 35 was enabled through the optimization and balance of critical properties including potency and pharmacokinetics (PK).

Drug-induced perturbations of the bile acid pool, cholestasis, and hepatotoxicity: mechanistic considerations beyond the direct inhibition of the bile salt export pump.

The bile salt export pump (BSEP) is located on the canalicular plasma membrane of hepatocytes and plays an important role in the biliary clearance of bile acids (BAs). Therefore, any drug or new chemical entity that inhibits BSEP has the potential to cause cholestasis and possibly liver injury. In reality, however, one must consider the complexity of the BA pool, BA enterohepatic recirculation (EHR), extrahepatic (renal) BA clearance, and the interplay of multiple participant transporters and enzymes (e.

A high-speed liquid chromatography/tandem mass spectrometry platform using multiplexed multiple-injection chromatography controlled by single software and its application in discovery ADME screening.

Rationale: Multiplexed liquid chromatography (LC) coupled with multiple-injection-chromatogram acquisition has emerged as the method of choice for high-speed discovery bioanalysis, because it significantly reduces injection-to-injection cycle time while maintaining the chromatography quality. Historically, systems utilizing this approach had been custom built, and therefore relied on custom software tools to communicate with multiple vendor software for system control, which lacked transferability, flexibility and robustness.

Methods: In this study, we refined a multiplexed bioanalytical system previously reported, by implementing open-deck auto-sampler manifold and multiple-injection-chromatogram acquisition, all on a commercially available system with single software control.

Identification of a potent and metabolically stable series of fluorinated diphenylpyridylethanamine-based cholesteryl ester transfer protein inhibitors.

A novel series of diphenylpyridylethanamine-based inhibitors of cholesteryl ester transfer protein is described. Optimization of the urea moiety, particularly by incorporation of fluorine, is explored to balance in vitro metabolic stability with CETP potency in the whole plasma assay.

Utility of high-resolution accurate MS to eliminate interferences in the bioanalysis of ribavirin and its phosphate metabolites.

Background: The polar nucleoside drug ribavirin (RBV) combined with IFN-α is a front-line treatment for chronic hepatitis C virus infection. RBV acts as a prodrug and exerts its broad antiviral activity primarily through its active phosphorylated metabolite ribavirin 5´-triphosphate (RTP), and also possibly through ribavirin 5´-monophosphate (RMP). To study RBV transport, diffusion, metabolic clearance and its impact on drug-metabolizing enzymes, a LC-MS method is needed to simultaneously quantify RBV and its phosphorylated metabolites (RTP, ribavirin 5´-diphosphate and RMP).

Evaluation of six proton pump inhibitors as inhibitors of various human cytochromes P450: focus on cytochrome P450 2C19.

Six proton pump inhibitors (PPIs), omeprazole, lansoprazole, esomeprazole, dexlansoprazole, pantoprazole, and rabeprazole, were shown to be weak inhibitors of cytochromes P450 (CYP3A4, -2B6, -2D6, -2C9, -2C8, and -1A2) in human liver microsomes. In most cases, IC₅₀ values were greater than 40 μM, except for dexlansoprazole and lansoprazole with CYP1A2 (IC₅₀ = ∼8 μM) and esomeprazole with CYP2C8 (IC₅₀ = 31 μM). With the exception of CYP2C19 inhibition by omeprazole and esomeprazole (IC₅₀ ratio, 2.

Discovery of 3-hydroxy-4-cyano-isoquinolines as novel, potent, and selective inhibitors of human 11β-hydroxydehydrogenase 1 (11β-HSD1).

Derived from the HTS hit 1, a series of hydroxyisoquinolines was discovered as potent and selective 11β-HSD1 inhibitors with good cross species activity. Optimization of substituents at the 1 and 4 positions of the isoquinoline group in addition to the core modifications, with a special focus on enhancing metabolic stability and aqueous solubility, resulted in the identification of several compounds as potent advanced leads.

Generation of 3,8-substituted 1,2,4-triazolopyridines as potent inhibitors of human 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD-1).

A series of pyridyl amide/sulfonamide inhibitors of 11β-HSD-1 were modified to incorporate a novel 1,2,4-triazolopyridine scaffold. Optimization of substituents at the 3 and 8 position of the TZP core, with a special focus on enhancing metabolic stability, resulted in the identification of compound 38 as a potent and metabolically stable inhibitor of the enzyme.

Ultrafast mass spectrometry based bioanalytical method for digoxin supporting an in vitro P-glycoprotein (P-gp) inhibition screen.

The evaluation of interactions between drug candidates and transporters such as P-glycoprotein (P-gp) has gained considerable interest in drug discovery and development. Inhibition of P-gp can be assessed by performing bi-directional permeability studies with in vitro P-gp-expressing cellular model systems such as Caco-2 (human colon carcinoma) cells, using digoxin as a substrate probe. Existing methodologies include either assaying (3)H-digoxin with liquid scintillation counting (LSC) detection or assaying non-labeled digoxin with liquid chromatography-tandem mass spectrometric (LC-MS/MS) analysis at a speed of several minutes per sample.

A high-throughput bioanalytical platform using automated infusion for tandem mass spectrometric method optimization and its application in a metabolic stability screen.

Liquid chromatography/tandem mass spectrometry (LC/MS/MS) is the bioanalytical method of choice to support plate-based, in vitro early ADME (Absorption, Distribution, Metabolism and Excretion) screens such as metabolic stability (Metstab) assessment. MS/MS method optimization has historically been the bottleneck in this environment, where samples from thousands of discrete compounds are analyzed on a monthly basis, mainly due to the lack of a high-quality commercially available platform to handle the necessary MS/MS method optimization steps for sample analysis by selected reaction monitoring (SRM) on triple quadrupole mass spectrometers. To address this challenge, we recently developed a highly automated bioanalytical platform by successfully integrating QuickQuan 2.