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. These efforts provided a potent BET inhibitor, 3-fluoropyridyl 12, that demonstrated robust efficacy in a multiple myeloma mouse tumor model at 1 mg/kg.

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. This work led to the identification of a potent BET inhibitor, (S)-2-{8-fluoro-5-[(3-fluoropyridin-2-yl)(oxan-4-yl)methyl]-7-[4-(2H3)methyl-1-methyl-1H-1,2,3-triazol-5-yl]-5H-pyrido[3,2-b]indol-3-yl}propan-2-ol (10), a compound with enhanced oral exposure in mice. Subsequent evaluation in a mouse triple-negative breast cancer tumor model revealed efficacy at 4 mg/kg of N9-carboline 10.

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-ß) is a pleiotropic cytokine that has a wide array of biological effects. For decades, tumor biology implicated TGF-ß 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-ß pathway. BMS-986260 is a small molecule, selective TGF-ßR1 kinase inhibitor that was under preclinical development for oncology. In vivo studies across mouse, rat, dog, and monkey and cryopreserved hepatocytes predicted human pharmacokinetics (PK) and distribution of BMS-986260. Efficacy studies of BMS-986260 were undertaken in the MC38 murine colon cancer model, and target engagement, as measured by phosphorylation of SMAD2/3, was assessed in whole blood to predict the clinical efficacious dose. The human clearance is predicted to be low, 4.25 ml/min/kg. BMS-986260 provided a durable and robust antitumor response at 3.75 mg/kg daily and 1.88 mg/kg twice-daily dosing regimens. Phosphorylation of SMAD2/3 was 3.5-fold less potent in human monocytes than other preclinical species. Taken together, the projected clinical efficacious dose was 600 mg QD or 210 mg BID for 3 days followed by a 4-day drug holiday. Mechanism-based cardiovascular findings in the rat ultimately led to the termination of BMS-986260. This study describes the preclinical PK characterization and pharmacodynamics-based efficacious dose projection of a novel small molecule TGF-ßR1 inhibitor.

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. An X-ray cocrystal structure of (S,S)-1 in the PXR ligand binding domain (LBD) allowed an examination of the potential of rational structural modifications designed to abrogate PXR. The introduction of bulky basic amines at the C-8 position provided macrocyclic IZP derivatives that displayed potent HIV-1 inhibitory activity in cell culture with no detectable PXR transactivation at the highest concentration tested.

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. 2. Therefore, the objective of this study was to evaluate 35 known inhibitors of human CYP3A using human (HLM) and cynomolgus monkey (CLM) liver microsomes. Midazolam was employed as substrate to generate IC50 values (concentration of inhibitor rendering 50% inhibition) in the absence and presence of a preincubation (30 mins) with NADPH. 3. In the absence of preincubation, the IC50 values generated with CLM were similar to those obtained with HLM (86% within 2-fold; 100% within 3-fold difference). However, significant differences (up to 48-fold) in preincubation IC50 were observed with 17% of the compounds (raloxifene, bergamottin, nicardipine, mibefradil, ritonavir, and diltiazem). 4. Our results indicate that in most cases the cynomolgus monkey can be a viable DDI model. However, significant species differences in time-dependent CYP3A inhibition can be observed for some compounds. In the case of raloxifene, such a difference can be ascribed to a specific CYP3A4 amino acid residue.

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.g., sulfotransferase 2A1, multidrug resistance-associated protein 2, 3, and 4). Moreover, BAs undergo extensive enzyme-catalyzed amidation and are subjected to metabolism by enterobacteria during EHR. Expression of the various enzymes and transporters described above is governed by nuclear hormone receptors (NHRs) that mount an adaptive response when intracellular levels of BAs are increased. The intracellular trafficking of transporters, and their ability to mediate the vectorial transport of BAs, is governed by specific kinases also. Finally, bile flow, micelle formation, canalicular membrane integrity, and BA clearance can be influenced by the inhibition of multidrug resistant protein 3- or ATPase-aminophospholipid transporter-mediated phospholipid flux. Consequently, when screening compounds in a discovery setting or conducting mechanistic studies to address clinical findings, one has to consider the direct (inhibitory) effect of the parent drug and metabolites on multiple BA transporters, as well as inhibition of BA sulfation and amidation and NHR function. Vectorial BA transport, in addition to BA EHR and homoeostasis, could also be impacted by drug-dependent modulation of kinases and enterobacteria.

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. RESULTS: As a result of these improvements, the developed LC/tandem mass spectrometry (MS/MS) method on the system was nearly three times faster than the previous method, while demonstrating comparable analytical accuracy, precision and robustness. This system has been evaluated for in vitro ADME screening assays including metabolic stability, CYP inhibition and Caco-2. The biological data generated on the developed system displayed good correlation with those from the previous LC/MS/MS approaches. CONCLUSIONS: The developed platform demonstrated applicability to the in vitro screening assays evaluated and has been successfully implemented to support the high-throughput metabolic stability assay, with a significantly improved bioanalytical throughput, capacity and data turnaround.

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, 2.5 to 5.9), there was no evidence for a marked time-dependent shift in IC50 (IC50 ratio, ≤ 2) after a 30-min preincubation with NADPH. In the absence of preincubation, lansoprazole (IC50 = 0.73 µM) and esomeprazole (IC50 = 3.7 µM) were the most potent CYP2C19 inhibitors, followed by dexlansoprazole and omeprazole (IC50 = ∼7.0 µM). Rabeprazole and pantoprazole (IC50 = ≥ 25 µM) were the weakest. A similar ranking was obtained with recombinant CYP2C19. Despite the IC50 ranking, after consideration of plasma levels (static and dynamic), protein binding, and metabolism-dependent inhibition, it is concluded that omeprazole and esomeprazole are the most potent CYP2C19 inhibitors. This was confirmed after the incubation of the individual PPIs with human primary hepatocytes (in the presence of human serum) and by monitoring their impact on diazepam N-demethylase activity at a low concentration of diazepam (2 µM). Data described herein are consistent with reports that PPIs are mostly weak inhibitors of cytochromes P450 in vivo. However, two members of the PPI class (esomeprazole and omeprazole) are more likely to serve as clinically relevant inhibitors of CYP2C19.

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.

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 in vitro profile. This work led to the identification of a potent BET inhibitor, 2-{8-fluoro-3-[4-(2H3)methyl-1-methyl-1H-1,2,3-triazol-5-yl]-5-[(S)-(oxan-4-yl)(phenyl)methyl]-5H-pyrido[3,2-b]indol-7-yl}propan-2-ol (15), which demonstrated reduced clearance and an improved pharmacokinetic (PK) profile across preclinical species. Importantly, no major metabolite was observed when 15 was incubated with human hepatocytes (hHEP) for 2 h. This study culminated with the evaluation of 15 in a mouse triple-negative breast cancer (TNBC) tumor model where it demonstrated robust efficacy at low doses.

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. However, it is not feasible to achieve a throughput high enough using these approaches to sustain an early liability screen that generates more than a thousand samples on a daily basis. To address this challenge, we developed an ultrafast (9 s per sample) bioanalytical method for digoxin analysis using RapidFire™, an on-line solid-phase extraction (SPE) system, with MS/MS detection. A stable isotope labeled analog, d3-digoxin, was used as internal standard to minimize potential ionization matrix effect during the RF-MS/MS analysis. The RF-MS/MS method was more than 16 times faster than the LC-MS/MS method but demonstrated similar sensitivity, selectivity, reproducibility, linearity and robustness. P-gp inhibition results of multiple validation compounds obtained with this RF-MS/MS method were in agreement with those generated by both the LC-MS/MS method and the (3)H-radiolabel assay. This method has been successfully deployed to assess P-gp inhibition potential as an important early liability screen for drug-transporter interaction.

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

A series of pyridyl amide/sulfonamide inhibitors of 11ß-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.

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

Derived from the HTS hit 1, a series of hydroxyisoquinolines was discovered as potent and selective 11ß-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.

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 21 is functionalized with alkyl substituents, which are shown to effectively modulate both potency and absorption, distribution, metabolism, and excretion (ADME) properties. The CF3Boc-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. The C7-fluoro, C6-CD3O substitution pattern of the P2* isoquinoline heterocycle of 21 was essential to securing the targeted potency, pharmacokinetic (PK), and toxicological profiles. The C6-CD3O redirected metabolism away from a problematic pathway, thereby circumventing the time-dependent cytochrome P (CYP) 450 inhibition observed with the C6-CH3O prototype.

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 2.0, a unique high-throughput solution featuring MS/MS method optimization by automated infusion, with a customized in-house software tool in support of a Metstab screen. In this platform, a dual-column setup running parallel chromatography was also implemented to reduce the bioanalytical cycle time for LC/MS/MS sample analysis. A set of 45 validation compounds was used to demonstrate the speed, quality and reproducibility of MS/MS method optimization, sample analysis, and data processing using this automated platform. Metstab results for the validation compounds in microsomes from multiple species (human, rat, mouse) showed good consistency within each batch, and also between batches conducted on different days. We have achieved and maintained a monthly throughput of 1300 compound assays representing 500 discrete compounds per instrument per month on this platform, and it has been used to generate metabolic stability data for more than 25 000 compounds to date with an overall success rate of more than 95%.

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 Aß1-42 and Aß1-40 in the plasma, brain, and cerebrospinal fluid of mice and rats. Consistent with the γ-secretase modulator mechanism, increases in Aß1-37 and Aß1-38 were observed, with no change in the total amount of Aß1-x produced. No Notch-based toxicity was observed, and the overall preclinical profile of BMS-932481 supported its further evaluation in human clinical trials.

An automated liquid chromatography-mass spectrometry process to determine metabolic stability half-life and intrinsic clearance of drug candidates by substrate depletion.

An automated process is described for the detailed assessment of the in vitro metabolic stability properties of drug candidates in support of pharmaceutical property profiling. Compounds are incubated with liver microsomes using a robotic liquid handler. Aliquots are taken at various time points, and the resulting samples are quantitatively analyzed by liquid chromatography-mass spectrometry utilizing ion trap mass spectrometers to determine the amount of compound remaining. From these data metabolism rates can be calculated. A high degree of automation is achieved through custom software, which is employed for instrument setup, data processing, and results reporting. The assay setup is highly configurable, allowing for any combination of up to six user-selected time points, variable substrate concentration, and microsomes or other biologically active media. The data, based on relative substrate depletion, affords an estimate of metabolic stability through the calculation of half-life (t(1/2)) and intrinsic clearance, which are used to differentiate and rank order drug leads. In general, t(1/2) is the time necessary for the metabolism, following first-order kinetics, of 50% of the initial compound. Intrinsic clearance is the proportionality constant between rate of metabolism of a compound and its concentration at the enzyme site. Described here is the setup of the assay, and data from assay test compounds are presented.

Synthesis of novel ketoconazole derivatives as inhibitors of the human Pregnane X Receptor (PXR; NR1I2; also termed SXR, PAR).

PXR, pregnane X receptor, in its activated state, is a validated target for controlling certain drug-drug interactions in humans. In this context, there is a paucity of inhibitors directed toward activated PXR. Using prior observations with ketoconazole as a PXR inhibitor, the target compound 3 was synthesized from (s)-glycidol with overall 56% yield. (+)-Glycidol was reacted with 4-bromophenol and potassium carbonate in DMF to yield the ring opened compound 6. This was then heated to reflux in benzene along with 2', 4'-difluoroacetophenone and catalytic amount of para-toluene sulfonic acid to yield 8. The resultant acetal 8 was then functionalized using Palladium chemistry to yield the target compound 3. The activity of the compound was compared with ketoconazole and UCL2158H. However, in contrast with ketoconazole (IC(50) approximately 0.020 microM; approximately 100% inhibition), 3 has negligible effects on inhibition of microsomal CYP450 (maximum approximately 20% inhibition) at concentrations >40 microM. In vitro, micromolar concentration of ketoconazole is toxic to passaged human cell lines, while 3 does not exhibit cytotoxicity up to concentrations approximately 100 microM (viability >85%). This is the first demonstration of a chemical analog of a PXR inhibitor that retains activity against activated PXR. Furthermore, in contrast with ketoconazole, 3 is less toxic in human cell lines and has negligible CYP450 activity.

An automated high throughput liquid chromatography-mass spectrometry process to assess the metabolic stability of drug candidates.

An automated high throughput process, termed the MetFast assay, is described to assess in vitro the general microsomal cytochrome P450 beta-nicotinamide adenine dinucleotide phosphate-mediated first-pass metabolic stability of potential drug candidates as a utility for pharmaceutical profiling. Utilizing robotic protocols with a multiprobe liquid handler, compounds are incubated with liver microsomes from different species. Samples are then analyzed by in-line liquid chromatography (LC)-mass spectrometry (MS) to determine the amount of compound remaining after a certain time, which allows calculation of metabolism rates. To quantitatively assess large numbers of structurally diverse compounds by LC-MS, a strategy based on an iterative two-step process was devised. Initially compounds are qualitatively analyzed by LC-ultraviolet (UV)/MS (step 1) to determine purity (UV detection) and structural integrity (MS detection). This step ensures that only correct and verified compounds with sufficient purity are being assayed to obtain reproducible high data quality. In addition, all necessary information is gathered to automatically generate specific quantitative methods for the subsequent bioanalytical analysis of metabolic stability samples by LC-UV/MS (step 2). In-house-developed, highly flexible and sophisticated data management software, termed SmartReport, is utilized for automated qualitative and quantitative LC-MS analysis set-up, data processing, and results reporting. The integration of key aspects, inherent "universal" collision-induced dissociation settings of ion trap mass spectrometers for tandem mass spectrometric scan functions utilized for compound-specific and sensitive quantitative MS methods, generic fast-LC conditions, generic MS instrument settings, and the functionality of SmartReport software resulted in an analytical process that routinely provides reproducible high-quality metabolic stability data on structurally diverse compounds. Described here is the setup of the MetFast assay, and metabolic stability data from assay validation compounds are given.

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. Major metabolic soft spots in liver microsomes (rodent and human) were generated in a batch mode, along with kinetics of parent disappearance and metabolite formation, typically within 1 week of incubation. RESULTS & CONCLUSION: A centralized metabolic soft spot identification assay has been developed and has successfully impacted discovery project teams in mitigating instability and establishing potential structure-metabolism relationships.