Matched and mixed cap derivatives in the tetracyclic indole class of HCV NS5A inhibitors.

A matched and mixed capping SAR study was conducted on the tetracyclic indole class of HCV NS5A inhibitors to examine the influence of modifications of this region on the overall HCV virologic resistance profiles.

Mechanism-based inactivation of human cytochrome P450 3A4 by two piperazine-containing compounds.

Human cytochrome P450 3A4 (CYP3A4) is responsible for the metabolism of more than half of pharmaceutic drugs, and inactivation of CYP3A4 can lead to adverse drug-drug interactions. The substituted imidazole compounds 5-fluoro-2-[4-[(2-phenyl-1H-imidazol-5-yl)methyl]-1-piperazinyl]pyrimidine (SCH 66712) and 1-[(2-ethyl-4-methyl-1H-imidazol-5-yl)methyl]-4-[4-(trifluoromethyl)-2-pyridinyl]piperazine (EMTPP) have been previously identified as mechanism-based inactivators (MBI) of CYP2D6. The present study shows that both SCH 66712 and EMTPP are also MBIs of CYP3A4. Inhibition of CYP3A4 by SCH 66712 and EMTPP was determined to be concentration, time, and NADPH dependent. In addition, inactivation of CYP3A4 by SCH 66712 was shown to be unaffected by the presence of electrophile scavengers. SCH 66712 displays type I binding to CYP3A4 with a spectral binding constant (Ks) of 42.9 ± 2.9 µM. The partition ratios for SCH 66712 and EMTPP were 11 and 94, respectively. Whole protein mass spectrum analysis revealed 1:1 binding stoichiometry of SCH 66712 and EMTPP to CYP3A4 and a mass increase consistent with adduction by the inactivators without addition of oxygen. Heme adduction was not apparent. Multiple mono-oxygenation products with each inactivator were observed; no other products were apparent. These are the first MBIs to be shown to be potent inactivators of both CYP2D6 and CYP3A4.

New class of azaheptapyridine FPT inhibitors as potential cancer therapy agents.

Tertiary hydroxyl class of C-imidazole bridgehead azaheptapyridine FPT inhibitors were prepared in an attempt to block in vivo oxidation of secondary hydroxyl series. One representative compound 5a exhibited potent enzyme (IC50=1.4 nM) and cellular activities (soft agar IC50=1.3 nM) with excellent oral pharmacokinetic profiles in rats, mice, monkeys and dogs. The in vivo study in wap-ras TG mouse models showed dose dependent tumor growth inhibition and regression.

Discovery of MK-8742: an HCV NS5A inhibitor with broad genotype activity.

The NS5A protein plays a critical role in the replication of HCV and has been the focus of numerous research efforts over the past few years. NS5A inhibitors have shown impressive in vitro potency profiles in HCV replicon assays, making them attractive components for inclusion in all oral combination regimens. Early work in the NS5A arena led to the discovery of our first clinical candidate, MK-4882 [2-((S)-pyrrolidin-2-yl)-5-(2-(4-(5-((S)-pyrrolidin-2-yl)-1H-imidazol-2-yl)phenyl)benzofuran-5-yl)-1H-imidazole]. While preclinical proof-of-concept studies in HCV-infected chimpanzees harboring chronic genotype 1 infections resulted in significant decreases in viral load after both single- and multiple-dose treatments, viral breakthrough proved to be a concern, thus necessitating the development of compounds with increased potency against a number of genotypes and NS5A resistance mutations. Modification of the MK-4882 core scaffold by introduction of a cyclic constraint afforded a series of tetracyclic inhibitors, which showed improved virologic profiles. Herein we describe the research efforts that led to the discovery of MK-8742, a tetracyclic indole-based NS5A inhibitor, which is currently in phase 2b clinical trials as part of an all-oral, interferon-free regimen for the treatment of HCV infection.

In Vivo Characterization of a Novel γ-Secretase Inhibitor SCH 697466 in Rodents and Investigation of Strategies for Managing Notch-Related Side Effects.

Substantial evidence implicates ß-amyloid (Aß) peptides in the etiology of Alzheimer's disease (AD). Aß is produced by the proteolytic cleavage of the amyloid precursor protein by ß- and γ-secretase suggesting that γ-secretase inhibition may provide therapeutic benefit for AD. Although many γ-secretase inhibitors have been shown to be potent at lowering Aß, some have also been shown to have side effects following repeated administration. All of these side effects can be attributed to altered Notch signaling, another γ-secretase substrate. Here we describe the in vivo characterization of the novel γ-secretase inhibitor SCH 697466 in rodents. Although SCH 697466 was effective at lowering Aß, Notch-related side effects in the intestine and thymus were observed following subchronic administration at doses that provided sustained and complete lowering of Aß. However, additional studies revealed that both partial but sustained lowering of Aßand complete but less sustained lowering of Aß were successful approaches for managing Notch-related side effects. Further, changes in several Notch-related biomarkers paralleled the side effect observations. Taken together, these studies demonstrated that, by carefully varying the extent and duration of Aß lowering by γ-secretase inhibitors, it is possible to obtain robust and sustained lowering of Aß without evidence of Notch-related side effects.

Development and validation of an LC-MS-MS method for the simultaneous determination of sulforaphane and its metabolites in rat plasma and its application in pharmacokinetic studies.

A highly sensitive and simple high-performance liquid chromatographic-tandem mass spectrometric (LC-MS-MS) assay is developed and validated for the quantification of sulforaphane and its metabolites in rat plasma. Sulforaphane (SFN) and its metabolites, sulforaphane glutathione (SFN-GSH) and sulforaphane N-acetyl cysteine (SFN-NAC) conjugates, are extracted from rat plasma by methanol-formic acid (100:0.1, v/v) and analyzed using a reversed-phase gradient elution on a Develosil 3 µm RP-Aqueous C(30) 140Å column. A 15-min linear gradient with acetonitrile-water (5:95, v/v), containing 10 mM ammonium acetate and 0.2% formic acid, as mobile phase A, and acetonitrile-water (95:5, v/v), containing 10 mM ammonium acetate and 0.2% formic acid as mobile phase B, is used. Sulforaphane and its metabolites are well separated. Sulforaphene is used as the internal standard. The lower limits of quantification are 1 ng/mL for SFN and 10 ng/mL for both SFN-NAC and SFN-GSH. The calibration curves are linear over the concentration range of 25-20,000 ng/mL of plasma for each analyte. This novel LC-MS-MS method shows satisfactory accuracy and precision and is sufficiently sensitive for the performance of pharmacokinetic studies in rats.

Substituted imidazole of 5-fluoro-2-[4-[(2-phenyl-1H-imidazol-5-yl)methyl]-1-piperazinyl]pyrimidine Inactivates cytochrome P450 2D6 by protein adduction.

5-Fluoro-2-[4-[(2-phenyl-1H-imidazol-5-yl)methyl]-1-piperazinyl]pyrimidine (SCH 66712) is a potent mechanism-based inactivator of human cytochrome P450 2D6 that displays type I binding spectra with a K(s) of 0.39 ± 0.10 µM. The partition ratio is ~3, indicating potent inactivation that addition of exogenous nucleophiles does not prevent. Within 15 min of incubation with SCH 66712 and NADPH, ∼90% of CYP2D6 activity is lost with only ~20% loss in ability to bind CO and ~25% loss of native heme over the same time. The stoichiometry of binding to the protein was 1.2:1. SDS-polyacrylamide gel electrophoresis with Western blotting and autoradiography analyses of CYP2D6 after incubations with radiolabeled SCH 66712 further support the presence of a protein adduct. Metabolites of SCH 66712 detected by mass spectrometry indicate that the phenyl group on the imidazole ring of SCH 66712 is one site of oxidation by CYP2D6 and could lead to methylene quinone formation. Three other metabolites were also observed. For understanding the metabolic pathway that leads to CYP2D6 inactivation, metabolism studies with CYP2C9 and CYP2C19 were performed because neither of these enzymes is significantly inhibited by SCH 66712. The metabolites formed by CYP2C9 and CYP2C19 are the same as those seen with CYP2D6, although in different abundance. Modeling studies with CYP2D6 revealed potential roles of various active site residues in the oxidation of SCH 66712 and inactivation of CYP2D6 and showed that the phenyl group of SCH 66712 is positioned at 2.2 Å from the heme iron.

Pharmacological characterization of a novel α2C-adrenoceptor agonist N-[3,4-dihydro-4-(1H-imidazol-4-ylmethyl)-2H-1, 4-benzoxazin-6-yl]-N-ethyl-N'-methylurea (compound A).

We define the pharmacological and pharmacokinetic profiles of a novel α(2C)-adrenoceptor agonist, compound A [N-[3,4-dihydro-4-(1H-imidazol-4-ylmethyl)-2H-1,4-benzoxazin-6-yl]-N-ethyl-N'-methylurea]. This compound has high affinity (K(i)) for the human α(2C)-adrenoceptor (K(i) = 12 nM), and 190- to 260-fold selectivity over the α(2A)- and α(2B)-adrenoceptor subtypes. In cell-based functional assays, compound A produced good agonist (EC(50) = 166 nM) and efficacy (E(max) = 64%) responses at the α(2C)-adrenoceptor, much lower potency and efficacy at the α(2A)-adrenoceptor (EC(50) = 1525 nM; E(max) = 8%) and α(2B)-adrenoceptor (EC(50) = 5814 nM; E(max) = 21%) subtypes, and low or no affinity and functional activity at the α(1A)-, α(1B)-, and α(1D)-adrenoceptor subtypes. In the human saphenous vein postjunctional α(2C)-adrenoceptor bioassay, compound A functions as a potent agonist (pD(2) = 6.3). In a real-time contraction bioassay of pig nasal mucosa, compound A preferentially constricted the veins (EC(50) = 108 nM), and the magnitude of arteriolar contraction reached only 50% of the maximum venular responses. Compound A exhibited no effect on locomotor activity, sedation, and body temperature in mice (up to 100 mg/kg) and did not cause hypertension and mydriasis (30 mg/kg) in conscious rats. Compound A is orally bioavailable (24%) with good plasma exposure. This compound is a substrate for the efflux P-glycoprotein transporter, resulting in very low central nervous system (CNS) penetration. In summary, compound A is a highly selective, orally active, and non-CNS-penetrating α(2C)-adrenoceptor agonist with desirable in vitro and in vivo pharmacological properties suitable for the treatment of nasal congestion.

Design, synthesis, and structure-activity relationship studies of N-arylsulfonyl morpholines as γ-secretase inhibitors.

Design and synthesis of cis-2,6-disubstituted N-arylsulfonyl morpholines as novel γ-secretase inhibitors for the potential treatment of Alzheimer's disease (AD) is reported. Several different small alkyl groups are installed on the left-hand side to lower the CYP3A4 liability while maintaining excellent in vitro potency.

Pharmacokinetics of pseudoephedrine in rats, dogs, monkeys and its pharmacokinetic-pharmacodynamic relationship in a feline model of nasal congestion.

The objectives of these studies were to characterize the pharmacokinetics (PK) of the nasal decongestant pseudoephedrine (PSE) in rats, dogs, and monkeys, and to evaluate its lower gastrointestinal tract regional bioavailability in rats. An LC-MS/MS assay with a lower limit of quantification (LLOQ) of 0.4 ng/mL of plasma was developed for the analysis of PSE in animal plasma. The total body clearance (CL) was the highest in rats (78 mL/min/kg), lowest in monkeys (15 mL/min/kg) and the dog averaged in between (33 mL/min/kg). The volume of distribution at steady state (Vdss) ranged from 3-5 L/kg in all species. In rats and dogs, the mean half-lives (t1/2) was ≈1.5 hr, while in monkeys the mean t1/2 was 4.6 hr, comparable to that observed in adult humans (4-8 hr). The oral bioavailability was 38, 58 and 78% in rats, dogs and monkeys. The bioavailability following intra-ileum or intra-colonic administration in rats was superior to that following oral dosing (66% and 78%, respectively) suggesting that colonic absorption may be compensating for the short half-life, thus enabling successful QD sustained release formulations of PSE. The pharmacokinetic/pharmacodynamic relationship (PK/PD) of PSE was also investigated in a feline model of nasal congestion to establish efficacious trough concentrations in cats for a comparison with that in humans. The PK/PD in the cat model followed a sigmoid Emax model with an EC50 (plasma concentration that elicits 50% of the maximum response) of 0.32 ±0.05 (SD) µM consistent with human plasma concentrations required for efficacy.

Protein binding-dependent decreases in hERG channel blocker potency assessed by whole-cell voltage clamp in serum.

In vitro hERG blocking potency is measured in drug discovery as part of an integrated cardiovascular risk assessment. Typically, the concentrations producing 50% inhibition are measured in protein-free saline solutions and compared with calculated free therapeutic in vivo Cmax values to estimate a hERG safety multiple. The free/unbound fraction is believed responsible for activity. We tested the validity of this approach with 12 compounds by determining potencies in voltage clamp studies conducted in the absence and presence of 100% dialyzed fetal bovine serum (FBS). Bath drug concentrations in saline solutions were measured to account for loss of compounds due to solubility, stability, and/or adsorption. Protein binding in dialyzed FBS was measured to enable predictions of serum IC50s based on the unbound fraction and the saline IC50. For 11 of 12 compounds, the measured potency in the presence of dialyzed FBS was within 2-fold of the predicted potency. The predicted IC50 in dialyzed FBS for one highly bound compound, amiodarone, was 9-fold higher than the measured serum IC50. These data suggest that for highly bound compounds, direct measurement of IC50s in the presence of 100% serum may provide a more accurate estimate of in vivo potencies than the approach based on calculated serum shifts.

Discovery of Dinaciclib (SCH 727965): A Potent and Selective Inhibitor of Cyclin-Dependent Kinases.

Inhibition of cyclin-dependent kinases (CDKs) has emerged as an attractive strategy for the development of novel oncology therapeutics. Herein is described the utilization of an in vivo screening approach with integrated efficacy and tolerability parameters to identify candidate CDK inhibitors with a suitable balance of activity and tolerability. This approach has resulted in the identification of SCH 727965, a potent and selective CDK inhibitor that is currently undergoing clinical evaluation.

Novel orally active morpholine N-arylsulfonamides gamma-secretase inhibitors with low CYP 3A4 liability.

A new class of 2,6-disubstituted morpholine N-arylsulfonamide gamma-secretase inhibitors was designed based on the introduction of a morpholine core in lieu or piperidine in our lead series. This resulted in compounds with improved CYP 3A4 profiles. Several analogs that were active at lowering Abeta levels in Tg CRND8 mice upon oral administration were identified.

Evaluation of CYP1A1 and CYP2B1/2 m-RNA induction in rat liver slices using the NanoString technology: a novel tool for drug discovery lead optimization.

Cytochrome P450 (CYP) induction in rodents and humans is considered a liability for new chemical entities (NCEs) in drug discovery. In particular, CYP1A1 and CYP2B1/2 have been associated with the induction of liver tumors in oncogenicity studies during safety evaluation studies of potential drugs. In our laboratory, real time PCR (Taqman) has been used to quantify the induction of rat hepatic CYP1A1 and CYP2B1/2 in precision -cut rat liver slices. A novel technology that does not require m-RNA isolation or RT-PCR, (developed by NanoString Technologies) has been investigated to quantify CYP1A1 and CYP2B1/2 induction in rat liver slices. Seventeen commercially available compounds were evaluated using both Taqman and NanoString technologies. Precision-cut rat liver slices were incubated with individual compounds for 24 hr at 37 degrees C in a humidified CO(2) incubator and CYP1A1 and CYP2B1/2 m-RNA were quantified. The results from the NanoString technology were similar to those of the Taqman(R) with a high degree of correlation for both CYP isoforms (r(2)>0.85). Therefore, NanoString provides an additional new technology to evaluate the induction of CYP1A1 and 2B1/2, as well as potentially other enzymes or transporters in rat liver slices.

High-throughput evaluation of CYP1A1 and 2B1 induction in rat liver slices using a semi-automated system.

Drug candidates with the propensity to induce rat CYP1A1 or 2B1 isoforms are believed to possess a greater tendency to induce hepatic tumors in oncogenicity studies. We have previously published on a manual rat liver slice assay that showed a satisfactory relationship between in vitro CYP2B1 m-RNA induction using real time PCR and the ex vivo pentoxyresorufin O-dealkylase (PROD) activity in liver microsomes prepared from rats treated daily via the oral route for 14 consecutive days with inducers or non-inducers. We now describe this automated in vitro high throughput liver slice technique to screen out drug candidates that are potent rodent CYP1A1 and/or CYP2B1 inducers. A good concordance between in vitro and in vivo data was observed for both CYP1A1 (100 %) and CYP2B1 (90%) isoforms. Automation of key steps has enabled us to increase the annual screening throughput from 200 (manual) to 1500 compounds. The increase in throughput allowed the quick development of structure-induction relationships (SIR's) for multiple drug discovery programs in a facile manner.

The conduct of in vitro studies to address time-dependent inhibition of drug-metabolizing enzymes: a perspective of the pharmaceutical research and manufacturers of America.

Time-dependent inhibition (TDI) of cytochrome P450 (P450) enzymes caused by new molecular entities (NMEs) is of concern because such compounds can be responsible for clinically relevant drug-drug interactions (DDI). Although the biochemistry underlying mechanism-based inactivation (MBI) of P450 enzymes has been generally understood for several years, significant advances have been made only in the past few years regarding how in vitro time-dependent inhibition data can be used to understand and predict clinical DDI. In this article, a team of scientists from 16 pharmaceutical research organizations that are member companies of the Pharmaceutical Research and Manufacturers of America offer a discussion of the phenomenon of TDI with emphasis on the laboratory methods used in its measurement. Results of an anonymous survey regarding pharmaceutical industry practices and strategies around TDI are reported. Specific topics that still possess a high degree of uncertainty are raised, such as parameter estimates needed to make predictions of DDI magnitude from in vitro inactivation parameters. A description of follow-up mechanistic experiments that can be done to characterize TDI are described. A consensus recommendation regarding common practices to address TDI is included, the salient points of which include the use of a tiered approach wherein abbreviated assays are first used to determine whether NMEs demonstrate TDI or not, followed by more thorough inactivation studies for those that do to define the parameters needed for prediction of DDI.

Estimation of the extent of oral absorption in animals from oral and intravenous pharmacokinetic data in drug discovery.

Oral administration is the most desirable route of drug delivery for systemically active drugs. Oral drugs must possess a certain level of oral bioavailability, which is a product of oral absorption and first-pass effect. Low oral bioavailability may be attributed to poor absorption and/or high first-pass hepatic elimination. In the lead optimization stage of drug discovery, if the relative contribution of oral absorption and metabolism could be discerned for poorly bioavailable compounds, a path forward for remedy would be possible. This report describes an approach utilizing oral/intravenous pharmacokinetic data to estimate oral absorption. The fraction of dose absorbed is calculated as the ratio of the actual bioavailable fraction to the maximum bioavailable fraction estimated from systemic clearance. An arbitrary classification was devised where low absorption encompasses compounds whose extent of absorption is or=70% absorption. There was approximately 78% concordance in rats, 65% in monkeys and almost complete concordance in dogs. This approach correctly identified the cause for low oral bioavailability for 11 out of 13 compounds evaluated, and therefore it could be used prospectively with nonradiolabeled compounds during the lead optimization process.

Discovery of a novel, potent and orally active series of gamma-lactams as selective NK1 antagonists.

Strategic replacement of the nitrogen of the lead compound 1 in the original cyclic urea series with a carbon resulted in the discovery of a novel, potent and orally more efficacious gamma-lactam series of selective NK(1) antagonists. Optimization of the lactam series culminated in the identification of compounds with high binding affinity and excellent oral CNS activity.

Posaconazole (Noxafil, SCH 56592), a new azole antifungal drug, was a discovery based on the isolation and mass spectral characterization of a circulating metabolite of an earlier lead (SCH 51048).

Posaconazole (SCH 56592) is a novel triazole antifungal drug that is marketed in Europe and the United States under the trade name 'Noxafil' for prophylaxis against invasive fungal infections. SCH 56592 was discovered as a possible active metabolite of SCH 51048, an earlier lead. Initial studies have shown that serum concentrations determined by a microbiological assay were higher than those determined by HPLC from animals dosed with SCH 51048. Subsequently, several animals species were dosed with (3)H-SCH 51048 and the serum was analyzed for total radioactivity, SCH 51048 concentration and antifungal activity. The antifungal activity was higher than that expected based on SCH 51048 serum concentrations, confirming the presence of active metabolite(s). Metabolite profiling of serum samples at selected time intervals pinpointed the peak that was suspected to be the active metabolite. Consequently, (3)H-SCH 51048 was administered to a large group of mice, the serum was harvested and the metabolite was isolated by extraction and semipreparative HPLC. LC-MS/MS analysis suggested that the active metabolite is a secondary alcohol with the hydroxyl group in the aliphatic side chain of SCH 51048. All corresponding monohydroxylated diastereomeric mixtures were synthesized and characterized. The HPLC retention time and LC-MS/MS spectra of the diastereomeric secondary alcohols of SCH 51048 were similar to those of the isolated active metabolite. Finally, all corresponding individual monohydroxylated diasteriomers were synthesized and evaluated for in vitro and in vivo antifungal potencies, as well as pharmacokinetics. SCH 56592 emerged as the candidate with the best overall profile.

Discovery of amide and heteroaryl isosteres as carbamate replacements in a series of orally active gamma-secretase inhibitors.

The design of amide and heteroaryl amide isosteres as replacements for the carbamate substructure in previously disclosed 2,6-disubstituted piperidine N-arylsulfonamides is described. In several cases, amides lessened CYP liabilities in this class of gamma-secretase inhibitors. Selected compounds showed significant reduction of Abeta levels upon oral dosing in a transgenic murine model of Alzheimer's disease.