Discovery of Novel 3,3-Disubstituted Piperidines as Orally Bioavailable, Potent, and Efficacious HDM2-p53 Inhibitors.

A new subseries of substituted piperidines as p53-HDM2 inhibitors exemplified by 21 has been developed from the initial lead 1. Research focused on optimization of a crucial HDM2 Trp23-ligand interaction led to the identification of 2-(trifluoromethyl)thiophene as the preferred moiety. Further investigation of the Leu26 pocket resulted in potent, novel substituted piperidine inhibitors of the HDM2-p53 interaction that demonstrated tumor regression in several human cancer xenograft models in mice. The structure of HDM2 in complex with inhibitors 3, 10, and 21 is described.

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.

Identification of PTC725, an orally bioavailable small molecule that selectively targets the hepatitis C Virus NS4B protein.

While new direct-acting antiviral agents for the treatment of chronic hepatitis C virus (HCV) infection have been approved, there is a continued need for novel antiviral agents that act on new targets and can be used in combination with current therapies to enhance efficacy and to restrict the emergence of drug-resistant viral variants. To this end, we have identified a novel class of small molecules, exemplified by PTC725, that target the nonstructural protein 4B (NS4B). PTC725 inhibited HCV 1b (Con1) replicons with a 50% effective concentration (EC50) of 1.7 nM and an EC90 of 9.6 nM and demonstrated a >1,000-fold selectivity window with respect to cytotoxicity. The compounds were fully active against HCV replicon mutants that are resistant to inhibitors of NS3 protease and NS5B polymerase. Replicons selected for resistance to PTC725 harbored amino acid substitutions F98L/C and V105M in NS4B. Anti-replicon activity of PTC725 was additive to synergistic in combination with alpha interferon or with inhibitors of HCV protease and polymerase. Immunofluorescence microscopy demonstrated that neither the HCV inhibitors nor the F98C substitution altered the subcellular localization of NS4B or NS5A in replicon cells. Oral dosing of PTC725 showed a favorable pharmacokinetic profile with high liver and plasma exposure in mice and rats. Modeling of dosing regimens in humans indicates that a once-per-day or twice-per-day oral dosing regimen is feasible. Overall, the preclinical data support the development of PTC725 for use in the treatment of chronic HCV infection.

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.

Dinaciclib (SCH 727965), a novel and potent cyclin-dependent kinase inhibitor.

Cyclin-dependent kinases (CDK) are key positive regulators of cell cycle progression and attractive targets in oncology. SCH 727965 inhibits CDK2, CDK5, CDK1, and CDK9 activity in vitro with IC(50) values of 1, 1, 3, and 4 nmol/L, respectively. SCH 727965 was selected as a clinical candidate using a functional screen in vivo that integrated both efficacy and safety parameters. Compared with flavopiridol, SCH 727965 exhibits superior activity with an improved therapeutic index. In cell-based assays, SCH 727965 completely suppressed retinoblastoma phosphorylation, which correlated with apoptosis onset and total inhibition of bromodeoxyuridine incorporation in >100 tumor cell lines of diverse origin and background. Moreover, short exposures to SCH 727965 were sufficient for long-lasting cellular effects. SCH 727965 induced regression of established solid tumors in a range of mouse models following intermittent scheduling of doses below the maximally tolerated level. This was associated with modulation of pharmacodynamic biomarkers in skin punch biopsies and rapidly reversible, mechanism-based effects on hematologic parameters. These results suggest that SCH 727965 is a potent and selective CDK inhibitor and a novel cytotoxic agent.

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.

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.

Pyrazolo[1,5-a]pyrimidines as orally available inhibitors of cyclin-dependent kinase 2.

Properly substituted pyrazolo[1,5-a]pyrimidines are potent and selective CDK2 inhibitors. Compound 15j is orally available and showed efficacy in a mouse A2780 xenograft model.

Utility of mass spectrometry for in-vitro ADME assays.

A balance between pharmacological activity, safety and drug metabolism and pharmacokinetics (DMPK) attributes determines the fate of a new chemical entity (NCE) in drug discovery. Because of the increased number of NCEs requiring DMPK evaluation, several in vitro higher-throughput screens and counter screens designed to evaluate DMPK attributes have been introduced in drug discovery. The DMPK screens evaluate NCEs for potential absorption, metabolism, drug-drug interactions, brain penetration, protein binding and pharmacokinetics. Higher-throughput analytical methodologies for the determination of either a common end product of a screen or the parent compound (and/or possible metabolites) are essential for successful DMPK screens. Because of its speed, sensitivity and specificity, liquid chromatography-tandem mass spectrometry (LC-MS/MS) has become the technology of choice for sample analysis. In this review, several in vitro screening assays that we employ in drug discovery are discussed with an emphasis on LC-MS/MS role in accelerating them.

Bridgehead modification of trihalocycloheptabenzopyridine leads to a potent farnesyl protein transferase inhibitor with improved oral metabolic stability.

Modification of the ethano bridge of the core structure of the antitumor agent, SARASAR (SCH66336) with concomitant introduction of a sulfonamide moiety off the distal piperidine afforded inhibitor 9-(S-), a compound with greatly improved PK profile. Other compounds with enhanced FPTase inhibitory activity were obtained as exemplified by amide 10-(S-) and urea 11-(S-): these compounds demonstrated activity in picomolar range.

Exploring the role of bromine at C(10) of (+)-4-[2-[4-(8-chloro-3,10-dibromo- 6,11-dihydro-5H-benzo[5,6]cyclohepta[1,2-b]pyridin-11(R)-yl)-1-piperidinyl]-2- oxoethyl]-1-piperidinecarboxamide (Sch-66336): the discovery of indolocycloheptapyridine inhibitors of farnesyl protein transferase.

The 10-bromobenzocycloheptapyridyl farnesyl transferase inhibitor (FTI) Sch-66336 (1) is currently under clinical evaluation for the treatment of human cancers. During structure-activity relationship development leading to 1, 10-bromobenzocycloheptapyridyl FTIs were found to be more potent than analogous compounds lacking the 10-Br substituent. This potency enhancement was believed to be due, in part, to an increase in conformational rigidity as the 10-bromo substituent could restrict the conformation of the appended C(11) piperidyl substituent in an axial orientation. A novel and potent class of FTIs, represented by indolocycloheptapyridine Sch-207758 [(+)-10a], have been designed based on this principle. Although structural and thermodynamic results suggest that entropy plays a crucial role in the increased potency observed with (+)-10a through conformational constraints and solvation effects, the results also indicate that the indolocycloheptapyridine moiety in (+)-10a provides increased hydrophobic interactions with the protein through the addition of the indole group. This report details the X-ray structure and the thermodynamic and pharmacokinetic profiles of (+)-10a, as well as the synthesis of indolocycloheptapyridine FTIs and their potencies in biochemical and biological assays.