Discovery of tetrahydroindazoles as a novel class of potent and in vivo efficacious gamma secretase modulators.

The identification and optimization of a novel series of centrally efficacious gamma secretase modulators (GSMs) offering an alternative to the privileged aryl imidazole motif is described. Chiral bicyclic tetrahydroindazolyl amine substituted triazolopyridines were identified as structurally distinct novel series of GSMs. Representative compound BI-1408 ((R)-42) was demonstrated to be centrally efficacious in rats at a 30 mg/kg oral dose.

Aza follow-ups to BI 207524, a thumb pocket 1 HCV NS5B polymerase inhibitor. Part 1: Mitigating the genotoxic liability of an aniline metabolite.

A series of heterocyclic aza-analogs of BI 207524 (2), a potent HCV NS5B polymerase thumb pocket 1 inhibitor, was investigated with the goal to reduce the liability associated with the release of a genotoxic aniline metabolite in vivo. Analog 4, containing a 2-aminopyridine aniline isostere that is negative in the Ames test was identified, and was found to provide comparable GT1a/1b potency to 2. Although the cross-species PK profile, poor predicted human liver distribution of analog 4 and allometry principles projected high doses to achieve a strong antiviral response in patients, this work has provided a path forward toward the design of novel thumb pocket 1 NS5B polymerase inhibitors with improved safety profiles.

A prodrug strategy for the oral delivery of a poorly soluble HCV NS5B thumb pocket 1 polymerase inhibitor using self-emulsifying drug delivery systems (SEDDS).

A prodrug approach was developed to address the low oral bioavailability of a poorly soluble (<0.1µg/mL in pH 6.8 buffer) but highly permeable thumb pocket 1 HCV NS5B polymerase inhibitor. Bioconversion rates of structurally diverse prodrug derivatives were evaluated in a panel of in vitro assays using microsomes, from either liver or intestinal tissues, simulated intestinal fluids, simulated gastric fluids or plasma. In vivo bioconversion of promising candidates was evaluated following oral administration to rats. The most successful strategy involved modification of the parent drug carboxylic acid moiety to glycolic amide esters which improved solubility in lipid-based self-emulsifying drug delivery systems (SEDDS). Crystalline prodrug analog 36 (mp 161°C) showed good solubility in individual SEDDS components (up to 80mg/mL) compared to parent 2 (<3mg/mL; mp 267°C) and cross-species bioconversions which correlated with in vitro stability in liver microsomes.

Multi-parameter optimization of aza-follow-ups to BI 207524, a thumb pocket 1 HCV NS5B polymerase inhibitor. Part 2: Impact of lipophilicity on promiscuity and in vivo toxicity.

We describe our efforts to identify analogs of thumb pocket 1 HCV NS5B inhibitor 1 (aza-analog of BI 207524) with improved plasma to liver partitioning and a predicted human half-life consistent with achieving a strong antiviral effect at a reasonable dose in HCV-infected patients. Compounds 3 and 7 were identified that met these criteria but exhibited off-target promiscuity in an in vitro pharmacology screen and in vivo toxicity in rats. High lipophilicity in this class was found to correlate with increased probability for promiscuous behavior and toxicity. The synthesis of an 8×11 matrix of analogs allowed the identification of C3, an inhibitor that displayed comparable potency to 1, improved partitioning to the liver and reduced lipophilicity. Although C3 displayed reduced propensity for in vitro off-target inhibition and the toxicity profile in rats was improved, the predicted human half-life of this compound was short, resulting in unacceptable dosing requirements to maintain a strong antiviral effect in patients.

Preclinical profile of BI 224436, a novel HIV-1 non-catalytic-site integrase inhibitor.

BI 224436 is an HIV-1 integrase inhibitor with effective antiviral activity that acts through a mechanism that is distinct from that of integrase strand transfer inhibitors (INSTIs). This 3-quinolineacetic acid derivative series was identified using an enzymatic integrase long terminal repeat (LTR) DNA 3'-processing assay. A combination of medicinal chemistry, parallel synthesis, and structure-guided drug design led to the identification of BI 224436 as a candidate for preclinical profiling. It has antiviral 50% effective concentrations (EC50s) of <15 nM against different HIV-1 laboratory strains and cellular cytotoxicity of >90 µM. BI 224436 also has a low, ∼2.1-fold decrease in antiviral potency in the presence of 50% human serum and, by virtue of a steep dose-response curve slope, exhibits serum-shifted EC95 values ranging between 22 and 75 nM. Passage of virus in the presence of inhibitor selected for either A128T, A128N, or L102F primary resistance substitutions, all mapping to a conserved allosteric pocket on the catalytic core of integrase. BI 224436 also retains full antiviral activity against recombinant viruses encoding INSTI resistance substitutions N155S, Q148H, and E92Q. In drug combination studies performed in cellular antiviral assays, BI 224436 displays an additive effect in combination with most approved antiretrovirals, including INSTIs. BI 224436 has drug-like in vitro absorption, distribution, metabolism, and excretion (ADME) properties, including Caco-2 cell permeability, solubility, and low cytochrome P450 inhibition. It exhibited excellent pharmacokinetic profiles in rat (clearance as a percentage of hepatic flow [CL], 0.7%; bioavailability [F], 54%), monkey (CL, 23%; F, 82%), and dog (CL, 8%; F, 81%). Based on the excellent biological and pharmacokinetic profile, BI 224436 was advanced into phase 1 clinical trials.

Phosphatidylinositol 4-kinase III beta is essential for replication of human rhinovirus and its inhibition causes a lethal phenotype in vivo.

Human rhinovirus (HRV) is the predominant cause of the common cold, but more importantly, infection may have serious repercussions in asthmatics and chronic obstructive pulmonary disorder (COPD) patients. A cell-based antiviral screen against HRV was performed with a subset of our proprietary compound collection, and an aminothiazole series with pan-HRV species and enteroviral activity was identified. The series was found to act at the level of replication in the HRV infectious cycle. In vitro selection and sequencing of aminothiazole series-resistant HRV variants revealed a single-nucleotide mutation leading to the amino acid change I42V in the essential HRV 3A protein. This same mutation has been previously implicated in resistance to enviroxime, a former clinical-stage antipicornavirus agent. Enviroxime-like compounds have recently been shown to target the lipid kinase phosphatidylinositol 4-kinase III beta (PI4KIIIß). A good correlation between PI4KIIIß activity and HRV antiviral potency was found when analyzing the data over 80 compounds of the aminothiazole series, covering a 750-fold potency range. The mechanism of action through PI4KIIIß inhibition was further demonstrated by small interfering RNA (siRNA) knockdown of PI4KB, which reduced HRV replication and also increased the potency of the PI4KIIIß inhibitors. Inhibitors from two different structural classes with promising pharmacokinetic profiles and with very good selectivity for PI4KIIIß were used to dissociate compound-related toxicity from target-related toxicity. Mortality was seen in all dosing groups of mice treated with either compound, therefore suggesting that short-term inhibition of PI4KIIIß is deleterious.

Human intestinal transporter database: QSAR modeling and virtual profiling of drug uptake, efflux and interactions.

PURPOSE: Membrane transporters mediate many biological effects of chemicals and play a major role in pharmacokinetics and drug resistance. The selection of viable drug candidates among biologically active compounds requires the assessment of their transporter interaction profiles. METHODS: Using public sources, we have assembled and curated the largest, to our knowledge, human intestinal transporter database (>5,000 interaction entries for >3,700 molecules). This data was used to develop thoroughly validated classification Quantitative Structure-Activity Relationship (QSAR) models of transport and/or inhibition of several major transporters including MDR1, BCRP, MRP1-4, PEPT1, ASBT, OATP2B1, OCT1, and MCT1. RESULTS: QSAR models have been developed with advanced machine learning techniques such as Support Vector Machines, Random Forest, and k Nearest Neighbors using Dragon and MOE chemical descriptors. These models afforded high external prediction accuracies of 71-100% estimated by 5-fold external validation, and showed hit retrieval rates with up to 20-fold enrichment in the virtual screening of DrugBank compounds. CONCLUSIONS: The compendium of predictive QSAR models developed in this study can be used for virtual profiling of drug candidates and/or environmental agents with the optimal transporter profiles.

Peptide backbone replacement of hepatitis C virus NS3 serine protease C-terminal cleavage product analogs: discovery of potent succinamide inhibitors.

A number of potent peptidic inhibitors of the NS3 protease have been described in the literature based on a substrate-based approach. In an on-going effort to reduce the peptidic character of this class of inhibitors, two novel series of analogs have been prepared in which the usual P3 amino acid residue is replaced by a succinamide fragment. This new backbone modification not only reduces the peptidic nature of traditional inhibitors but also provides new SAR opportunities for the capping group. Optimization of each of these two series resulted in inhibitors with sub-nanomolar potencies.

Anthranilic acid-based Thumb Pocket 2 HCV NS5B polymerase inhibitors with sub-micromolar potency in the cell-based replicon assay.

Optimization efforts on the anthranilic acid-based Thumb Pocket 2 HCV NS5B polymerase inhibitors 1 and 2 resulted in the identification of multiple structural elements that contributed to improved cell culture potency. The additive effect of these elements resulted in compound 46, an inhibitor with enzymatic (IC50) and cell culture (EC50) potencies of less than 100 nanomolar.

Optimization of a 1,5-dihydrobenzo[b][1,4]diazepine-2,4-dione series of HIV capsid assembly inhibitors 2: structure-activity relationships (SAR) of the C3-phenyl moiety.

Detailed structure-activity relationships of the C3-phenyl moiety that allow for the optimization of antiviral potency of a series of 1,5-dihydrobenzo[b][1,4]diazepine-2,4-dione inhibitors of HIV capsid (CA) assembly are described. Combination of favorable substitutions gave additive SAR and allowed for the identification of the most potent compound in the series, analog 27. Productive SAR also transferred to the benzotriazepine and spirobenzodiazepine scaffolds, providing a solution to the labile stereocenter at the C3 position. The molecular basis of how compound 27 inhibits mature CA assembly is rationalized using high-resolution structural information. Our understanding of how compound 27 may inhibit immature Gag assembly is also discussed.

Synthesis and optimization of a novel series of HCV NS3 protease inhibitors: 4-arylproline analogs.

In this report we describe the synthesis and evaluation of diverse 4-arylproline analogs as HCV NS3 protease inhibitors. Introduction of this novel P2 moiety opened up new SAR and, in combination with a synthetic approach providing a versatile handle, allowed for efficient exploitation of this novel series of NS3 protease inhibitors. Multiple structural modifications of the aryl group at the 4-proline, guided by structural analysis, led to the identification of analogs which were very potent in both enzymatic and cell based assays. The impact of this systematic SAR on different drug properties is reported.

Design, synthesis and biological evaluation of novel aminothiazoles as antiviral compounds acting against human rhinovirus.

We describe here the design, synthesis and biological evaluation of antiviral compounds acting against human rhinovirus (HRV). A series of aminothiazoles demonstrated pan-activity against the HRV genotypes screened and productive structure-activity relationships. A comprehensive investigational library was designed and performed allowing the identification of potent compounds with lower molecular weight and improved ADME profile. 31d-1, 31d-2, 31f showed good exposures in CD-1 mice. The mechanism of action was discovered to be a host target: the lipid kinase phosphatidylinositol 4-kinase III beta (PI4KIIIß). The identification of the pan-HRV active compound 31f combined with a structurally distinct literature compound T-00127-HEV1 allowed the assessment of target related tolerability of inhibiting this kinase for a short period of time in order to prevent HRV replication.

Identification of potent and orally bioavailable nucleotide competing reverse transcriptase inhibitors: in vitro and in vivo optimization of a series of benzofurano[3,2-d]pyrimidin-2-one derived inhibitors.

Recently, a new class of HIV reverse transcriptase (HIV-RT) inhibitors has been reported. The novel mechanism of inhibition by this class involves competitive binding to the active site of the RT enzyme and has been termed Nucleotide-Competing Reverse Transcriptase Inhibitors (NcRTIs). In this publication we describe the optimization of a novel benzofurano[3,2-d]pyrimidin-2-one series of NcRTIs. The starting point for the current study was inhibitor 2, which had high biochemical and antiviral potency but only moderate permeability in a Caco-2 assay and high B-to-A efflux, resulting in moderate rat bioavailability and low Cmax. We present herein the results and strategies we employed to optimize both the potency as well as the permeability, metabolic stability and pharmacokinetic profile of this series. One of the key observations of the present study was the importance of shielding polar functionality, at least in the context of the current chemotype, to enhance permeability. These studies led to the identification of inhibitors 39 and 45, which display sub-nanomolar antiviral potency in a p24 ELISA assay with significantly reduced efflux ratios (ratios <1.5). These inhibitors also display excellent rat pharmacokinetic profiles with high bioavailabilities and low clearance.

Assessment of CYP3A-mediated drug-drug interaction potential for victim drugs using an in vivo rat model.

The present study aims to determine if an in vivo rat model of drug-drug interaction (DDI) could be useful to discriminate a sensitive (buspirone) from a 'non-sensitive' (verapamil) CYP3A substrate, using ketoconazole and ritonavir as perpetrator drugs. Prior to in vivo studies, ketoconazole and ritonavir were shown to inhibit midazolam hydroxylation with IC50 values of 350 ± 60 nm and 11 ± 3 nm, respectively, in rat liver microsomes (RLM). Buspirone and verapamil were also shown to be substrates of recombinant rat CYP3A1/3A2. In the rat model, the mean plasma AUC0-inf of buspirone (10 mg/kg, p.o.) was increased by 7.4-fold and 12.8-fold after co-administration with ketoconazole and ritonavir (20 mg/kg, p.o.), respectively. The mean plasma AUC0-inf of verapamil (10 mg/kg, p.o.) was increased by 3.0-fold and 4.8-fold after co-administration with ketoconazole and ritonavir (20 mg/kg, p.o.), respectively. Thus, the rat DDI model correctly identified buspirone as a sensitive CYP3A substrate (>5-fold AUC change) in contrast to verapamil. In addition, for both victim drugs, the extent of DDI when co-administered was greater with ritonavir compared with ketoconazole, in line with their in vitro CYP3A inhibition potency in RLM. In conclusion, our study extended the rat DDI model applicability to two additional victim/perpetrator pairs. In addition, we suggest that use of this model would increase our confidence in estimation of the DDI potential for victim drugs in early discovery.

The liver partition coefficient-corrected inhibitory quotient and the pharmacokinetic-pharmacodynamic relationship of directly acting anti-hepatitis C virus agents in humans.

Pharmacokinetic-pharmacodynamic (PK-PD) data analyses from early hepatitis C virus (HCV) clinical trials failed to show a good correlation between the plasma inhibitory quotient (IQ) and antiviral activity of different classes of directly acting antiviral agents (DAAs). The present study explored whether use of the liver partition coefficient-corrected IQ (LCIQ) could improve the PK-PD relationship. Animal liver partition coefficients (Kp(liver)) were calculated from liver to plasma exposure ratios. In vitro hepatocyte partition coefficients (Kp(hep)) were determined by the ratio of cellular to medium drug concentrations. Human Kp(liver) was predicted using an in vitro-in vivo proportionality method: the species-averaged animal Kp(liver) multiplied by the ratio of human Kp(hep) over those in animals. LCIQ was calculated using the IQ multiplied by the predicted human Kp(liver). Our results demonstrated that the in vitro-in vivo proportionality approach provided the best human Kp(liver) prediction, with prediction errors of <45% for all 5 benchmark drugs evaluated (doxorubicin, verapamil, digoxin, quinidine, and imipramine). Plasma IQ values correlated poorly (r(2) of 0.48) with maximum viral load reduction and led to a corresponding 50% effective dose (ED(50)) IQ of 42, with a 95% confidence interval (CI) of 0.1 to 148534. In contrast, the LCIQ-maximum VLR relationship fit into a typical sigmoidal curve with an r(2) value of 0.95 and an ED(50) LCIQ of 121, with a 95% CI of 83 to 177. The present study provides a novel human Kp(liver) prediction model, and the LCIQ correlated well with the viral load reductions observed in short-term HCV monotherapy of different DAAs and provides a valuable tool to guide HCV drug discovery.

Inhibition of HIV-1 capsid assembly: optimization of the antiviral potency by site selective modifications at N1, C2 and C16 of a 5-(5-furan-2-yl-pyrazol-1-yl)-1H-benzimidazole scaffold.

A uHTS campaign led to the discovery of a 5-(5-furan-2-ylpyrazol-1-yl)-1H-benzimidazole series that inhibits assembly of HIV-1 capsid. Synthetic manipulations at N1, C2 and C16 positions improved the antiviral potency by a . The X-ray structure of 33 complexed with the capsid N-terminal domain allowed identification of major interactions between the inhibitor and the protein.

Cross-species absorption, metabolism, distribution and pharmacokinetics of BI 201335, a potent HCV genotype 1 NS3/4A protease inhibitor.

The present study describes the cross-species absorption, metabolism, distribution and pharmacokinetics of BI 201335, a potent HCV protease inhibitor currently in phase III clinical trials. BI 201335 showed a good Caco-II permeability (8.7 × 10(-6) cm/sec) and in vitro metabolic stability (predicted hepatic clearence (CL(hep)) <19% Q(h) in all species tested). Single dose PK revealed a clearance of 17, 3.0 and 2.6 mL/min/kg in rat, monkey and dog respectively, with a corresponding oral bioavailability of 29.1, 25.5 and 35.6%. Comparative plasma and liver PK profile in rodents showed a high liver Kp in the rat (42-fold), suggesting high target tissue distribution. Simple allometry based on animal PK predicted a human oral CL/F of 168 mL/min, within two-fold of the observed value (118 mL/min) at 240 mg in healthy volunteers. Allometry of volume of distribution generated a low exponent of 0.59, and a much lower predicted Vss/F (5-fold less than observed). Several different approaches of Vss/F prediction were evaluated and compared with the value observed in human. The averaged Vss/F from preclinical animals provides the best estimation of the observed human value (169 L vs. 175 L). Corresponding human "effective" t(1/2) values were also compared. The predicted human t(1/2) based on the CL from allometry with metabolic corrections and the averaged animal Vss represented the best estimation of the clinical data (12.1 vs. 17.2 hr). The present study demonstrated that the good preclinical ADMEPK profile of BI 201335 is consistent with that observed in the clinic. While preclinical data accurately predicted the human CL, the prediction of human Vss seems to be more challenging. The averaged Vss/F from all tested preclinical animals provided the best prediction of human Vss and the resulting "effective" t(1/2).

Sema3A Antibody BI-X Prevents Cell Permeability and Cytoskeletal Collapse in HRMECs and Increases Tip Cell Density in Mouse Oxygen-Induced Retinopathy.

Purpose: Semaphorin 3A (Sema3A) is an axonal guidance molecule that inhibits angiogenesis by vasorepulsion and blocks revascularization in the ischemic retina. BI-X is an intravitreal anti-Sema3A agent under clinical investigation in patients with proliferative diabetic retinopathy (PDR) and diabetic macular ischemia (DMI). Methods: Surface plasmon resonance was used to determine binding affinity of BI-X to human and murine Sema3A. In vitro, human retinal microvascular endothelial cells (HRMECs) were used to assess effects of BI-X on cell permeability and cytoskeletal collapse induced by Sema3A. In vivo, intravitreal BI-X or an anti-trinitrophenol control antibody was administered in both eyes in mice with oxygen-induced retinopathy (OIR). Retinal flat mounts were prepared, and avascular area and tip cell density were determined using confocal laser-scanning microscopy. Results: Dissociation constants for BI-X binding to human and murine Sema3A were 29 pM and 27 pM, respectively. In vitro, BI-X prevented HRMEC permeability and cytoskeletal collapse induced by Sema3A. In vivo, BI-X increased tip cell density by 33% (P < 0.001) and reduced avascular area by 12% (not significant). A significant negative correlation was evident between avascular area and tip cell density (r2 = 0.4205, P < 0.0001). Conclusions: BI-X binds to human Sema3A with picomolar affinity and prevents cell permeability and cytoskeletal collapse in HRMECs. BI-X also enhances revascularization in mice with OIR. Translational Relevance: BI-X is a potent inhibitor of human Sema3A that improves revascularization in a murine model of OIR; BI-X is currently being investigated in patients with laser-treated PDR and DMI.

From benzimidazole to indole-5-carboxamide Thumb Pocket I inhibitors of HCV NS5B polymerase. Part 1: indole C-2 SAR and discovery of diamide derivatives with nanomolar potency in cell-based subgenomic replicons.

Replacement of the benzimidazole core of allosteric Thumb Pocket 1 HCV NS5B finger loop inhibitors by more lipophilic indole derivatives provided up to 30-fold potency improvements in cell-based subgenomic replicon assays. Optimization of C-2 substitution on the indole core led to the identification of analogs with EC(50)<100 nM and modulated the pharmacokinetic properties of the inhibitors based on preliminary data from in vitro ADME profiles and in vivo rat PK.

Indole 5-carboxamide Thumb Pocket I inhibitors of HCV NS5B polymerase with nanomolar potency in cell-based subgenomic replicons (part 2): central amino acid linker and right-hand-side SAR studies.

In this part 2, new indole 5-carboxamide Thumb Pocket 1 inhibitors of HCV NS5B polymerase are described. Structure-activity relationships (SAR) were explored at the central amino acid linker position and the right-hand-side of the molecule in an attempt to identify molecules with a balanced overall profile of potency (EC(50)<100 nM), physicochemical properties and ADME characteristics.