Discovery of Potent and Selective Quinoxaline-Based Protease-Activated Receptor 4 (PAR4) Antagonists for the Prevention of Arterial Thrombosis.

PAR4 is a promising antithrombotic target with potential for separation of efficacy from bleeding risk relative to current antiplatelet therapies. In an effort to discover a novel PAR4 antagonist chemotype, a quinoxaline-based HTS hit 3 with low µM potency was identified. Optimization of the HTS hit through the use of positional SAR scanning and the design of conformationally constrained cores led to the discovery of a quinoxaline-benzothiazole series as potent and selective PAR4 antagonists. The lead compound 48, possessing a 2 nM IC50 against PAR4 activation by γ-thrombin in platelet-rich plasma (PRP) and greater than 2500-fold selectivity versus PAR1, demonstrated robust antithrombotic efficacy and minimal bleeding in the cynomolgus monkey models.

Discovery of 12 (BMS-986172) as a Highly Potent MGAT2 Inhibitor that Achieved Targeted Efficacious Exposures at a Low Human Dose for the Treatment of Metabolic Disorders.

A series of dihydropyridinone (DHP) compounds was prepared and evaluated for MGAT2 activity. The efforts led to the identification of novel tetrazolones with potent MGAT2 inhibitory activity and favorable in vitro profiles. Further tests of select analogues in mouse models revealed significant reduction in food intake and body weight. Subsequent studies in MGAT2 knockout mice with the lead candidate 12 (BMS-986172) showed on-target- and mechanism-based pharmacology. Moreover, its favorable pharmacokinetic (PK) profile and the lack of species variability in the glucuronidation potential resulted in a greater confidence level in the projection of a low dose for achieving targeted efficacious exposures in humans. Consistent with these projections, PK data from a phase 1 trial confirmed that targeted efficacious exposures could be achieved at a low dose in humans, which supported compound 12 as our second and potentially superior development candidate for the treatment of various metabolic disorders.

Screening Hit to Clinical Candidate: Discovery of BMS-963272, a Potent, Selective MGAT2 Inhibitor for the Treatment of Metabolic Disorders.

MGAT2 inhibition is a potential therapeutic approach for the treatment of metabolic disorders. High-throughput screening of the BMS internal compound collection identified the aryl dihydropyridinone compound 1 (hMGAT2 IC50 = 175 nM) as a hit. Compound 1 had moderate potency against human MGAT2, was inactive vs mouse MGAT2 and had poor microsomal metabolic stability. A novel chemistry route was developed to synthesize aryl dihydropyridinone analogs to explore structure-activity relationship around this hit, leading to the discovery of potent and selective MGAT2 inhibitors 21f, 21s, and 28e that are stable to liver microsomal metabolism. After triaging out 21f due to its inferior in vivo potency, pharmacokinetics, and structure-based liabilities and tetrazole 28e due to its inferior channel liability profile, 21s (BMS-963272) was selected as the clinical candidate following demonstration of on-target weight loss efficacy in the diet-induced obese mouse model and an acceptable safety and tolerability profile in multiple preclinical species.

Rapid Compound Integrity Assessment for High-Throughput Screening Hit Triaging.

Hits from high-throughput screening (HTS) assays are typically evaluated using cheminformatics and/or empirical approaches before a decision for follow-up (activity confirmation and/or sample resynthesis) is made. However, the compound integrity (i.e., identity and purity) of these hits often remains largely unknown at this stage, since many compounds in the screening collection could undergo various changes such as degradation, polymerization, and precipitation during storage over time. When compound integrity is actually assessed for HTS hits postassay to address this issue, the process often increases the overall cycle time by weeks due to the reacquisition of the samples and the lengthy liquid chromatography-ultraviolet/mass spectrometric analysis time. Here we present a novel approach where compound integrity data are collected concurrently with the concentration-response curve (CRC) stage of HTS, with both assays occurring either in parallel on two distributions from the same liquid sample or serially using the original source liquid sample. The rapid generation of compound integrity data has been enabled by a high-speed ultra-high-pressure liquid chromatography-ultraviolet/mass spectrometric platform capable of analyzing ~2000 samples per instrument per week. From this parallel approach, both compound integrity and CRC potency results for screening hits become available to medicinal chemists at the same time, which has greatly enhanced the decision-making process for hit follow-up and progression. In addition, the compound integrity results from recent hits provide a real-time and representative "snapshot" of the sample integrity of the entire compound collection, and the data can be used for in-depth analyses of the screening collection.

Discovery of Potent Protease-Activated Receptor 4 Antagonists with in Vivo Antithrombotic Efficacy.

In an effort to identify novel antithrombotics, we have investigated protease-activated receptor 4 (PAR4) antagonism by developing and evaluating a tool compound, UDM-001651, in a monkey thrombosis model. Beginning with a high-throughput screening hit, we identified an imidazothiadiazole-based PAR4 antagonist chemotype. Detailed structure-activity relationship studies enabled optimization to a potent, selective, and orally bioavailable PAR4 antagonist, UDM-001651. UDM-001651 was evaluated in a monkey thrombosis model and shown to have robust antithrombotic efficacy and no prolongation of kidney bleeding time. This combination of excellent efficacy and safety margin strongly validates PAR4 antagonism as a promising antithrombotic mechanism.

Blockade of protease-activated receptor-4 (PAR4) provides robust antithrombotic activity with low bleeding.

Antiplatelet agents are proven efficacious treatments for cardiovascular and cerebrovascular diseases. However, the existing drugs are compromised by unwanted and sometimes life-threatening bleeding that limits drug usage or dosage. There is a substantial unmet medical need for an antiplatelet drug with strong efficacy and low bleeding risk. Thrombin is a potent platelet agonist that directly induces platelet activation via the G protein (heterotrimeric guanine nucleotide-binding protein)-coupled protease-activated receptors PAR1 and PAR4. A PAR1 antagonist is approved for clinical use, but its use is limited by a substantial bleeding risk. Conversely, the potential of PAR4 as an antiplatelet target has not been well characterized. Using anti-PAR4 antibodies, we demonstrated a low bleeding risk and an effective antithrombotic profile with PAR4 inhibition in guinea pigs. Subsequently, high-throughput screening and an extensive medicinal chemistry effort resulted in the discovery of BMS-986120, an orally active, selective, and reversible PAR4 antagonist. In a cynomolgus monkey arterial thrombosis model, BMS-986120 demonstrated potent and highly efficacious antithrombotic activity. BMS-986120 also exhibited a low bleeding liability and a markedly wider therapeutic window compared to the standard antiplatelet agent clopidogrel tested in the same nonhuman primate model. These preclinical findings define the biological role of PAR4 in mediating platelet aggregation. In addition, they indicate that targeting PAR4 is an attractive antiplatelet strategy with the potential to treat patients at a high risk of atherothrombosis with superior safety compared with the current standard of care.

Triazolopyrimidines identified as reversible myeloperoxidase inhibitors.

Myeloperoxidase, a mammalian peroxidase involved in the immune system as an anti-microbial first responder, can produce hypochlorous acid in response to invading pathogens. Myeloperoxidase has been implicated in several chronic pathological diseases due to the chronic production of hypochlorous acid, as well as other reactive radical species. A high throughput screen and triaging protocol was developed to identify a reversible inhibitor of myeloperoxidase toward the potential treatment of chronic diseases such as atherosclerosis. The identification and characterization of a reversible myeloperoxidase inhibitor, 7-(benzyloxy)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-amine is described.

A Small Molecule Inhibitor Selectively Induces Apoptosis in Cells Transformed by High Risk Human Papilloma Viruses.

A phenotypic high-throughput cell culture screen was performed to identify compounds that prevented proliferation of the human Papilloma virus type 16 (HPV-16) transformed cell line Ca Ski. A series of quinoxaline compounds exemplified by Compound 1 was identified. Testing against a panel of cell lines demonstrated that Compound 1 selectively inhibited replication of all HPV-16, HPV-18, and HPV-31 transformed cell lines tested with 50% Inhibitory Concentration (IC50) values of 2 to 8 µM relative to IC50 values of 28 to 73 µM in HPV-negative cell lines. Treatment with Compound 1 resulted in a cascade of multiple apoptotic events, including selective activation of effector caspases 3 and 7, fragmentation of cellular DNA, and PARP (poly(ADP-ribose) polymerase) cleavage in HPV-positive cells relative to HPV-negative cells. Unregulated proliferation of HPV transformed cells is dependent on the viral oncogenes, E6 and E7. Treatment with Compound 1 resulted in a decrease in HPV E7 protein in Ca Ski cells. However, the timing of this reduction relative to other effects of compound treatment suggests that this was a consequence, rather than a cause, of the apoptotic cascade. Likewise, compound treatment resulted in no obvious effects on the E6- and E7- mediated down regulation of p53 and Rb, or their downstream effectors, p21 or PCNA. Further investigation of apoptotic signals induced by Compound 1 revealed cleavage of Caspase-8 in HPV-positive cells as early as 2 hours post-treatment, suggesting the compound initiates apoptosis through the extrinsic, death receptor-mediated, pathway of cell death. These studies provide proof of concept that cells transformed by oncogenic Papillomaviruses can be selectively induced to undergo apoptosis by compound treatment.

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-ß peptide (Aß), particularly the 42-amino acid Aß1-42, in the brain. Aß1-42 levels can be decreased by γ-secretase modulators (GSM), which are small molecules that modulate γ-secretase, an enzyme essential for Aß production. BMS-869780 is a potent GSM that decreased Aß1-42 and Aß1-40 and increased Aß1-37 and Aß1-38, without inhibiting overall levels of Aß peptides or other APP processing intermediates. BMS-869780 also did not inhibit Notch processing by γ-secretase and lowered brain Aß1-42 without evidence of Notch-related side effects in rats. Human pharmacokinetic (PK) parameters were predicted through allometric scaling of PK in rat, dog, and monkey and were combined with the rat pharmacodynamic (PD) parameters to predict the relationship between BMS-869780 dose, exposure and Aß1-42 levels in human. Off-target and safety margins were then based on comparisons to the predicted exposure required for robust Aß1-42 lowering. Because of insufficient safety predictions and the relatively high predicted human daily dose of 700 mg, further evaluation of BMS-869780 as a potential clinical candidate was discontinued. Nevertheless, BMS-869780 demonstrates the potential of the GSM approach for robust lowering of brain Aß1-42 without Notch-related side effects.

In vitro Characterization of a small molecule inhibitor of the alanine serine cysteine transporter -1 (SLC7A10).

NMDA receptor hypofunction is hypothesized to contribute to cognitive deficits associated with schizophrenia. Since direct activation of NMDA receptors is associated with serious adverse effects, modulation of the NMDA co-agonists, glycine or D-serine, represents a viable alternative therapeutic approach. Indeed, clinical trials with glycine and D-serine have shown positive results, although concerns over toxicity related to the high-doses required for efficacy remain. Synaptic concentrations of D-serine and glycine are regulated by the amino acid transporter alanine serine cysteine transporter-1 (asc-1). Inhibition of asc-1 would increase synaptic D-serine and possibly glycine, eliminating the need for high-dose systemic D-serine or glycine treatment. In this manuscript, we characterize Compound 1 (BMS-466442), the first known small molecule inhibitor of asc-1. Compound 1 selectively inhibited asc-1 mediated D-serine uptake with nanomolar potency in multiple cellular systems. Moreover, Compound 1 inhibited asc-1 but was not a competitive substrate for this transporter. Compound 1 is the first reported selective inhibitor of the asc-1 transporter and may provide a new path for the development of asc-1 inhibitors for the treatment of schizophrenia.

Identification of small molecules that selectively inhibit diacylglycerol lipase-α activity.

Recent genetic evidence suggests that the diacylglycerol lipase (DAGL-α) isoform is the major biosynthetic enzyme for the most abundant endocannabinoid, 2-arachidonoyl-glycerol (2-AG), in the central nervous system. Revelation of its essential role in regulating retrograde synaptic plasticity and adult neurogenesis has made it an attractive therapeutic target. Therefore, it has become apparent that selective inhibition of DAGL-α enzyme activity with a small molecule could be a strategy for the development of novel therapies for the treatment of disease indications such as depression, anxiety, pain, and cognition. In this report, the authors present the identification of small-molecule inhibitor chemotypes of DAGL-α, which were selective (≥10-fold) against two other lipases, pancreatic lipase and monoacylglycerol lipase, via high-throughput screening of a diverse compound collection. Seven chemotypes of interest from a list of 185 structural clusters, which included 132 singletons, were initially selected for evaluation and characterization. Selection was based on potency, selectivity, and chemical tractability. One of the chemotypes, the glycine sulfonamide series, was prioritized as an initial lead for further medicinal chemistry optimization.

Discovery of (S,E)-3-(2-fluorophenyl)-N-(1-(3-(pyridin-3-yloxy)phenyl)ethyl)-acrylamide as a potent and efficacious KCNQ2 (Kv7.2) opener for the treatment of neuropathic pain.

Acrylamide (S)-6, a potent and efficacious KCNQ2 (Kv7.2) opener, demonstrated significant activity in two models of neuropathic pain and in the formalin test, suggesting that KCNQ2 openers may be useful in the treatment of neuropathic pain including diabetic neuropathy.

Discovery of a novel series of quinolone α7 nicotinic acetylcholine receptor agonists.

High throughput screening led to the identification of a novel series of quinolone α7 nicotinic acetylcholine receptor (nAChR) agonists. Optimization of an HTS hit (1) led to 4-phenyl-1-(quinuclidin-3-ylmethyl)quinolin-2(1H)-one, which was found to be potent and selective. Poor brain penetrance in this series was attributed to transporter-mediated efflux, which was in turn due to high pKa. A novel 4-fluoroquinuclidine significantly lowered the pKa of the quinuclidine moiety, reducing efflux as measured by a Caco-2 assay.

Preliminary SAR studies on non-apamin-displacing 4-(aminomethylaryl)pyrrazolopyrimidine K(Ca) channel blockers.

An exploratory SAR study on a series of potent, non-apamin-displacing 4-(aminomethylaryl)pyrazolopyrimidine K(Ca) channel blockers is described and their selectivity against K(Ca) channel subtypes is reported. The most potent analog, 5-chloro-N-(thiophen-2-ylmethyl)pyrazolo[1,5-a]pyrimidin-7-amine (24) displayed sub-micromolar activity in both a thallium flux and whole-cell electrophysiology assay and did not displace apamin in a competitive binding study.

Initial SAR studies on apamin-displacing 2-aminothiazole blockers of calcium-activated small conductance potassium channels.

An initial SAR study on a series of apamin-displacing 2-aminothiazole K(Ca)2 channel blockers is described. Potent inhibitors such as N-(4-methylpyridin-2-yl)-4-(pyridin-2-yl)thiazol-2-amine (13) are disclosed, and for select members of the series, the relationship between the observed activity in a thallium flux, a binding and a whole-cell electrophysiology assay is presented.

Characterization of the 5-HT2b receptor in evaluation of aequorin detection of calcium mobilization for miniaturized GPCR high-throughput screening.

Fluorescent detection of calcium mobilization has been used successfully to identify modulators of G-protein-coupled receptors (GPCRs); however, inherent issues with fluorescence may limit its potential for high-throughput screening miniaturization. The data presented here demonstrate that the calcium-sensitive photoprotein aequorin (AequoScreen), when compared with FLUO-4 in the same cellular background, allows for miniaturization of functional kinetic calcium flux assays, in which the rank order of potency and efficacy was maintained for a series of diverse small-molecule modulators. Small-volume (<10 microL) 384- and 1536-well aequorin assays were implemented by integration of acoustic dispensing (Echo 550) and kinetic flash luminometry (CyBi Lumax). The enhanced high signal-to-background ratios observed relative to fluorescence were readily manipulated by altering per-well cell densities and yielded acceptable screening statistics in miniaturized format for both agonist and antagonist screening scenarios. In addition, the authors demonstrate the feasibility of using agonist concentrations less than EC(50) in a miniaturized antagonist assay. These features, coupled with improved sample handling, should enhance sensitivity and provide the benefits of miniaturization including cost reduction and throughput gains.

BL-1249 [(5,6,7,8-tetrahydro-naphthalen-1-yl)-[2-(1H-tetrazol-5-yl)-phenyl]-amine]: a putative potassium channel opener with bladder-relaxant properties.

BL-1249 [(5,6,7,8-tetrahydro-naphthalen-1-yl)-[2-(1H-tetrazol-5-yl)-phenyl]-amine] produced a concentration-dependent membrane hyperpolarization of cultured human bladder myocytes, assessed as either a reduction in fluorescence of the voltage-sensitive dye bis-(1,2-dibutylbarbituric acid)trimethine oxonol (EC50 = 1.26 +/- 0.6 microM) or by direct electrophysiological measurement (EC50 = 1.49 +/- 0.08 microM). BL-1249 also produced a membrane hyperpolarization of acutely dissociated rat bladder myocytes. Voltage-clamp studies in human bladder cells revealed that BL-1249 activated an instantaneous, noninactivating current that reversed near E(K). The BL-1249-evoked outward K+ current was insensitive to blockade by glyburide, tetraethylammonium, iberiotoxin, 4-aminopyridine, apamin, or Mg2+. However, the current was inhibited by extracellular Ba2+ (10 mM). In in vitro organ bath experiments, BL-1249 produced a concentration-dependent relaxation of 30 mM KCl-induced contractions in rat bladder strips (EC50 = 1.12 +/- 0.37 microM), yet had no effect on aortic strips up to the highest concentration tested (10 microM). The bladder relaxation produced by BL-1249 was partially blocked by Ba2+ (1 and 10 mM) but not by apamin, iberiotoxin, 4-aminopyridine, glyburide, or tetraethylammonium. In an anesthetized rat model, BL-1249 (1 mg/kg i.v.) decreased the number of isovolumic contractions, without significantly affecting blood pressure. Thus, BL-1249 behaves as a potassium channel activator that exhibits bladder versus vascular selectivity both in vitro and in vivo. A survey of potassium channels exhibiting sensitivity to extracellular Ba2+ at millimolar concentration revealed that the expression of the K2P2.1 (TREK-1) channel was relatively high in human bladder cells versus human aortic cells, suggesting this channel as a possible candidate target for BL-1249.

(S,E)-N-[1-(3-heteroarylphenyl)ethyl]-3-(2-fluorophenyl)acrylamides: synthesis and KCNQ2 potassium channel opener activity.

Replacement of the morpholinyl moiety in (S,E)-N-[1-(3-morpholinophenyl)ethyl]-3-phenylacrylamide (1) with heteroaryl groups led to the identification of (S,E)-N-1-[3-(6-fluoropyridin-3-yl)phenyl]ethyl-3-(2-fluorophenyl)acrylamide (5) as a potent KCNQ2 potassium channel opener. Among this series of heteroaryl substituted acrylamides, (S,E)-N-1-[3-(1H-pyrazol-1-yl)phenyl]ethyl-3-(2-fluorophenyl)acrylamide (9) exhibits balanced potency and efficacy. The syntheses and the KCNQ2 opener activity of this series of acrylamides are described.

Synthesis and KCNQ2 opener activity of N-(1-benzo[1,3]dioxol-5-yl-ethyl, N-[1-(2,3-dihydro-benzofuran-5-yl)-ethyl, and N-[1-(2,3-dihydro-1H-indol-5-yl)-ethyl acrylamides.

Bioisosteric replacement studies led to the identification of N-(1-benzo[1,3]dioxol-5-yl-ethyl)-3-(2-chloro-phenyl)-acrylamide ((S)-3) as a highly potent KCNQ2 opener, and 3-(2,6-difluoro-phenyl)-N-[1-(2,3-dihydro-benzofuran-5-yl)-ethyl]-acrylamide ((S)-4), and N-[1-(2,3-dihydro-1H-indol-5-yl)-ethyl]-3-(2-fluoro-phenyl)-acrylamide ((S)-5) as highly efficacious KCNQ2 openers. In contrast, their respective R enantiomers showed significantly less or no appreciable KCNQ2 opener activity even at the highest concentration tested (10 microM). Because of its high potency and moderate efficacy as well as its convenient synthesis, (+/-)-3 was selected as a reference compound for analyzing efficacies of KCNQ openers in electrophysiology studies. Compounds (S)-4 and (S)-5 demonstrated significant activity in reducing neuronal hyperexcitability in rat hippocampal slices. The synthesis and the KCNQ2 opener activity of these acrylamides are described.

(S)-N-[1-(4-cyclopropylmethyl-3,4-dihydro-2H-benzo[1,4]oxazin-6-yl)-ethyl]-3-(2-fluoro-phenyl)-acrylamide is a potent and efficacious KCNQ2 opener which inhibits induced hyperexcitability of rat hippocampal neurons.

(S)-N-[1-(4-Cyclopropylmethyl-3,4-dihydro-2H-benzo[1,4]oxazin-6-yl)-ethyl]-3-(2-fluoro-phenyl)-acrylamide ((S)-2) was identified as a potent and efficacious KCNQ2 opener. This compound demonstrated significant activity in reducing neuronal hyperexcitability in rat hippocampal slices, and the inhibition mediated by (S)-2 was reversed by the KCNQ blocker linopirdine.