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.

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.

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.

(S)-N-[1-(3-morpholin-4-ylphenyl)ethyl]- 3-phenylacrylamide: an orally bioavailable KCNQ2 opener with significant activity in a cortical spreading depression model of migraine.

(S)-N-[1-(3-Morpholin-4-ylphenyl)ethyl]-3-phenylacrylamide (2) was synthesized as an orally bioavailable KCNQ2 potassium channel opener. In a rat model of migraine, 2 demonstrated significant oral activity in reducing the total number of cortical spreading depressions induced by potassium chloride.