Discovery of BAY-390, a Selective CNS Penetrant Chemical Probe as Transient Receptor Potential Ankyrin 1 (TRPA1) Antagonist.

Transient receptor potential ankyrin 1 (TRPA1) is a voltage-dependent, ligand-gated ion channel, and activation thereof is linked to a variety of painful conditions. Preclinical studies have demonstrated the role of TRPA1 receptors in a broad range of animal models of acute, inflammatory, and neuropathic pain. In addition, a clinical study using the TRPA1 antagonist GRC-17536 (Glenmark Pharmaceuticals) demonstrated efficacy in a subgroup of patients with painful diabetic neuropathy. Consequently, there is an increasing interest in TRPA1 inhibitors as potential analgesics. Herein, we report the identification of a fragment-like hit from a high-throughput screening (HTS) campaign and subsequent optimization to provide a novel and brain-penetrant TRPA1 inhibitor (compound 18, BAY-390), which is now being made available to the research community as an open-source in vivo probe.

Discovery and Characterization of Selective and Ligand-Efficient DYRK Inhibitors.

Dual-specificity tyrosine-regulated kinase 1A (DYRK1A) regulates the proliferation and differentiation of neuronal progenitor cells during brain development. Consequently, DYRK1A has attracted interest as a target for the treatment of neurodegenerative diseases, including Alzheimer's disease (AD) and Down's syndrome. Recently, the inhibition of DYRK1A has been investigated as a potential treatment for diabetes, while DYRK1A's role as a mediator in the cell cycle has garnered interest in oncologic indications. Structure-activity relationship (SAR) analysis in combination with high-resolution X-ray crystallography leads to a series of pyrazolo[1,5-b]pyridazine inhibitors with excellent ligand efficiencies, good physicochemical properties, and a high degree of selectivity over the kinome. Compound 11 exhibited good permeability and cellular activity without P-glycoprotein liability, extending the utility of 11 in an in vivo setting. These pyrazolo[1,5-b]pyridazines are a viable lead series in the discovery of new therapies for the treatment of diseases linked to DYRK1A function.

Discovery and optimization of pyridyl-cycloalkyl-carboxylic acids as inhibitors of microsomal prostaglandin E synthase-1 for the treatment of endometriosis.

Here we report on novel and potent pyridyl-cycloalkyl-carboxylic acid inhibitors of microsomal prostaglandin E synthase-1 (PTGES). PTGES produces, as part of the prostaglandin pathway, prostaglandin E2 which is a well-known driver for pain and inflammation. This fact together with the observed upregulation of PTGES during inflammation suggests that blockade of the enzyme might provide a beneficial treatment option for inflammation related conditions such as endometriosis. Compound 5a, a close analogue of the screening hit, potently inhibited PTGES in vitro, displayed excellent PK properties in vitro and in vivo and demonstrated efficacy in a CFA-induced pain model in mice and in a rat dyspareunia endometriosis model and was therefore selected for further studies.

Identification of a Benzimidazolecarboxylic Acid Derivative (BAY 1316957) as a Potent and Selective Human Prostaglandin E2 Receptor Subtype 4 (hEP4-R) Antagonist for the Treatment of Endometriosis.

The presence and growth of endometrial tissue outside the uterine cavity in endometriosis patients are primarily driven by hormone-dependent and inflammatory processes-the latter being frequently associated with severe, acute, and chronic pelvic pain. The EP4 subtype of prostaglandin E2 (PGE2) receptors (EP4-R) is a particularly promising anti-inflammatory and antinociceptive target as both this receptor subtype and the pathways forming PGE2 are highly expressed in endometriotic lesions. High-throughput screening resulted in the identification of benzimidazole derivatives as novel hEP4-R antagonists. Careful structure-activity relationship investigation guided by rational design identified a methyl substitution adjacent to the carboxylic acid as an appropriate means to accomplish favorable pharmacokinetic properties by reduction of glucuronidation. Further optimization led to the identification of benzimidazolecarboxylic acid BAY 1316957, a highly potent, specific, and selective hEP4-R antagonist with excellent drug metabolism and pharmacokinetics properties. Notably, treatment with BAY 1316957 can be expected to lead to prominent and rapid pain relief and significant improvement of the patient's quality of life.

Cobalt versus Osmium: Control of Both trans and cis Selectivity in Construction of the EFG Rings of Pectenotoxin†4.

Catalytic oxidative cyclisation reactions have been employed for the synthesis of the E and F rings of the complex natural product target pectenotoxin 4. The choice of metal catalyst (cobalt- or osmium-based) allowed for the formation of THF rings with either trans or cis stereoselectivity. Fragment union using a modified Julia reaction then enabled the synthesis of an advanced synthetic intermediate containing the EF and G rings of the target.

Metabolism and pharmacokinetics of JM6 in mice: JM6 is not a prodrug for Ro-61-8048.

Understanding whether regulation of tryptophan metabolites can ameliorate neurodegeneration is of high interest to investigators. A recent publication describes 3,4-dimethoxy-N-(4-(3-nitrophenyl)-5-(piperidin-1-ylmethyl)thiazol-2-yl)benzenesulfonamide (JM6) as a novel prodrug for the kynurenine 3-monooxygenase (KMO) inhibitor 3,4-dimethoxy-N-(4-(3-nitrophenyl)thiazol-2-yl)benzenesulfonamide (Ro-61-8048) that elicits therapeutic effects in mouse models of Huntington's and Alzheimer's diseases (Cell 145:863-874, 2011). Our evaluation of the metabolism and pharmacokinetics of JM6 and Ro-61-8048 indicate instead that Ro-61-8048 concentrations in mouse plasma after JM6 administration originate from a Ro-61-8048 impurity (<0.1%) in JM6. After a 0.05 mg/kg Ro-61-8048 oral dose alone or coadministered with 10 mg/kg JM6 to mice, the Ro-61-8048 areas under the concentration-time curves (AUCs) from 0 to infinity were similar (4300 and 4900 nM × h, respectively), indicating no detectable contributions of JM6 metabolism to the Ro-61-8048 AUCs. JM6 was stable in incubations under acidic conditions and Ro-61-8048 was not a product of JM6 metabolism in vitro (plasma, blood, or hepatic models). Species differences in the quantitative rate of oxidative metabolism indicate that major circulating JM6 metabolite(s) in mice are unlikely to be major in humans: JM6 is rapidly metabolized via the piperidyl moiety in mouse (forming an iminium ion reactive intermediate) but is slowly metabolized in human (in vitro), primarily via O-dealkylation at the phenyl ring. Our data indicate that JM6 is not a prodrug for Ro-61-8048 and is not a potent KMO inhibitor.

The neuroprotective effect of a specific P2X7 receptor antagonist derives from its ability to inhibit assembly of the NLRP3 inflammasome in glial cells.

Release of interleukin (IL)-1ß from immunocompetent cells requires formation of the NACHT, LLR and PYD domains-containing protein 3 (NLRP3) inflammasome and caspase 1 activation. Adenosine 5'-triphosphate (ATP), acting on the P2X(7) receptor, is one factor that stimulates inflammasome assembly. We show that a novel specific P2X(7) receptor antagonist, GSK1370319A, inhibits ATP-induced increase in IL-1ß release and caspase 1 activation in lipopolysaccharide (LPS)-primed mixed glia by blocking assembly of the inflammasome in a pannexin 1-dependent manner. GSK1370319A also inhibits ATP-induced subregion-specific neuronal loss in hippocampal organotypic slice cultures, which is dependent on its ability to prevent inflammasome assembly in glia. Significantly, GSK1370319A attenuates age-related deficits in long-term potentiation (LTP) and inhibits the accompanying age-related caspase 1 activity. We conclude that inhibiting P2X(7) receptor-activated NLRP3 inflammasome formation and the consequent IL-1ß release from glia preserve neuronal viability and synaptic activity.

The influence of exocyclic stereochemistry on the tethered aminohydroxylation reaction.

A new strategy that employs an exocyclic stereocenter to effect diastereocontrol in the tethered aminohydroxylation (TA) reaction is applied to the stereoselective synthesis of a range of amino alcohols in good to excellent yields, and with anti selectivities of up to 20:1. The influence of the reaction conditions and substrate parameters on the level of diastereocontrol is described. Furthermore, an "inside alkoxy" model is employed to rationalize the sense and degree of stereoselectivity observed in these systems.

Identification of 2-oxo-N-(phenylmethyl)-4-imidazolidinecarboxamide antagonists of the P2X(7) receptor.

A backup molecule to compound 2 was sought by targeting the most likely metabolically vulnerable site in this molecule. Compound 18 was subsequently identified as a potent P2X(7) antagonist with very low in vivo clearance and high oral bioavailability in all species examined. Some evidence to support the role of P2X(7) in the etiology of pain is also presented.

A novel oxidative cyclisation onto vinyl silanes.

A novel osmium-catalysed oxidative cyclisation of 1,2-diols bearing a pendant vinyl silane affords THFs that contain silicon functionality at the ring junction. When the cyclisation occurs onto a vinyl benzyldimethylsilyl group, the resulting silyl group can act as a masked hydroxyl group and undergo a Fleming-Tamao type oxidation at a later stage to form the corresponding lactol. The scope of this reaction can also be extended beyond 1,2-diols and applied to the cyclisation of α-hydroxy-sulfonamides and α-hydroxy-amides.

Discovery and structure-activity relationships of a series of pyroglutamic acid amide antagonists of the P2X7 receptor.

A computational lead-hopping exercise identified compound 4 as a structurally distinct P2X(7) receptor antagonist. Structure-activity relationships (SAR) of a series of pyroglutamic acid amide analogues of 4 were investigated and compound 31 was identified as a potent P2X(7) antagonist with excellent in vivo activity in animal models of pain, and a profile suitable for progression to clinical studies.

Synthesis and biological activity of a series of tetrasubstituted-imidazoles as P2X(7) antagonists.

A series of analogues of the pyrazole lead 1 were synthesized in which the heterocyclic core was replaced with an imidazole. A number of potent antagonists were identified and structure-activity relationships (SAR) were investigated both with respect to activity at the P2X(7) receptor and in vitro metabolic stability. Compound 10 was identified as a potent P2X(7) antagonist with reduced in vitro metabolism and high solubility.

BACE-1 hydroxyethylamine inhibitors using novel edge-to-face interaction with Arg-296.

Inhibition of the aspartyl protease BACE-1 has the potential to deliver a disease-modifying therapy for Alzheimer's disease. Herein, is described a series of potent inhibitors based on an hydroxyethylamine (HEA) transition state mimetic template. These inhibitors interact with the non prime side of the enzyme using a novel edge-to-face interaction with Arg-296.

Structure-activity relationships and in vivo activity of (1H-pyrazol-4-yl)acetamide antagonists of the P2X(7) receptor.

Structure-activity relationships (SAR) of analogues of lead compound 1 were investigated and compound 16 was selected for further study in animal models of pain. Compound 16 was shown to be a potent antihyperalgesic agent in both the rat acute complete Freund's adjuvant (CFA) model of inflammatory pain [Iadarola, M. J.; Douglass, J.; Civelli, O.; Naranjo, J. R. rain Res.1988, 455, 205] and the knee joint model of chronic inflammatory pain [Wilson, A. W.; Medhurst, S. J.; Dixon, C. I.; Bontoft, N. C.; Winyard, L. A.; Brackenborough, K. T.; De Alba, J.; Clarke, C. J.; Gunthorpe, M. J.; Hicks, G. A.; Bountra, C.; McQueen, D. S.; Chessell, I. P. Eur. J. Pain2006, 10, 537].

Synthesis and structure-activity relationships of a series of (1H-pyrazol-4-yl)acetamide antagonists of the P2X7 receptor.

High-throughput screening identified compound 1 as a potent P2X(7) receptor antagonist suitable for lead optimisation. Structure-activity relationships (SAR) of a series of (1H-pyrazol-4-yl)acetamides were investigated and compound 32 was identified as a potent P2X(7) antagonist with enhanced potency and favourable physicochemical and pharmacokinetic properties.

Second generation of BACE-1 inhibitors part 2: Optimisation of the non-prime side substituent.

Our first generation of hydroxyethylamine transition-state mimetic BACE-1 inhibitors allowed us to validate BACE-1 as a key target for Alzheimer's disease by demonstrating amyloid lowering in an animal model, albeit at rather high doses. Finding a molecule from this series which was active at lower oral doses proved elusive and demonstrated the need to find a novel series of inhibitors with improved pharmacokinetics. This Letter describes the discovery of such inhibitors.

Second generation of BACE-1 inhibitors. Part 1: The need for improved pharmacokinetics.

Inhibition of the aspartyl protease BACE-1 has the potential to deliver a disease-modifying therapy for Alzheimer's disease. We have recently disclosed a series of transition-state mimetic BACE-1 inhibitors showing nanomolar potency in cell-based assays. Amongst them, GSK188909 (compound 2) had favorable pharmacokinetics and was the first orally bioavailable inhibitor reported to demonstrate brain amyloid lowering in an animal model. In this Letter, we describe the reasons that led us to favor a second generation of inhibitors for further in vivo studies.

Second generation of BACE-1 inhibitors part 3: Towards non hydroxyethylamine transition state mimetics.

Our first generation of hydroxyethylamine BACE-1 inhibitors proved unlikely to provide molecules that would lower amyloid in an animal model at low oral doses. This observation led us to the discovery of a second generation of inhibitors having nanomolar activity in a cell-based assay and with the potential for improved pharmacokinetic profiles. In this Letter, we describe our successful strategy for the optimization of oral bioavailability and also give insights into the design of compounds with the potential for improved brain penetration.

Mechanism of action of species-selective P2X(7) receptor antagonists.

BACKGROUND AND PURPOSE: AZ11645373 and N-{2-methyl-5-[(1R, 5S)-9-oxa-3,7-diazabicyclo[3.3.1]non-3-ylcarbonyl]phenyl}-2-tricyclo[3.3.1.13,7]dec-1-ylacetamide hydrochloride (compound-22) are recently described P2X(7) receptor antagonists. In this study we have further characterized these compounds to determine their mechanism of action and interaction with other species orthologues. EXPERIMENTAL APPROACH: Antagonist effects at recombinant and chimeric P2X(7) receptors were assessed by ethidium accumulation and radioligand-binding studies. KEY RESULTS: AZ11645373 and compound-22 were confirmed as selective non-competitive antagonists of human or rat P2X(7) receptors respectively. Both compounds were weak antagonists of the mouse and guinea-pig P2X(7) receptors and, for each compound, their potency estimates at human and dog P2X(7) receptors were similar. The potency of compound-22 was moderately temperature-dependent while that of AZ11645373 was not. The antagonist effects of both compounds were slowly reversible and were not prevented by decavanadate, suggesting that they were allosteric antagonists. Indeed, the compounds competed for binding sites labelled by an allosteric radio-labelled P2X(7) receptor antagonist. The species selectivity of AZ11645373, but not compound-22, was influenced by the nature of the amino acid at position 95 of the P2X(7) receptor. N(2)-(3,4-difluorophenyl)-N(1)-[2-methyl-5-(1-piperazinylmethyl)phenyl]glycinamide dihydrochloride, a positive allosteric modulator of the rat receptor, reduced the potency of compound-22 at the rat receptor but had little effect on the actions of AZ11645373. CONCLUSIONS: AZ11645373 and compound-22 are allosteric antagonists of human and rat P2X(7) receptors respectively. The differential interaction of the two compounds with the receptor suggests there may be more than one allosteric regulatory site on the P2X(7) receptor at which antagonists can bind and affect receptor function.

A Lewis acid promoted oxidative cyclization.

Replacing trifluoroacetic acid with a catalytic amount of Lewis acid in the osmium mediated oxidative cyclization results in higher yielding reactions that can proceed nearly an order of magnitude faster. The osmium loading can also be reduced to as little as 0.2 mol %. Furthermore, these mildly acidic conditions are capable of tolerating a wide range of acid sensitive protecting groups that are incompatible with previous cyclization conditions.