Discovery of a potent M5 antagonist with improved clearance profile. Part 2: Pyrrolidine amide-based antagonists.

This Letter describes our ongoing effort to improve the clearance of selective M5 antagonists. Herein, we report the replacement of the previously disclosed piperidine amide (4, disclosed in Part 1) with a pyrrolidine amide core. Several compounds within this series provided good potency, subtype selectivity, and low to moderate clearance profiles. Interestingly, the left-hand side SAR for this series diverged from our earlier efforts.

Discovery of a potent M5 antagonist with improved clearance profile. Part 1: Piperidine amide-based antagonists.

The lack of potent and selective tool compounds with pharmaceutically favorable properties limits the in vivo understanding of muscarinic acetylcholine receptor subtype 5 (M5) biology. Previously, we presented a highly potent and selective M5 antagonist VU6019650 with a suboptimal clearance profile as our second-generation tool compound. Herein, we disclose our ongoing efforts to generate next-generation M5 antagonists with improved clearance profiles. A mix and match approach between VU6019650 (lead) and VU0500325 (HTS hit) generated a piperidine amide-based novel M5 antagonist series. Several analogs within this series, including 29f, provided good on-target potency with improved clearance profiles, though room for improvement remains.

Development and profiling of mGlu7 NAMs with a range of saturable inhibition of agonist responses in vitro.

We describe here a series of metabotropic glutamate receptor 7 (mGlu7) negative allosteric modulators (NAMs) with a saturable range of activity in inhibiting responses to an orthosteric agonist in two distinct in vitro pharmacological assays. The range of inhibition among compounds in this scaffold provides highly structurally related ligands with differential degrees of receptor blockade that can be used to understand inhibitory efficacy profiles in native tissue or in vivo.

Development of VU6019650: A Potent, Highly Selective, and Systemically Active Orthosteric Antagonist of the M5 Muscarinic Acetylcholine Receptor for the Treatment of Opioid Use Disorder.

The muscarinic acetylcholine receptor (mAChR) subtype 5 (M5) represents a novel potential target for the treatment of multiple addictive disorders, including opioid use disorder. Through chemical optimization of several functional high-throughput screening hits, VU6019650 (27b) was identified as a novel M5 orthosteric antagonist with high potency (human M5 IC50 = 36 nM), M5 subtype selectivity (>100-fold selectivity against human M1-4) and favorable physicochemical properties for systemic dosing in preclinical addiction models. In acute brain slice electrophysiology studies, 27b blocked the nonselective muscarinic agonist oxotremorine-M-induced increases in neuronal firing rates of midbrain dopamine neurons in the ventral tegmental area, a part of the mesolimbic dopaminergic reward circuitry. Moreover, 27b also inhibited oxycodone self-administration in male Sprague-Dawley rats within a dose range that did not impair general motor output.

Discovery and Optimization of a Novel Series of Competitive and Central Nervous System-Penetrant Protease-Activated Receptor 4 (PAR4) Inhibitors.

The detailed pharmacology and therapeutic potential of the central PAR4 receptors are poorly understood due to a lack of potent, selective, and brain-penetrant tool compounds. Despite this, robust data with biochemical and genetic tools show the therapeutic potential of PAR4 antagonists in traumatic brain injury, Alzheimer's disease, Parkinson's disease, and other neurodegenerative disorders with a neuroinflammatory component. Thus, we performed a functional HTS campaign, identified a fundamentally new PAR4 competitive inhibitor chemotype, optimized this new series (increased potency >45-fold), discovered enantiospecific activity (though opposing preference for human versus mouse PAR4), and engendered high central nervous system penetration (rat Kp's of 0.52 to 4.2 and Kp,uu's of 0.52 to 1.2).

Discovery of a novel class of heteroaryl-pyrrolidinones as positive allosteric modulators of the muscarinic acetylcholine receptor M1.

This Letter describes the synthesis and optimization of a series of heteroaryl-pyrrolidinone positive allosteric modulators (PAMs) of the muscarinic acetylcholine receptor M1 (mAChR M1). Through the continued optimization of M1 PAM tool compound VU0453595, with a focus on replacement of the 6,7-dihydro-5H-pyrrolo[3,4-b]pyridin-5-one with a wide variety of alternative 4,5-dihydropyrrolo-fused heteroaromatics, the generation of M1 PAMs with structurally novel chemotypes is disclosed. Two compounds from these subseries, 8b (VU6005610) and 20a (VU6005852), show robust selectivity for the M1 mAChR, and no M1 agonism. Both compounds have favorable preliminary PK profiles in vitro;8b additionally demonstrates high brain exposure in a rodent IV cassette model.

Discovery and optimization of a novel CNS penetrant series of mGlu4 PAMs based on a 1,4-thiazepane core with in vivo efficacy in a preclinical Parkinsonian model.

A high throughput screen (HTS) identified a novel, but weak (EC50 = 6.2 µM, 97% Glu Max) mGlu4 PAM chemotype based on a 1,4-thiazepane core, VU0544412. Reaction development and chemical optimization delivered a potent mGlu4 PAM VU6022296 (EC50 = 32.8 nM, 108% Glu Max) with good CNS penetration (Kp = 0.45, Kp,uu = 0.70) and enantiopreference. Finally, VU6022296 displayed robust, dose-dependent efficacy in reversing Haloperidol-Induced Catalepsy (HIC), a rodent preclinical Parkinson's disease model.

Modulation of arousal and sleep/wake architecture by M1 PAM VU0453595 across young and aged rodents and nonhuman primates.

Degeneration of basal forebrain cholinergic circuitry represents an early event in the development of Alzheimer's disease (AD). These alterations in central cholinergic function are associated with disruptions in arousal, sleep/wake architecture, and cognition. Changes in sleep/wake architecture are also present in normal aging and may represent a significant risk factor for AD. M1 muscarinic acetylcholine receptor (mAChR) positive allosteric modulators (PAMs) have been reported to enhance cognition across preclinical species and may also provide beneficial effects for age- and/or neurodegenerative disease-related changes in arousal and sleep. In the present study, electroencephalography was conducted in young animals (mice, rats and nonhuman primates [NHPs]) and in aged mice to examine the effects of the selective M1 PAM VU0453595 in comparison with the acetylcholinesterase inhibitor donepezil, M1/M4 agonist xanomeline (in NHPs), and M1 PAM BQCA (in rats) on sleep/wake architecture and arousal. In young wildtype mice, rats, and NHPs, but not in M1 mAChR KO mice, VU0453595 produced dose-related increases in high frequency gamma power, a correlate of arousal and cognition enhancement, without altering duration of time across all sleep/wake stages. Effects of VU0453595 in NHPs were observed within a dose range that did not induce cholinergic-mediated adverse effects. In contrast, donepezil and xanomeline increased time awake in rodents and engendered dose-limiting adverse effects in NHPs. Finally, VU0453595 attenuated age-related decreases in REM sleep duration in aged wildtype mice. Development of M1 PAMs represents a viable strategy for attenuating age-related and dementia-related pathological disturbances of sleep and arousal.

Synthesis and SAR of a series of mGlu7 NAMs based on an ethyl-8-methoxy-4-(4-phenylpiperazin-1-yl)quinoline carboxylate core.

A High-Throughput Screening (HTS) campaign identified a fundamentally new mGlu7 NAM chemotype, based on an ethyl-8-methoxy-4-(4-phenylpiperazin-1-yl)quinolone carboxylate core. The initial hit, VU0226390, was a potent mGlu7 NAM (IC50 = 647 nM, 6% L-AP4 min) with selectivity versus the other group III mGlu receptors (>30 µM vs. mGlu4 and mGlu8). A multi-dimensional optimization effort surveyed all regions of this new chemotype, and found very steep SAR, reminiscent of allosteric modulators, and unexpected piperazine mimetics (whereas classical bioisosteres failed). While mGlu7 NAM potency could be improved (IC50s ~ 350 nM), the necessity of the ethyl ester moiety and poor physiochemical and DMPK properties precluded optimization towards in vivo tool compounds or clinical candidates. Still, this hit-to-lead campaign afforded key medicinal chemistry insights and new opportunities.

Discovery of VU6027459: A First-in-Class Selective and CNS Penetrant mGlu7 Positive Allosteric Modulator Tool Compound.

Herein, we report the discovery of the first selective and CNS penetrant mGlu7 PAM (VU6027459) derived from a "molecular switch" within a selective mGlu7 NAM chemotype. VU6027459 displayed CNS penetration in both mice (Kp = 2.74) and rats (Kp= 4.78), it was orally bioavailable in rats (%F = 69.5), and undesired activity at DAT was ablated.

Discovery, synthesis and characterization of a series of 7-aryl-imidazo[1,2-a]pyridine-3-ylquinolines as activin-like kinase (ALK) inhibitors.

The activin-like kinases are a family of kinases that play important roles in a variety of disease states. Of this class of kinases, ALK2, has been shown by a gain-of-function to be the primary driver of the childhood skeletal disease fibrodysplasia ossificans progressiva (FOP) and more recently the pediatric cancer diffuse intrinsic pontine glioma (DIPG). Herein, we report our efforts to identify a novel imidazo[1,2-a]pyridine scaffold as potent inhibitors of ALK2 with good in vivo pharmacokinetic properties suitable for future animal studies.

Discovery of VU6015929: A Selective Discoidin Domain Receptor 1/2 (DDR1/2) Inhibitor to Explore the Role of DDR1 in Antifibrotic Therapy.

Herein, we report the discovery of a potent and selective dual DDR1/2 inhibitor, 7e (VU6015929), displaying low cytotoxicity, good kinome selectivity, and possessing an acceptable in vitro DMPK profile with good rodent in vivo pharmacokinetics. VU6015929 potently blocks collagen-induced DDR1 activation and collagen-IV production, suggesting DDR1 inhibition as an exciting target for antifibrotic therapy.

Activation of the mGlu1 metabotropic glutamate receptor has antipsychotic-like effects and is required for efficacy of M4 muscarinic receptor allosteric modulators.

Recent clinical and preclinical studies suggest that selective activators of the M4 muscarinic acetylcholine receptor have potential as a novel treatment for schizophrenia. M4 activation inhibits striatal dopamine release by mobilizing endocannabinoids, providing a mechanism for local effects on dopamine signaling in the striatum but not in extrastriatal areas. G protein-coupled receptors (GPCRs) typically induce endocannabinoid release through activation of Gαq/11-type G proteins whereas M4 transduction occurs through Gαi/o-type G proteins. We now report that the ability of M4 to inhibit dopamine release and induce antipsychotic-like effects in animal models is dependent on co-activation of the Gαq/11-coupled mGlu1 subtype of metabotropic glutamate (mGlu) receptor. This is especially interesting in light of recent findings that multiple loss of function single nucleotide polymorphisms (SNPs) in the human gene encoding mGlu1 (GRM1) are associated with schizophrenia, and points to GRM1/mGlu1 as a gene within the "druggable genome" that could be targeted for the treatment of schizophrenia. Herein, we report that potentiation of mGlu1 signaling following thalamo-striatal stimulation is sufficient to inhibit striatal dopamine release, and that a novel mGlu1 positive allosteric modulator (PAM) exerts robust antipsychotic-like effects through an endocannabinoid-dependent mechanism. However, unlike M4, mGlu1 does not directly inhibit dopamine D1 receptor signaling and does not reduce motivational responding. Taken together, these findings highlight a novel mechanism of cross talk between mGlu1 and M4 and demonstrate that highly selective mGlu1 PAMs may provide a novel strategy for the treatment of positive symptoms associated with schizophrenia.

Further exploration of an N-aryl phenoxyethoxy pyridinone-based series of mGlu3 NAMs: Challenging SAR, enantiospecific activity and in vivo efficacy.

This letter describes the further optimization of a series of mGlu3 NAMs based on an N-aryl phenoxyethoxy pyridinone core. A multidimensional optimization campaign, with focused matrix libraries, quickly established challenging SAR, enantiospecific activity, differences in assay read-outs (Ca2+ flux via a promiscuous G protein (Gα15) versus native coupling to GIRK channels), identified both full and partial mGlu3 NAMs and a new in vivo tool compound, VU6017587. This mGlu3 NAM showed efficacy in tail suspension, elevated zero maze and marble burying, suggesting selective inhibition of mGlu3 affords anxiolytic-like and antidepressant-like phenotypes in mice.

Structure-Activity Relationships, Pharmacokinetics, and Pharmacodynamics of the Kir6.2/SUR1-Specific Channel Opener VU0071063.

Glucose-stimulated insulin secretion from pancreatic ß-cells is controlled by ATP-regulated potassium (KATP) channels composed of Kir6.2 and sulfonylurea receptor 1 (SUR1) subunits. The KATP channel-opener diazoxide is FDA-approved for treating hyperinsulinism and hypoglycemia but suffers from off-target effects on vascular KATP channels and other ion channels. The development of more specific openers would provide critically needed tool compounds for probing the therapeutic potential of Kir6.2/SUR1 activation. Here, we characterize a novel scaffold activator of Kir6.2/SUR1 that our group recently discovered in a high-throughput screen. Optimization efforts with medicinal chemistry identified key structural elements that are essential for VU0071063-dependent opening of Kir6.2/SUR1. VU0071063 has no effects on heterologously expressed Kir6.1/SUR2B channels or ductus arteriole tone, indicating it does not open vascular KATP channels. VU0071063 induces hyperpolarization of ß-cell membrane potential and inhibits insulin secretion more potently than diazoxide. VU0071063 exhibits metabolic and pharmacokinetic properties that are favorable for an in vivo probe and is brain penetrant. Administration of VU0071063 inhibits glucose-stimulated insulin secretion and glucose-lowering in mice. Taken together, these studies indicate that VU0071063 is a more potent and specific opener of Kir6.2/SUR1 than diazoxide and should be useful as an in vitro and in vivo tool compound for investigating the therapeutic potential of Kir6.2/SUR1 expressed in the pancreas and brain.

Discovery of VU2957 (Valiglurax): An mGlu4 Positive Allosteric Modulator Evaluated as a Preclinical Candidate for the Treatment of Parkinson's Disease.

Herein, we report the discovery of a novel potent, selective, CNS penetrant, and orally bioavailable mGlu4 PAM, VU0652957 (VU2957, Valiglurax). VU2957 possessed attractive in vitro and in vivo pharmacological and DMPK properties across species. To advance toward the clinic, a spray-dried dispersion (SDD) formulation of VU2957 was developed to support IND-enabling toxicology studies. Based on its overall profile, VU2957 was evaluated as a preclinical development candidate for the treatment of Parkinson's disease.

Surveying heterocycles as amide bioisosteres within a series of mGlu7 NAMs: Discovery of VU6019278.

This letter describes a diversity-oriented library approach to rapidly assess diverse heterocycles as bioisosteric replacements for a metabolically labile amide moiety within a series of mGlu7 negative allosteric modulators (NAMs). SAR rapidly honed in on either a 1,2,4- or 1,3,4-oxadizaole ring system as an effective bioisostere for the amide. Further optimization of the southern region of the mGlu7 NAM chemotype led to the discovery of VU6019278, a potent mGlu7 NAM (IC50 = 501 nM, 6.3% L-AP4 Min) with favorable plasma protein binding (rat fu = 0.10), low predicted hepatic clearance (rat CLhep = 27.7 mL/min/kg) and high CNS penetration (rat Kp = 4.9, Kp,uu = 0.65).

Discovery of an Orally Bioavailable and Central Nervous System (CNS) Penetrant mGlu7 Negative Allosteric Modulator (NAM) in Vivo Tool Compound: N-(2-(1 H-1,2,4-triazol-1-yl)-5-(trifluoromethoxy)phenyl)-4-(cyclopropylmethoxy)-3-methoxybenzamide (VU6012962).

Herein, we report the discovery of a new, orally bioavailable and CNS-penetrant metabotropic glutamate receptor 7 (mGlu7) negative allosteric modulator (NAM) that achieves exposure in cerebral spinal fluid (CSF) 2.5× above the in vitro IC50 at minimum effective doses (MEDs) of 3 mg/kg in preclinical anxiety models.

Discovery of Tricyclic Triazolo- and Imidazopyridine Lactams as M1 Positive Allosteric Modulators.

This Letter describes the chemical optimization of a new series of muscarinic acetylcholine receptor subtype 1 (M1) positive allosteric modulators (PAMs) based on novel tricyclic triazolo- and imidazopyridine lactam cores, devoid of M1 agonism, e.g., no M1 ago-PAM activity, in high expressing recombinant cell lines. While all the new tricyclic congeners afforded excellent rat pharmacokinetic (PK) properties (CLp < 8 mL/min/kg and t1/2 > 5 h), regioisomeric triazolopyridine analogues were uniformly not CNS penetrant ( Kp < 0.05), despite a lack of hydrogen bond donors. However, removal of a single nitrogen atom to afford imidazopyridine derivatives proved to retain the excellent rat PK and provide high CNS penetration ( Kp > 2), despite inclusion of a basic nitrogen. Moreover, 24c was devoid of M1 agonism in high expressing recombinant cell lines and did not induce cholinergic seizures in vivo in mice. Interestingly, all of the new M1 PAMs across the diverse tricyclic heterocyclic cores possessed equivalent CNS MPO scores (>4.5), highlighting the value of both "medicinal chemist's eye" and experimental data, e.g., not sole reliance (or decision bias) on in silico calculated properties, for parameters as complex as CNS penetration.

Discovery of Novel Central Nervous System Penetrant Metabotropic Glutamate Receptor Subtype 2 (mGlu2) Negative Allosteric Modulators (NAMs) Based on Functionalized Pyrazolo[1,5- a]pyrimidine-5-carboxamide and Thieno[3,2- b]pyridine-5-carboxamide Cores.

A scaffold hopping exercise from a monocyclic mGlu2 NAM with poor rodent PK led to two novel heterobicyclic series of mGlu2 NAMs based on either a functionalized pyrazolo[1,5- a]pyrimidine-5-carboxamide core or a thieno[3,2- b]pyridine-5-carboxamide core. These novel analogues possess enhanced rodent PK, while also maintaining good mGlu2 NAM potency, selectivity (versus mGlu3 and the remaining six mGlu receptors), and high CNS penetration. Interestingly, SAR was divergent between the new 5,6-heterobicyclic systems.