Discovery of VU6016235: A Highly Selective, Orally Bioavailable, and Structurally Distinct Tricyclic M4 Muscarinic Acetylcholine Receptor Positive Allosteric Modulator (PAM).

Herein, we report structure-activity relationship (SAR) studies to develop novel tricyclic M4 PAM scaffolds with improved pharmacological properties. This endeavor involved a "tie-back" strategy to replace a 5-amino-2,4-dimethylthieno[2,3-d]pyrimidine-6-carboxamide core, which led to the discovery of two novel tricyclic cores. While both tricyclic cores displayed low nanomolar potency against both human and rat M4 and were highly brain-penetrant, the 2,4-dimethylpyrido[4',3':4,5]thieno[2,3-d]pyrimidine tricycle core provided lead compound, VU6016235, with an overall superior pharmacological and drug metabolism and pharmacokinetics (DMPK) profile, as well as efficacy in a preclinical antipsychotic animal model.

Development of VU6036864: A Triazolopyridine-Based High-Quality Antagonist Tool Compound of the M5 Muscarinic Acetylcholine Receptor.

While the muscarinic acetylcholine receptor mAChR subtype 5 (M5) has been studied over decades, recent findings suggest that more in-depth research is required to elucidate a thorough understanding of its physiological function related to neurological and psychiatric disorders. Our efforts to identify potent, selective, and pharmaceutically favorable next-generation M5 antagonist tool compounds have led to the discovery of a novel triazolopyridine-based series. In particular, VU6036864 (45) showed exquisite potency (human M5 IC50 = 20 nM), good subtype selectivity (>500 fold selectivity against human M1-4), desirable brain exposure (Kp = 0.68, Kp,uu = 0.65), and high oral bioavailability (%F > 100%). VU6036864 (45) and its close analogues will support further studies of M5 as advanced antagonist tool compounds and play an important role in the emerging biology of M5.

Discovery of VU6007496: Challenges in the Development of an M1 Positive Allosteric Modulator Backup Candidate.

Herein we report progress toward a backup clinical candidate to the M1 positive allosteric modulator (PAM) VU319/ACP-319. Scaffold-hopping from the pyrrolo[2,3-b]pyridine-based M1 PAM VU6007477 to isomeric pyrrolo[3,2-b]pyridine and thieno[3,2-b]pyridine congeners identified several backup contenders. Ultimately, VU6007496, a pyrrolo[3,2-b]pyridine, advanced into late stage profiling, only to be plagued with unanticipated, species-specific metabolism and active/toxic metabolites which were identified in our phenotypic seizure liability in vivo screen, preventing further development. However, VU6007496 proved to be a highly selective and CNS penetrant M1 PAM, with minimal agonism, that displayed excellent multispecies IV/PO pharmacokinetics (PK), CNS penetration, no induction of long-term depression (or cholinergic toxicity) and robust efficacy in novel object recognition (minimum effective dose = 3 mg/kg p.o.). Thus, VU6007496 can serve as another valuable in vivo tool compound in rats and nonhuman primates, but not mouse, to study selective M1 activation.

Discovery of VU6008677: A Structurally Distinct Tricyclic M4 Positive Allosteric Modulator with Improved CYP450 Profile.

This Letter details our efforts to develop novel tricyclic muscarinic acetylcholine receptor subtype 4 (M4) positive allosteric modulator (PAM) scaffolds with improved pharmacological properties. This endeavor involved a "tie-back" strategy to replace the 3-amino-5-chloro-4,6-dimethylthieno[2,3-b]pyridine-2-carboxamide core, which led to the discovery of two novel tricyclic cores: an 8-chloro-9-methylpyrido[3',2':4,5]thieno[3,2-d]pyrimidin-4-amine core and 8-chloro-7,9-dimethylpyrido[3',2':4,5]furo[3,2-d]pyrimidin-4-amine core. Both tricyclic cores displayed low nanomolar potency against human M4 and greatly reduced cytochrome P450 inhibition when compared with parent compound ML253.

Development of a Selective and High Affinity Radioligand, [3H]VU6013720, for the M4 Muscarinic Receptor.

M4 muscarinic receptors are highly expressed in the striatum and cortex, brain regions that are involved in diseases such as Parkinson's disease, schizophrenia, and dystonia. Despite potential therapeutic advantages of specifically targeting the M4 receptor, it has been historically challenging to develop highly selective ligands, resulting in undesired off-target activity at other members of the muscarinic receptor family. Recently, we have reported first-in-class, potent, and selective M4 receptor antagonists. As an extension of that work, we now report the development and characterization of a radiolabeled M4 receptor antagonist, [3H]VU6013720, with high affinity (pKd of 9.5 ± 0.2 at rat M4, 9.7 at mouse M4, and 10 ± 0.1 at human M4 with atropine to define nonspecific binding) and no significant binding at the other muscarinic subtypes. Binding assays using this radioligand in rodent brain tissues demonstrate loss of specific binding in Chrm4 knockout animals. Dissociation kinetics experiments with various muscarinic ligands show differential effects on the dissociation of [3H]VU6013720 from M4 receptors, suggesting a binding site that is overlapping but may be distinct from the orthosteric site. Overall, these results demonstrate that [3H]VU6013720 is the first highly selective antagonist radioligand for the M4 receptor, representing a useful tool for studying the basic biology of M4 as well for the support of M4 receptor-based drug discovery. SIGNIFICANCE STATEMENT: This manuscript describes the development and characterization of a novel muscarinic (M) acetylcholine subtype 4 receptor antagonist radioligand, [3H]VU6013720. This ligand binds to or overlaps with the acetylcholine binding site, providing a highly selective radioligand for the M4 receptor that can be used to quantify M4 protein expression in vivo and probe the selective interactions of acetylcholine with M4 versus the other members of the muscarinic receptor family.

Differential Activity of Orthosteric Agonists and Allosteric Modulators at Metabotropic Glutamate Receptor 7.

Metabotropic glutamate receptor 7 (mGlu7) is a G protein coupled receptor that has demonstrated promise as a therapeutic target across a number of neurologic and psychiatric diseases. Compounds that modulate the activity of mGlu7, such as positive and negative allosteric modulators, may represent new therapeutic strategies to modulate receptor activity. The endogenous neurotransmitter associated with the mGlu receptor family, glutamate, exhibits low efficacy and potency in activating mGlu7, and surrogate agonists, such as the compound L-(+)-2-Amino-4-phosphonobutyric acid (L-AP4), are often used for receptor activation and compound profiling. To understand the implications of the use of such agonists in the development of positive allosteric modulators (PAMs), we performed a systematic evaluation of receptor activation using a system in which mutations can be made in either protomer of the mGlu7 dimer; we employed mutations that prevent interaction with the orthosteric site as well as the G-protein coupling site of the receptor. We then measured increases in calcium levels downstream of a promiscuous G protein to assess the effects of mutations in one of the two protomers in the presence of two different agonists and three positive allosteric modulators. Our results reveal that distinct PAMs, for example N-[3-Chloro-4-[(5-chloro-2-pyridinyl)oxy]phenyl]-2-pyridinecarboxamide (VU0422288) and 3-(2,3-Difluoro-4-methoxyphenyl)-2,5-dimethyl-7-(trifluoromethyl)pyrazolo[1,5-a]pyrimidine (VU6005649), do exhibit different maximal levels of potentiation with L-AP4 versus glutamate, but there appear to be common stable receptor conformations that are shared among all of the compounds examined here. SIGNIFICANCE STATEMENT: This manuscript describes the systematic evaluation of the mGlu7 agonists glutamate and L-(+)-2-Amino-4-phosphonobutyric acid (L-AP4) in the presence and absence of three distinct potentiators examining possible mechanistic differences. These findings demonstrate that mGlu7 potentiators display subtle variances in response to glutamate versus L-AP4.

Persistent challenges in the development of an mGlu7 PAM in vivo tool compound: The discovery of VU6046980.

Herein, we report on the further chemical optimization of the first reported mGlu7 positive allosteric modulator (PAM), VU6027459. Replacement of the quinoline core by a cinnoline scaffold increased mGlu7 PAM potency by âˆ¼ 10-fold, and concomitant introduction of a chiral tricyclic motif led to potent mGlu7 PAMs with enantioselective mGlu receptor selectivity profiles. Of these, VU6046980 emerged as a putative in vivo tool compound with excellent CNS penetration (Kp = 4.1; Kp,uu = 0.7) and efficacy in preclinical models. However, either off-target activity at the sigma-1 receptor or activity at a target not elucidated by large ancillary pharmacology panels led to sedation not driven by activation of mGlu7 (validated in Grm7 knockout mice). Thus, despite a significant advance, a viable mGlu7 PAM in vivo tool remains elusive.

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.

Synthesis and characterization of chiral 6-azaspiro[2.5]octanes as potent and selective antagonists of the M4 muscarinic acetylcholine receptor.

In this manuscript, we report a series of chiral 6-azaspiro[2.5]octanes and related spirocycles as highly potent and selective antagonists of the muscarinic acetylcholine receptor subtype 4 (mAChR4). Chiral separation and subsequent X-ray crystallographic analysis of early generation analogs revealed the R enantiomer to possess excellent human and rat M4 potency, and further structure-activity relationship (SAR) studies on this chiral scaffold led to the discovery of VU6015241 (compound 19). Compound 19 is characterized by high M4 potency and selectivity across multiple species, excellent aqueous solubility, and moderate brain exposure in rodents after intraperitoneal administration.

Discovery of structurally distinct tricyclic M4 positive allosteric modulator (PAM) chemotypes - Part 2.

This Letter details our efforts to develop novel tricyclic M4 PAM scaffolds with improved pharmacological properties. This endeavor involved a "tie-back" strategy to replace the 3-amino-4,6-dimethylthieno[2,3-b]pyridine-2-carboxamide core which lead to the discovery of two novel tricyclic cores: a 7,9-dimethylpyrido[3',2':4,5]thieno[3,2-d]pyrimidine core and 2,4-dimethylthieno[2,3-b:5,4-c']dipyridine core. Both tricyclic cores displayed low nanomolar potency against the human M4 receptor.

Discovery of VU6028418: A Highly Selective and Orally Bioavailable M4 Muscarinic Acetylcholine Receptor Antagonist.

Herein, we report the SAR leading to the discovery of VU6028418, a potent M4 mAChR antagonist with high subtype-selectivity and attractive DMPK properties in vitro and in vivo across multiple species. VU6028418 was subsequently evaluated as a preclinical candidate for the treatment of dystonia and other movement disorders. During the characterization of VU6028418, a novel use of deuterium incorporation as a means to modulate CYP inhibition was also discovered.

Discovery of the First Selective M4 Muscarinic Acetylcholine Receptor Antagonists with in Vivo Antiparkinsonian and Antidystonic Efficacy.

Nonselective antagonists of muscarinic acetylcholine receptors (mAChRs) that broadly inhibit all five mAChR subtypes provide an efficacious treatment for some movement disorders, including Parkinson's disease and dystonia. Despite their efficacy in these and other central nervous system disorders, antimuscarinic therapy has limited utility due to severe adverse effects that often limit their tolerability by patients. Recent advances in understanding the roles that each mAChR subtype plays in disease pathology suggest that highly selective ligands for individual subtypes may underlie the antiparkinsonian and antidystonic efficacy observed with the use of nonselective antimuscarinic therapeutics. Our recent work has indicated that the M4 muscarinic acetylcholine receptor has several important roles in opposing aberrant neurotransmitter release, intracellular signaling pathways, and brain circuits associated with movement disorders. This raises the possibility that selective antagonists of M4 may recapitulate the efficacy of nonselective antimuscarinic therapeutics and may decrease or eliminate the adverse effects associated with these drugs. However, this has not been directly tested due to lack of selective antagonists of M4. Here, we utilize genetic mAChR knockout animals in combination with nonselective mAChR antagonists to confirm that the M4 receptor activation is required for the locomotor-stimulating and antiparkinsonian efficacy in rodent models. We also report the synthesis, discovery, and characterization of the first-in-class selective M4 antagonists VU6013720, VU6021302, and VU6021625 and confirm that these optimized compounds have antiparkinsonian and antidystonic efficacy in pharmacological and genetic models of movement disorders.

Positive allosteric modulators (PAMs) of the group II metabotropic glutamate receptors: Design, synthesis, and evaluation as ex-vivo tool compounds.

This letter describes synthesis and evaluation of two series of dual mGlu2/mGlu3 positive allosteric modulators with moderate mGlu3 potency and robust mGlu2 potency in thallium flux assays. These compounds were profiled their ability to modulate mGlu3-mediated signaling in central neurons by co-application of a selective mGlu2 NAM to isolate mGlu3-selective effects. Using acute mouse brain slices from the prefrontal cortex, potentiation of group II mGlu receptor agonist Ca2+ signaling in PFC pyramidal cells with either the dual mGlu2/mGlu3 PAM 16e or 23d demonstrated effects mediated selectively via mGlu3.

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.

Discovery of "Molecular Switches" within a Series of mGlu5 Allosteric Ligands Driven by a "Magic Methyl" Effect Affording Both PAMs and NAMs with In Vivo Activity, Derived from an M1 PAM Chemotype.

In the course of optimizing an M1 PAM chemotype, introduction of an ether moiety unexpectedly abolished M1 PAM activity while engendering a "molecular switch" to afford a weak, pure mGlu5 PAM. Further optimization was able to deliver a potent (mGlu5 EC50 = 520 nM, 63% Glu Max), centrally penetrant (Kp = 0.83), MPEP-site binding mGlu5 PAM 17a (VU6036486) that reversed amphetamine-induced hyperlocomotion. A pronounced "magic methyl" effect was noted with a regioisomeric methyl congener, leading to a change in pharmacology to afford a potent (mGlu5 IC50 = 110 nM, 3% Glu Min), centrally penetrant (Kp = 0.94), MPEP-site binding NAM 28d (VU6044766) that displayed anxiolytic activity in a mouse marble burying assay. These data further support the growing body of literature concerning the existence of G protein-coupled receptor (GPCR) allosteric privileged structures, and the value and impact of subtle methyl group walks, as well as the highly productive fluorine walk, around allosteric ligand cores to stabilize unique GPCR conformations.

Lead optimization of the VU0486321 series of mGlu1 PAMs. Part 4: SAR reveals positive cooperativity across multiple mGlu receptor subtypes leading to subtype unselective PAMs.

Further optimization of the VU0486321 series of highly selective and CNS-penetrant mGlu1 PAMs identified unique 'molecular switches' on the central aromatic ring that engendered positive cooperativity with multiple mGlu subtypes across the receptor family, resulting in compounds with comparable activity at Group I (mGlu1/5) and Group III (mGlu4/6/7/8) mGlu receptors, receptors. These exciting data suggests this PAM chemotype appears to bind to multiple mGlu receptors, and that subtype selectivity is dictated by the degree of cooperativity, not a subtype selective, unique allosteric binding site. Moreover, there is interesting therapeutic potential for mGlu1/4/7/8 PAMs, as well as the first report of a GPCR allosteric 'privileged structure'.