Challenges in the development of an M4 PAM preclinical candidate: The discovery, SAR, and in vivo characterization of a series of 3-aminoazetidine-derived amides.

This letter details the continued chemical optimization of a novel series of M4 positive allosteric modulators (PAMs) based on a 5-amino-thieno[2,3-c]pyridazine core by incorporating a 3-amino azetidine amide moiety. The analogs described within this work represent the most potent M4 PAMs reported for this series to date. The SAR to address potency, clearance, subtype selectivity, CNS exposure, and P-gp efflux are described. This work culminated in the discovery of VU6000918, which demonstrated robust efficacy in a rat amphetamine-induced hyperlocomotion reversal model at a minimum efficacious dose of 0.3mg/kg.

Challenges in the development of an M4 PAM preclinical candidate: The discovery, SAR, and biological characterization of a series of azetidine-derived tertiary amides.

Herein we describe the continued optimization of M4 positive allosteric modulators (PAMs) within the 5-amino-thieno[2,3-c]pyridazine series of compounds. In this letter, we disclose our studies on tertiary amides derived from substituted azetidines. This series provided excellent CNS penetration, which had been challenging to consistently achieve in other amide series. Efforts to mitigate high clearance, aided by metabolic softspot analysis, were unsuccessful and precluded this series from further consideration as a preclinical candidate. In the course of this study, we found that potassium tetrafluoroborate salts could be engaged in a tosyl hydrazone reductive cross coupling reaction, a previously unreported transformation, which expands the synthetic utility of the methodology.

Challenges in the development of an M4 PAM in vivo tool compound: The discovery of VU0467154 and unexpected DMPK profiles of close analogs.

This letter describes the chemical optimization of a novel series of M4 positive allosteric modulators (PAMs) based on a 5-amino-thieno[2,3-c]pyridazine core, developed via iterative parallel synthesis, and culminating in the highly utilized rodent in vivo tool compound, VU0467154 (5). This is the first report of the optimization campaign (SAR and DMPK profiling) that led to the discovery of VU0467154, and details all of the challenges faced in allosteric modulator programs (steep SAR, species differences in PAM pharmacology and subtle structural changes affecting CNS penetration).

Optimization of M4 positive allosteric modulators (PAMs): The discovery of VU0476406, a non-human primate in vivo tool compound for translational pharmacology.

This letter describes the further chemical optimization of the 5-amino-thieno[2,3-c]pyridazine series (VU0467154/VU0467485) of M4 positive allosteric modulators (PAMs), developed via iterative parallel synthesis, culminating in the discovery of the non-human primate (NHP) in vivo tool compound, VU0476406 (8p). VU0476406 is an important in vivo tool compound to enable translation of pharmacodynamics from rodent to NHP, and while data related to a Parkinson's disease model has been reported with 8p, this is the first disclosure of the optimization and discovery of VU0476406, as well as detailed pharmacology and DMPK properties.

Discovery and SAR of a novel series of potent, CNS penetrant M4 PAMs based on a non-enolizable ketone core: Challenges in disposition.

This Letter describes the chemical optimization of a novel series of M4 PAMs based on a non-enolizable ketone core, identified from an MLPCN functional high-throughput screen. The HTS hit was potent, selective and CNS penetrant; however, the compound was highly cleared in vitro and in vivo. SAR provided analogs for which M4 PAM potency and CNS exposure were maintained; yet, clearance remained high. Metabolite identification studies demonstrated that this series was subject to rapid, and near quantitative, reductive metabolism to the corresponding secondary alcohol metabolite that was devoid of M4 PAM activity.

Discovery and optimization of a novel series of highly CNS penetrant M4 PAMs based on a 5,6-dimethyl-4-(piperidin-1-yl)thieno[2,3-d]pyrimidine core.

This Letter describes the chemical optimization of a novel series of M4 positive allosteric modulators (PAMs) based on a 5,6-dimethyl-4-(piperidin-1-yl)thieno[2,3-d]pyrimidine core, identified from an MLPCN functional high-throughput screen. The HTS hit was potent and selective, but not CNS penetrant. Potency was maintained, while CNS penetration was improved (rat brain:plasma Kp=0.74), within the original core after several rounds of optimization; however, the thieno[2,3-d]pyrimidine core was subject to extensive oxidative metabolism. Ultimately, we identified a 6-fluoroquinazoline core replacement that afforded good M4 PAM potency, muscarinic receptor subtype selectivity and CNS penetration (rat brain:plasma Kp>10). Moreover, this campaign provided fundamentally distinct M4 PAM chemotypes, greatly expanding the available structural diversity for this exciting CNS target.

Selective actions of novel allosteric modulators reveal functional heteromers of metabotropic glutamate receptors in the CNS.

Metabotropic glutamate (mGlu) receptors play important roles in regulating CNS function and are known to function as obligatory dimers. Although recent studies have suggested heterodimeric assembly of mGlu receptors in vitro, the demonstration that distinct mGlu receptor proteins can form heterodimers or hetero-complexes with other mGlu subunits in native tissues, such as neurons, has not been shown. Using biochemical and pharmacological approaches, we demonstrate here that mGlu2 and mGlu4 form a hetero-complex in native rat and mouse tissues which exhibits a distinct pharmacological profile. These data greatly extend our current understanding of mGlu receptor interaction and function and provide compelling evidence that mGlu receptors can function as heteromers in intact brain circuits.

Discovery and SAR of muscarinic receptor subtype 1 (M1) allosteric activators from a molecular libraries high throughput screen. Part 1: 2,5-dibenzyl-2H-pyrazolo[4,3-c]quinolin-3(5H)-ones as positive allosteric modulators.

Results from a 2012 high-throughput screen of the NIH Molecular Libraries Small Molecule Repository (MLSMR) against the human muscarinic receptor subtype 1 (M1) for positive allosteric modulators is reported. A content-rich screen utilizing an intracellular calcium mobilization triple-addition protocol allowed for assessment of all three modes of pharmacology at M1, including agonist, positive allosteric modulator, and antagonist activities in a single screening platform. We disclose a dibenzyl-2H-pyrazolo[4,3-c]quinolin-3(5H)-one hit (DBPQ, CID 915409) and examine N-benzyl pharmacophore/SAR relationships versus previously reported quinolin-3(5H)-ones and isatins, including ML137. SAR and consideration of recently reported crystal structures, homology modeling, and structure-function relationships using point mutations suggests a shared binding mode orientation at the putative common allosteric binding site directed by the pendant N-benzyl substructure.

Acyl dihydropyrazolo[1,5-a]pyrimidinones as metabotropic glutamate receptor 5 positive allosteric modulators.

We report the optimization of a series of metabotropic glutamate receptor 5 (mGlu5) positive allosteric modulators (PAMs) from an acyl dihydropyrazolo[1,5-a]pyrimidinone class. Investigation of exocyclic amide transpositions with this unique 5,6-bicyclic core were conducted in attempt to modulate physicochemical properties and identify a suitable backup candidate with a reduced half-life. A potent and selective PAM, 1-(2-(phenoxymethyl)-6,7-dihydropyrazolo[1,5-a]pyrimidin-4(5H)-yl)ethanone (9a, VU0462807), was identified with superior solubility and efficacy in the acute amphetamine-induced hyperlocomotion (AHL) rat model with a minimum effective dose of 3mg/kg. Attempts to mitigate oxidative metabolism of the western phenoxy of 9a through extensive modification and profiling are described.

Discovery and SAR of novel series of imidazopyrimidinones and dihydroimidazopyrimidinones as positive allosteric modulators of the metabotropic glutamate receptor 5 (mGlu5).

We report the discovery and SAR of two novel series of imidazopyrimidinones and dihydroimidazopyrimidinones as metabotropic glutamate receptor 5 (mGlu5) positive allosteric modulators (PAMs). Exploration of several structural features in the western and eastern part of the imidazopyrimidinone core and combinations thereof, revealed compound 4a as a mGlu5 PAM with good in vitro potency and efficacy, acceptable drug metabolism and pharmacokinetic (DMPK) properties and in vivo efficacy in an amphetamine-based model of psychosis. However, the presence of CNS-mediated adverse effects in preclinical species precluded any further in vivo evaluation.

Discovery and SAR of a novel series of metabotropic glutamate receptor 5 positive allosteric modulators with high ligand efficiency.

We report the optimization of a series of novel metabotropic glutamate receptor 5 (mGlu5) positive allosteric modulators (PAMs) from a 5,6-bicyclic class of dihydropyrazolo[1,5-a]pyridin-4(5H)-ones containing a phenoxymethyl linker. Studies focused on a survey of non-amide containing hydrogen bond accepting (HBA) pharmacophore replacements. A highly potent and selective PAM, 2-(phenoxymethyl)-6,7-dihydropyrazolo[1,5-a]pyridin-4(5H)-one (11, VU0462054), bearing a simple ketone moiety, was identified (LE=0.52, LELP=3.2). In addition, hydroxyl, difluoro, ether, and amino variations were examined. Despite promising lead properties and exploration of alternative core heterocycles, linkers, and ketone replacements, oxidative metabolism and in vivo clearance remained problematic for the series.

Novel allosteric agonists of M1 muscarinic acetylcholine receptors induce brain region-specific responses that correspond with behavioral effects in animal models.

M(1) muscarinic acetylcholine receptors (mAChRs) represent a viable target for treatment of multiple disorders of the central nervous system (CNS) including Alzheimer's disease and schizophrenia. The recent discovery of highly selective allosteric agonists of M(1) receptors has provided a major breakthrough in developing a viable approach for the discovery of novel therapeutic agents that target these receptors. Here we describe the characterization of two novel M(1) allosteric agonists, VU0357017 and VU0364572, that display profound differences in their efficacy in activating M(1) coupling to different signaling pathways including Ca(2+) and ß-arrestin responses. Interestingly, the ability of these agents to differentially activate coupling of M(1) to specific signaling pathways leads to selective actions on some but not all M(1)-mediated responses in brain circuits. These novel M(1) allosteric agonists induced robust electrophysiological effects in rat hippocampal slices, but showed lower efficacy in striatum and no measureable effects on M(1)-mediated responses in medial prefrontal cortical pyramidal cells in mice. Consistent with these actions, both M(1) agonists enhanced acquisition of hippocampal-dependent cognitive function but did not reverse amphetamine-induced hyperlocomotion in rats. Together, these data reveal that M(1) allosteric agonists can differentially regulate coupling of M(1) to different signaling pathways, and this can dramatically alter the actions of these compounds on specific brain circuits important for learning and memory and psychosis.

Biotransformation of a novel positive allosteric modulator of metabotropic glutamate receptor subtype 5 contributes to seizure-like adverse events in rats involving a receptor agonism-dependent mechanism.

Activation of metabotropic glutamate receptor subtype 5 (mGlu5) represents a novel strategy for therapeutic intervention into multiple central nervous system disorders, including schizophrenia. Recently, a number of positive allosteric modulators (PAMs) of mGlu5 were discovered to exhibit in vivo efficacy in rodent models of psychosis, including PAMs possessing varying degrees of agonist activity (ago-PAMs), as well as PAMs devoid of agonist activity. However, previous studies revealed that ago-PAMs can induce seizure activity and behavioral convulsions, whereas pure mGlu5 PAMs do not induce these adverse effects. We recently identified a potent and selective mGlu5 PAM, VU0403602, that was efficacious in reversing amphetamine-induced hyperlocomotion in rats. The compound also induced time-dependent seizure activity that was blocked by coadministration of the mGlu5 antagonist, 2-methyl-6-(phenylethynyl) pyridine. Consistent with potential adverse effects induced by ago-PAMs, we found that VU0403602 had significant allosteric agonist activity. Interestingly, inhibition of VU0403602 metabolism in vivo by a pan cytochrome P450 (P450) inactivator completely protected rats from induction of seizures. P450-mediated biotransformation of VU0403602 was discovered to produce another potent ago-PAM metabolite-ligand (M1) of mGlu5. Electrophysiological studies in rat hippocampal slices confirmed agonist activity of both M1 and VU0403602 and revealed that M1 can induce epileptiform activity in a manner consistent with its proconvulsant behavioral effects. Furthermore, unbound brain exposure of M1 was similar to that of the parent compound, VU0403602. These findings indicate that biotransformation of mGlu5 PAMs to active metabolite-ligands may contribute to the epileptogenesis observed after in vivo administration of this class of allosteric receptor modulators.

Functional impact of allosteric agonist activity of selective positive allosteric modulators of metabotropic glutamate receptor subtype 5 in regulating central nervous system function.

Positive allosteric modulators (PAMs) of metabotropic glutamate receptor subtype 5 (mGlu(5)) have emerged as an exciting new approach for the treatment of schizophrenia and other central nervous system (CNS) disorders. Of interest, some mGlu(5) PAMs act as pure PAMs, only potentiating mGlu(5) responses to glutamate whereas others [allosteric agonists coupled with PAM activity (ago-PAMs)] potentiate responses to glutamate and have intrinsic allosteric agonist activity in mGlu(5)-expressing cell lines. All mGlu(5) PAMs previously shown to have efficacy in animal models act as ago-PAMs in cell lines, raising the possibility that allosteric agonist activity is critical for in vivo efficacy. We have now optimized novel mGlu(5) pure PAMs that are devoid of detectable agonist activity and structurally related mGlu(5) ago-PAMs that activate mGlu(5) alone in cell lines. Studies of mGlu(5) PAMs in cell lines revealed that ago-PAM activity is dependent on levels of mGlu(5) receptor expression in human embryonic kidney 293 cells, whereas PAM potency is relatively unaffected by levels of receptor expression. Furthermore, ago-PAMs have no agonist activity in the native systems tested, including cortical astrocytes and subthalamic nucleus neurons and in measures of long-term depression at the hippocampal Schaffer collateral-CA1 synapse. Finally, studies with pure PAMs and ago-PAMs chemically optimized to provide comparable CNS exposure revealed that both classes of mGlu(5) PAMs have similar efficacy in a rodent model predictive of antipsychotic activity. These data suggest that the level of receptor expression influences the ability of mGlu(5) PAMs to act as allosteric agonists in vitro and that ago-PAM activity observed in cell-based assays may not be important for in vivo efficacy.

Investigating metabotropic glutamate receptor 5 allosteric modulator cooperativity, affinity, and agonism: enriching structure-function studies and structure-activity relationships.

Drug discovery programs increasingly are focusing on allosteric modulators as a means to modify the activity of G protein-coupled receptor (GPCR) targets. Allosteric binding sites are topographically distinct from the endogenous ligand (orthosteric) binding site, which allows for co-occupation of a single receptor with the endogenous ligand and an allosteric modulator that can alter receptor pharmacological characteristics. Negative allosteric modulators (NAMs) inhibit and positive allosteric modulators (PAMs) enhance the affinity and/or efficacy of orthosteric agonists. Established approaches for estimation of affinity and efficacy values for orthosteric ligands are not appropriate for allosteric modulators, and this presents challenges for fully understanding the actions of novel modulators of GPCRs. Metabotropic glutamate receptor 5 (mGlu(5)) is a family C GPCR for which a large array of allosteric modulators have been identified. We took advantage of the many tools for probing allosteric sites on mGlu(5) to validate an operational model of allosterism that allows quantitative estimation of modulator affinity and cooperativity values. Affinity estimates derived from functional assays fit well with affinities measured in radioligand binding experiments for both PAMs and NAMs with diverse chemical scaffolds and varying degrees of cooperativity. We observed modulation bias for PAMs when we compared mGlu(5)-mediated Ca(2+) mobilization and extracellular signal-regulated kinase 1/2 phosphorylation data. Furthermore, we used this model to quantify the effects of mutations that reduce binding or potentiation by PAMs. This model can be applied to PAM and NAM potency curves in combination with maximal fold-shift data to derive reliable estimates of modulator affinities.

Development of a more highly selective M(1) antagonist from the continued optimization of the MLPCN Probe ML012.

This Letter describes the continued optimization of an MLPCN probe molecule (ML012) through an iterative parallel synthesis approach. After exploring extensive modifications throughout the parent structure, we arrived at a more highly M(1)-selective antagonist, compound 13l (VU0415248). Muscarinic subtype selectivity across all five human and rat receptors for 13l, along with rat selectivity for the lead compound (ML012), is presented.

Chemical lead optimization of a pan Gq mAChR M1, M3, M5 positive allosteric modulator (PAM) lead. Part II: development of a potent and highly selective M1 PAM.

This Letter describes a chemical lead optimization campaign directed at VU0119498, a pan G(q) mAChR M(1), M(3), M(5) positive allosteric modulator (PAM) with the goal of developing a selective M(1) PAM. An iterative library synthesis approach delivered a potent (M(1) EC(50)=830 nM) and highly selective M(1) PAM (>30 microM vs M(2)-M(5)).

Immediate and delayed consequences of xanomeline wash-resistant binding at the M3 muscarinic receptor.

Xanomeline is thought to be a M1/M4 functionally selective agonist at muscarinic receptors. We have previously demonstrated that it binds in a unique manner at the M1 receptor. In the current study, we examined the ability of xanomeline to bind to the M3 receptor and determined the long-term consequences of this mode of binding in Chinese hamster ovary cells expressing M3 receptors. Xanomeline binds in a reversible and wash-resistant manner at the M3 receptor and elicits a functional response under both conditions. Long-term exposure to xanomeline resulted in changes in the binding profile of [(3)H]NMS and a decrease in cell-surface receptor density. Additionally, pretreatment with xanomeline was associated with antagonism of the functional response to subsequent stimulation by conventional agonists. Our results indicate that xanomeline binds to and activates the M3 muscarinic receptor in a wash-resistant manner, and that this type of binding results in time-dependent receptor regulation.

Mechanisms of M3 muscarinic receptor regulation by wash-resistant xanomeline binding.

BACKGROUND/AIMS: Xanomeline has been shown to bind in a unique manner at M1 and M3 muscarinic receptors, with interactions at both the orthosteric site and an allosteric site. We have previously shown that brief exposure of Chinese hamster ovary cells that express the M3 receptor to xanomeline followed by removal of free agonist results in a delayed decrease in radioligand binding and receptor response to agonists. In the current study, we were interested in determining the mechanisms of this effect. METHODS: Cells were treated with carbachol, pilocarpine or xanomeline for 1 h followed by washing and either used immediately or after waiting for 23 h. Control groups included cells that were not exposed to agonists and cells that were treated with agonists for 24 h. Radioligand binding and functional assays were conducted to determine the effects of agonist treatments. RESULTS: The above treatment protocol with xanomeline resulted in similar effects of the binding of [(3)H]NMS and [(3)H]QNB. When receptor function is blocked using a variety of methods, the long-term effects of xanomeline binding were absent. CONCLUSION: Our data indicate that xanomeline wash-resistant binding at the receptor allosteric site leads to receptor downregulation and that receptor activation is necessary for these effects.