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This Letter describes the synthesis and structure activity relationship (SAR) studies of structurally novel M4 antagonists, based on a 4,6-disubstituted core, identified from a high-throughput screening campaign. A multi-dimensional optimization effort enhanced potency at both human and rat M4 (IC50s < 300 nM), with no substantial species differences noted. Moreover, CNS penetration proved attractive for this series (brain:plasma Kp,uu = 0.87), while other DMPK attributes were addressed in the course of the optimization effort, providing low in vivo clearance in rat (CLp = 5.37 mL/min/kg). Surprisingly, this series displayed pan-muscarinic antagonist activity across M1–5, despite the absence of the prototypical basic or quaternary amine moiety, thus offering a new chemotype from which to develop a next generation of pan-muscarinic antagonist agents.

Although selective activation of the M(1) muscarinic acetylcholine receptor (mAChR) subtype has been shown to improve cognitive function in animal models of neuropsychiatric disorders, recent evidence suggests that enhancing M(4) mAChR function can also improve memory performance. Positive allosteric modulators (PAMs) targeting the M(4) mAChR subtype have shown therapeutic potential for the treatment of multiple symptoms observed in schizophrenia, including positive and cognitive symptoms when assessed in acute pre-clinical dosing paradigms. Since the cholinergic system has been implicated in multiple stages of learning and memory, we evaluated the effects of repeated dosing with the highly selective M(4) PAM VU0467154 on either acquisition and/or consolidation of learning and memory when dosed alone or after pharmacologic challenge with the N-methyl-D-aspartate subtype of glutamate receptors (NMDAR) antagonist MK-801. In animals, MK-801 challenge represents a well-documented model of NMDAR hypofunction that is thought to underlie some of the positive and cognitive symptoms observed in schizophrenia. In wildtype mice, 10-day, once-daily dosing of VU0467154 either prior to, or immediately after daily testing enhanced the rate of learning in a touchscreen visual pairwise discrimination task; these effects were absent in M(4) mAChR knockout mice. Following a similar 10-day, once-daily dosing regimen of VU0467154, we also observed 1) improved acquisition of memory in a cue-mediated conditioned freezing paradigm, 2) attenuation of MK-801-induced disruptions in the acquisition of memory in a context-mediated conditioned freezing paradigm and 3) reversal of MK-801-induced hyperlocomotion. Comparable efficacy and plasma and brain concentrations of VU0467154 were observed after repeated dosing as those previously reported with an acute, single dose administration of this M(4) PAM. Together, these studies are the first to demonstrate that cognitive enhancing and antipsychotic-like activity are not subject to the development of tolerance following repeated dosing with a selective M(4) PAM in mice and further suggest that activation of M(4) mAChRs may modulate both acquisition and con-solidation of memory functions.

Alzheimer’s disease (AD) is the leading cause of dementia worldwide, and currently no disease-modifying therapy is available to slow or prevent AD, underscoring the urgent need for neuroprotective therapies. Selective M1 muscarinic acetylcholine receptor (mAChR) activation is an attractive mechanism for AD therapy since M1 mediates key effects on memory, cognition, and behavior and has potential for disease-modifying effects on Aβ formation and tau phosphorylation. To validate M1 as a neuroprotective treatment target for AD, the M1-selective agonist, VU0364572, was chronically dosed to 5XFAD mice from a young age preceding Aβ pathology (2 months) to an age where these mice are known to display memory impairments (6 months). Chronic M1 activation prevented mice from becoming memory-impaired, as measured by Morris water maze (MWM) testing at 6 months of age. Additionally, M1 activation significantly reduced levels of soluble and insoluble Aβ40,42 in the cortex and hippocampus of these animals, as measured by ELISA and immunohistochemistry. Moreover, soluble hippocampal Aβ42 levels were strongly correlated with MWM memory impairments and M1 activation with VU0364572 abolished this correlation. Finally, VU0364572 significantly decreased oligomeric (oAβ) levels in the cortex, suggesting one mechanism whereby VU0364572 may be exerting its neuroprotective effects is by reducing the available oAβ pool in the brain. These findings suggest that chronic M1 activation has neuroprotective potential for preventing memory impairments and reducing neuropathology in AD. M1 activation therefore represents a promising avenue for preventative treatment, as well as a promising opportunity to combine symptomatic and disease-modifying effects for early AD treatment.

This letter describes the continued optimization of M5 NAM ML375 (VU0483253). While a valuable in vivo tool compound, ML375 has an excessively long elimination half-life in rat (t1/2 = 80 hours), which can be problematic in certain rodent addiction paradigms (e.g., reinstatement). Thus, we required an M5 NAM of comparable potency to ML375, but with a rat t1/2 of less than 4 hours. Steep SAR plagued this chemotype, and here we detail aniline replacements that offered some improvements over ML375, but failed to advance. Ultimately, incorporation of a single methyl group to the 9b-phenyl ring acted as a metabolic shunt, providing (S)-11 (VU6008667), an equipotent M5 NAM, with high CNS penetration, excellent selectivity versus M1–4 and the desired short half-life (t1/2 = 2.3 hours) in rat.

A functional high throughput screen identified a novel chemotype for the positive allosteric modulation (PAM) of the muscarinic acetylcholine receptor (mAChR) subtype 5 (M5). Application of rapid analog, iterative parallel synthesis efficiently optimized M5 potency to arrive at the most potent M5 PAMs prepared to date and provided tool compound 8n (ML380) demonstrating modest CNS penetration (human M5 EC50 = 190 nM, rat M5 EC50 = 610 nM, brain to plasma ratio (Kp) of 0.36).

Positive allosteric modulators (PAMs) of the M4 muscarinic acetylcholine receptor (mAChR) represent a novel approach for the treatment of psychotic symptoms associated with schizophrenia and other neuropsychiatric disorders. We recently reported that the selective M4 PAM VU0152100 produced an antipsychotic drug-like profile in rodents after amphetamine challenge. Previous studies suggest that enhanced cholinergic activity may also improve cognitive function and reverse deficits observed with reduced signaling through the N-methyl-d-aspartate subtype of the glutamate receptor (NMDAR) in the central nervous system. Prior to this study, the M1 mAChR subtype was viewed as the primary candidate for these actions relative to the other mAChR subtypes. Here we describe the discovery of a novel M4 PAM, VU0467154, with enhanced in vitro potency and improved pharmacokinetic properties relative to other M4 PAMs, enabling a more extensive characterization of M4 actions in rodent models. We used VU0467154 to test the hypothesis that selective potentiation of M4 receptor signaling could ameliorate the behavioral, cognitive, and neurochemical impairments induced by the noncompetitive NMDAR antagonist MK-801. VU0467154 produced a robust dose-dependent reversal of MK-801-induced hyperlocomotion and deficits in preclinical models of associative learning and memory functions, including the touchscreen pairwise visual discrimination task in wild-type mice, but failed to reverse these stimulant-induced deficits in M4 KO mice. VU0467154 also enhanced the acquisition of both contextual and cue-mediated fear conditioning when administered alone in wild-type mice. These novel findings suggest that M4 PAMs may provide a strategy for addressing the more complex affective and cognitive disruptions associated with schizophrenia and other neuropsychiatric disorders.

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 nonamide 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.

Of the five muscarinic receptor subtypes, the M5receptor is the only one detectable in midbrain dopaminergic neurons, making it an attractive potential therapeutic target for treating disorders in which dopaminergic signaling is disrupted. However, developing an understanding of the role of M5in regulating midbrain dopamine neuron function has been hampered by a lack of subtype-selective compounds. Here, we extensively characterize the novel compound VU0238429 and demonstrate that it acts as a positive allosteric modulator with unprecedented selectivity for the M5receptor. We then used VU0238429, along with M5knock-out mice, to elucidate the role of this receptor in regulating substantia nigra pars compacta (SNc) neuron physiology in both mice and rats. In sagittal brain slices that isolate the SNc soma from their striatal terminals, activation of muscarinic receptors induced Ca2+mobilization and inward currents in SNc dopamine neurons, both of which were potentiated by VU0238429 and absent in M5knock-out mice. Activation of M5also increased the spontaneous firing rate of SNc neurons, suggesting that activation of somatodendritic M5increases the intrinsic excitability of SNc neurons. However, in coronal slices of the striatum, potentiation of M5with VU0238429 resulted in an inhibition in dopamine release as monitored with fast scan cyclic voltammetry. Accordingly, activation of M5can lead to opposing physiological outcomes depending on the location of the receptor. Although activation of somatodendritic M5receptors on SNc neurons leads to increased neuronal firing, activation of M5receptors in the striatum induces an inhibition in dopamine release.

Herein, we report the structure-activity relationships within a series of potent, selective, and orally bioavailable muscarinic acetylcholine receptor 4 (M

A functional high throughput screen and subsequent multidimensional, iterative parallel synthesis effort identified the first muscarinic acetylcholine receptor (mAChR) negative allosteric modulator (NAM) selective for the M5 subtype. ML375 is a highly selective M5 NAM with submicromolar potency (human M5 IC50 = 300 nM, rat M5 IC50 = 790 nM, M1–M4 IC50 > 30 μM), excellent multispecies PK, high CNS penetration, and enantiospecific inhibition.

Positive allosteric modulators (PAMs) of metabotropic glutamate receptor 5 (mGlu5) represent a promising therapeutic strategy for the treatment of schizophrenia. Both allosteric agonism and high glutamate fold-shift have been implicated in the neurotoxic profile of some mGlu5 PAMs; however, these hypotheses remain to be adequately addressed. To develop tool compounds to probe these hypotheses, the structure-activity relationship of allosteric agonism was examined within an acetylenic series of mGlu5 PAMs exhibiting allosteric agonism in addition to positive allosteric modulation (ago-PAMs). PAM 38t, a low glutamate fold-shift allosteric ligand (maximum fold-shift ~ 3.0), was selected as a potent PAM with no agonism in the in vitro system used for compound characterization and in two native electrophysiological systems using rat hippocampal slices. PAM 38t (ML254) will be useful to probe the relative contribution of cooperativity and allosteric agonism to the adverse effect liability and neurotoxicity associated with this class of mGlu5 PAMs.

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.

Herein we report a next generation muscarinic receptor 4 (M(4)) positive allosteric modulator (PAM), ML253 which exhibits nanomolar activity at both the human (EC(50)=56 nM) and rat (EC(50)=176 nM) receptors and excellent efficacy by the left-ward shift of the ACh concentration response curve (fold shift, human=106; rat=50). In addition, ML253 is selective against the four other muscarinic subtypes, displays excellent CNS exposure and is active in an amphetamine-induced hyperlocomotion assay.

Utilizing a combination of high-throughput and multi-step synthesis, SAR in a novel series of M1 acetylcholine receptor antagonists was rapidly established. The efforts led to the discovery the highly potent M1 antagonists 6 (VU0431263), and 8f (VU0433670). Functional Schild analysis and radioligand displacement experiments demonstrated the competitive, orthosteric binding of these compounds; human selectivity data are presented.

We previously reported the discovery of VU0364572 and VU0357017 as M(1)-selective agonists that appear to activate M(1) through actions at an allosteric site. Previous studies have revealed that chemical scaffolds for many allosteric modulators contain molecular switches that allow discovery of allosteric antagonists and allosteric agonists or positive allosteric modulators (PAMs) based on a single chemical scaffold. Based on this, we initiated a series of studies to develop selective M(1) allosteric antagonists based on the VU0364572 scaffold. Interestingly, two lead antagonists identified in this series, VU0409774 and VU0409775, inhibited ACh-induced Ca(2+) responses at rat M(1-5) receptor subtypes, suggesting they are nonselective muscarinic antagonists. VU0409774 and VU0409775 also completely displaced binding of the nonselective radioligand [(3)H]-NMS at M(1) and M(3) mAChRs with affinities similar to their functional IC(50) values. Finally, Schild analysis revealed that these compounds inhibit M(1) responses through a fully competitive interaction at the orthosteric binding site. This surprising finding prompted further studies to determine whether agonist activity of VU0364572 and VU0357017 may also engage in previously unappreciated actions at the orthosteric site on M(1). Surprisingly, both VU0364572 and VU0357017 completely displaced [(3)H]-NMS binding to the orthosteric site of M(1)-M(5) receptors at high concentrations. Furthermore, evaluation of agonist activity in systems with varying levels of receptor reserve and Furchgott analysis using a cell line expressing M(1) under control of an inducible promotor was consistent with an action of these compounds as weak orthosteric partial agonists of M(1). However, consistent with previous studies suggesting actions at a site that is distinct from the orthosteric binding site, VU0364572 or VU0357017 slowed the rate of [(3)H]-NMS dissociation from CHO-rM(1) membranes. Together, these results suggest that VU0364572 and VU0357017 act as bitopic ligands and that novel antagonists in this series act as competitive orthosteric site antagonists.

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.

Allosteric modulation of GPCRs has initiated a new era of basic and translational discovery, filled with therapeutic promise yet fraught with caveats. Allosteric ligands stabilize unique conformations of the GPCR that afford fundamentally new receptors, capable of novel pharmacology, unprecedented subtype selectivity, and unique signal bias. This review provides a comprehensive overview of the basics of GPCR allosteric pharmacology, medicinal chemistry, drug metabolism, and validated approaches to address each of the major challenges and caveats. Then, the review narrows focus to highlight recent advances in the discovery of allosteric ligands for metabotropic glutamate receptor subtypes 1–5 and 7 (mGlu 1–57) highlighting key concepts (“molecular switches”, signal bias, heterodimers) and practical solutions to enable the development of tool compounds and clinical candidates. The review closes with a section on late-breaking new advances with allosteric ligands for other GPCRs and emerging data for endogenous allosteric modulators.

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.

Significance Huntington’s disease (HD) is a devastating neurodegenerative genetic disorder characterized by progressive decline of motor control. Although the genetic mutation responsible for the syndrome associated with HD has been clearly identified, a specific treatment for HD is not yet available. Therefore, there is a tremendous need for new therapeutic approaches and new molecular therapeutic targets. Using HD mouse models, we show age-dependent alterations of corticostriatal transmission paralleled by alterations of M 4 muscarinic acetylcholine receptor (mAChR)-mediated control of corticostriatal glutamate signaling. Also, chronic treatment with the selective M 4 mAChR positive allosteric modulator VU0467154 improves motor and synaptic deficits in 5-mo-old YAC128 mice. This suggests that M 4 may represent a therapeutic target for the treatment of HD.

This Letter describes a chemical lead optimization campaign directed at VU0108370, a weak M(1) PAM hit with a novel chemical scaffold from a functional HTS screen within the MLPCN. An iterative parallel synthesis approach rapidly established SAR for this series and afforded VU0405652 (ML169), a potent, selective and brain penetrant M(1) PAM with an in vitro profile comparable to the prototypical M(1) PAM, BQCA, but with an improved brain to plasma ratio.

This Letter describes a chemical lead optimization campaign directed at VU0238429, the first M(5)-preferring positive allosteric modulator (PAM), discovered through analog work around VU0119498, a pan G(q) mAChR M(1), M(3), M(5) PAM. An iterative parallel synthesis approach was employed to incorporate basic heterocycles to improve physiochemical properties.

Purpose Current intra-arterial chemotherapy (IAC) drug regimens for retinoblastoma have ocular and vascular toxicities. No small-animal model of IAC exists to test drug efficacy and toxicity in vivo for IAC drug discovery. The purpose of this study was to develop a small-animal model of IAC and to analyze the ocular tissue penetration, distribution, pharmacokinetics, and treatment efficacy. Methods Following selective ophthalmic artery (OA) catheterization, melphalan (0.4 to 1.2 mg/kg) was injected. For pharmacokinetic studies, rabbits were euthanized at 0.5, 1, 2, 4, or 6 hours following intra-OA infusion. Drug levels were determined in vitreous, retina, and blood by liquid chromatography tandem mass spectrometry. To assess toxicity, angiograms, photography, fluorescein angiography, and histopathology were performed. For in situ tissue drug distribution, matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) was performed. The tumor model was created by combined subretinal/intravitreal injection of human WERI-Rb1 retinoblastoma cells; the tumor was treated in vivo with intra-arterial melphalan or saline; and induction of tumor death was measured by cleaved caspase-3 activity. Results OA was selectively catheterized for 79 of 79 (100%) eyes in 47 of 47 (100%) rabbits, and melphalan was delivered successfully in 31 of 31 (100%) eyes, without evidence of vascular occlusion or retinal damage. For treated eyes, maximum concentration (Cmax) in the retina was 4.95 μM and area under the curve (AUC0→∞) was 5.26 μM·h. Treated eye vitreous Cmax was 2.24 μM and AUC0→∞ was 4.19 μM·h. Vitreous Cmax for the treated eye was >100-fold higher than for the untreated eye (P = 0.01), and AUC0→∞ was ∼50-fold higher (P = 0.01). Histology-directed MALDI-IMS revealed highest drug localization within the retina. Peripheral blood Cmax was 1.04 μM and AUC0→∞ was 2.07 μM·h. Combined subretinal/intravitreal injection of human retinoblastoma cells led to intra-retinal tumors and subretinal/vitreous seeds, which could be effectively killed in vivo with intra-arterial melphalan. Conclusions This first small-animal model of IAC has excellent vitreous and retinal tissue drug penetration, achieving levels sufficient to kill human retinoblastoma cells, facilitating future IAC drug discovery.

J. Phillip Kennedy[a],+, Thomas M. Bridges[b],+, Patrick R. Gentry[b], John T. Brogan[a], Alexander S. Kane[b], Carrie K. Jones[b],[c], Ashley E. Brady[b], Jana K. Shirey[b], P. Jeffrey Conn [Prof.][b], and Craig W. Lindsley [Prof.]*,[a],[b] [a] Department of Chemistry, Vanderbilt University 12475 MRB4, Nashville, TN 37232-6600 (USA) [b] Vanderbilt Program in Drug Discovery, Department of Pharmacology Vanderbilt University Medical Center 12475 MRB4, Nashville, TN 37232-6600 (USA)

Cholinergic transmission in the forebrain is mediated primarily by five subtypes of muscarinic acetylcholine receptors (mAChRs), termed M(1)-M(5). Of the mAChR subtypes, M(1) is among the most heavily expressed in regions that are critical for learning and memory, and has been viewed as the most critical mAChR subtype for memory and attention mechanisms. Unfortunately, it has been difficult to develop selective activators of M(1) and other individual mAChR subtypes, which has prevented detailed studies of the functional roles of selective activation of M(1). Using a functional HTS screen and subsequent diversity-oriented synthesis approach we have discovered a novel series of highly selective M(1) allosteric agonists. These compounds activate M(1) with EC(50) values in the 150 nM to 500 nM range and have unprecedented, clean ancillary pharmacology (no substantial activity at 10μM across a large panel of targets). Targeted mutagenesis revealed a potentially novel allosteric binding site in the third extracellular loop of the M(1) receptor for these allosteric agonists. Optimized compounds, such as VU0357017, provide excellent brain exposure after systemic dosing and have robust in vivo efficacy in reversing scopolamine-induced deficits in a rodent model of contextual fear conditioning. This series of selective M(1) allosteric agonists provides critical research tools to allow dissection of M(1)-mediated effects in the CNS and potential leads for novel treatments for Alzheimer's disease and schizophrenia.

This Letter describes the first account of the synthesis and SAR, developed through an iterative analogue library approach, of analogues of the highly selective M1 allosteric agonist TBPB. With slight structural changes, mAChR selectivity was maintained, but the degree of partial M1 agonism varied considerably.

This Letter describes the further synthesis and SAR, developed through an iterative analogue library approach, of analogues of the highly selective M1 allosteric agonist TBPB by deletion of the distal basic piperidine nitrogen by the formation of amides, sulfonamides and ureas. Despite the large change in basicity and topology, M1 selectivity was maintained.

Previous clinical and animal studies suggest that selective activators of M(1) and/or M(4) muscarinic acetylcholine receptors (mAChRs) have potential as novel therapeutic agents for treatment of schizophrenia and Alzheimer's disease. However, highly selective centrally penetrant activators of either M(1) or M(4) have not been available, making it impossible to determine the in vivo effects of selective activation of these receptors. We previously identified VU10010 [3-amino-N-(4-chlorobenzyl)-4, 6-dimethylthieno[2,3-b]pyridine-2-carboxamide] as a potent and selective allosteric potentiator of M(4) mAChRs. However, unfavorable physiochemical properties prevented use of this compound for in vivo studies. We now report that chemical optimization of VU10010 has afforded two centrally penetrant analogs, VU0152099 [3-amino-N-(benzo[d][1,3]dioxol-5-ylmethyl)-4,6-dimethylthieno[2,3-b]pyridine carboxamide] and VU0152100 [3-amino-N-(4-methoxybenzyl)-4,6-dimethylthieno[2,3-b]pyridine carboxamide], that are potent and selective positive allosteric modulators of M(4). VU0152099 and VU0152100 had no agonist activity but potentiated responses of M(4) to acetylcholine. Both compounds were devoid of activity at other mAChR subtypes or at a panel of other GPCRs. The improved physiochemical properties of VU0152099 and VU0152100 allowed in vivo dosing and evaluation of behavioral effects in rats. Interestingly, these selective allosteric potentiators of M(4) reverse amphetamine-induced hyperlocomotion in rats, a model that is sensitive to known antipsychotic agents and to nonselective mAChR agonists. This is consistent with the hypothesis that M(4) plays an important role in regulating midbrain dopaminergic activity and raises the possibility that positive allosteric modulation of M(4) may mimic some of the antipsychotic-like effects of less selective mAChR agonists.

The science that established combinatorial chemistry as important new discipline in the 1980s and 1990s has re-emerged and infiltrated every sub-discipline within modern drug discovery and has profound impact. The application of combinatorial chemistry science has revolutioned high-throughput screening paradigms, chemical lead optimization, library purification, and post-purification sample handling, as well as in vitro and in vivo drug metabolism and pharmacokinetic assays. Although no longer in the spotlight and heralded as the savior of the drug industry, “combinatorial chemistry” is alive and well; in fact, combinatorial chemistry science is more prevalent and wide-spread than ever before.

Accumulating evidence indicates direct relationships between sleep abnormalities and the severity and prevalence of other symptom clusters in schizophrenia. Assessment of potential state-dependent alterations in sleep architecture and arousal relative to antipsychotic-like activity is critical for the development of novel antipsychotic drugs (APDs). Recently, we reported that VU0467154, a selective positive allosteric modulator (PAM) of the M4 muscarinic acetylcholine receptor (mAChR), exhibits robust APD-like and cognitive enhancing activity in rodents. However, the state-dependent effects of VU0467154 on sleep architecture and arousal have not been examined. Using polysomnography and quantitative electroencephalographic recordings from subcranial electrodes in rats, we evaluated the effects of VU0467154, in comparison with the atypical APD clozapine and the M1/M4-preferring mAChR agonist xanomeline. VU0467154 induced state-dependent alterations in sleep architecture and arousal by delaying Rapid Eye Movement (REM) sleep onset, selectively increased cumulative duration of total and Non-Rapid Eye Movement (NREM) sleep, and increased arousal during waking periods. Clozapine decreased arousal during wake, increased cumulative NREM, and decreased REM sleep. In contrast, xanomeline increased time awake and arousal during wake, but reduced slow wave activity during NREM sleep. Additionally, in combination with the N-methyl-d-aspartate subtype of glutamate receptor (NMDAR) antagonist MK-801, modeling NMDAR hypofunction thought to underlie many symptoms in schizophrenia, both VU0467154 and clozapine attenuated MK-801-induced elevations in high frequency gamma power consistent with an APD-like mechanism of action. These findings suggest that selective M4 PAMs may represent a novel mechanism for treating multiple symptoms of schizophrenia, including disruptions in sleep architecture without a sedative profile.

Herein, we report the structure–activity relationship of a novel series of (2(phenoxymethyl)-6,7-dihydrooxazolo[5,4-c]pyridine-5(4H)-yl(aryl)methanones as potent, selective, and orally bioavailable metabotropic glutamate receptor subtype 5 (mGlu5) positive allosteric modulators (PAMs). On the basis of its robust in vitro potency and in vivo efficacy in multiple preclinical models of multiple domains of schizophrenia, coupled with a good DMPK profile and an acceptable therapeutic window, 17a (VU0409551/JNJ-46778212) was selected as a candidate for further development.

This Letter describes the continued optimization of the MLPCN probe ML375, a highly selective M5 negative allosteric modulator (NAM), through a combination of matrix libraries and iterative parallel synthesis. True to certain allosteric ligands, SAR was shallow, and the matrix library approach highlighted the challenges with M5 NAM SAR within in this chemotype. Once again, enantiospecific activity was noted, and potency at rat and human M5 were improved over ML375, along with slight enhancement in physiochemical properties, certain in vitro DMPK parameters and CNS distribution. Attempts to further enhance pharmacokinetics with deuterium incorporation afforded mixed results, but pretreatment with a pan-P450 inhibitor (1-aminobenzotriazole; ABT) provided increased plasma exposure.

Approaches to efficiently and accurately define the pharmacokinetics (PK) of large sets of small molecules in rodents have been previously described. Likewise, a variety of methods for determining brain tissue distribution (BTD) have been reported for use in the discovery of therapeutics targeting the central nervous system (CNS). Herein we describe a novel cassette approach to efficiently obtain concurrent PK and BTD data from a dose of up to five compounds in one rat over 24 h. In conjunction with fraction unbound (fu) data obtained in plasma and brain homogenate, this approach serves as an efficient means to determine compound unbound brain:unbound plasma partition coefficients (K p,uu), thereby providing insight to compounds bearing poor permeability and/or active transporter activity impacting their permeation of the blood-brain barrier (BBB). This integrated approach was utilized in a lead optimization effort towards the discovery of CNS-penetrant allosteric modulators of a seven-transmembrane (7TM) receptor target. Rat PK and brain distribution was rapidly obtained for 70 compounds and correlated to data obtained from in vitro assessments. Two compounds that were evaluated in cassette and discrete studies, displayed agreement in PK (compound 1: cassette CLp = 1.6 mL min(-1) kg(-1), discrete CLp = 1.6 mL min(-1) kg(-1); compound 2: cassette CLp = 11 mL min(-1) kg(-1), discrete CLp = 8.1 mL min(-1) kg(-1)) and BTD (compound 1: cassette K p = 0.11, discrete K p = 0.09; compound 2: cassette K p0.05, discrete K p = 0.04). The resulting data were used to guide medicinal chemistry efforts and to enable the progression of optimized compounds to in vivo pharmacodynamic assessments.

Recent findings indicate that selective activation of the M4 muscarinic acetylcholine receptor (M4 mAChR) may represent a novel treatment approach for the psychotic symptoms and cognitive disturbances observed in several neuropsychiatric disorders. In the present studies, cognitive performance of M4 mAChR knock-out mice (M4-KO mice) was examined across a series of computer touchscreen-based pairwise discrimination tasks assessing susceptibility to interference from previously learned competing sets of information. While M4-KO mice acquired the initial discrimination task in a similar number of sessions as wildtype mice, the M4-KO mice showed impaired acquisition on all subsequent visual discriminations. In addition, the novel M4 positive allosteric modulator VU0467154 reversed disruptions in pairwise discrimination induced by MK-801, an antagonist of the N-methyl-D-aspartate subtype of glutamate receptors. These data reveal that selective M4 mAChR activation is critical for modulation of normal associativ...

Of the five G-protein-coupled muscarinic acetylcholine receptors (mAChRs; M1–M5), M5 is the least explored and understood due to a lack of mAChR subtype-selective ligands. We recently performed a high-throughput functional screen and identified a number of weak antagonist hits that are selective for the M5 receptor. Here, we report an iterative parallel synthesis and detailed molecular pharmacologic profiling effort that led to the discovery of the first highly selective, central nervous system (CNS)-penetrant M5-orthosteric antagonist, with sub-micromolar potency (hM5 IC50=450 nM, hM5 Ki=340 nM, M1–M4 IC50 >30 μM), enantiospecific inhibition, and an acceptable drug metabolism and pharmacokinetics (DMPK) profile for in vitro and electrophysiology studies. This compound will be a powerful tool and molecular probe for the further investigation into the role of M5 in addiction and other diseases.

Accumulating evidence suggests that selective M4 muscarinic acetylcholine receptor (mAChR) activators may offer a novel strategy for the treatment of psychosis. However, previous efforts to develop selective M4 activators were unsuccessful because of the lack of M4 mAChR subtype specificity and off-target muscarinic adverse effects. We recently developed VU0152100, a highly selective M4 positive allosteric modulator (PAM) that exerts central effects after systemic administration. We now report that VU0152100 dose-dependently reverses amphetamine-induced hyperlocomotion in rats and wild-type mice, but not in M4 KO mice. VU0152100 also blocks amphetamine-induced disruption of the acquisition of contextual fear conditioning and prepulse inhibition of the acoustic startle reflex. These effects were observed at doses that do not produce catalepsy or peripheral adverse effects associated with non-selective mAChR agonists. To further understand the effects of selective potentiation of M4 on region-specific brain activation, VU0152100 alone and in combination with amphetamine were evaluated using pharmacologic magnetic resonance imaging (phMRI). Key neural substrates of M4-mediated modulation of the amphetamine response included the nucleus accumbens (NAS), caudate-putamen (CP), hippocampus, and medial thalamus. Functional connectivity analysis of phMRI data, specifically assessing correlations in activation between regions, revealed several brain networks involved in the M4 modulation of amphetamine-induced brain activation, including the NAS and retrosplenial cortex with motor cortex, hippocampus, and medial thalamus. Using in vivo microdialysis, we found that VU0152100 reversed amphetamine-induced increases in extracellular dopamine levels in NAS and CP. The present data are consistent with an antipsychotic drug-like profile of activity for VU0152100. Taken together, these data support the development of selective M4 PAMs as a new approach to the treatment of psychosis and cognitive impairments associated with psychiatric disorders such as schizophrenia.

Allosteric modulation of G protein-coupled receptors has gained considerable attention in the drug discovery arena because it opens avenues to achieve greater selectivity over orthosteric ligands. We recently identified a series of positive allosteric modulators (PAMs) of metabotropic glutamate receptor 5 (mGlu(5)) for the treatment of schizophrenia that exhibited robust heterotropic activation of CYP3A4 enzymatic activity. The prototypical compound from this series, 5-(4-fluorobenzyl)-2-((3-fluorophenoxy)methyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazine (VU0448187), was found to activate CYP3A4 to100% of its baseline intrinsic midazolam (MDZ) hydroxylase activity in vitro; activation was CYP3A substrate specific and mGlu(5) PAM dependent. Additional studies revealed the concentration-dependence of CYP3A activation by VU0448187 in multispecies hepatic and intestinal microsomes and hepatocytes, as well as a diminished effect observed in the presence of ketoconazole. Kinetic analyses of the effect of VU0448187 on MDZ metabolism in recombinant P450 or human liver microsomes resulted in a significant increase in V(max) (minimal change in K(m)) and required the presence of cytochrome b5. The atypical kinetics translated in vivo, as rats receiving an intraperitoneal administration of VU0448187 prior to MDZ treatment demonstrated a significant increase in circulating 1- and 4-hydroxy- midazolam (1-OH-MDZ, 4-OH-MDZ) levels compared with rats administered MDZ alone. The discovery of a potent substrate-selective activator of rodent CYP3A with an in vitro to in vivo translation serves to illuminate the impact of increasing intrinsic enzymatic activity of hepatic and extrahepatic CYP3A in rodents, and presents the basis to build models capable of framing the clinical relevance of substrate-dependent heterotropic activation.

Impaired transmission through glutamatergic circuits has been postulated to play a role in the underlying pathophysiology of schizophrenia. Furthermore, inhibition of the N-methyl-d-aspartate (NMDA) subtype of ionotropic glutamate receptors (NMDAR) induces a syndrome that recapitulates many of the symptoms observed in patients with schizophrenia. Selective activation of metabotropic glutamate receptor subtype 5 (mGlu5) may provide a novel therapeutic approach for treatment of symptoms associated with schizophrenia through facilitation of transmission through central glutamatergic circuits. Here, we describe the characterization of two novel N-aryl piperazine mGlu5 positive allosteric modulators (PAMs): 2-(4-(2-(benzyloxy)acetyl)piperazin-1-yl)benzonitrile (VU0364289) and 1-(4-(2,4-difluorophenyl)piperazin-1-yl)-2-((4-fluorobenzyl)oxy)ethanone (DPFE). VU0364289 and DPFE induced robust leftward shifts in the glutamate concentration-response curves for Ca(2+) mobilization and extracellular signal-regulated kinases 1 and 2 phosphorylation. Both PAMs displayed micromolar affinity for the common mGlu5 allosteric binding site and high selectivity for mGlu5. VU0364289 and DPFE possessed suitable pharmacokinetic properties for dosing in vivo and produced robust dose-related effects in reversing amphetamine-induced hyperlocomotion, a preclinical model predictive of antipsychotic-like activity. In addition, DPFE enhanced acquisition of contextual fear conditioning in rats and reversed behavioral deficits in a mouse model of NMDAR hypofunction. In contrast, DPFE had no effect on reversing apomorphine-induced disruptions of prepulse inhibition of the acoustic startle reflex. These mGlu5 PAMs also increased monoamine levels in the prefrontal cortex, enhanced performance in a hippocampal-mediated memory task, and elicited changes in electroencephalogram dynamics commensurate with procognitive effects. Collectively, these data support and extend the role for the development of novel mGlu5 PAMs for the treatment of psychosis and cognitive deficits observed in individuals with schizophrenia.

A functional high throughput screen and subsequent multi-dimensional, iterative parallel synthesis effort identified the first muscarinic acetylcholine receptor (mAChR) negative allosteric modulator (NAM) selective for the M5 subtype. ML375 is a highly selective M5 NAM with sub-micromolar potency (human M5 IC50 = 300 nM, rat M5 IC50 = 790 nM, M1–4 IC50 >30 μM), excellent multi-species PK, high CNS penetration, and enantiospecific inhibition.

This letter describes the further chemical optimization of the M5 PAM MLPCN probes ML129 and ML172. A multi-dimensional iterative parallel synthesis effort quickly explored isatin replacements and a number of southern heterobiaryl variations with no improvement over ML129 and ML172. An HTS campaign identified several weak M5 PAMs (M5 EC50 >10 μM) with a structurally related isatin core that possessed a southern phenethyl ether linkage. While SAR within the HTS series was very shallow and unable to be optimized, grafting the phenethyl ether linkage onto the ML129/ML172 cores led to the first sub-micromolar M5 PAM, ML326 (VU0467903), (human and rat M5 EC50s of 409 nM and 480 nM, respectively) with excellent mAChR selectivity (M1-M4 EC50s

Ser-64, an autophosphorylation site in the autoinhibitory subdomain of cGMP-dependent protein kinase type I-alpha (PKGI-alpha), lowers affinity for cGMP and suppresses catalytic activity (1). Using the structure of homologous cAMP-dependent protein kinase as a model, three conserved residues (Gln-401, His-404, Cys-518) in the PKGI-alpha catalytic site are predicted to be juxtaposed to Ser-64 (2). Individual point mutants (Q401A, H404A and C518A) and a double mutant (S64A/H404A) have been generated. cGMP or cAMP affinities (K(a)) of each mutant protein for phosphotransferase activation and allosteric (3H)cGMP-binding affinity (K(D)) of each mutant protein are significantly improved over those of wild-type (WT) PKGI-alpha. However, affinities (K(m)) of the mutant PKGs for peptide substrates or ATP are unaltered. Kinase activity ratio (-GMP/+cGMP) of H404A is greater than that for WT, Q401A, or C518A, and similar to that for S64A and S64A/H404A. These results reveal a unique mechanism whereby catalytic domain residues predicted to be spatially close to Ser-64 of the regulatory domain weaken the intrinsically high affinity of PKGI-alpha for cGMP and provide for autoinhibition of catalytic activity.

This Letter describes the further optimization of an MLPCN probe molecule (ML137) through the introduction of 5- and 6-membered spirocycles in place of the isatin ketone. Interestingly divergent structure–activity relationships, when compared to earlier M1 PAMs, are presented. These novel spirocycles possess improved efficacy relative to ML137, while also maintaining high selectivity for the human and rat muscarinic M1 receptor subtype.

This Letter describes the continued optimization of an MLPCN probe molecule (ML137) with a focused effort on the replacement/modification of the isatin moiety present in this highly selective M(1) PAM. A diverse range of structures were validated as viable replacements for the isatin, many of which engendered sizeable improvements in their ability to enhance the potency and efficacy of acetylcholine when compared to ML137. Muscarinic receptor subtype selectivity for the M(1) receptor was also maintained.

Herein we report a next generation muscarinic receptor 4 (M4) positive allosteric modulator (PAM), ML253 which exhibits nanomolar activity at both the human (EC50 = 56 nM) and rat (EC50 = 176 nM) receptors and excellent efficacy by the left-ward shift of the ACh concentration response curve (Fold Shift, human = 106; rat = 50). In addition, ML253 is selective against the four other muscarinic subtypes, displays excellent CNS exposure and is active in an amphetamine-induced hyperlocomotion assay.

Negative allosteric modulation (NAM) of metabotropic glutamate receptor subtype 5 (mGlu5) represents a therapeutic strategy for the treatment of childhood developmental disorders, such as fragile X syndrome and autism. VU0409106 emerged as a lead compound within a biaryl ether series, displaying potent and selective inhibition of mGlu5. Despite its high clearance and short half-life, VU0409106 demonstrated efficacy in rodent models of anxiety after extravascular administration. However, lack of a consistent correlation in rat between in vitro hepatic clearance and in vivo plasma clearance for the biaryl ether series prompted an investigation into the biotransformation of VU0409106 using hepatic subcellular fractions. An in vitro appraisal in rat, monkey, and human liver S9 fractions indicated that the principal pathway was NADPH-independent oxidation to metabolite M1 (+16 Da). Both raloxifene (aldehyde oxidase inhibitor) and allopurinol (xanthine oxidase inhibitor) attenuated the formation of M1, thus implicating the contribution of both molybdenum hydroxylases in the biotransformation of VU0409106. The use of 18O-labeled water in the S9 experiments confirmed the hydroxylase mechanism proposed, because 18O was incorporated into M1 (+18 Da) as well as in a secondary metabolite (M2; +36 Da), the formation of which was exclusively xanthine oxidase-mediated. This unusual dual and sequential hydroxylase metabolism was confirmed in liver S9 and hepatocytes of multiple species and correlated with in vivo data because M1 and M2 were the principal metabolites detected in rats administered VU0409106. An in vitro-in vivo correlation of predicted hepatic and plasma clearance was subsequently established for VU0409106 in rats and nonhuman primates.

Utilizing a combination of high-throughput and multi-step synthesis, SAR in a novel series of M(1) acetylcholine receptor antagonists was rapidly established. The efforts led to the discovery the highly potent M(1) antagonists 6 (VU0431263), and 8f (VU0433670). Functional Schild analysis and radioligand displacement experiments demonstrated the competitive, orthosteric binding of these compounds; human selectivity data are presented.

The M(1) muscarinic acetylcholine receptor is thought to play an important role in memory and cognition, making it a potential target for the treatment of Alzheimer's disease (AD) and schizophrenia. Moreover, M(1) interacts with BACE1 and regulates its proteosomal degradation, suggesting selective M(1) activation could afford both palliative cognitive benefit as well as disease modification in AD. A key challenge in targeting the muscarinic acetylcholine receptors is achieving mAChR subtype selectivity. Our lab has previously reported the M(1) selective positive allosteric modulator ML169. Herein we describe our efforts to further optimize this lead compound by preparing analogue libraries and probing novel scaffolds. We were able to identify several analogues that possessed submicromolar potency, with our best example displaying an EC(50) of 310 nM. The new compounds maintained complete selectivity for the M(1) receptor over the other subtypes (M(2)-M(5)), displayed improved DMPK profiles, and potentiated the carbachol (CCh)-induced excitation in striatal MSNs. Selected analogues were able to potentiate CCh-mediated nonamyloidogenic APPsα release, further strengthening the concept that M(1) PAMs may afford a disease-modifying role in the treatment of AD.

Background Metabotropic glutamate receptor subtype 5 (mGlu 5 ) activators have emerged as a novel approach to the treatment of schizophrenia. Positive allosteric modulators (PAMs) of mGlu 5 have generated tremendous excitement and fueled major drug discovery efforts. Although mGlu 5 PAMs have robust efficacy in preclinical models of schizophrenia, preliminary reports suggest that these compounds may induce seizure activity. Prototypical mGlu 5 PAMs do not activate mGlu 5 directly but selectively potentiate activation of mGlu 5 by glutamate. This mechanism may be critical to maintaining normal activity-dependence of mGlu 5 activation and achieving optimal in vivo effects. Methods Using specially engineered mGlu 5 cell lines incorporating point mutations within the allosteric and orthosteric binding sites, as well as brain slice electrophysiology and in vivo electroencephalography and behavioral pharmacology, we found that some mGlu 5 PAMs have intrinsic allosteric agonist activity in the absence of glutamate. Results Both in vitro mutagenesis and in vivo pharmacology studies demonstrate that VU0422465 is an agonist PAM that induces epileptiform activity and behavioral convulsions in rodents. In contrast, VU0361747, an mGlu 5 PAMs optimized to eliminate allosteric agonist activity, has robust in vivo efficacy and does not induce adverse effects at doses that yield high brain concentrations. Conclusions Loss of the absolute dependence of mGlu 5 PAMs on glutamate release for their activity can lead to severe adverse effects. The finding that closely related mGlu 5 PAMs can differ in their intrinsic agonist activity provides critical new insights that is essential for advancing these molecules through clinical development for treatment of schizophrenia.

This Paper describes the continued optimization of an MLPCN probe molecule M(1) antagonist (ML012) through an iterative parallel synthesis approach. After several rounds of modifications of the parent compound, we arrived at a new azetidine scaffold that displayed improved potency while maintaining a desirable level of selectivity over other muscarinic receptor subtypes. Data for representative molecules 7w (VU0452865) and 12a (VU0455691) are presented.