This Letter describes the discovery, SAR and in vitro and in vivo pharmacological profile of a novel non-MPEP derived mGlu5 positive allosteric modulator (PAM) based upon an N-aryl piperazine chemotype. This mGlu5 chemotype exhibits the ability to act as either a non-competitive antagonist/negative allosteric modulator (NAM) or potentiator of the glutamate response depending on the identity of the amide substituent, i.e., a ‘molecular switch’. A rapidly optimized PAM, 10e (VU0364289), was shown to be potent and specific for the rat mGlu5 receptor and subsequently demonstrated to be efficacious in a clinically relevant rodent model predictive of anti-psychotic activity, thus providing the first example of a centrally active mGluR5 PAM optimized from an HTS-derived mGluR5 competitive antagonist.

Muscarinic and nicotinic acetylcholine (ACh) receptors (mAChRs and nAChRs) are emerging as important targets for the development of novel treatments for the symptoms associated with schizophrenia. Preclinical and early proof-of-concept clinical studies have provided strong evidence that activators of specific mAChR (M1 and M4) and nAChR (α7 and α2β4) subtypes are effective in animal models of antipsychotic-like activity and/or cognitive enhancement, and in the treatment of positive and cognitive symptoms in patients with schizophrenia. While early attempts to develop selective mAChR and nAChR agonists provided important preliminary findings, these compounds have ultimately failed in clinical development due to a lack of true subtype selectivity and subsequent dose-limiting adverse effects. In recent years, there have been major advances in the discovery of highly selective activators for the different mAChR and nAChR subtypes with suitable properties for optimization as potential candidates for clinical trials. One novel strategy has been to identify ligands that activate a specific receptor subtype through actions at sites that are distinct from the highly conserved ACh-binding site, termed allosteric sites. These allosteric activators, both allosteric agonists and positive allosteric modulators, of mAChR and nAChR subtypes demonstrate unique mechanisms of action and high selectivity in vivo, and may provide innovative treatment strategies for schizophrenia.

T-Type Ca2+ channel inhibitors hold tremendous therapeutic potential for the treatment of pain, epilepsy, sleep disorders, essential tremor, and other neurological disorders; however, a lack of truly selective tools has hindered basic research, and selective tools from the pharmaceutical industry are potentially burdened with intellectual property (IP) constraints. Thus, an MLPCN high-throughput screen (HTS) was conducted to identify novel T-type Ca2+ channel inhibitors free from IP constraints, and freely available through the MLPCN, for use by the biomedical community to study T-type Ca2+ channels. While the HTS provided numerous hits, these compounds could not be optimized to the required level of potency to be appropriate tool compounds. Therefore, a scaffold hopping approach, guided by SurflexSim, ultimately afforded ML218 (CID 45115620), a selective T-type Ca2+ (Cav3.1, Cav3.2, Cav3.3) inhibitor (Cav3.2, IC50 = 150 nM in Ca2+ flux; Cav3.2 IC50 = 310 nM; and Cav3.3 IC50 = 270 nM, respectively in patch clamp electrophysiology) with good DMPK properties, acceptable in vivo rat PK, and excellent brain levels. Electrophysiology studies in subthalamic nucleus (STN) neurons demonstrated robust effects of ML218 on the inhibition of T-type calcium current, inhibition of low threshold spike, and rebound burst activity. Based on the basal ganglia circuitry in Parkinson’s disease (PD), the effects of ML218 in STN neurons suggest a therapeutic role for T-type Ca2+ channel inhibitors, and ML218 was found to be orally efficacious in haloperidol-induced catalepsy, a preclinical PD model, with comparable efficacy to an A2A antagonist, a clinically validated PD target. ML218 proves to be a powerful new probe to study T-type Ca2+ function in vitro and in vivo, and freely available.

This Letter describes the hit-to-lead progression and SAR of a series of biphenyl acetylene compounds derived from an HTS screening campaign targeting the mGlu5 receptor. ‘Molecular switches’ were identified that modulated modes of pharmacology, and several compounds within this series were shown to be efficacious in reversal of amphetamine induced hyperlocomotion in rats after i.p. dosing, a preclinical model that shows similar positive effects with known antipsychotic agents.

There is an increasing amount of literature data showing the positive effects on preclinical anti-Parkinsonian rodent models with selective positive allosteric modulators of metabotropic glutamate receptor 4 (mGlu4).1 However, most of the data generated utilize compounds that have not been optimized for drug-like properties and, as a consequence, they exhibit poor pharmacokinetic properties and thus do not cross the blood-brain barrier. Herein, we report on a series of N-4-(2,5-dioxopyrrolidin-1-yl)-phenylpicolinamides with improved PK properties with excellent potency and selectivity as well as improved brain exposure in rodents. Finally, ML182 was shown to be orally active in the haloperidol induced catalepsy model, a well-established anti-Parkinsonian model.

Glutamate is the major excitatory transmitter in the mammalian CNS, exerting its effects through both ionotropic and metabotropic glutamate receptors. The metabotropic glutamate receptors (mGlus) belong to family C of the G-protein-coupled receptors (GPCRs). The eight mGlus identified to date are classified into three groups based on their structure, preferred signal transduction mechanisms, and pharmacology (Group I: mGlu1 and mGlu5; Group II: mGlu2 and mGlu3; Group III: mGlu4, mGlu6, mGlu7, and mGlu8). Non-competitive antagonists, also known as negative allosteric modulators (NAMs), of mGlu5 offer potential therapeutic applications in diseases such as pain, anxiety, gastroesophageal reflux disease (GERD), Parkinson's disease (PD), fragile X syndrome, and addiction. The development of SAR in a (3-cyano-5-fluorophenyl)biaryl series using our functional cell-based assay is described in this communication. Further characterization of a selected compound, 3-fluoro-5-(2-methylbenzo[d]thiazol-5-yl)benzonitrile, in additional cell based assays as well as in vitro assays designed to measure its metabolic stability and protein binding indicated its potential utility as an in vivo tool. Subsequent evaluation of the same compound in a pharmacokinetic study using intraperitoneal dosing in mice showed good exposure in both plasma and brain samples. The compound was efficacious in a mouse marble burying model of anxiety, an assay known to be sensitive to mGlu5 antagonists. A new operant model of addiction termed operant sensation seeking (OSS) was chosen as a second behavioral assay. The compound also proved efficacious in the OSS model and constitutes the first reported example of efficacy with a small molecule mGlu5 NAM in this novel assay.

Glutamate is the major excitatory transmitter in the mammalian central nervous system (CNS), exerting its effects through both ionotropic and metabotropic glutamate receptors. The metabotropic glutamate receptors (mGlus) belong to family C of the G-protein-coupled receptors (GPCRs). The eight mGlus identified to date are classified into three groups based on their structure, preferred signal transduction mechanisms, and pharmacology (group I: mGlu1 and mGlu5; group II: mGlu2 and mGlu3; group III: mGlu4, mGlu6, mGlu7, and mGlu8). Noncompetitive antagonists, also known as negative allosteric modulators (NAMs), of mGlu5 offer potential therapeutic applications in diseases such as pain, anxiety, gastresophageal reflux disease (GERD), Parkinson’s disease (PD), fragile X syndrome, and addiction. The development of structure−activity relationships (SAR) in a (3-cyano-5-fluorophenyl)biaryl series using our functional cell-based assay is described in this communication. Further characterization of a selected compound, 3-fluoro-5-(2-methylbenzo[d]thiazol-5-yl)benzonitrile, in additional cell based assays as well as in vitro assays designed to measure its metabolic stability and protein binding indicated its potential utility as an in vivo tool. Subsequent evaluation of the same compound in a pharmacokinetic study using intraperitoneal dosing in mice showed good exposure in both plasma and brain samples. The compound was efficacious in a mouse marble burying model of anxiety, an assay known to be sensitive to mGlu5 antagonists. A new operant model of addiction termed operant sensation seeking (OSS) was chosen as a second behavioral assay. The compound also proved efficacious in the OSS model and constitutes the first reported example of efficacy with a small molecule mGlu5 NAM in this novel assay.

This Letter describes a chemical lead optimization campaign directed at VU0108370, a weak M1 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 M1 PAM with an in vitro profile comparable to the prototypical M1 PAM, BQCA, but with an improved brain to plasma ratio.

Herein we report the discovery, synthesis and evaluation of a series of N-(4-acetamido)-phenylpicolinamides as positive allosteric modulators of mGlu4.a Compounds from the series show submicromolar potency at both human and rat mGlu4. In addition, pharmacokinetic studies utilizing subcutaneous dosing demonstrated good brain exposure in rats.

This Letter describes the synthesis and SAR of a series of analogs of the mGlu5 partial antagonist 5-(phenylethynyl)pyrimidine. New molecular switches are identified that modulate the pharmacological activity of the lead compound. Slight structural modifications around the proximal pyrimidine ring change activity of the partial antagonist lead to that of potent and selective full negative allosteric modulators and positive allosteric modulators, that demonstrate in vivo efficacy in rodent models for anxiolytic activity and antipsychotic, respectively.

Cholinergic transmission in the forebrain is mediated primarily by five subtypes of muscarinic acetylcholine receptors (mAChRs), termed M1-M5. Of the mAChR subtypes, M1 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 M1 and other individual mAChR subtypes, which has prevented detailed studies of the functional roles of selective activation of M1. Using a functional HTS screen and subsequent diversity-oriented synthesis approach we have discovered a novel series of highly selective M1 allosteric agonists. These compounds activate M1 with EC50 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 M1 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 M1 allosteric agonists provides critical research tools to allow dissection of M1-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.

Development of SAR in a 3-cyano-5-fluoro-N-arylbenzamide series of non-competitive antagonists of mGlu5 using a functional cell-based assay is described in this letter. Further characterization of selected potent compounds in in vitro assays designed to measure their metabolic stability and protein binding is also presented. Subsequent evaluation of two new compounds in pharmacokinetic studies using intraperitoneal dosing in rats demonstrated good exposure in both plasma and brain samples.

Cholinergic transmission in the forebrain is mediated primarily by five subtypes of muscarinic acetylcholine receptors (mAChRs), termed M1−M5. Of the mAChR subtypes, M1 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 M1 and other individual mAChR subtypes, which has prevented detailed studies of the functional roles of selective activation of M1. Using a functional high-throughput screening and subsequent diversity-oriented synthesis approach, we have discovered a novel series of highly selective M1 allosteric agonists. These compounds activate M1 with EC50 values in the 150−500 nM range and have unprecedented, clean ancillary pharmacology (no substantial activity at 10 μM across a large panel of targets). Targeted mutagenesis revealed a potentially novel allosteric binding site in the third extracellular loop of the M1 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 M1 allosteric agonists provides critical research tools to allow dissection of M1-mediated effects in the CNS and potential leads for novel treatments for Alzheimer’s disease and schizophrenia.

[18F]fallypride PET studies can be used to estimate the non-displaceable binding potential (BPND) in vivo of dopamine D2/D3 receptor-rich regions of the brain. These studies often take considerable time, up to two or more hours, limiting the throughput. In this work, we investigated whether limited-duration scans performed subsequent to tracer administration yielded stable BPND estimates. In particular, we applied a modified version of the Logan plot method on the last 60 min of 120 min data and compared the results to those from analysis of the full data set.

Methods

Fourteen male Sprague-Dawley rats were injected with [18F]fallypride intravenously while under isoflurane anesthesia and dynamic data were acquired on the microPET Focus 220 for 120 min. The distribution volume ratio (DVR = BPND + 1) was calculated from a Logan plot using 120 min of data and from a modified version using only the last 60 min. Three of these rats were imaged again on a second day to test the reproducibility. A two-tissue compartment model also was used to fit the time activity curves (TACs) of the 120 min scans to estimate the parameters K1, k2, kon, k4, and Bmax. These parameters then were used to simulate similar TACs while changing kon to reflect changes in the dopaminergic system. The simulated TACs were used as a means for exploring the differences in DVR estimates between the last 60 min only and the full 120 min of simulated data.

Results

The average DVR from the full 120 min scans was 13.8 ± 0.9 whereas the average distribution volume ratio estimated from only the last 60 min of data (DVR′) was 16.3 ± 1.0. The distribution volume ratio estimates showed good reproducibility in the three rats (mean DVR = 13.8 ± 1.5 on Day 1 and DVR = 13.8 ± 0.9 on Day 2). The simulations showed that the relationship between DVR′ and DVR estimates follows a semi-linear form with varying kon.

Conclusion

Although the BPND estimates are slightly overestimated in a delayed scan mode (i.e. no initial radiotracer uptake measurements) compared to a full scan, this overestimation depends primarily on k3 (≈ kon × Bmax) and has been evaluated in this work for a wide range of kon values using simulated TACs. In particular, the sensitivity of DVR′ to changes in kon is similar to that of DVR. This method of delayed scans eliminates the necessity of imaging during the initial uptake of the radiotracer and, thus, can be used to increase the throughput of studies.

In recent years, the metabotropic glutamate (mGlu) receptors have emerged as potential new drug targets for treatment of a range of CNS disorders. Some of the most compelling advances have been made in targeting specific mGlu receptor subtypes as a fundamentally new approach to the treatment of schizophrenia. Recent animal and clinical studies provide strong evidence that agonists of group II mGlu receptors (mGluR2 and mGluR3) are effective in the treatment of the positive symptoms of schizophrenia, and animal studies suggest that mGluR5 agonists could provide a novel approach for the treatment of all major symptom domains (positive, negative, and cognitive) of this disorder. Although the discovery of selective agonists of these receptors is a challenge, there have been recent advances in the discovery of highly selective positive allosteric modulators (PAMs) of mGluR2 and mGluR5. These mGlu receptor-selective PAMs have properties needed for optimization as clinical candidates and have robust effects in animal models that predict efficacy in treatment of schizophrenia.

Muscarinic acetylcholine receptors (mAChRs) have long been viewed as viable targets for novel therapeutic agents for the treatment of Alzheimer’s disease (AD) and other disorders involving impaired cognitive function. More recent evidence indicates that mAChR activators might also have utility in treating psychosis and other symptoms associated with schizophrenia and other central nervous system (CNS) disorders. Efforts to develop mAChR subtype-selective agonists have been hampered by difficulty in achieving high selectivity for individual mAChR subtypes important for CNS function (M1 and M4) and adverse effects due to activation of peripheral mAChRs (especially M2 and M3). Major advances have now been achieved in the discovery of allosteric agonists and positive allosteric modulators of M1 and M4 that show greater selectivity for individual mAChR subtypes than do previous mAChR agonists. Early studies indicate that these allosteric mAChR activators have properties needed for optimization as potential clinical candidates and have robust effects in animal models that predict efficacy in the treatment of AD, schizophrenia and related disorders.

Parkinson's disease (PD) is caused by the death of dopamine neurons in the basal ganglia and results in motor symptoms such as tremor and bradykinesia. Activation of metabotropic glutamate receptor 4 (mGluR4) has been shown to modulate neurotransmission in the basal ganglia and results in antiparkinsonian effects in rodent PD models. PHCCC is a positive allosteric modulator (PAM) of mGluR4 which has been used to further validate the role of mGluR4 in PD, but the compound suffers from a lack of selectivity, relatively low potency and poor solubility. Via high-throughput screening, we discovered over 400 novel PAMs of mGluR4. Compounds derived from a novel chemical scaffold were characterized in vitro at both rat and human mGluR4 using two distinct assays of mGluR4 function. The lead compound was approximately 8-fold more potent than PHCCC, enhanced the potency of glutamate at mGluR4 by 8-fold, and did not show any significant potentiator or antagonist activity at other mGluR subtypes. Resolution of the regioisomers of the lead revealed that the cis regioisomer, VU0155041, contained the majority of the mGluR4 PAM activity and also exhibited partial agonist activity at mGluR4 at a site that was distinct from the glutamate binding site, suggesting that this compound is a mixed allosteric agonist/PAM of mGluR4. VU0155041 was soluble in an aqueous vehicle and intracerebroventricular administration of 31 to 316 nmol of VU0155041 dose-dependently decreased haloperidol-induced catalepsy and reserpine-induced akinesia in rats. These exciting results provide continued support for mGluR4 as a therapeutic target in PD.

Previous clinical and animal studies suggest that selective activators of M1 and/or M4 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 M1 or M4 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 M4 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 M4. VU0152099 and VU0152100 had no agonist activity but potentiated responses of M4 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 M4 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 M4 plays an important role in regulating midbrain dopaminergic activity and raises the possibility that positive allosteric modulation of M4 may mimic some of the antipsychotic-like effects of less selective mAChR agonists.

Recent studies suggest that subtype selective activators of M1/M4 muscarinic acetylcholine receptors (mAChRs) may offer a novel approach for the treatment of psychotic symptoms associated with schizophrenia and Alzheimer’s disease. Previously developed muscarinic agonists have provided clinical data in support of this hypothesis but failed in clinical development due to a lack of true subtype specificity and adverse effects associated with activation of other mAChR subtypes. We now report characterization of a novel highly selective agonist for the M1 receptor with no agonist activity on any of the other mAChR subtypes, termed TBPB. Mutagenesis and molecular pharmacology studies revealed that TBPB activates M1 through an allosteric site rather than the orthosteric ACh binding site, which is likely critical for this unprecedented selectivity. Whole cell patch clamp recordings demonstrated that activation of M1 by TBPB potentiates NMDA receptor currents in hippocampal pyramidal cells, but does not alter excitatory or inhibitory synaptic transmission, responses thought to be mediated by M2 and M4. TBPB was efficacious in models predictive of antipsychotic-like activity in rats at doses that did not produce catalepsy or peripheral adverse effects of other mAChR agonists. Finally, TBPB had effects on the processing of the amyloid precursor protein towards the non-amyloidogenic pathway and decreased Aβ production in vitro. Taken together, these data suggest that selective activation of M1 may provide a novel approach for the treatment of symptoms associated with schizophrenia and Alzheimer’s disease.

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 M1-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.

T-type Ca2+ channel inhibitors hold tremendous therapeutic potential for the treatment of pain, epilepsy, sleep disorders, essential tremor and other neurological disorders; however, a lack of truly selective tools has hindered basic research, and selective tools from the pharmaceutical industry are potentially burdened with intellectual property (IP) constraints. Thus, an MLPCN high-throughput screen (HTS) was conducted to identify novel T-type Ca2+ channel inhibitors free from IP constraints, and freely available through the MLPCN, for use by the biomedical community to study T-type Ca2+ channels. While the HTS provided numerous hits, these compounds could not be optimized to the required level of potency to be appropriate tool compounds. Therefore, a scaffold hopping approach, guided by SurflexSim, ultimately afforded ML218 (CID 45115620) a selective T-Type Ca2+ (Cav3.1, Cav3.2, Cav3.3) inhibitor (Cav3.2, IC50 = 150 nM in Ca2+ flux; Cav3.2 IC50 = 310 nM and Cav3.3 IC50 = 270 nM, respectively in patch clamp electrophysiology) with good DMPK properties, acceptable in vivo rat PK and excellent brain levels. Electrophysiology studies in subthalamic nucleus (STN) neurons demonstrated robust effects of ML218 on the inhibition of T-Type calcium current, inhibition of low threshold spike and rebound burst activity. Based on the basal ganglia circuitry in Parkinson’s disease (PD), the effects of ML218 in STN neurons suggest a therapeutic role for T-type Ca2+ channel inhibitors, and ML218 was found to be orally efficacious in haloperidol-induced catalepsy, a preclinical PD model, with comparable efficacy to an A2A antagonist, a clinically validated PD target. ML218 proves to be a powerful new probe to study T-Type Ca2+ function in vitro and in vivo, and freely available.