Synthesis and SAR of a novel Kir6.2/SUR1 channel opener scaffold identified by HTS.

Kir6.2/SUR1 is an ATP-regulated potassium channel that acts as an intracellular metabolic sensor, controlling insulin and appetite-stimulatory neuropeptides secretion. In this Letter, we present the SAR around a novel Kir6.2/SUR1 channel opener scaffold derived from an HTS screening campaign. New series of compounds with tractable SAR trends and favorable potencies are reported.

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

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

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

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

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

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

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

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

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

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

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

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

Discovery of a novel 2,3-dimethylimidazo[1,2-a]pyrazine-6-carboxamide M4 positive allosteric modulator (PAM) chemotype.

This Letter details our efforts to discover structurally unique M4 PAMs containing 5,6-heteroaryl ring systems. In an attempt to improve the DMPK profiles of the 2,3-dimethyl-2H-indazole-5-carboxamide and 1-methyl-1H-benzo[d][1,2,3]triazole-6-carboxamide cores, we investigated a plethora of core replacements. This exercise identified a novel 2,3-dimethylimidazo[1,2-a]pyrazine-6-carboxamide core that provided improved M4 PAM activity and CNS penetration.

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

This Letter details our efforts to develop new M4 PAM scaffolds with improved pharmacological properties. This endeavor involved replacing the 3,4-dimethylpyridazine core with two novel cores: a 2,3-dimethyl-2H-indazole-5-carboxamide core or a 1-methyl-1H-benzo[d][1,2,3]triazole-6-carboxamide core. Due to shallow SAR, these cores were further evolved into two unique tricyclic cores: an 8,9-dimethyl-8H-pyrazolo[3,4-h]quinazoline core and an 1-methyl-1H-[1,2,3]triazolo[4,5-h]quinazoline core. Both tricyclic cores displayed low nanomolar potency against both human and rat M4.

Evaluation of 1,2,3-Triazoles as Amide Bioisosteres In Cystic Fibrosis Transmembrane Conductance Regulator Modulators VX-770 and VX-809.

The 1,2,3-triazole has been successfully utilized as an amide bioisostere in multiple therapeutic contexts. Based on this precedent, triazole analogues derived from VX-809 and VX-770, prominent amide-containing modulators of the cystic fibrosis transmembrane conductance regulator (CFTR), were synthesized and evaluated for CFTR modulation. Triazole 11, derived from VX-809, displayed markedly reduced efficacy in F508del-CFTR correction in cellular TECC assays in comparison to VX-809. Surprisingly, triazole analogues derived from potentiator VX-770 displayed no potentiation of F508del, G551D, or WT-CFTR in cellular Ussing chamber assays. However, patch clamp analysis revealed that triazole 60 potentiates WT-CFTR similarly to VX-770. The efficacy of 60 in the cell-free patch clamp experiment suggests that the loss of activity in the cellular assay could be due to the inability of VX-770 triazole derivatives to reach the CFTR binding site. Moreover, in addition to the negative impact on biological activity, triazoles in both structural classes displayed decreased metabolic stability in human microsomes relative to the analogous amides. In contrast to the many studies that demonstrate the advantages of using the 1,2,3-triazole, these findings highlight the negative impacts that can arise from replacement of the amide with the triazole and suggest that caution is warranted when considering use of the 1,2,3-triazole as an amide bioisostere.

Discovery of a novel 3,4-dimethylcinnoline carboxamide M4 positive allosteric modulator (PAM) chemotype via scaffold hopping.

This Letter details our efforts to replace the 2,4-dimethylquinoline carboxamide core of our previous M4 PAM series, which suffered from high predicted hepatic clearance and protein binding. A scaffold hopping exercise identified a novel 3,4-dimethylcinnoline carboxamide core that provided good M4 PAM activity and improved clearance and protein binding profiles.

SAR inspired by aldehyde oxidase (AO) metabolism: Discovery of novel, CNS penetrant tricyclic M4 PAMs.

This letter describes progress towards an M4 PAM preclinical candidate inspired by an unexpected aldehyde oxidase (AO) metabolite of a novel, CNS penetrant thieno[2,3-c]pyridine core to an equipotent, non-CNS penetrant thieno[2,3-c]pyrdin-7(6H)-one core. Medicinal chemistry design efforts yielded two novel tricyclic cores that enhanced M4 PAM potency, regained CNS penetration, displayed favorable DMPK properties and afforded robust in vivo efficacy in reversing amphetamine-induced hyperlocomotion in rats.

Discovery of a novel, CNS penetrant M4 PAM chemotype based on a 6-fluoro-4-(piperidin-1-yl)quinoline-3-carbonitrile core.

This Letter details the discovery and subsequent optimization of a novel M4 PAM scaffold based on an 6-fluoro-4-(piperidin-1-yl)quinoline-3-carbonitrile core, which represents a distinct departure from the classical M4 PAM chemotypes. Optimized compounds in this series demonstrated improved M4 PAM potency on both human and rat M4 (4 to 5-fold relative to HTS hit), and displayed attractive physicochemical and DMPK profiles, including good CNS penetration (rat brain:plasma Kp=5.3, Kp,uu=2.4; MDCK-MDR1 (P-gp) ER=1.1).

Discovery of a novel 2,4-dimethylquinoline-6-carboxamide M4 positive allosteric modulator (PAM) chemotype via scaffold hopping.

This Letter details our efforts to replace the 3-amino moiety, an essential pharmacophore for M4 PAM activity in most M4 PAMs to date, within the thieno[2,3-b]pyridine core, as the ß-amino carboxamide motif has been shown to engender poor solubility, varying degrees of P-gp efflux and represents a structural alert. A scaffold hopping exercise identified a novel 2,4-dimethylquinoline carboxamide core that provided M4 PAM activity and good CNS penetration without an amino moiety. In addition, MacMillan photoredox catalysis chemistry was essential for construction of the 2,4-dimethylquinoline core.

Discovery and optimization of 3-(4-aryl/heteroarylsulfonyl)piperazin-1-yl)-6-(piperidin-1-yl)pyridazines as novel, CNS penetrant pan-muscarinic antagonists.

This letter describes the synthesis and structure activity relationship (SAR) studies of structurally novel M4 antagonists, based on a 3-(4-aryl/heteroarylsulfonyl)piperazin-1-yl)-6-(piperidin-1-yl)pyridazine core, identified from a high-throughput screening campaign. A multi-dimensional optimization effort enhanced potency at human M4 (hM4 IC50s<200nM), with only moderate species differences noted, and with enantioselective inhibition. Moreover, CNS penetration proved attractive for this series (rat brain:plasma Kp=2.1, Kp,uu=1.1). Despite the absence of the prototypical mAChR antagonist basic or quaternary amine moiety, this series displayed pan-muscarinic antagonist activity across M1-5 (with 9- to 16-fold functional selectivity at best). This series further expands the chemical diversity of mAChR antagonists.

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.

Further optimization of the M1 PAM VU0453595: Discovery of novel heterobicyclic core motifs with improved CNS penetration.

This Letter describes the continued chemical optimization of the VU0453595 series of M1 positive allosteric modulators (PAMs). By surveying alternative 5,6- and 6,6-heterobicylic cores for the 6,7-dihydro-5H-pyrrolo[3,4-b]pyridine-5-one core of VU453595, we found new cores that engendered not only comparable or improved M1 PAM potency, but significantly improved CNS distribution (Kps 0.3-3.1). Moreover, this campaign provided fundamentally distinct M1 PAM chemotypes, greatly expanding the available structural diversity for this valuable CNS target, devoid of hydrogen-bond donors.