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GPR84 was first identified as an open reading frame encoding an orphan Class A G protein coupled receptor in 2001. Gpr84 mRNA is expressed in a limited number of cell types with the highest levels of expression being in innate immune cells, M1 polarised macrophages and neutrophils. The first reported ligands for this receptor were medium chain fatty acids with chain lengths between 9 and 12 carbons. Subsequently, a series of synthetic agonists that signal via the GPR84 receptor were identified. Radioligand binding assays and molecular modelling with site‐directed mutagenesis suggest the presence of three ligand binding sites on the receptor, but the physiological agonist(s) of the receptor remain unidentified. Here, we review the effects of GPR84 agonists on innate immune cells following a series of chemical discoveries since 2001. The development of highly biased agonists has helped to probe receptor function in vitro, and the remaining challenge is to follow the effects of biased signalling to the physiological functions of innate immune cell types. LINKED ARTICLES: This article is part of a themed issue GPR84 Pharmacology. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v181.10/issuetoc [ABSTRACT FROM AUTHOR]

GPR84 is an orphan G-protein coupled receptor (GPCR) linked to inflammation. Strategies targeting GPR84 to prevent excessive inflammation in disease are hampered by a lack of understanding of its precise functional role. We have developed heterologous cell lines with low GPR84 expression levels that phenocopy the response of primary cells in a label-free cell electrical impedance (CEI) sensing system that measures cell morphology and adhesion. We then investigated the signalling profile and membrane localisation of GPR84 upon treatment with 6-OAU and DL-175, two agonists known to differentially influence immune cell function. When compared to 6-OAU, DL-175 was found to exhibit a delayed impedance response, a delayed and suppressed activation of Akt, which together correlated with an impaired ability to internalise GPR84 from the plasma membrane. The signalling differences were transient and occurred only at early time points in the low expressing cell lines, highlighting the importance of receptor number and kinetic readouts when evaluating signalling bias. Our findings open new ways to understand GPR84 signalling and evaluate the effect of newly developed agonists. [Display omitted] [ABSTRACT FROM AUTHOR]

GPR84 is an inflammation-induced receptor highly expressed on immune cells, yet its endogenous ligand is still unknown. This makes any interpretation of its physiological activity in vivo difficult. However, experiments with potent synthetic agonists have highlighted what the receptor can do, namely, enhance proinflammatory signaling and macrophage effector functions such as phagocytosis. Developing drugs to block these effects has attracted interest from the scientific community with the aim of decreasing disease activity in inflammatory disorders or enhancing inflammation resolution. In this review, we critically reassess the widely held belief that the major role of GPR84 is that of being a medium-chain fatty acid (MCFA) receptor. While MCFAs have been shown to activate GPR84, it remains to be demonstrated that they are present in relevant tissues at appropriate concentrations. In contrast to four other "full-time" free fatty acid receptor subtypes, GPR84 is not expressed by enteroendocrine cells and has limited expression in the gastrointestinal tract. Across multiple tissues and cell types, the highest expression levels of GPR84 are observed hours after exposure to an inflammatory stimulus. These factors obscure the relationship between ligand and receptor in the human body and do not support the exclusive physiological pairing of MCFAs with GPR84. To maximize the chances of developing efficacious drugs for inflammatory diseases, we must advance our understanding of GPR84 and what it does in vivo. [ABSTRACT FROM AUTHOR]

This letter describes the synthesis and structure activity relationship (SAR) studies of structurally novel M 4 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 M 4 (hM 4 IC 50 s < 200 nM), with only moderate species differences noted, and with enantioselective inhibition. Moreover, CNS penetration proved attractive for this series (rat brain:plasma K p = 2.1, K p,uu = 1.1). Despite the absence of the prototypical mAChR antagonist basic or quaternary amine moiety, this series displayed pan-muscarinic antagonist activity across M 1-5 (with 9- to 16-fold functional selectivity at best). This series further expands the chemical diversity of mAChR antagonists. [ABSTRACT FROM AUTHOR]

This letter describes the synthesis and structure activity relationship (SAR) studies of structurally novel M 4 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 M 4 (IC 50 s < 300 nM), with no substantial species differences noted. Moreover, CNS penetration proved attractive for this series (brain:plasma K p,uu = 0.87), while other DMPK attributes were addressed in the course of the optimization effort, providing low in vivo clearance in rat (CL p = 5.37 mL/min/kg). Surprisingly, this series displayed pan -muscarinic antagonist activity across M 1–5 , 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. [ABSTRACT FROM AUTHOR]

[Display omitted] This Letter describes the synthesis and optimization of a series of heteroaryl-pyrrolidinone positive allosteric modulators (PAMs) of the muscarinic acetylcholine receptor M 1 (mAChR M 1). Through the continued optimization of M 1 PAM tool compound VU0453595, with a focus on replacement of the 6,7-dihydro-5 H -pyrrolo[3,4- b ]pyridin-5-one with a wide variety of alternative 4,5-dihydropyrrolo-fused heteroaromatics, the generation of M 1 PAMs with structurally novel chemotypes is disclosed. Two compounds from these subseries, 8b (VU6005610) and 20a (VU6005852), show robust selectivity for the M 1 mAChR, and no M 1 agonism. Both compounds have favorable preliminary PK profiles in vitro; 8b additionally demonstrates high brain exposure in a rodent IV cassette model. [ABSTRACT FROM AUTHOR]

This letter describes progress towards an M 4 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(6 H)-one core. Medicinal chemistry design efforts yielded two novel tricyclic cores that enhanced M 4 PAM potency, regained CNS penetration, displayed favorable DMPK properties and afforded robust in vivo efficacy in reversing amphetamine-induced hyperlocomotion in rats. [ABSTRACT FROM AUTHOR]

• First disclosure of VU6005806/AZN-00016130, an M 4 PAM profiled as a putative candidate. • Novel SAR findings based on novel variations to the C3 position on the pyridazine core. • Minimum effective dose of 0.56 mg/kg po in rat AHL. This letter describes progress towards an M 4 PAM preclinical candidate that resulted in the discovery of VU6005806/AZN-00016130. While the thieno[2,3- c ]pyridazine core has been a consistent feature of key M 4 PAMs, no work had previously been reported with respect to alternate functionality at the C3 position of the pyridazine ring. Here, we detail new chemistry and analogs that explored this region, and quickly led to VU6005806/AZN-00016130, which was profiled as a putative candidate. While, the β-amino carboxamide moiety engendered solubility limited absorption in higher species precluding advancement (or requiring extensive pharmaceutical sciences formulation), VU6005806/AZN-00016130 represents a new, high quality preclinical in vivo probe. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • First description of the role of the β-aminocarboxamide moiety within M 4 PAM scaffolds. • Novel des -amino and tricyclic variant M 4 PAMs. • The role of the IMHB motif was established, and could be re-enforced by tricycles. Abstract This letter describes a focused exercise to explore the role of the β-amino carboxamide moiety found in all of the first generation M 4 PAMs and question if the NH 2 group served solely to stabilize an intramolecular hydrogen bond (IMHB) and enforce planarity. To address this issue (and to potentially find a substitute for the β-amino carboxamide that engendered P-gp and contributed to solubility liabilities), we removed the NH 2 , generating des -amino congeners and surveyed other functional groups in the β-position. These modifications led to weak M 4 PAMs with poor DMPK properties. Cyclization of the β-amino carboxamide moiety by virtue of a pyrazole ring re-enforced the IMHB, led to potent (and patented) M 4 PAMs, many as potent as the classical bicyclic β-amino carboxamide analogs, but with significant CYP1A2 inhibition. Overall, this exercise indicated that the β-amino carboxamide moiety most likely facilitates an IMHB, and is essential for M 4 PAM activity within classical bicyclic M 4 PAM scaffolds. [ABSTRACT FROM AUTHOR]

Herein we describe the continued optimization of M 4 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. [ABSTRACT FROM AUTHOR]

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

This letter describes the chemical optimization of a novel series of M 4 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). [ABSTRACT FROM AUTHOR]

This Letter details our efforts to replace the 3-amino moiety, an essential pharmacophore for M 4 PAM activity in most M 4 PAMs to date, within the thieno[2,3- b ]pyridine core, as the β-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 M 4 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. [ABSTRACT FROM AUTHOR]

[Display omitted] This Letter details our efforts to develop novel tricyclic M 4 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 M 4 receptor. [ABSTRACT FROM AUTHOR]

• Discovery of novel 2,3-dimethylimidazo[1,2- a ]pyrazine-6-carboxamide-based M 4 PAMs. • Broad survey of diverse heterocyclic cores. • Balanced human and rat M 4 PAM potency with excellent CNS penetration. This Letter details our efforts to develop new M 4 PAM scaffolds with improved pharmacological properties. This endeavor involved replacing the 3,4-dimethylpyridazine core with two novel cores: a 2,3-dimethyl-2 H -indazole-5-carboxamide core or a 1-methyl-1 H -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-1 H -[1,2,3]triazolo[4,5- h ]quinazoline core. Both tricyclic cores displayed low nanomolar potency against both human and rat M 4. [ABSTRACT FROM AUTHOR]

• Discovery of two novel tricyclic-based M 4 PAMs. • Utility of scaffold hopping to improve potency/properties/DMPK. • Balanced human and rat M 4 PAM potency. • Rare 8,9-dimethyl-8 H -pyrazolo[3,4- h ]quinazoline and 1-methyl-1 H -[1,2,3]triazolo[4,5- h ]quinazoline cores. This Letter details our efforts to discover structurally unique M 4 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-1 H -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 M 4 PAM activity and CNS penetration. [ABSTRACT FROM AUTHOR]

• Novel 2.4-dimehtylcinnoline-based M 4 PAMs. • Utility of scaffold hopping to improve properties/DMPK. • Balanced human and rat M 4 PAM potency. This Letter details our efforts to replace the 2,4-dimethylquinoline carboxamide core of our previous M 4 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 M 4 PAM activity and improved clearance and protein binding profiles. [ABSTRACT FROM AUTHOR]

Preclinical evidence in support of the potential utility of mGlu5 NAMs for the treatment of a variety of psychiatric and neurodegenerative disorders is extensive, and multiple such molecules have entered clinical trials. Despite some promising results from clinical studies, no small molecule mGlu5 NAM has yet to reach market. Here we present the discovery and evaluation of N-(5-fluoropyridin-2-yl)-6-methyl-4-(pyrimidin-5-yloxy)picolinamide (27, VU0424238), a compound selected for clinical evaluation. Compound 27 is more than 900-fold selective for mGlu5 versus the other mGlu receptors, and binding studies established a Ki value of 4.4 nM at a known allosteric binding site. Compound 27 had a clearance of 19.3 and 15.5 mL/min/kg in rats and cynomolgus monkeys, respectively. Imaging studies using a known mGlu5 PET ligand demonstrated 50% receptor occupancy at an oral dose of 0.8 mg/kg in rats and an intravenous dose of 0.06 mg/kg in baboons. [ABSTRACT FROM AUTHOR]