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This publication details the discovery of a series of selective transient receptor potential cation channel subfamily M member 5 (TRPM5) agonists culminating with the identification of the lead compound (1R, 3R)-1-(3-chloro-5-fluorophenyl)-3-(hydroxymethyl)-1,2,3,4-tetrahydroisoquinoline-6-carbonitrile (39). We describe herein our biological rationale for agonism of the target, the examination of the then current literature tool molecules, and finally the process of our discovery starting with a high throughput screening hit through lead development. We also detail the selectivity of the lead compound 39 versus related family members TRPA1, TRPV1, TRPV4, TRPM4 and TRPM8, the drug metabolism and pharmacokinetics (DMPK) profile and in vivo efficacy in a mouse model of gastrointestinal motility., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: The authors are current or former employees of Takeda Pharmaceutical Company or contracted and funded (by Takeda) in the case of Aptuit (now an Evotec Company). All research described in this manuscript was funded solely by Takeda Pharmaceutical Company., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

A stepwise chemical modification from human neurokinin-3 receptor (hNK-3R)-selective antagonists to potent and combined hNK-3R and hNK-2R antagonists using the same 2-phenylquinoline template is described. Docking studies with 3-D models of the hNK-3 and hNK-2 receptors were used to drive the chemical design and speed up the identification of potent and combined antagonsits at both receptors. (S)-(+)-N-(1-Cyclohexylethyl)-3-[(4-morpholin-4-yl)piperidin-1-yl]methyl-2-phenylquinoline-4-carboxamide (compound 25, SB-400238: hNK-3R binding affinity, K(i) = 0.8 nM; hNK-2R binding affinity, K(i) = 0.8 nM) emerged as the best example in this approach. Further studies led to the identification of (S)-(+)-N-(1,2,2-trimethylpropyl)-3-[(4-piperidin-1-yl)piperidin-1-yl]methyl-2-phenylquinoline-4-carboxamide (compound 28, SB-414240: hNK-3R binding affinity, K(i) = 193 nM; hNK-2R binding affinity, K(i) = 1.0 nM) as the first hNK-2R-selective antagonist belonging to the 2-phenylquinoline chemical class. Since some members of this chemical series showed a significant binding affinity for the human mu-opioid receptor (hMOR), docking studies were also conducted on a 3-D model of the hMOR, resulting in the identification of a viable chemical strategy to avoid any significant micro-opioid component. Compounds 25 and 28 are therefore suitable pharmacological tools in the tachykinin area to elucidate further the pathophysiological role of NK-3 and NK-2 receptors and the therapeutic potential of selective NK-2 (28) or combined NK-3 and NK-2 (25) receptor antagonists.

Several non-peptidic opioids have been synthesized recently as part of a program to develop selective delta receptor agonists. In this study, the affinities of a set of compounds for cloned delta and mu opioid receptors expressed in HEK 293 cell lines were determined by competition analysis of [3H]bremazocine binding to membrane preparations. All compounds studied exhibited high affinity and selectivity, with apparent dissociation constants in the range of 0.6-1.7 nM for the delta opioid receptor and 240-1165 nM for the mu opioid receptor. We next sought to determine which domain of the delta receptor was critical for mediating the highly selective binding by analysis of ligand affinities for mu/delta receptor chimeras. Receptor binding profiles suggested that a critical site of receptor/ligand interaction was located between transmembrane domain 5 (TM5) and TM7 of the delta receptor. Substitution of tryptophan 284, located at the extracellular surface of TM6, with lysine, which is found at the equivalent position in the mu opioid receptor, led to a spectrum of effects on affinities, depending on the ligand tested. Affinities of SB 219825 and SB 222941 were particularly sensitive to the substitution, displaying a 50-fold and 70-fold decrease in affinity, respectively. Activities of the delta receptor-selective agonists were tested in two functional assays. Brief exposure of HEK 293 cells expressing delta opioid receptors with selective ligands induced phosphorylation of MAP kinase, although the non-peptidic ligands were less efficacious than the enkephalin derivative DADL (Tyr-D-Ala-Gly-Phe-D-Leu). Similarly, chronic exposure of HEK 293 cells expressing delta opioid receptors with selective, non-peptidic ligands, with the exception of SB 206848, caused receptor down-regulation, however, the SB compounds were less efficacious than DADL.

[formula: see text] Synthesis of four novel thieno derivatives 4-7 featuring the codeine skeletal backbone is reported. Characterization by 1H and 13C NMR is also discussed, along with binding profile for opioid receptors.

Results from a medicinal chemistry approach aimed at replacing the quinoline ring system in the potent and selective human neurokinin-3 (hNK-3) receptor antagonists 1-4 of general formula I are discussed. The data give further insight upon the potential NK-3 pharmacophore. In particular, it is highlighted that both the benzene-condensed ring and the quinoline nitrogen are crucial determinants for optimal binding affinity to the hNK-3 receptor. Some novel compounds maintained part of the binding affinity to the receptor (5, 6, 10 and 13) and compound 5, featuring the naphthalene ring system, appears to be suitable for further modifications; it offers the option to introduce electron-withdrawing groups at position 2 and 4, conferring on the ring an overall electron-deficiency similar to that of the quinoline.

Optimization of the previously reported 2-phenyl-4-quinolinecarboxamide NK-3 receptor antagonist 14, with regard to potential metabolic instability of the ester moiety and affinity and selectivity for the human neurokinin-3 (hNK-3) receptor, is described. The ester functionality could be successfully replaced by the ketone (31) or by lower alkyl groups (Et, 21, or n-Pr, 24). Investigation of the substitution pattern of the quinoline ring resulted in the identification of position 3 as a key position to enhance hNK-3 binding affinity and selectivity for the hNK-3 versus the hNK-2 receptor. All of the chemical groups introduced at this position, with the exception of halogens, increased the hNK-3 binding affinity, and compounds 53 (3-OH, SB 223412, hNK-3-CHO binding Ki = 1.4 nM) and 55 (3-NH2, hNK-3-CHO binding Ki = 1.2 nM) were the most potent compounds of this series. Selectivity studies versus the other neurokinin receptors (hNK-2-CHO and hNK-1-CHO) revealed that 53 is about 100-fold selective for the hNK-3 versus hNK-2 receptor, with no affinity for the hNK-1 at concentrations up to 100 microM. In vitro studies demonstrated that 53 is a potent functional antagonist of the hNK-3 receptor (reversal of senktide-induced contractions in rabbit isolated iris sphincter muscles and reversal of NKB-induced Ca2+ mobilization in CHO cells stably expressing the hNK-3 receptor), while in vivo this compound showed oral and intravenous activity in NK-3 receptor-driven models (senktide-induced behavioral responses in mice and senktide-induced miosis in rabbits). Overall, the biological data indicate that (S)-N-(1-phenylpropyl)-3-hydroxy-2-phenylquinoline-4-carboxamide (53, SB 223412) may serve as a pharmacological tool in animal models of disease to assess the functional and pathophysiological role of the NK-3 receptor and to establish therapeutic indications for non-peptide NK-3 receptor antagonists.

A novel class of potent and selective non-peptide neurokinin-3 (NK-3) receptor antagonists, featuring the 4-quinolinecarboxamide framework, has been designed based upon chemically diverse NK-1 receptor antagonists. The novel compounds 33-76, prompted by chemical modifications of the prototype 4, have been characterized by binding analysis using a membrane preparation of chinese hamster ovary (CHO) cells expressing the human neurokinin-3 receptors (hNK-3-CHO), and clear structure-activity relationships (SARs) have been established. From SARs, (R)-N-[alpha-(methoxycarbonyl)benzyl]-2-phenylquinoline-4-carboxamide (65, SB 218795, hNK-3-CHO binding Ki = 13 nM) emerged as one of the most potent compounds of this novel class. Selectivity studies versus the other neurokinin receptors (hNK-2-CHO and hNK-1-CHO) revealed that 65 is about 90-fold selective for hNK-3 versus hNK-2 receptors (hNK-2-CHO binding Ki = 1221 nM) and over 7000-fold selective versus hNK-1 receptors (hNK-1-CHO binding Ki = > 100 microM). In vitro functional studies in rabbit isolated iris sphincter muscle preparation demonstrated that 65 is a competitive antagonist of the contractile response induced by the potent and selective NK-3 receptor agonist senktide with a Kb = 43 nM. Overall, the data indicate that 65 is a potent and selective hNK-3 receptor antagonist and a useful lead for further chemical optimization.

We previously demonstrated that [3H]beta-funaltrexamine ([3H]beta-FNA) labeled the rat mu opioid receptor expressed in Chinese hamster ovary cells with high specificity, and [3H]beta-FNA-labeled receptors migrated as one broad band with a mass of 80 kDa. In this study, we determined the region and then the amino acid residue of the mu receptor involved in the covalent binding of [3H]beta-FNA. [3H]beta-FNA-labeled receptors were solubilized and purified to approximately 10% purity by immunoaffinity chromatography with antibodies against a C-terminal domain peptide. The site of covalent bond formation was determined to be within Ala206-Met243 by CNBr cleavage of partially purified labeled mu receptors and determinations of sizes of labeled receptor fragments. The amino acid residue of beta-FNA covalent incorporation was then determined by site-directed mutagenesis studies within this region. Mutation of Lys233 to Ala, Arg, His, and Leu completely eliminated covalent binding of [3H]beta-FNA, although these mutants bound beta-FNA with high affinity. Mutations of other amino acid residues did not affect covalent binding of [3H]beta-FNA. These results indicate that [3H]beta-FNA binds covalently to Lys233. Since [3H]beta-FNA is a rigid molecule, the information will be very useful for molecular modeling of interaction between morphinans and the mu receptor.