Indolyl and dihydroindolyl N-glycinamides as potent and in vivo active NPY5 antagonists.

A novel series of indolyl and dihydroindolyl glycinamides were identified as potent NPY5 antagonists with in vivo activity from screen hit 1. The dihydroindolyl glycinamide 10a significantly inhibits NPY5 agonist induced feeding at a dose of 0.1 mg/kg. The indolyl glycinamide 12c also inhibits NPY5 agonist induced feeding at a dose of 1 mg/kg. Both compounds 10a and 12c represent potential tools for further investigation into the biology of the NPY5 receptor.

5-(2'-Pyridyl)-2-aminothiazoles: alkyl amino sulfonamides and sulfamides as potent NPY(5) antagonists.

Synthesis, SAR and physico-chemical properties of an alkyl aminothiazole series 8 and 16 are described. 2-Pyridylaminothiazole based compounds such as 8c and 16a exhibit high affinity at the NPY(5) receptor with desirable cLogPs and solubilities. However, they also suffer from high in vitro and in vivo clearance. Compound 16a partially inhibits the feeding behavior elicited by i.c.v. injection of the selective NPY(5) agonist [cPP(1-7), NPY(19-23), Ala(31), Aib(32), Gln(34)]-human pancreatic polypeptide polypeptide (cPP).

Discovery of Lu AA33810: a highly selective and potent NPY5 antagonist with in vivo efficacy in a model of mood disorder.

The structure-activity relationship of a series of tricyclic-sulfonamide compounds 11-32 culminating in the discovery of N-[trans-4-(4,5-dihydro-3,6-dithia-1-aza-benzo[e]azulen-2-ylamino)-cyclohexylmethyl]-methanesulfonamide (15, Lu AA33810) is reported. Compound 15 was identified as a selective and high affinity NPY5 antagonist with good oral bioavailability in mice (42%) and rats (92%). Dose dependent inhibition of feeding was observed after i.c.v. injection of the selective NPY5 agonist ([cPP(1-7),NPY(19-23),Ala(31),Aib(32),Gln(34)]-hPP). In addition, ip administration of Lu AA33810 (10 mg/kg) produced antidepressant-like effects in a rat model of chronic mild stress.

N-Heteroaryl glycinamides and glycinamines as potent NPY5 antagonists.

Subtype specific ligands are needed to evaluate the therapeutic potential of modulating the brain's neuropeptide Y system. The benzothiazepine glycinamide 1a was identified as an NPY5 antagonist lead. While having acceptable solubility, the compound was found to suffer from high clearance and poor exposure. Optimization efforts are described targeting improvements in potency, microsomal stability, and PK properties. The low microsomal stability and poor PK properties were addressed through the optimization of the sulfonyl urea and replacement of the benzothiazepinone with other N-heteroaryl glycinamides. For example, the analogous benzoxazine glycinamide 2e has improvements in both affinity (human Y5 K(i) 4 nM vs 1a 27 nM) and microsomal stability (human CL(int) 2.5 L/min vs 1a 35L/min). However the brain penetration (B/P 43/430 nM at 10 mg/kg PO) remained an unresolved issue. Further optimization by decreasing the hydrogen bond donating properties and PSA provided potent and brain penetrant NPY5 antagonists such as 5f (human Y5 K(i) 9 nM, B/P 520/840 nM 10 mg/kg PO).

Maintenance of Y receptor dimers in epithelial cells depends on interaction with G-protein heterotrimers.

Treatment of CHO cells expressing human Y receptors (Y(1), Y(2) or Y4 subtype) with pertussis toxin results in a large decrease in functional receptors, with a preferential loss of heteropentameric assemblies of receptor dimers and G-protein trimers. This occurs in parallel to inactivation of the nucleotide site of Gi α subunits, with a half period of about 4 h. The loss could be mainly due to proteolysis at the level of recycling/perinuclear endosomes, and of receptor completion in the ER, since it is reduced by co-treatment with ammonium chloride, an inhibitor of particulate proteinases. Antagonists do not strongly decrease the heteropentameric fraction. These findings indicate that the upkeep of Y receptor dimers in epithelial cell lines depends on the association of receptor oligomers with functional Gi α subunits. This interaction could use the juxtamembrane helix 8 in the fourth intracellular domain, and could also be supported by the C-terminal helix of the third intracellular loop, as outlined in the companion review (Parker et al., Amino Acids, doi: 10.1007/s00726-010-0616-1 , 2010).

The novel neuropeptide Y Y5 receptor antagonist Lu AA33810 [N-[[trans-4-[(4,5-dihydro[1]benzothiepino[5,4-d]thiazol-2-yl)amino]cyclohexyl]methyl]-methanesulfonamide] exerts anxiolytic- and antidepressant-like effects in rat models of stress sensitivity.

Neuropeptide Y (NPY) regulates physiological processes via receptor subtypes (Y(1), Y(2), Y(4), Y(5), and y(6)). The Y(5) receptor is well known for its role in appetite. Based on expression in the limbic system, we hypothesized that the Y(5) receptor might also modulate stress sensitivity. We identified a novel Y(5) receptor-selective antagonist, Lu AA33810 [N-[[trans-4-[(4,5-dihydro[1]-benzothiepino[5,4-d]thiazol-2-yl)amino]cyclohexyl]methyl]-methanesulfonamide], that bound to cloned rat Y(5) receptors (K(i) = 1.5 nM) and antagonized NPY-evoked cAMP and calcium mobilization in vitro. Lu AA33810 (3-30 mg/kg p.o.) blocked feeding elicited by intracerebroventricular injection of the Y(5) receptor-selective agonist [cPP(1-7),NPY(19-23),Ala(31),Aib(32),Gln(34)]-hPancreatic Polypeptide in Sprague-Dawley rats. In vivo effects of Lu AA33810 were correlated with brain exposure > or = 50 ng/g and ex vivo Y(5) receptor occupancy of 22 to 95%. Lu AA33810 was subsequently evaluated in models of stress sensitivity. In Fischer 344 rats, Lu AA33810 (30 mg/kg p.o.) attenuated increases in plasma ACTH and corticosterone elicited by intracerebroventricular injection of [cPP(1-7),NPY(19-23),Ala(31),Aib(32),Gln(34)]-hPancreatic Polypeptide. In Sprague-Dawley rats subjected to the social interaction test, Lu AA33810 (3-30 mg/kg p.o.) produced anxiolytic-like effects after acute or chronic treatment. In Flinders sensitive line rats, chronic dosing of Lu AA33810 (10 mg/kg/day i.p.) produced anxiolytic-like effects in the social interaction test, plus antidepressant-like effects in the forced swim test. In Wistar rats exposed to chronic mild stress, chronic dosing of Lu AA33810 (3 and 10 mg/kg/day i.p.) produced antidepressant-like activity, i.e., normalization of stress-induced decrease in sucrose consumption. We propose that Y(5) receptors may function as part of an endogenous stress-sensing system to mediate social anxiety and reward or motivational deficits in selected rodent models.

3-arylimino-2-indolones are potent and selective galanin GAL3 receptor antagonists.

A series of 3-imino-2-indolones are the first published, high-affinity antagonists of the galanin GAL3 receptor. One example, 1,3-dihydro-1-phenyl-3-[[3-(trifluoromethyl)phenyl]imino]-2H-indol-2-one (9), was shown to have high affinity for the human GAL3 receptor (Ki=17 nM) and to be highly selective for GAL3 over a broad panel of targets, including GAL1 and GAL2. Compound 9 was also shown to be an antagonist in a human GAL3 receptor functional assay (Kb=29 nM).

Amino substituted analogs of 1-phenyl-3-phenylimino-2-indolones with potent galanin Gal3 receptor binding affinity and improved solubility.

A series of amino analogs of 1,3-dihydro-1-phenyl-3-[[3-(trifluoromethyl)phenyl]imino]-2H-indol-2-one (1) were synthesized to improve aqueous solubility, while retaining high affinity for the human galanin Gal3 receptor. A very potent analog (9e, 1,3-dihydro-1-[3-(2-pyrrolidinylethoxy)phenyl]-3-[[3-(trifluoromethyl)phenyl]imino]-2H-indol-2-one, Ki=5 nM) shows good selectivity and solubility of 48 microg/mL at pH 7.4.

Anxiolytic- and antidepressant-like profiles of the galanin-3 receptor (Gal3) antagonists SNAP 37889 and SNAP 398299.

The neuropeptide galanin mediates its effects through the receptor subtypes Gal(1), Gal(2), and Gal(3) and has been implicated in anxiety- and depression-related behaviors. Nevertheless, the receptor subtypes relevant to these behaviors are not known because of the lack of available galanin-selective ligands. In this article, we use behavioral, neurochemical, and electrophysiological approaches to investigate the anxiolytic- and antidepressant-like effects of two potent small-molecule, Gal(3)-selective antagonists, SNAP 37889 and the more soluble analog SNAP 398299. Acute administration of SNAP 37889 or SNAP 398299 enhanced rat social interaction. Furthermore, acute SNAP 37889 was also shown to reduce guinea pig vocalizations after maternal separation, to attenuate stress-induced hyperthermia in mice, to increase punished drinking in rats, and to decrease immobility and increase swimming time during forced swim tests with rats. Moreover, SNAP 37889 increased the social interaction time after 14 days of treatment and maintained its antidepressant effects during forced swim tests with rats after 21 days of treatment. In microdialysis studies, SNAP 37889 partially antagonized the galanin-evoked reduction in hippocampal serotonin (5-hydroxytryptamine, 5-HT), as did the 5-HT(1A) receptor antagonist WAY100635. Their combination produced a complete reversal of the effect of galanin. SNAP 398299 partially reversed the galanin-evoked inhibition of dorsal raphe cell firing and galanin-evoked hyperpolarizing currents. These results indicate that Gal(3)-selective antagonists produce anxiolytic- and antidepressant-like effects, possibly by attenuating the inhibitory influence of galanin on 5-HT transmission at the level of the dorsal raphe nucleus.

Use of Caenorhabditis elegans G{alpha}q chimeras to detect G-protein-coupled receptor signals.

G-protein-coupled receptors (GPCRs) activate heterotrimeric G-proteins (G(i)-, G(s)-, G(q)-, or G(12)-like) to generate specific intracellular responses, depending on the receptor/G-protein coupling. The aim was to enable a majority of GPCRs to generate a predetermined output by signaling through a single G-protein-supported pathway. The authors focused on calcium responses as the output, then engineered Galpha(q) to promote promiscuous receptor interactions. Starting with a human Galpha(q) containing 5 Galpha(z) residues in the C-terminal receptor recognition domain (hGalpha(q/z5)), they evaluated agonist-stimulated calcium responses for 33 diverse GPCRs (G(i)-, G(s)-, and G(q)-coupled) and found 20 of 33 responders. In parallel, they tested Caenorhabditis elegans Galpha(q) containing 5 or 9 C-terminal Galpha(z) residues (cGalpha(q/z5), cGalpha(q/z9)). Signal detection was enhanced with cGalpha(q/z5) and cGalpha(q/z9) (yielding 25/33 and 26/33 responders, respectively). In a separate study of Galpha(s)-coupled receptors, the authors compared hGalpha(q/s5) versus hGalpha(q/s9), cGalpha(q/s9), andcGalphaq/s21 and observed optimal function with cGalpha(q/s9). Cotransfection of an engineered Galpha(q) "cocktail" (cGalpha(q/z5) plus cGalpha(q/s9)) provided a powerful and efficient screening platform. When the chimeras included N-terminal myristoylation sites (to promote membrane localization), calcium responses were sustained or improved, depending on the receptor. This approach toward a "universal functional assay" is particularly useful for orphan GPCRs whose signaling pathways are unknown.

Discovery of potent and selective small molecule NPY Y5 receptor antagonists.

The discovery of a new class of sulfonamide NPY Y5 receptor antagonists is described. Optimization of this series led to the identification of compounds with high affinity for the hY5 subtype and excellent selectivity over the other NPY receptor subtypes. The SAR for this series was examined and a model for understanding the ligand-receptor interactions was developed.

High-throughput synthesis optimization of sulfonamide NPY Y5 antagonists.

A series of sulfonamide neuropeptide Y Y5 antagonists was optimized by preparation of sets of analogues using high-throughput synthesis and purification techniques. Testing of these compounds for their ability to bind to the human NPY Y5 receptor revealed separate SAR trends for sulfonamide amides versus sulfonamide ureas versus sulfonamide amines. By understanding these SAR trends, potent compounds were identified in all three series.