Brain-penetrant cyanoindane and cyanotetralin inhibitors of G2019S-LRRK2 kinase activity.

The G2019S variant of LRRK2, which causes an increase in kinase activity, is associated with the occurrence of Parkinson's disease (PD). Potent, mutation-selective, and brain penetrant inhibitors of LRRK2 can suppress the biological effects specific to G2019S-LRRK2 that cause pathogenicity. We report the discovery of a series of cyanoindane and cyanotetralin kinase inhibitors culminating in compound 34 that demonstrated selective inhibition of phosphorylation of LRRK2 in the mouse brain. These novel inhibitors may further enable the precision medicine path for future PD therapeutics.

Selective Inhibitors of G2019S-LRRK2 Kinase Activity.

Pathogenic variants in the leucine-rich repeat kinase 2 (LRRK2) gene have been identified that increase the risk for developing Parkinson's disease in a dominantly inherited fashion. These pathogenic variants, of which G2019S is the most common, cause abnormally high kinase activity, and compounds that inhibit this activity are being pursued as potentially disease-modifying therapeutics. Because LRRK2 regulates important cellular processes, developing inhibitors that can selectively target the pathogenic variant while sparing normal LRRK2 activity could offer potential advantages in heterozygous carriers. We conducted a high-throughput screen and identified a single selective compound that preferentially inhibited G2019S-LRRK2. Optimization of this scaffold led to a series of novel, potent, and highly selective G2019S-LRRK2 inhibitors.

GSK1614343, a novel ghrelin receptor antagonist, produces an unexpected increase of food intake and body weight in rodents and dogs.

Ghrelin is a 28-amino-acid polypeptide expressed in the stomach and hypothalamus that stimulates GH secretion, increases food intake (FI) and promotes body weight (BW) gain most likely via activation of the growth hormone secretagogue receptor type 1a (GHSR1a). GSK1614343 is a novel selective and potent GHSR antagonist with no partial agonist properties, recently characterized as GH secretion inhibitor by Sabbatini et al. [Chem Med Chem 2010;5:1450-1455]. In the present study, GSK1614343 (10 mg/kg) was not able to antagonize ghrelin-induced food consumption in rat, but unexpectedly stimulated FI and BW gain in both rats and dogs, a profile associated with decreased ghrelin plasma level. Interestingly, GSK1614343 selectively reduced the pro-opiomelanocortin mRNA levels in rat hypothalami chronically treated with the compound. To better understand the observed effects, we administered GSK1614343 (30 mg/kg) to Ghsr null mice and measured body mass components (fat, lean and free fluid) by using a NMR spectrometer. The increases of FI and BW were abolished in Ghsr null mice, while fat and lean masses increased in wild-type mice. Taken together, these results indicate that the orexigenic effect of GSK1614343 is mediated by GHSR1a and that the weight gain could be attributed to the increase of both adiposity and muscle mass, but not to fluid retention. The observed dissociation between effects on GH secretion and effects on FI/BW is inconsistent with a simple hormone-receptor model, suggesting unknown underlying regulations of the ghrelin system whose understanding require further investigation.

Pharmacological characterization of the ghrelin receptor antagonist, GSK1614343 in rat RC-4B/C cells natively expressing GHS type 1a receptors.

A novel growth hormone secretagogues type 1a (GHS1a) receptors antagonist (2R)-N'-[3,5- bis(trifluoromethyl)phenyl]-2-[(8aR)-hexahydropyrrolo[1,2-a]pyrazin-2(1H)-yl]-2-(3- pyridinyl)ethanohydrazide (GSK1614343) was functionally characterised in rat pituitary adenoma cell line, RC-4B/C endogenously expressing GHS1a receptors. The antagonism profile of GSK1614343 was compared with that of 6-[(4-fluorophenyl)oxy]-2-methyl-3-{[(3S)-1-(1-methylethyl)-3- piperidinyl]methyl}-4(3H)-quinazolinone (YIL-781) another ghrelin receptor antagonist recently published. The activity of both compounds was also evaluated at rat recombinant GHS1a receptors. The characterization of the two antagonists was performed by intracellular calcium mobilization measurements by using fluorometric imaging plate reader (FLIPR) technology and inositol phosphate (IP) turnover measurements by [3H]-IP accumulation assay. RC-4B/C and U2-OS cells transiently transduced with rat GHS1a receptors virus were used. In RC-4B/C cells, GSK1614343 and YIL-781, depressed the ghrelin maximal response in FLIPR assay as result of hemi-equilibria phenomenon. When using the [3H]-IP accumulation assay both compounds behaved as competitive antagonist with pKB values of 8.03 for GSK1614343 and 7.54 for YIL-781. In rat recombinant receptor, GSK1614343 and YIL-781 inhibited the calcium response induced by ghrelin with pIC50 values of 7.90 and 8.27, respectively. GSK1614343 and YIL-781 did not show intrinsic activity in both endogenously expressed and recombinant rat GHS1a receptors. The new ghrelin receptor antagonist GSK1614343 is a potent competitive antagonist in rat pituitary RC-4B/C cells endogenously expressing GHS1a receptors when equilibrium conditions between ligand and receptor are reached in the test assay. GSK1614343 represents a useful tool to investigate the physiological relevance of the ghrelin system in rat models.

Design, synthesis and SAR of a novel series of benzimidazoles as potent NPY Y5 antagonists.

A novel class of benzimidazole NPY Y5 receptor antagonists was prepared exploiting a privileged spirocarbamate moiety. The structure-activity relationship of this series and efforts to achieve a profile suitable for further development and an appropriate pharmacokinetic profile in rat are described. Optimisation led to the identification of the brain penetrant, orally bioavailable Y5 antagonist 9b which significantly inhibited the food intake induced by a Y5 selective agonist with a minimal effective dose of 30mg/kg po.

Synthesis and pharmacological characterization of constrained analogues of Vestipitant as in vitro potent and orally active NK(1) receptor antagonists.

A focused exploration targeting conformationally restricted analogues of Vestipitant, resulted in the discovery of novel, in vitro potent NK(1) antagonists. In particular, two of the compounds reported exhibited a good pharmacokinetic (PK) profile and produced anxiolytic-like effects in the gerbil foot tapping (GFT) in vivo model.

Synthesis and pharmacological characterization of novel druglike corticotropin-releasing factor 1 antagonists.

To identify new CRF(1) receptor antagonists, an attempt to modify the bis-heterocycle moiety present in the top region of the dihydropyrrole[2,3]pyridine template was made following new pharmacophoric hypothesis on the CRF(1) receptor antagonists binding pocket. In particular, the 2-thiazole ring, present in the previous series of compounds, was replaced by more hydrophilic non aromatic heterocycles able to make appropriate H-bond interactions with amino acid residues Thr192 and Tyr195. This exploration, followed by an accurate analysis of the substitution of the pendant aryl ring, enabled to identify in vitro potent compounds showing excellent pharmacokinetics and outstanding in vivo activity in animal models of anxiety, both in rodents and primates.

Dihydropyrrole[2,3-d]pyridine derivatives as novel corticotropin-releasing factor-1 antagonists: mapping of the receptor binding pocket by in silico docking studies.

In an effort to discover novel CRF-1 receptor antagonists exhibiting improved physicochemical properties, a dihydropirrole[2,3]pyridine scaffold was designed and explored in terms of the SAR of the substitution at the pendent phenyl ring and the nature of the heterocyclic moieties present in the upper region of the molecule. Selective and potent compounds have been discovered endowed with reduced ClogP with respect to compounds known in the literature. Of particular relevance was the finding that the in vitro affinity of the series was maintained by reducing the overall lipophilicity. The results achieved by this exploration enabled the formulation of a novel hypothesis on the nature of the receptor binding pocket of this class of CRF-1 receptor antagonists, making use of in silico docking studies of the putative nonpeptidic antagonist binding site set up in house by homology modeling techniques.

Novel substituted tetrahydrotriazaacenaphthylene derivatives as potent CRF1 receptor antagonists.

Corticotropin-releasing factor (CRF), a 41 amino acid peptide neurohormone synthesised by specific hypothalamic nuclei in the brain, is implicated in stress-related function. Antagonism of CRF(1) receptors is an attractive therapeutic approach for the treatment of depression and anxiety. Unsaturated tetrahydrotriazaacenaphthylenes of general structure 3 have been identified as potent and selective CRF(1) receptor antagonists with a suitable oral pharmacokinetic profile.

Cyclopenta[d]pyrimidines and dihydropyrrolo[2,3-d]pyrimidines as potent and selective corticotropin-releasing factor 1 receptor antagonists.

Two new classes of potent and selective CRF(1) receptor antagonists are presented. Exploration of general templates 3 and 4 through modifications of the top amine and bottom phenyl substituents led to optimization of the in vitro affinity and pharmacokinetic profiles. The typical alkyl chains present in the top region of CRF(1) antagonists were replaced by substituted heteroaryl moieties, leading to a dramatic improvement of the metabolic stability. This improvement was apparent when the compounds were dosed in vivo: several compounds exhibited low plasma clearance, good oral bioavailability, and high brain penetration. As a consequence of their outstanding pharmacokinetic profiles, these CRF(1) antagonists, as exemplified by compound 4 fi (4-(4-bromo-3-methyl-1H-pyrazol-1-yl)-7-(2,4-dichlorophenyl)-2-methyl-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidine), produced a dose-dependent "anxiolytic-like" effect when administered orally, decreasing the vocalization of rat pups.

From pyrroles to pyrrolo[1,2-a]pyrazinones: a new class of mGluR1 antagonists.

Exploiting the SAR of the known pyrrole derivatives, a new class of mGluR1 antagonists was developed through a cyclization of the C-2 position on the pyrrole N-1 nitrogen. The resulting pyrrolo[1,2-a]pyrazinones are potent and noncompetitive antagonists.

3-Methyl pyrrole-2,4-dicarboxylic acid 2-propyl ester 4-(1,2,2-trimethyl-propyl) ester: an exploration of the C-2 position. Part I.

Following the recent disclosure of 3-methyl pyrrole-2,4-dicarboxylic acid 2-propyl ester 4-(1,2,2-trimethyl-propyl) ester, a potent and selective mGluR1 non-competitive antagonist, we report here a detailed exploration of the C-2 position of this scaffold with the preparation of differently substituted amides. Great improvement of the pharmacokinetic properties has been achieved through this exercise.

3-Methyl pyrrole-2,4-dicarboxylic acid 2-propyl ester 4-(1,2,2-trimethyl-propyl) ester: an exploration of the C-2 position. Part II, A solid-phase approach.

Following the recent disclosure (Part I of this paper) of 3-methyl pyrrole-2,4-dicarboxylic acid 2-propyl ester 4-(1,2,2-trimethyl-propyl) amides and of their improved pharmacokinetic profile with respect to the originally reported esters, a further exploration of the C-2 position through a solid-phase approach is reported here.

Enantiomerically pure tetrahydroquinoline derivatives as in vivo potent antagonists of the glycine binding site associated to the NMDA receptor.

To identify neuroprotective agents after stroke, new substituted tetrahydroquinoline derivatives were designed as antagonists of the glycine binding site associated to the NMDA receptor, satisfying the key pharmacophoric requirements. In particular, the racemate 3c exhibited outstanding in vivo activity in the MCAo model in rats, when given iv both pre- and post-ischemia. Pure enantiomers 3c-(+) and 3c-(-) have been prepared following an original synthetic route. Despite the significant difference of activity observed in vitro, they shown similar neuroprotective profile in the MCAo model in rats.

Benzoazepine derivative as potent antagonists of the glycine binding site associated to the NMDA receptor.

A series of benzoazepine derivatives, bearing suitable substituents at the C-3 position, was designed and evaluated by superimposition with the pharmacophore model of the glycine binding site. To fully explore the SAR of this class of compounds and to allow the preparation of new different compounds at the C-3 position, appropriate synthetic routes were set up. The benzoazepines were evaluated in terms of in vitro affinity using [3H]glycine binding assay and in vivo potency by inhibition of convulsions induced by N-methyl-D-aspartate (NMDA) in mice. This further analysis confirmed the preliminary results previously reported and that compound 27 is the most promising compound (Ki=32 nM, ED(50)=0.09 mg/kg, i.v.) in this series. Significant neuroprotective effect was observed after both pre- and post-ischaemia administration in the MCAo model. In particular, after post-ischaemia administration, it was found to be still effective when the administration was delayed up to 6 h after occlusion of the middle cerebral artery.

2,4-Dicarboxy-pyrroles as selective non-competitive mGluR1 antagonists: an exploration of the role of the pyrrolic scaffold.

Following the disclosure of 3-(1,2,2-trimethylpropyl) 4-[3,5-dimethyl-2-propyloxycarbonyl]pyrrolecarboxylate as a potent and selective mGluR1 non-competitive antagonist, the role and the importance of the pyrrole template were investigated. Different aromatic moieties were investigated as possible bio-isosteric replacement of the original scaffold and some of them were shown to be partially able to mimic the properties of the original pyrrole ring.

2,4-Dicarboxy-pyrroles as selective non-competitive mGluR1 antagonists: further characterization of 3,5-dimethyl pyrrole-2,4-dicarboxylic acid 2-propyl ester 4-(1,2,2-trimethyl-propyl) ester and structure-activity relationships.

Following the disclosure of 3,5-dimethyl pyrrole-2,4-dicarboxylic acid 2-propyl ester 4-(1,2,2-trimethyl-propyl) ester [3,5-dimethyl PPP] as a potent and selective mGluR1 non-competitive antagonist, we report here further in vivo characterization of this important tool and disclose the investigation of the C-5 position, which led to very potent compounds.