Discovery of 2-(Anilino)pyrimidine-4-carboxamides as Highly Potent, Selective, and Orally Active Glycogen Synthase Kinase-3 (GSK-3) Inhibitors.

Glycogen synthase kinase-3 (GSK-3) is a serine/threonine kinase that serves as an important regulator of a broad range of cellular functions. It has been linked to Alzheimer's disease as well as various other diseases, including mood disorders, type 2 diabetes, and cancer. There is considerable evidence indicating that GSK-3ß in the central nervous system plays a role in the production of abnormal, hyperphosphorylated, microtubule-associated tau protein found in neurofibrillary tangles associated with Alzheimer's disease. A series of analogues containing a pyrimidine-based hinge-binding heterocycle was synthesized and evaluated, leading to the identification of highly potent GSK-3 inhibitors with excellent kinase selectivity. Further evaluation of 34 and 40 in vivo demonstrated that these compounds are orally bioavailable, brain-penetrant GSK-3 inhibitors that lowered levels of phosphorylated tau in a triple-transgenic mouse Alzheimer's disease model.

Design, Structure-Activity Relationships, and In Vivo Evaluation of Potent and Brain-Penetrant Imidazo[1,2-b]pyridazines as Glycogen Synthase Kinase-3ß (GSK-3ß) Inhibitors.

Glycogen synthase kinase-3 (GSK-3) is a serine/threonine kinase that regulates numerous cellular processes, including metabolism, proliferation, and cell survival. Due to its multifaceted role, GSK-3 has been implicated in a variety of diseases, including Alzheimer's disease, type 2 diabetes, cancer, and mood disorders. GSK-3ß has been linked to the formation of the neurofibrillary tangles associated with Alzheimer's disease that arise from the hyperphosphorylation of tau protein. The design and synthesis of a series of imidazo[1,2-b]pyridazine derivatives that were evaluated as GSK-3ß inhibitors are described herein. Structure-activity relationship studies led to the identification of potent GSK-3ß inhibitors. In vivo studies with 47 in a triple-transgenic mouse Alzheimer's disease model showed that this compound is a brain-penetrant, orally bioavailable GSK-3ß inhibitor that significantly lowered levels of phosphorylated tau.

Bicyclic Heterocyclic Replacement of an Aryl Amide Leading to Potent and Kinase-Selective Adaptor Protein 2-Associated Kinase 1 Inhibitors.

Adaptor protein 2-associated kinase 1 (AAK1) is a serine/threonine kinase that was identified as a therapeutic target for the potential treatment of neuropathic pain. Inhibition of AAK1 in the central nervous system, particularly within the spinal cord, was found to be the relevant site for achieving an antinociceptive effect. We previously reported that compound 7 is a brain-penetrant, AAK1 inhibitor that showed efficacy in animal models for neuropathic pain. One approach we took to improve upon the potency of 7 involved tying the amide back into the neighboring phenyl ring to form a bicyclic heterocycle. Investigation of the structure-activity relationships (SARs) of substituents on the resultant quinazoline and quinoline ring systems led to the identification of (S)-31, a brain-penetrant, AAK1-selective inhibitor with improved enzyme and cellular potency compared to 7. The synthesis, SAR, and in vivo evaluation of a series of quinazoline and quinoline-based AAK1 inhibitors are described herein.

Discovery, Structure-Activity Relationships, and In Vivo Evaluation of Novel Aryl Amides as Brain Penetrant Adaptor Protein 2-Associated Kinase 1 (AAK1) Inhibitors for the Treatment of Neuropathic Pain.

Effective treatment of chronic pain, in particular neuropathic pain, without the side effects that often accompany currently available treatment options is an area of significant unmet medical need. A phenotypic screen of mouse gene knockouts led to the discovery that adaptor protein 2-associated kinase 1 (AAK1) is a potential therapeutic target for neuropathic pain. The synthesis and optimization of structure-activity relationships of a series of aryl amide-based AAK1 inhibitors led to the identification of 59, a brain penetrant, AAK1-selective inhibitor that proved to be a valuable tool compound. Compound 59 was evaluated in mice for the inhibition of µ2 phosphorylation. Studies conducted with 59 in pain models demonstrated that this compound was efficacious in the phase II formalin model for persistent pain and the chronic-constriction-injury-induced model for neuropathic pain in rats. These results suggest that AAK1 inhibition is a promising approach for the treatment of neuropathic pain.

Synthesis and evaluation of prodrugs of corticotropin-releasing factor-1 (CRF1) receptor antagonist BMS-665053 leading to improved oral bioavailability.

A series of phosphate and ester-based prodrugs of anilinopyrazinone 1 (BMS-665053) containing either a methylene or an (acyloxy)alkoxy linker was prepared and evaluated in rat pharmacokinetic studies with the goal of improving the oral bioavailability of the parent (1). The prodrugs, in general, had improved aqueous solubility and oral bioavailability compared to 1. Prodrug 12, which contains an (acyloxy)alkoxy linker, showed the greatest improvement in the oral bioavailability relative to the parent (1), with a seven-fold increase (from 5% to 36%) in rat pharmacokinetic studies.

Synthesis and evaluation of carbamate and aryl ether substituted pyrazinones as corticotropin releasing factor-1 (CRF1) receptor antagonists.

A series of pyrazinone-based compounds incorporating either carbamate or aryl ether groups was synthesized and evaluated as corticotropin-releasing factor-1 (CRF1) receptor antagonists. Structure-activity relationship studies led to the identification of highly potent CRF1 receptor antagonists 14a (IC50=0.74 nM) and 14b (IC50=1.9 nM). The synthesis, structure-activity relationships and in vitro metabolic stability properties of compounds in this series will be described.

Potential CRF1R PET imaging agents: 1-fluoroalkylsubstituted 5-halo-3-(arylamino)pyrazin-2(1H)-ones.

A series of pyrazinones were prepared and evaluated as potential CRF(1)R PET imaging agents. Optimization of their CRF(1)R binding potencies and octanol-phosphate buffer phase distribution coefficients are discussed herein.

[18F](R)-5-chloro-1-(1-cyclopropyl-2-methoxyethyl)-3-(4-(2-fluoroethoxy)-2,5-dimethyl phenylamino)pyrazin-2(1H)-one: introduction of N3-phenylpyrazinones as potential CRF-R1 PET imaging agents.

Based on a favorable balance between CRF-R1 affinity, lipophilicity and metabolic stability, compound 10 was evaluated for potential development as PET radioligand. Compound [(18)F]10 was prepared with high radiochemical purity and showed promising binding properties in rat brain imaging experiments.

Chiral separation of potent corticotropin-releasing factor-1 receptor antagonists by supercritical fluid chromatography.

Pyrazinones bearing an N-1-alkyl chain with a chiral center have been reported as potent antagonists of the corticotropin-releasing factor-1 receptor (CRF1R). Separation of individual enantiomers for preclinical testing was an important aspect of lead optimization. To evaluate the applicability and efficiency of supercritical fluid chromatography (SFC) for enantiomeric resolution of this class of compounds, enantiomeric pairs of eight pyrazinones with different structural characteristics were tested under an array of SFC conditions. The results showed that pyrazinones with a 1-cyclopropyl-2-methoxyethyl substituent were readily separated with a Chiralpak AD-H or Chiralcel OD-H column with ethanol as the modifier. On the other hand, analogs with a less polar alkyl substituent were not amenable to the general method and required further optimization of the chromatographic conditions. In addition, structural variations on the pyrazinone core and aromatic moiety had an impact on the chiral resolution of this class of compounds. This investigation led to the development of efficient chiral SFC methods for separating all eight pyrazinone enantiomeric pairs encompassing an array of structural variations.

Synthesis and structure-activity relationships of N3-pyridylpyrazinones as corticotropin-releasing factor-1 (CRF1) receptor antagonists.

A series of N(3)-pyridylpyrazinones was investigated as corticotropin-releasing factor-1 receptor antagonists. It was observed that the binding affinity of analogues containing a pyridyl group was influenced not only by the substitution pattern on the pyridyl group, but also by the pK(a) of the pyridyl nitrogen. Analogues containing a novel 6-(difluoromethoxy)-2,5-dimethylpyridin-3-amine group were among the most potent N(3)-pyridylpyrazinones synthesized. The synthesis and SAR of N(3)-pyridylpyrazinones is described herein.

Comparative biotransformation of pyrazinone-containing corticotropin-releasing factor receptor-1 antagonists: minimizing the reactive metabolite formation.

(S)-5-Chloro-1-(1-cyclopropylethyl)-3-(2,6-dichloro-4-(trifluoromethyl)phenylamino)pyrazin-2(1H)-one (BMS-665053), a pyrazinone-containing compound, is a potent and selective antagonist of corticotropin-releasing factor receptor-1 (CRF-R1) that showed efficacy in the defensive withdrawal model for anxiety in rats, suggesting its use as a potential treatment for anxiety and depression. In vitro metabolism studies of BMS-665053 in rat and human liver microsomes revealed cytochrome P450-mediated oxidation of the pyrazinone moiety, followed by ring opening, as the primary metabolic pathway. Detection of a series of GSH adducts in trapping experiments suggested the formation of a reactive intermediate, probably as a result of epoxidation of the pyrazinone moiety. In addition, BMS-665053 (20 mg/kg i.v.) underwent extensive metabolism in bile duct-cannulated (BDC) rats. The major drug-related materials in rat plasma were the pyrazinone oxidation products. In rat bile and urine (0-7 h), only a trace amount of the parent drug was recovered, whereas significant levels of the pyrazinone epoxide-derived metabolites and GSH-related conjugates were detected. Further evidence suggested that GSH-related conjugates also formed at the dichloroarylamine moiety possibly via an epoxide or a quinone imine intermediate. Other major metabolites in BDC rat bile and urine included glucuronide conjugates. To reduce potential liability due to metabolic activation of BMS-665053, a number of pyrazinone analogs with different substituents were synthesized and investigated for reactive metabolite formation, leading to the discovery of a CRF-R1 antagonist with diminished in vitro metabolic activation.

A strategy to minimize reactive metabolite formation: discovery of (S)-4-(1-cyclopropyl-2-methoxyethyl)-6-[6-(difluoromethoxy)-2,5-dimethylpyridin-3-ylamino]-5-oxo-4,5-dihydropyrazine-2-carbonitrile as a potent, orally bioavailable corticotropin-releasing factor-1 receptor antagonist.

Detailed metabolic characterization of 8, an earlier lead pyrazinone-based corticotropin-releasing factor-1 (CRF(1)) receptor antagonist, revealed that this compound formed significant levels of reactive metabolites, as measured by in vivo and in vitro biotransformation studies. This was of particular concern due to the body of evidence suggesting that reactive metabolites may be involved in idiosyncratic drug reactions. Further optimization of the structure-activity relationships and in vivo properties of pyrazinone-based CRF(1) receptor antagonists and studies to assess the formation of reactive metabolites led to the discovery of 19e, a high affinity CRF(1) receptor antagonist (IC(50) = 0.86 nM) wherein GSH adducts were estimated to be only 0.1% of the total amount of drug-related material excreted through bile and urine, indicating low levels of reactive metabolite formation in vivo. A novel 6-(difluoromethoxy)-2,5-dimethylpyridin-3-amine group in 19e contributed to the potency and improved in vivo properties of this compound and related analogues. 19e had excellent pharmacokinetic properties in rats and dogs and showed efficacy in the defensive withdrawal model of anxiety in rats. The lowest efficacious dose was 1.8 mg/kg. The results of a two-week rat safety study with 19e indicated that this compound was well-tolerated.

In vitro intrinsic clearance-based optimization of N3-phenylpyrazinones as corticotropin-releasing factor-1 (CRF1) receptor antagonists.

A series of pyrazinone-based heterocycles was identified as potent and orally active corticotropin-releasing factor-1 (CRF(1)) receptor antagonists. Selected compounds proved efficacious in an anxiety model in rats; however, pharmacokinetic properties were not optimal. In this article, we describe an in vitro intrinsic clearance-based approach to the optimization of pyrazinone-based CRF(1) receptor antagonists wherein sites of metabolism were identified by incubation with human liver microsomes. It was found that the rate of metabolism could be decreased by incorporation of appropriate substituents at the primary sites of metabolism. This led to the discovery of compound 12x, a highly potent (IC(50) = 1.0 nM) and selective CRF(1) receptor antagonist with good oral bioavailability (F = 52%) in rats and efficacy in the defensive withdrawal anxiety test in rats.

Synthesis, structure-activity relationships, and in vivo evaluation of N3-phenylpyrazinones as novel corticotropin-releasing factor-1 (CRF1) receptor antagonists.

Evidence suggests that corticotropin-releasing factor-1 (CRF(1)) receptor antagonists may offer therapeutic potential for the treatment of diseases associated with elevated levels of CRF such as anxiety and depression. A pyrazinone-based chemotype of CRF(1) receptor antagonists was discovered. Structure-activity relationship studies led to the identification of numerous potent analogues including 12p, a highly potent and selective CRF(1) receptor antagonist with an IC(50) value of 0.26 nM. The pharmacokinetic properties of 12p were assessed in rats and Cynomolgus monkeys. Compound 12p was efficacious in the defensive withdrawal test (an animal model of anxiety) in rats. The synthesis, structure-activity relationships and in vivo properties of compounds within the pyrazinone chemotype are described.

Design, synthesis, and biological evaluation of 1,2,3,7-tetrahydro-6h-purin-6-one and 3,7-dihydro-1h-purine-2,6-dione derivatives as corticotropin-releasing factor(1) receptor antagonists.

A growing body of evidence suggests that CRF(1) receptor antagonism offers considerable therapeutic potential in the treatment of diseases resulting from elevated levels of CRF, such as anxiety and depression. A series of novel 1,2,3,7-tetrahydro-6H-purin-6-one and 3,7-dihydro-1H-purine-2,6-dione derivatives was synthesized and evaluated as corticotropin releasing factor-1 (CRF(1)) receptor antagonists. Compounds within this series, represented by compound 12d (IC(50) = 5.4 nM), were found to be highly potent CRF(1) receptor antagonists. In addition, compounds 12d and 12j were determined to be selective CRF(1) antagonists. The synthesis, structure-activity relationships and pharmacokinetic properties of compounds within this series is presented.