Vijay T. Ahuja

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.

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.

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.

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.

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.

Design, synthesis, and evaluation of phenylglycinols and phenyl amines as agonists of GPR88.

Small molecule modulators of GPR88 activity (agonists, antagonists, or modulators) are of interest as potential agents for the treatment of a variety of psychiatric disorders including schizophrenia. A series of phenylglycinol and phenylamine analogs have been prepared and evaluated for their GPR88 agonist activity and pharmacokinetic (PK) properties.

[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 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.