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Introduction: A radioligand for measuring the density of corticotropin-releasing factor subtype-1 receptors (CRF1 receptors) in living animal and human brain with positron emission tomography (PET) would be a useful tool for neuropsychiatric investigations and the development of drugs intended to interact with this target. This study was aimed at discovery of such a radioligand from a group of CRF1 receptor ligands based on a core 3-(phenylamino)-pyrazin-2(1H)-one scaffold., Methods: CRF1 receptor ligands were selected for development as possible PET radioligands based on their binding potency at CRF1 receptors (displacement of [(125)I]CRF from rat cortical membranes), measured lipophilicity, autoradiographic binding profile in rat and rhesus monkey brain sections, rat biodistribution, and suitability for radiolabeling with carbon-11 or fluorine-18. Two identified candidates (BMS-721313 and BMS-732098) were labeled with fluorine-18. A third candidate (BMS-709460) was labeled with carbon-11 and all three radioligands were evaluated in PET experiments in rhesus monkey. CRF1 receptor density (Bmax) was assessed in rhesus brain cortical and cerebellum membranes with the CRF1 receptor ligand, [(3)H]BMS-728300., Results: The three ligands selected for development showed high binding affinity (IC50 values, 0.3-8nM) at CRF1 receptors and moderate lipophilicity (LogD, 2.8-4.4). [(3)H]BMS-728300 and the two (18)F-labeled ligands showed region-specific binding in rat and rhesus monkey brain autoradiography, namely higher binding density in the frontal and limbic cortex, and cerebellum than in thalamus and brainstem. CRF1 receptor Bmax in rhesus brain was found to be 50-120 fmol/mg protein across cortical regions and cerebellum. PET experiments in rhesus monkey showed that the radioligands [(18)F]BMS-721313, [(18)F]BMS-732098 and [(11)C]BMS-709460 gave acceptably high brain radioactivity uptake but no indication of the specific binding as seen in vitro., Conclusions: Candidate CRF1 receptor PET radioligands were identified but none proved to be effective for imaging monkey brain CRF1 receptors. Higher affinity radioligands are likely required for successful PET imaging of CRF1 receptors., (Copyright © 2014 Elsevier Inc. All rights reserved.)

BMS-763534 is a potent (CRF(1) IC(50) = 0.4 nM) and selective (>1000-fold selectivity vs. all other sites tested) CRF(1) receptor antagonist (pA2 = 9.47 vs. CRF(1)-mediated cAMP production in Y79 cells). BMS-763534 accelerated the dissociation of (125)I-o-CRF from rat frontal cortex membrane CRF(1) receptors consistent with a negative allosteric modulation of CRF binding. BMS-763534 produced dose-dependent increases in CRF(1) receptor occupancy and anxiolytic efficacy; lowest effective anxiolytic dose = 0.56 mg/kg, PO, which was associated with 71 ± 5% CRF(1) receptor occupancy of frontoparietal CRF(1) receptors. Sedative/ataxic effects of BMS-763534 were only observed at high dose multiples (54-179×) relative to the lowest dose required for anxiolytic efficacy. At doses of 5- to 18-fold higher than the lowest efficacious dose in the anxiety assay, BMS-763534 shared subjective effects with the benzodiazepine chlordiazepoxide. Interestingly BMS-790318, the O-demethylated metabolite of BMS-763534, showed weak affinity for the TBOB site of the GABA(A) receptor (67% inhibition at 10 μM) and augmented GABA evoked currents (EC(50) = 1.6 μM). Thus, the unanticipated signal in the drug discrimination assay may have resulted from an interaction of the metabolite BMS-790318 with the TBOB site on the GABA(A) channel where it appears to behave as an allosteric potentiator of GABA evoked currents., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

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.

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.

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.

Corticotropin releasing factor (CRF) is the primary regulator of the hypothalamus-pituitary-adrenal (HPA) axis, coordinating the endocrine, behavioral, and autonomic responses to stress. It has been postulated that small molecules that can antagonize the binding of CRF1 to its receptor may serve as a treatment for anxiety-related and/or affective disorders. Members within a series of 3,4-dihydro-1H-pyrido[2,3-b]pyrazin-2-ones, exemplified by compound 2 (IC50 = 0.70 nM), were found to be very potent antagonists of CRF1. Compound 8w showed high CRF1 receptor binding affinity and was examined further in vivo. The compound was efficacious in a defensive withdrawal model of anxiety in rats and had a long half-life and reasonable oral bioavailability in dog pharmacokinetic studies.