Thaddeus F. Molski

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.

Ex vivo assessment of binding site occupancy of monoamine reuptake inhibitors: Methodology and biological significance

The goal of this study was to develop and validate ex vivo binding assays for serotonin (SERT), norepinephrine (NET) and dopamine (DAT) transporters, and to use these assays to evaluate the binding site occupancy of triple and double monoamine reuptake inhibitors in rat brains. This study demonstrated that while autoradiographic methods provided anatomic precision and regional resolution, the homogenate binding method for site occupancy assessment yielded comparable sensitivity with markedly improved throughput. For ex vivo binding assays, the reduction of temperature and time during the in vitro process (primarily incubation with a radioligand) markedly decreased the dissociation of test agents from binding sites in brain tissues. This reduction, in turn, minimized the potential for underestimation of site occupancy in vivo especially for test compounds with affinity >10nM. The ratios of measured occupancy ED(50) values (doses at which 50% occupancy occurs) among SERT, NET and DAT sites for duloxetine, venlafaxine, nomifensine, indatraline, DOV 21,947 and DOV 216,303 were consistent with the ratios of the in vitro affinities between these target binding sites. The biological relevance of the monoamine transporter occupancy for these compounds is discussed.

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.

Conformationally restricted homotryptamines. Part 5: 3-(trans-2-aminomethylcyclopentyl)indoles as potent selective serotonin reuptake inhibitors

A series of racemic 3-(trans-2-aminomethylcyclopentyl)indoles was synthesized and found to have potent binding to the human serotonin transporter (hSERT). The most active analog was synthesized stereospecifically and the active enantiomer was shown to have high affinity binding to hSERT.

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.

Neurochemical, pharmacokinetic, and behavioral effects of the novel selective serotonin reuptake inhibitor BMS-505130

BMS-505130 is a potent and selective serotonin transport inhibitor; K(i) for binding to the serotonin transporter = 0.18 nM (K(i) values for binding to the norepinephrine and dopamine transporters = 4.6 and 2.1 microM, respectively). In platelet serotonin uptake studies BMS-505130 (5 mg/kg, p.o.) produced a robust inhibition of serotonin uptake. In microdialysis studies oral dosing with BMS-505130 produced a dose-dependent increase in cortical serotonin levels that reached a maximal effect of 200% above baseline at a dose of 1 mg/kg, p.o.; the peak serotonin response was transient in nature. Following oral administration, peak plasma concentrations of BMS-505130 reached Tmax at 1.6 +/- 0.7 h and then declined to concentrations <10% of Cmax within the following 6 h; plasma half-life following i.v. dosing was 0.46 +/- 0.02 h. Parallel microdialysis and pharmacokinetic studies revealed that changes in serotonin levels in the cortex mirrored changes in the brain concentration of BMS-505130. In a behavioral assay known to be sensitive to selective serotonin reuptake inhibitors (SSRIs), mouse tail suspension, BMS-505130 produced a robust response after either oral or intraperitoneal dosing. BMS-505130 exhibits a pharmacological, neurochemical and behavioral profile consistent with a potent SSRI. Moreover, BMS-505130s short half-life may be advantageous for the treatment of premature ejaculation where an acute effect to delay ejaculation followed by a relatively rapid fall in SSRI plasma concentrations might be desirable.

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.

Discovery of D1 Dopamine Receptor Positive Allosteric Modulators: Characterization of Pharmacology and Identification of Residues that Regulate Species Selectivity

The present studies represent the first published report of a dopamine D1 positive allosteric modulator (PAM). D1 receptors have been proposed as a therapeutic target for the treatment of cognitive deficits associated with schizophrenia. However, the clinical utility of orthosteric agonist compounds is limited by cardiovascular side effects, poor pharmacokinetics, lack of D1 selectivity, and an inverted dose response. A number of these challenges may be overcome by utilization of a selective D1 PAM. The current studies describe two chemically distinct D1 PAMs: Compound A [1-((rel-1S,3R,6R)-6-(benzo[d][1,3]dioxol-5-yl)bicyclo[4.1.0]heptan-3-yl)-4-(2-bromo-5-chlorobenzyl)piperazine] and Compound B [rel-(9R,10R,12S)-N-(2,6-dichloro-3-methylphenyl)-12-methyl-9,10-dihydro-9,10-ethanoanthracene-12-carboxamide]. Compound A shows pure PAM activity, with an EC50 of 230 nM and agonist activity at the D2 receptor in D2-expressing human embryonic kidney cells. Compound B shows superior potency (EC50 of 43 nM) and selectivity for D1 versus D2 dopamine receptors. Unlike Compound A, Compound B is selective for human and nonhuman primate D1 receptors, but lacks activity at the rodent (rat and mouse) D1 receptors. Using molecular biology techniques, a single amino acid was identified at position 130, which mediates the species selectivity of Compound B. These data represent the first described D1-selective PAMs and define critical amino acids that regulate species selectivity.

Synthesis and evaluation of candidate PET radioligands for corticotropin-releasing factor type-1 receptors

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.

Inhibition of AAK1 Kinase as a Novel Therapeutic Approach to Treat Neuropathic Pain

To identify novel targets for neuropathic pain, 3097 mouse knockout lines were tested in acute and persistent pain behavior assays. One of the lines from this screen, which contained a null allele of the adapter protein-2 associated kinase 1 (AAK1) gene, had a normal response in acute pain assays (hot plate, phase I formalin), but a markedly reduced response to persistent pain in phase II formalin. AAK1 knockout mice also failed to develop tactile allodynia following the Chung procedure of spinal nerve ligation (SNL). Based on these findings, potent, small-molecule inhibitors of AAK1 were identified. Studies in mice showed that one such inhibitor, LP-935509, caused a reduced pain response in phase II formalin and reversed fully established pain behavior following the SNL procedure. Further studies showed that the inhibitor also reduced evoked pain responses in the rat chronic constriction injury (CCI) model and the rat streptozotocin model of diabetic peripheral neuropathy. Using a nonbrain-penetrant AAK1 inhibitor and local administration of an AAK1 inhibitor, the relevant pool of AAK1 for antineuropathic action was found to be in the spinal cord. Consistent with these results, AAK1 inhibitors dose-dependently reduced the increased spontaneous neural activity in the spinal cord caused by CCI and blocked the development of windup induced by repeated electrical stimulation of the paw. The mechanism of AAK1 antinociception was further investigated with inhibitors of α2 adrenergic and opioid receptors. These studies showed that α2 adrenergic receptor inhibitors, but not opioid receptor inhibitors, not only prevented AAK1 inhibitor antineuropathic action in behavioral assays, but also blocked the AAK1 inhibitor–induced reduction in spinal neural activity in the rat CCI model. Hence, AAK1 inhibitors are a novel therapeutic approach to neuropathic pain with activity in animal models that is mechanistically linked (behaviorally and electrophysiologically) to α2 adrenergic signaling, a pathway known to be antinociceptive in humans.