Discovery of Imidazo[1,2-a]pyrazines and Pyrazolo[1,5-c]pyrimidines as TARP γ-8 Selective AMPAR Negative Modulators.

This report discloses the discovery and characterization of imidazo[1,2-a]pyrazines and pyrazolo[1,5-c]pyrimidines as selective negative modulators of α-amino-3-hydroxy-5-methylisoxazole-4-propionate receptors (AMPARs) associated with transmembrane AMPAR regulatory protein γ-8. Imidazopyrazine 5 was initially identified as a promising γ-8 selective high-throughput screening hit, and subsequent structure-activity relationship optimization yielded subnanomolar, brain penetrant leads. Replacement of the imidazopyrazine core with an isosteric pyrazolopyrimidine scaffold improved microsomal stability and efflux liabilities to provide 26, JNJ-61432059. Following oral administration, 26 exhibited time- and dose-dependent AMPAR/γ-8 receptor occupancy in mouse hippocampus, which resulted in robust seizure protection in corneal kindling and pentylenetetrazole (PTZ) anticonvulsant models.

Lead Optimization of 5-Aryl Benzimidazolone- and Oxindole-Based AMPA Receptor Modulators Selective for TARP γ-8.

Glutamate mediates fast excitatory neurotransmission via ionotropic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. The trafficking and gating properties of AMPA receptors (AMPARs) can be amplified by transmembrane AMPAR regulatory proteins (TARPs), which are often expressed in localized brain regions. Herein, we describe the discovery, lead optimization, and preclinical characterization of 5-arylbenzimidazolone and oxindole-based negative modulators of AMPARs associated with TARP γ-8, the primary TARP found in hippocampus. High-throughput screen lead 4 was optimized for potency and brain penetration to provide benzimidazolone 3, JNJ-55511118.1 Replacement of the benzimidazolone core in 3 with an oxindole mitigated reactive metabolite formation and led to the identification of 18 (GluA1/γ-8 pIC50 = 9.7). Following oral dosing in rats, 18 demonstrated robust target engagement in hippocampus as assessed by ex vivo autoradiography (ED50 = 0.6 mg/kg, plasma EC50 = 9 ng/mL).

4-Methyl-6,7-dihydro-4H-triazolo[4,5-c]pyridine-Based P2X7 Receptor Antagonists: Optimization of Pharmacokinetic Properties Leading to the Identification of a Clinical Candidate.

The synthesis and preclinical characterization of novel 4-(R)-methyl-6,7-dihydro-4H-triazolo[4,5-c]pyridines that are potent and selective brain penetrant P2X7 antagonists are described. Optimization efforts based on previously disclosed unsubstituted 6,7-dihydro-4H-triazolo[4,5-c]pyridines, methyl substituted 5,6,7,8-tetrahydro[1,2,4]triazolo[4,3-a]pyrazines, and several other series lead to the identification of a series of 4-(R)-methyl-6,7-dihydro-4H-triazolo[4,5-c]pyridines that are selective P2X7 antagonists with potency at the rodent and human P2X7 ion channels. These novel P2X7 antagonists have suitable physicochemical properties, and several analogs have an excellent pharmacokinetic profile, good partitioning into the CNS and show robust in vivo target engagement after oral dosing. Improvements in metabolic stability led to the identification of JNJ-54175446 (14) as a candidate for clinical development. The drug discovery efforts and strategies that resulted in the identification of the clinical candidate are described herein.

Identification of (R)-(2-Chloro-3-(trifluoromethyl)phenyl)(1-(5-fluoropyridin-2-yl)-4-methyl-6,7-dihydro-1H-imidazo[4,5-c]pyridin-5(4H)-yl)methanone (JNJ 54166060), a Small Molecule Antagonist of the P2X7 receptor.

The synthesis and SAR of a series of 4,5,6,7-tetrahydro-imidazo[4,5-c]pyridine P2X7 antagonists are described. Addressing P2X7 affinity and liver microsomal stability issues encountered with this template afforded methyl substituted 4,5,6,7-tetrahydro-imidazo[4,5-c]pyridines ultimately leading to the identification of 1 (JNJ 54166060). 1 is a potent P2X7 antagonist with an ED50 = 2.3 mg/kg in rats, high oral bioavailability and low-moderate clearance in preclinical species, acceptable safety margins in rats, and a predicted human dose of 120 mg of QD. Additionally, 1 possesses a unique CYP profile and was found to be a regioselective inhibitor of midazolam CYP3A metabolism.

Novel benzamide-based histamine h3 receptor antagonists: the identification of two candidates for clinical development.

The preclinical characterization of novel phenyl(piperazin-1-yl)methanones that are histamine H3 receptor antagonists is described. The compounds described are high affinity histamine H3 antagonists. Optimization of the physical properties of these histamine H3 antagonists led to the discovery of several promising lead compounds, and extensive preclinical profiling aided in the identification of compounds with optimal duration of action for wake promoting activity. This led to the discovery of two development candidates for Phase I and Phase II clinical trials.

PET brain imaging of neuropeptide Y2 receptors using N-11C-methyl-JNJ-31020028 in pigs.

UNLABELLED: Neuropeptide Y2 (NPY2) receptors are implicated in diverse brain disorders, but no suitable PET radiotracers are currently available for studying NPY2 receptors in the living brain. We developed a novel positron-emitting radioligand based on the NPY2 receptor antagonist JNJ-31020028 (N-(4-(4-[2-(diethylamino)-2-oxo-1-phenylethyl]piperazin-1-yl)-3-fluorophenyl)-2-pyridin-3-ylbenzamide) and used the radiotracer for PET brain imaging in pigs. METHODS: In vitro receptor autoradiography studies were performed to establish the anatomic distribution of NPY2 receptors in the pig brain. In vivo, baseline 90-min PET recordings of N-(11)C-methyl-JNJ-31020028 were conducted in anesthetized Yorkshire x Landrace pigs, concurrent with arterial blood sampling. Postchallenge scans were conducted after injection of unlabeled JNJ-31020028 as a pharmacologic intervention. Cyclosporine A was used to enhance levels of the PET radiotracer in the brain. The PET images were manually coregistered to a MR imaging atlas of the pig brain. Maps of the N-(11)C-methyl-JNJ-31020028 volume of distribution in the brain were prepared, and regional binding potentials of NPY2 receptors toward the radioligand were calculated using the simplified reference tissue method. RESULTS: In autoradiography studies, N-(11)C-methyl-JNJ-31020028 receptor binding sites were observed primarily in the hippocampus and were inhibited by unlabeled JNJ-31020028. In PET studies, N-(11)C-methyl-JNJ-31020028 was metabolized slowly in the bloodstream, with 25% of the (11)C-labeled parent compound remaining 30 min after injection. PET imaging showed baseline binding potentials of 0.64 ± 0.07 in the thalamus, 0.55 ± 0.02 in the caudate, and 0.49 ± 0.03 in the hippocampus. Graphical reference region analyses demonstrated that N-(11)C-methyl-JNJ-31020028 binding was reversible; infusion of unlabeled JNJ-31020028 markedly displaced the PET radioligand from binding sites in the hippocampus, thalamus, caudate nucleus, and cerebellum but not in the corpus callosum, which served as reference region for nonspecific binding. CONCLUSION: N-(11)C-methyl-JNJ-31020028 has several suitable properties for PET neuroimaging of NPY2 receptors. First, it is metabolized slowly in the bloodstream of pigs. Second, using cyclosporine, the target-to-background ratio of N-(11)C-methyl-JNJ-31020028 is sufficient for estimating pharmacokinetic parameters. Third, N-(11)C-methyl-JNJ-31020028 binds reversibly and competitively to cerebral sites known to contain relatively high numbers of NPY2 receptors, such as the hippocampus, thalamus, caudate nucleus, and cerebellum. Fourth, white matter such as corpus callosum, known to contain negligible numbers of NPY2 receptors, can serve as a reference region for estimating binding potentials in brain regions. To our knowledge, there is no other radioligand with these favorable properties and with this specificity for NPY2 receptors, which makes N-(11)C-methyl-JNJ-31020028 the first candidate radioligand for PET investigations of NPY2 receptors in the living brain.

The discovery and synthesis of JNJ 31020028, a small molecule antagonist of the Neuropeptide Y Y2 receptor.

A series of small molecules based on a chemotype identified from our compound collection were synthesized and tested for binding affinity (IC(50)) at the human Neuropeptide Y Y(2) receptor (NPY Y(2)). Six of the 23 analogs tested possessed an NPY Y(2) IC(50) ≤ 15 nM. One member of this series, JNJ 31020028, is a selective, high affinity, receptor antagonist existing as a racemic mixture. As such a synthetic route to the desired enantiomer was designed starting from commercially available (S)-(+)-mandelic acid.

In vitro and in vivo characterization of JNJ-31020028 (N-(4-{4-[2-(diethylamino)-2-oxo-1-phenylethyl]piperazin-1-yl}-3-fluorophenyl)-2-pyridin-3-ylbenzamide), a selective brain penetrant small molecule antagonist of the neuropeptide Y Y(2) receptor.

RATIONALE: The lack of potent, selective, brain penetrant Y(2) receptor antagonists has hampered in vivo functional studies of this receptor. OBJECTIVE: Here, we report the in vitro and in vivo characterization of JNJ-31020028 (N-(4-{4-[2-(diethylamino)-2-oxo-1-phenylethyl]piperazin-1-yl}-3-fluorophenyl)-2-pyridin-3-ylbenzamide), a novel Y(2) receptor antagonist. METHODS: The affinity of JNJ-31020028 was determined by inhibition of the PYY binding to human Y(2) receptors in KAN-Ts cells and rat Y(2) receptors in rat hippocampus. The functional activity was determined by inhibition of PYY-stimulated calcium responses in KAN-Ts cells expressing a chimeric G protein Gqi5 and in the rat vas deferens (a prototypical Y(2) bioassay). Ex vivo receptor occupancy was revealed by receptor autoradiography. JNJ-31020028 was tested in vivo with microdialysis, in anxiety models, and on corticosterone release. RESULTS: JNJ-31020028 bound with high affinity (pIC(50) = 8.07 +/- 0.05, human, and pIC(50) = 8.22 +/- 0.06, rat) and was >100-fold selective versus human Y(1), Y(4), and Y(5) receptors. JNJ-31020028 was demonstrated to be an antagonist (pK(B) = 8.04 +/- 0.13) in functional assays. JNJ-31020028 occupied Y(2) receptor binding sites (approximately 90% at 10 mg/kg) after subcutaneous administration in rats. JNJ-31020028 increased norepinephrine release in the hypothalamus, consistent with the colocalization of norepinephrine and neuropeptide Y. In a variety of anxiety models, JNJ-31020028 was found to be ineffective, although it did block stress-induced elevations in plasma corticosterone, without altering basal levels, and normalized food intake in stressed animals without affecting basal food intake. CONCLUSION: These results suggest that Y(2) receptors may not be critical for acute behaviors in rodents but may serve modulatory roles that can only be elucidated under specific situational conditions.

Heterocyclic replacement of the central phenyl core of diamine-based histamine H3 receptor antagonists.

A series of small molecules consisting of a heterocyclic core flanked by two basic functionalities were synthesized and screened for in vitro affinity at the human histamine H(3) receptor (hH(3)R). Nine of the twenty-eight compounds tested were found to possess a hH(3)R K(i) of less than 5 nM and consisted of a diverse range of central hetero-aromatic linkers (pyridine, pyrazine, oxazole, isoxazole, thiazole, furan, thiophene, and pyrrole). One member of this series, (4-isopropyl-piperazin-1-yl)-(6-piperidin-1-ylmethyl-pyridin-3-yl)-methanone (37), was found to be a high affinity, selective antagonist that crosses the blood-brain barrier and occupies H(3) receptors after oral administration in the rat.

Pharmacology and antitussive efficacy of 4-(3-trifluoromethyl-pyridin-2-yl)-piperazine-1-carboxylic acid (5-trifluoromethyl-pyridin-2-yl)-amide (JNJ17203212), a transient receptor potential vanilloid 1 antagonist in guinea pigs.

Transient receptor potential vanilloid 1 (TRPV1) plays an integral role in modulating the cough reflex, and it is an attractive antitussive drug target. The purpose of this study was to characterize a TRPV1 antagonist, 4-(3-trifluoromethyl-pyridin-2-yl)-piperazine-1-carboxylic acid (5-trifluoromethyl-pyridin-2-yl)-amide (JNJ17203212), against the guinea pig TRPV1 receptor in vitro followed by a proof-of-principle study in an acid-induced model of cough. The affinity of JNJ17203212 for the recombinant guinea pig TRPV1 receptor was estimated by radioligand binding, and it was functionally characterized by antagonism of low-pH and capsaicin-induced activation of the ion channel (fluorometric imaging plate reader and electrophysiology). The nature of antagonism was further tested against the native channel in isolated guinea pig tracheal rings. Following pharmacokinetic characterization of JNJ17203212 in guinea pigs, pharmacodynamic and efficacy studies were undertaken to establish the antitussive efficacy of the TRPV1 antagonist. The pK(i) of JNJ17203212 for recombinant guinea pig TRPV1 was 7.14 +/- 0.06. JNJ17203212 inhibited both pH (pIC(50) of 7.23 +/- 0.05) and capsaicin (pIC(50) of 6.32 +/- 0.06)-induced channel activation. In whole-cell patch clamp, the pIC(50) for inhibition of guinea pig TRPV1 was 7.3 +/- 0.01. JNJ17203212 demonstrated surmountable antagonism in isolated trachea, with a pK(B) value of 6.2 +/- 0.1. Intraperitoneal administration of 20 mg/kg JNJ17203212 achieved a maximal plasma exposure of 8.0 +/- 0.4 microM, and it attenuated capsaicin evoked coughs with similar efficacy to codeine (25 mg/kg). Last, JNJ17203212 dose-dependently produced antitussive efficacy in citric acid-induced experimental cough in guinea pigs. Our data provide preclinical support for developing TRPV1 antagonists for the treatment of cough.

Aplysamine-1 and related analogs as histamine H3 receptor antagonists.

Aplysamine-1 (1), a marine natural product, was synthesized and screened for in vitro activity at the human and rat histamine H3 receptors. Aplysamine-1 (1) was found to possess a high binding affinity for the human H3 receptor (Ki = 30+/-4 nM). Synthetic analogs of 1, including des-bromoaplysamine-1 (10) and dimethyl-{2-[4-(3-piperidin-1-yl-propoxy)-phenyl]-ethyl}-amine (13), were potent H3 antagonists.

Identification and biological evaluation of 4-(3-trifluoromethylpyridin-2-yl)piperazine-1-carboxylic acid (5-trifluoromethylpyridin-2-yl)amide, a high affinity TRPV1 (VR1) vanilloid receptor antagonist.

High throughput screening using the recombinant human TRPV1 receptor was used to identify a series of pyridinylpiperazine ureas (3) as TRPV1 vanilloid receptor ligands. Exploration of the structure-activity relationships by parallel synthesis identified the essential pharmacophoric elements for antagonism that permitted further optimization via targeted synthesis to provide a potent orally bioavailable and selective TRPV1 modulator 41 active in several in vivo models.

Selective blockade of the capsaicin receptor TRPV1 attenuates bone cancer pain.

Cancer colonization of bone leads to the activation of osteoclasts, thereby producing local tissue acidosis and bone resorption. This process may contribute to the generation of both ongoing and movement-evoked pain, resulting from the activation of sensory neurons that detect noxious stimuli (nociceptors). The capsaicin receptor TRPV1 (transient receptor potential vanilloid subtype 1) is a cation channel expressed by nociceptors that detects multiple pain-producing stimuli, including noxious heat and extracellular protons, raising the possibility that it is an important mediator of bone cancer pain via its capacity to detect osteoclast- and tumor-mediated tissue acidosis. Here, we show that TRPV1 is present on sensory neuron fibers that innervate the mouse femur and that, in an in vivo model of bone cancer pain, acute or chronic administration of a TRPV1 antagonist or disruption of the TRPV1 gene results in a significant attenuation of both ongoing and movement-evoked nocifensive behaviors. Administration of the antagonist had similar efficacy in reducing early, moderate, and severe pain-related responses, suggesting that TRPV1 may be a novel target for pharmacological treatment of chronic pain states associated with bone cancer metastasis.