Jeffrey W. Cramer

3-(4-Chloro-2-Morpholin-4-yl-Thiazol-5-yl)-8-(1-Ethylpropyl)-2,6-Dimethyl-Imidazo[1,2-b]Pyridazine: A Novel Brain-Penetrant, Orally Available Corticotropin-Releasing Factor Receptor 1 Antagonist with Efficacy in Animal Models of Alcoholism

We describe a novel corticotropin-releasing factor receptor 1 (CRF1) antagonist with advantageous properties for clinical development, and its in vivo activity in preclinical alcoholism models. 3-(4-Chloro-2-morpholin-4-yl-thiazol-5-yl)-8-(1-ethylpropyl)-2,6-dimethyl-imidazo[1,2-b]pyridazine (MTIP) inhibited 125I-sauvagine binding to rat pituitary membranes and cloned human CRF1 with subnanomolar affinities, with no detectable activity at the CRF2 receptor or other common drug targets. After oral administration to rats, MTIP inhibited 125I-sauvagine binding to rat cerebellar membranes ex vivo with an ED50 of ∼1.3 mg/kg and an oral bioavailability of 91.1%. Compared with R121919 (2,5-dimethyl-3-(6-dimethyl-4-methylpyridin-3-yl)-7-dipropylamino-pyrazolo[1,5-a]pyrimidine) and CP154526 (N-butyl-N-ethyl-4,9-dimethyl-7-(2,4,6-trimethylphenyl)-3,5,7-triazabicyclo[4.3.0]nona-2,4,8,10-tetraen-2-amine), MTIP had a markedly reduced volume of distribution and clearance. Neither open-field activity nor baseline exploration of an elevated plus-maze was affected by MTIP (1–10 mg/kg). In contrast, MTIP dose-dependently reversed anxiogenic effects of withdrawal from a 3 g/kg alcohol dose. Similarly, MTIP blocked excessive alcohol self-administration in Wistar rats with a history of dependence, and in a genetic model of high alcohol preference, the msP rat, at doses that had no effect in nondependent Wistar rats. Also, MTIP blocked reinstatement of stress-induced alcohol seeking both in postdependent and in genetically selected msP animals, again at doses that were ineffective in nondependent Wistar rats. Based on these findings, MTIP is a promising candidate for treatment of alcohol dependence.

LY2456302 is a novel, potent, orally-bioavailable small molecule kappa-selective antagonist with activity in animal models predictive of efficacy in mood and addictive disorders

Kappa opioid receptors and their endogenous neuropeptide ligand, dynorphin A, are densely localized in limbic and cortical areas comprising the brain reward system, and appear to play a key role in modulating stress and mood. Growing literature indicates that kappa receptor antagonists may be beneficial in the treatment of mood and addictive disorders. However, existing literature on kappa receptor antagonists has used extensively JDTic and nor-BNI which exhibit long-lasting pharmacokinetic properties that complicate experimental design and interpretation of results. Herein, we report for the first time the in vitro and in vivo pharmacological profile of a novel, potent kappa opioid receptor antagonist with excellent selectivity over other receptors and markedly improved drug-like properties over existing research tools. LY2456302 exhibits canonical pharmacokinetic properties that are favorable for clinical development, with rapid absorption (t(max): 1-2 h) and good oral bioavailability (F = 25%). Oral LY2456302 administration selectively and potently occupied central kappa opioid receptors in vivo (ED₅₀ = 0.33 mg/kg), without evidence of mu or delta receptor occupancy at doses up to 30 mg/kg. LY2456302 potently blocked kappa-agonist-mediated analgesia and disruption of prepulse inhibition, without affecting mu-agonist-mediated effects at doses >30-fold higher. Importantly, LY2456302 did not block kappa-agonist-induced analgesia one week after administration, indicating lack of long-lasting pharmacodynamic effects. In contrast to the nonselective opioid antagonist naltrexone, LY2456302 produced antidepressant-like effects in the mouse forced swim test and enhanced the effects of imipramine and citalopram. LY2456302 reduced ethanol self-administration in alcohol-preferring (P) rats and, unlike naltrexone, did not exhibit significant tolerance upon 4 days of repeated dosing. LY2456302 is a centrally-penetrant, potent, kappa-selective antagonist with pharmacokinetic properties favorable for clinical development and activity in animal models predictive of efficacy in mood and addictive disorders.

Central H3R activation by thioperamide does not affect energy balance

The central histamine 3 receptor (H3R) is a presynaptic autoreceptor that regulates neuronal release and synthesis of histamine, and is thought to play a key role in controlling numerous central nervous system (CNS)-mediated parameters, including energy homeostasis. Thioperamide, the prototypical selective H3R antagonist, was used to examine the role that H3R plays in regulating energy balance in vivo. Thioperamide was administered either intraperitoneally or orally to rats and the pharmacokinetic parameters were examined along with central H3R binding and histaminergic system activation. Food intake and metabolic parameters of either route of thioperamide administration were likewise examined. In a dose-dependent manner, both the intraperitoneal and oral route of administration resulted in similar ex vivo binding curves and tele-methylhistamine dose-response curves despite the route of administration. However, only intraperitoneal administration of 30 mg/kg thioperamide resulted in a significant decrease in 24-h food intake (60% lower than control) and respiratory quotient (RQ), while the oral route of delivery did not. Moreover, the decrease in RQ with the 30 mg/kg ip administration also decreased energy expenditure (EE) thus resulting in an unchanged energy balance. The decrease in food intake and EE was coupled with a conditioned taste aversion with the 30-mg/kg ip administration. These data indicate that the activation of the central H3R system by thioperamide does not play a direct role in decreasing food intake or altering energy homeostasis.

Bactericidal Activity of an Imidazo[1, 2-a]pyridine Using a Mouse M. tuberculosis Infection Model

Tuberculosis remains a global threat due in part to the long treatment regimen and the increased prevalence of drug resistant M. tuberculosis strains. Therefore, new drug regimens are urgently required to combat this deadly disease. We previously synthesized and evaluated a series of new anti-tuberculosis compounds which belong to the family of imidazo[1,2-a]pyridines. This family of compounds showed low nM MIC (minimal inhibitory concentration) values against M. tuberculosis in vitro. In this study, a derivative of imidazo[1,2-a]pyridines, (N-(4-(4-chlorophenoxy)benzyl)-2,7-dimethylimidazo[1,2-a]pyridine-3-carboxamide) (ND-09759), was selected as a promising lead compound to determine its protective efficacy using a mouse infection model. Pharmacokinetic analysis of ND-09759 determined that at a dosage of 30 mg/kg mouse body weight (PO) gave a maximum serum drug concentration (Cmax) of 2.9 µg/ml and a half-life of 20.1 h. M. tuberculosis burden in the lungs and spleens was significantly decreased in mice treated once daily 6 days per week for 4-weeks with ND-09759 compared to untreated mice and this antibiotic activity was equivalent to isoniazid (INH) and rifampicin (RMP), two first-line anti-TB drugs. We observed slightly higher efficacy when using a combination of ND-09759 with either INH or RMP. Finally, the histopathological analysis revealed that infected mice treated with ND-09759 had significantly reduced inflammation relative to untreated mice. In conclusion, our findings indicate ND-09759 might be a potent candidate for the treatment of active TB in combination with current standard anti-TB drugs.

Identification of Phenoxyalkylbenzimidazoles with Antitubercular Activity.

We conducted an evaluation of the phenoxyalkylbenzimidazole series based on the exemplar 2-ethyl-1-(3-phenoxypropyl)-1H-benzo[d]imidazole for its antitubercular activity. Four segments of the molecule were examined systematically to define a structure-activity relationship with respect to biological activity. Compounds had submicromolar activity against Mycobacterium tuberculosis; the most potent compound had a minimum inhibitory concentration (MIC) of 52 nM and was not cytotoxic against eukaryotic cells (selectivity index = 523). Compounds were selective for M. tuberculosis over other bacterial species, including the closely related Mycobacterium smegmatis. Compounds had a bacteriostatic effect against aerobically grown, replicating M. tuberculosis, but were bactericidal against nonreplicating bacteria. Representative compounds had moderate to high permeability in MDCK cells, but were rapidly metabolized in rodents and human liver microsomes, suggesting the possibility of rapid in vivo hepatic clearance mediated by oxidative metabolism. These results indicate that the readily synthesized phenoxyalkylbenzimidazoles are a promising class of potent and selective antitubercular agents, if the metabolic liability can be solved.

Synthesis and evaluation of the 2,4-diaminoquinazoline series as anti-tubercular agents.

The 2,4-diaminoquinazoline class of compounds has previously been identified as an effective inhibitor of Mycobacterium tuberculosis growth. We conducted an extensive evaluation of the series for its potential as a lead candidate for tuberculosis drug discovery. Three segments of the representative molecule N-(4-fluorobenzyl)-2-(piperidin-1-yl)quinazolin-4-amine were examined systematically to explore structure-activity relationships influencing potency. We determined that the benzylic amine at the 4-position, the piperidine at 2-position and the N-1 (but not N-3) are key activity determinants. The 3-deaza analog retained similar activity to the parent molecule. Biological activity was not dependent on iron or carbon source availability. We demonstrated through pharmacokinetic studies in rats that good in vivo compound exposure is achievable. A representative compound demonstrated bactericidal activity against both replicating and non-replicating M. tuberculosis. We isolated and sequenced M. tuberculosis mutants resistant to this compound and observed mutations in Rv3161c, a gene predicted to encode a dioxygenase, suggesting that the compound may act as a pro-drug.

The synthesis and evaluation of triazolopyrimidines as anti-tubercular agents

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Imidazo[1,2-a]Pyridine-3-Carboxamides Are Active Antimicrobial Agents against Mycobacterium avium Infection In Vivo

ABSTRACT A panel of six imidazo[1,2-a]pyridine-3-carboxamides (IAPs) were shown to have low-micromolar activity against Mycobacterium avium strains. Compound ND-10885 (compound 2) showed significant activity in the lung, spleen, and liver in a mouse M. avium infection model. A combined regimen consisting of ND-10885 (compound 2) and rifampin was additive in its anti-M. avium activity in the lung. Our data indicate that IAPs represent a new class of antibiotics that are active against M. avium and could potentially serve as an effective addition to a combined treatment regimen.

Bioisosteres of ethyl 8-ethynyl-6-(pyridin-2-yl)-4 H -benzo[ f ]imidazo [1,5- a ][1,4]diazepine-3-carboxylate (HZ-166) as novel alpha 2,3 selective potentiators of GABA A receptors: Improved bioavailability enhances anticonvulsant efficacy

&NA; HZ‐166 has previously been characterized as an &agr;2,3‐selective GABAA receptor modulator with anticonvulsant, anxiolytic, and anti‐nociceptive properties but reduced motor effects. We discovered a series of ester bioisosteres with reduced metabolic liabilities, leading to improved efficacy as anxiolytic‐like compounds in rats. In the present study, we evaluated the anticonvulsant effects KRM‐II‐81 across several rodent models. In some models we also evaluated key structural analogs. KRM‐II‐81 suppressed hyper‐excitation in a network of cultured cortical neurons without affecting the basal neuronal activity. KRM‐II‐81 was active against electroshock‐induced convulsions in mice, pentylenetetrazole (PTZ)‐induced convulsions in rats, elevations in PTZ‐seizure thresholds, and amygdala‐kindled seizures in rats with efficacies greater than that of diazepam. KRM‐II‐81 was also active in the 6 Hz seizure model in mice. Structural analogs of KRM‐II‐81 but not the ester, HZ‐166, were active in all models in which they were evaluated. We further evaluated KRM‐II‐81 in human cortical epileptic tissue where it was found to significantly‐attenuate picrotoxin‐ and AP‐4‐induced increases in firing rate across an electrode array. These molecules generally had a wider margin of separation in potencies to produce anticonvulsant effects vs. motor impairment on an inverted screen test than did diazepam. Ester bioisosters of HZ‐166 are thus presented as novel agents for the potential treatment of epilepsy acting via selective positive allosteric amplification of GABAA signaling through &agr;2/&agr;3‐containing GABA receptors. The in vivo data from the present study can serve as a guide to dosing parameters that predict engagement of central GABAA receptors. HighlightsKRM‐II‐81, a positive modulator of &agr; 2,3 GABAA receptors was characterized.KRM‐II‐81 was efficacious in multiple rodent anticonvulsant models.KRM‐II‐81 was also active in human cortical epileptic tissue.KRM‐II‐81 had a reduced motor impairing effects vs diazepam.KRM‐II‐81 and analogs are valuable new research tools.

In Vitro Evaluation of Novel Nitazoxanide Derivatives against Mycobacterium tuberculosis

Nitazoxanide has antiparasitic and antibiotic activities including activity against Mycobacterium tuberculosis. We prepared and evaluated a set of its analogues to determine the structure–activity relationship, and identified several amide- and urea-based analogues with low micromolar activity against M. tuberculosis in vitro. Pharmacokinetics in the rat suggested a path forward to obtain bioavailable compounds. The series had a good microbiological profile with bactericidal activity in vitro against replicating and nonreplicating M. tuberculosis. Analogues had limited activity against other Gram-positive bacteria but no activity against Gram-negative bacteria. Our studies identified the key liability in this series as cytotoxicity. Future work concentrating on identifying the target(s) could assist in removing activity against eukaryotic cells.