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  • Further SAR was explored with the imidazole series to achiev

    2022-08-05

    Further SAR was explored with the imidazole series to achieve better enzyme inhibition activity (). In comparison to published earlier, replacing the amide with sulfonamide and reverse sulfonamide maintained the GSNOR inhibitory activity. It is clear that exchanging the phenyl ring (, and ) by thienyl ( and ) improved the GSNOR inhibition activity 4–10-fold. Within the phenyl series –, the reverse sulfonamide demonstrated the best activity, followed by the reverse amides , and . Compounds with basic functionalities such as amine () and aminomethyl () resulted in a substantial loss in GSNOR inhibitory activity. Within the methyl imidazole series –, 2,4-substituted thienyl analog is less active than the 2,5-disubstituted analog . In the Sodium Nitroprusside phenol series –, , and , where R=OH, des-methyl analog seems more active than the corresponding methyl analog , this was not observed in the other amide replacement compounds. Methyl imidazole is also less active than its des-methyl imidazole analog . Selected GSNOR inhibitors were screened for potential off-target activity with a panel of 55 transmembrane and soluble receptors, ion channels, and monoamine transporters involved in maintaining homeostasis of critical organ systems. Typical binding assays were performed with a minimum of 6-control wells with/without vehicle for soluble compounds. Inhibition of 50% or greater was considered a positive response. Off-target effects were estimated from the percent inhibition of receptor radio-ligand binding in the presence of 10μM of test compound. Compound and did not show any off-target activity in the Cerep receptor/ion channel panel, by Sodium Nitroprusside with as reported earlier. Compounds –, , , and were also screened for cytotoxicity towards the A549 epithelial lung cell line. The IC values for all compounds tested were >150μM. Compounds and were selected for pharmacokinetic studies in mice. Oral bioavailability of these compounds was 3.9% and 6.8%, respectively, compared to 4.4% for reported previously. The plasma clearance (CL) after intravenous (IV) administration was 23.9 and 37.1ml/min/kg for and , respectively, which is comparable to 37.7ml/min/kg for . Compound was tested in a 5-day mouse toxicity study with intravenous QD dosing at 1, 10, or 50mg/kg. Surprisingly, despite a better off-target activity profile of this compound compared to , the treatment of male CD-1 mice with for 5days resulted in significant adverse effects. In particular, histological findings demonstrated toxicity to the liver, spleen, and thymus of treated animals. The NOAEL for from the study was determined to be 1mg/kg/day. The efficacy of GSNOR inhibitors was assessed in an animal model of asthma, a disease influenced by dysregulated GSNOR and altered function of NO, GSNO, and SNOs. Asthma was induced by exposure of mice to OVA. was given as a single 1mg/kg IV dose 24h prior to challenge with aerosolized methacholine (MCh). Other groups of mice were treated with 3 inhaled doses of Combivent (5.2mg/kg albuterol and 0.9mg/kg ipratropium per dose at 48, 24, and 1h prior to MCh) or a single iv administration of PBS vehicle as study controls. Efficacy was assessed by measuring attenuation of the MCh-induced bronchoconstriction using whole body plethysmography (Buxco) and attenuation of the eosinophil infiltration into the bronchoalveolar lavage fluid (BALF). Values are means±SEM of 10 mice per group. Compound attenuated methacholine-induced bronchoconstriction (airway hyper-responsiveness) and eosinophil infiltration into the lungs following a single IV dose administered 24h prior to the methacholine challenge. Significant efficacy was observed for compound at dose 1mg/kg (, ). In conclusion, the carboxamide substituent on the pendant -phenyl ring of pyrrole based GSNOR inhibitors can be replaced by a number of functional groups such as hydroxyl, sulfonamide, reverse amide, and reverse sulfonamide without losing significant GSNOR inhibition activity. The thienyl analogs are generally more potent than their phenyl counter parts. Compound demonstrated potent inhibitory activity, while having no off-target activities in the Cerep receptor/ion channel panel screening and a clean profile in cytotoxicity assay. In vivo efficacy was achieved with in the OVA induced asthma model in mice. Compound was well tolerated when administered IV in 5-day toxicity evaluations in mice up to 50mg/kg. However, this compound had a less desirable safety profile with a NOAEL of 1mg/kg as compared to with a NOAEL of 30mg/kg.