Cyanine 5-dUTP mg br Acknowledgments This research was suppo

Acknowledgments This research was supported by the Czech Ministry of Education, Youth and Sports by grants MSM LH11013 and Czech Grant Agency (501/12/0590). David Kopečný and Martina Kopečná were supported by the grant LO1204 from the National Program of Sustainability I by the Ministry of Education, Youth and Sports, Czech Republic. We thank Eva Dejmková for her technical assistance to isothermal titration calorimetry measurements performed in CEITEC – open access project, ID number LM2011020, funded by the Ministry of Education, Youth and Sports of the Czech Republic under the activity “Projects of Major Infrastructures for Research, Development and Innovations”.
Nitric oxide (NO) is synthesized from -arginine by nitric oxide synthases (NOS)., -nitrosoglutathione (GSNO), an adduct of NO and glutathione, exists in equilibrium with other low molecular weight and protein-bound -nitrosothiols (SNOs). GSNO and SNOs serve as more stable reservoirs for bioavailable NO, in comparison to NO itself. -nitrosoglutathione reductase (GSNOR, also known as alcohol dehydrogenase 3) catalyzes the reduction of GSNO, to the unstable intermediate -(-hydroxyamino)glutathione which spontaneously rearranges to glutathione sulfinamide or reacts with glutathione (GSH) to form glutathione disulfide and hydroxylamine., , , , At low pH, the glutathione sulfinamide is readily hydrolyzed to sulfinic Cyanine 5-dUTP mg and ammonia. Therefore GSNOR indirectly controls intracellular levels of SNOs and thus, NO ()., , , , , , , GSNOR knockout mice have been shown to have increased lung SNOs and were protected from airway hyperresponsiveness after methacholine or allergen challenge, suggesting that GSNOR is a crucial modulator of airway tone., Given such findings, GSNOR has been recognized as a potential therapeutic target for the treatment of a broad range of diseases due to the important role that GSNO plays in the biological systems., , , , , We recently reported the discovery of , a potent GSNOR inhibitor that is in clinical development for the treatment of acute asthma. Following this communication, we also disclosed the structure–activity relationship of the pyrrole based GSNOR inhibitors related to including the identification of pyrrole regioisomer and potent GSNOR inhibitor with reduced CYP inhibition, as shown in . In this Letter, we discuss the synthesis and structure–activity relationship of the pyrrole based GSNOR inhibitors mainly focusing on the replacement and modification of the carboxamide, in an attempt to further understand the structure–activity relationship and improve enzyme inhibitory potency and ADME properties. The general synthetic route of GSNOR inhibitors is outlined in . The synthesis started from either commercially available ketones or the ketones prepared according to the procedures described in the . In , condensation of ketones and 2-furanaldehyde provided intermediate in good yield. Furan ring opening of intermediates by hydrogen bromide in ethanol under reflux conditions provided diketones . Pyrrole formation was achieved by condensation of the diketones with anilines under acidic conditions to afford compounds . The synthesis of compounds –, where the X is bromo or methoxy, was accomplished by hydrolysis of compounds in aqueous lithium hydroxide. Compounds – were synthesized using substituted imidazoles as starting materials to couple with intermediates (X=Br) using -proline as a catalyst in the presence of copper iodide (I) and potassium carbonate in dimethylsulfoxide followed by hydrolysis of the ester in aqueous lithium hydroxide., The synthesis of key compounds is described in the and the other compounds were prepared in the similar manners as detailed in our earlier publications., , To examine the SAR of the amide replacement, we kept the rest of the molecule the same, X=OMe, and R=H or Me () except compound , where X is bromo. Within the des-methyl series –, where R=H, the hydroxyl analog is the most potent inhibitor followed by the amide analog Methylation of the hydroxyl analog () resulted in a 4–5-fold loss in GSNOR inhibition activity. Replacing the hydroxyl group with bromide also diminished the binding affinity to the enzyme. The reversed amide lost 10-fold GSNOR inhibitory activity. Spacing the amide from the phenyl ring with either methylene or NH (urea) caused >10-fold loss in the GSNOR inhibitory activity. More extensive SAR was explored with the methyl series, where R=Me. Sulfonamide achieved the best activity with IC=330nM followed by the sulfonyl diamide . Interestingly, the hydroxyl analog was not as potent as the des-methyl comparator and O-methylation also resulted in only a minor loss in activity. Substituted amide analogs and were much less active than the primary amide reported earlier (X=OMe, R=CONH, R=Me, IC=210nM). However, introducing a methoxyethyl group or hydroxyethyl group recovered some of the loss in GSNOR inhibition. Furthermore, we prepared the heterocyclic amides – in an attempt to pick up more binding to the enzyme. The 4-pyridyl amide demonstrated an IC of 170nM, which is the best within the series. The bromo analog of achieved double digit nanomolar IC (61nM).