Most of the GSNOR inhibitors presented here were

Most of the GSNOR inhibitors presented here were synthesized according to . The synthesis started from either commercially available ketones or the ketones prepared as described in the . In , condensation of ketone and 2-furanaldehyde provided intermediate in good yield. Furan ring opening of intermediate by HBr in ethanol under reflux conditions provided diketone . Pyrrole formation was achieved by condensation of the diketone and 4-amino-3-methylbenzamide under acidic conditions to afford intermediate . For the synthesis of compounds –, where the Ar azd6244 australia phenyl, compounds – were synthesized either using the Suzuki coupling conditions for the C–C connected aromatic heterocyclic analogs, or coupling of heterocycles such as morpholine () with intermediate in the presence of copper iodide (I) and potassium carbonate in DMSO to form N–C connected compounds. The N–C connected analogs can also be synthesized using similar conditions, but using amides instead. Compounds – were prepared using substituted imidazoles as starting materials to couple with intermediate either using -proline or ,-dimethyl-cyclohexane-1,2-diamine as a catalyst in the presence of copper iodide(I) and potassium carbonate in DMSO followed by hydrolysis of ester in aqueous lithium hydroxide. The synthesis of compounds – are described in detail in the . One issue we encountered during the lead optimization of GSNOR inhibitors was cytochrome P450 enzyme inhibition with compounds, such as , containing a nitrogen linked imidazole moiety. CYP inhibitions may be a concern in the drug development due to potential drug–drug interaction. Our initial approach was to replace the imidazole ring with other heterocycles in an attempt to avoid the CYP inhibition. A large set of five-membered heterocyclic analogs was synthesized and the GSNOR inhibitory activity was evaluated (). Overall, none of the new analogs exhibited better GSNOR inhibition than although a number of compounds (, , and ) achieved sub-micromolar activity. To our surprise, the nitrogen containing analogs – exhibited substantially decreased GSNOR inhibitory activity. The thiadiazole analog was completely devoid of enzyme inhibition activity while the thiazole comparator achieved sub-micromolar potency. Substitution of imidazole at 5-position () resulted in greater than a 2-log of loss in GSNOR inhibition activity. The best compound in the series was compound having GSNOR IC=0.51μM. Next, six-membered ring heterocycles (–) were also used to replace imidazole. However, all of these compounds exhibited markedly decreased GSNOR inhibitory activity as shown in . As a second approach to circumvent the CYP inhibition, we evaluated the crystal structure of the antifungal agent, ketoconazole, bound to CYP 3A4 in comparison with the crystal structure of bound to the GSNOR enzyme. We hypothesized that substitution at the 2-position of imidazole may hinder the binding of imidazole to CYP 3A4 enzyme, with the assumption that the imidazole moiety of binds similarly to CYP 3A4. In addition, there is space in the -GSNOR crystal structure to fit small groups. We started the SAR exploration with substitution of the imidazole ring at 2-position by a series of lower alkyl groups (). The overall CYP activity, including activity for CYP 3A4 and 2C19, was significantly reduced with 2-methyl () and 2-ethyl () substituted analogs as compared to non-substituted . However, the GSNOR inhibition activity was also reduced by substitution, particularly with a larger group such as cyclopropyl () and isopropyl (). Hydroxymethyl () and trifluoromethyl () substitution at 2-postion of imidazole diminished the GSNOR inhibitory activity. In order to maintain or improve the GSNOR inhibition activity and remove CYP inhibition, the phenyl ring connecting to the imidazole was replaced with a variety of heterocycles (). Replacement of phenyl ring on with thiophene (, ) maintained the GSNOR inhibition activity, but the CYP inhibition issue remained (). Substitution at 2-position of the imidazole with a methyl group () not only maintained the GSNOR inhibition activity, also reduced the CYP inhibition to <30% as compared to >50% inhibition for (). The thiophene regioisomer of () also achieved a similar result. Heterocyclic analogs other than thiophene, such as furan (), thiazole (), pyridines (, ) and pyrimidine () lost significant GSNOR inhibitory activity.