In search of new Nitric Oxide Synthase (NOS) inhibitor agents, two isosteric series of derivatives with an imidamide scaffold (one of them with a hydroxyl group and the other with a carbonyl one) were synthesized and evaluated on inducible (iNOS) and neuronal (nNOS) isoforms. These compounds have been designed by combining a kynurenamine framework with an amidine moiety in order to improve selectivity for the inducible isoform. In general, the in vitro inhibitory assays exhibited better inhibition values on the iNOS isoform, being the N-(3-(2-amino-5-methoxyphenyl)-3-hydroxypropyl)-4-(trifluoromethyl)benzimidamide 4i the most active inhibitor with the highest iNOS selectivity, without inhibiting eNOS. Docking studies on the two most active compounds suggest a different binding mode on both isozymes, supporting the experimentally observed selectivity towards the inducible isoform. Physicochemical in silico studies suggest that these compounds possess good drug-likeness properties.

The synthesis and biological evaluation of new types of N,N′-disubstituted thiourea and urea derivatives as inhibitors of both neuronal nitric oxide synthase (nNOS) and inducible nitric oxide synthase (iNOS) are described. These compounds have been designed by reduction of the carbonyl group in the thiourea and urea kynurenamine derivatives 3 previously synthesized by our research group. The synthetic route performed to this new family also allows us to obtain the molecules 3 with less synthetic steps and higher global yield. Regarding the biological results, in general, the new derivatives 4a–q inhibit the neuronal NOS isoform better than the inducible one. Furthermore, thioureas exhibit higher inhibition than ureas for both isoenzymes. Among all the tested compounds, 4g shows significant nNOS (80.6%) and iNOS (76.6%) inhibition values without inhibiting eNOS. This molecule could be an interesting starting point for the design of new inhibitors with application in neurological disorders where both isoenzymes are implicated such as Parkinson's disease.

Nitric oxide (NO), which is produced by oxidation of L-arginine to L-citrulline in a process catalyzed by different isoforms of nitric oxide synthase (NOS), exhibits diverse roles in several physiological processes, including neurotransmission, blood pressure regulation and immunological defense mechanisms. On the other hand, an overproduction of NO is related with several disorders as Alzheimer’s disease, Huntington´s disease and the amyotrophic lateral sclerosis. Taking melatonin as a model, our research group has designed and synthesized several families of compounds that act as NOS inhibitors, and their effects on the excitability of N-methyl-D-aspartate (NMDA)-dependent neurons in rat striatum, and on the activity on both nNOS and iNOS were evaluated. Structural comparison between the three most representative families of compounds (kynurenines, kynurenamines and 4,5-dihydro-1H-pyrazole derivatives) allows the establishment of structure-activity relationships for the inhibition of nNOS, and a pharmacophore model that fulfills all of the observed SARs were developed. This model could serve as a template for the design of other potential nNOS inhibitors. The last family of compounds, pyrrole derivatives, shows moderate in vitro NOS inhibition, but some of these compounds show good iNOS/nNOS selectivity. Two of these compounds, 5-(2-aminophenyl)-1H-pyrrole-2-carboxylic acid methylamide and cyclopentylamide, have been tested as regulators of the in vivo nNOS and iNOS activity. Both compounds prevented the increment of the inducible NOS activity in both cytosol (iNOS) and mitochondria (i-mtNOS) observed in a MPTP model of Parkinson´s disease.