N-aryl N′-hydroxyguanidines, a new class of NO-donors after selective oxidation by nitric oxide synthases: Structure-activity relationship

Abstract

The formation of nitric oxide (NO) was followed during the oxidation of 37 N-hydroxyguanidines or related derivatives, including 18 new N-aryl N′ -hydroxyguanidines, by recombinant inducible nitric oxide synthase (NOS II). Several N-aryl N′-hydroxyguanidines bearing a relatively small, electron-donating para subtituent, such as H, F, Cl, CH3, OH, OCH3, and NH2, led to NO formation rates between 8 and 41% of that of NO formation from the natural NOS substrate, Nω-hydroxy-L-arginine (NOHA). The characteristics of these reactions were very similar to those previously reported for the oxidation of NOHA by NOS: (i) the strict requirement of NOS containing (6R)-5,6,7,8-tetrahydro-L-biopterin, reduced nicotinamide adenine dinucleotide phosphate, and O2 for the oxidation to occur, (ii) the formation of NO and the corresponding urea in a 1:1 molar ratio, and (iii) a strong inhibitory effect of the classical NOS inhibitors such as Nω-nitro-L-arginine and S-ethyl-iso-thiourea. Structure-activity relationship studies showed that two structural factors are crucial for NO formation from compounds containing a C=NOH function. The first one is the presence of a monosubstituted N-hydroxyguanidine function, since disubstituted N-hydroxyguanidines, amidoximes, ketoximes, and aldoximes failed to produce NO. The second one is the presence of a N-phenyl ring bearing a relatively small, not electron-withdrawing para substituent that could favorably interact with a hydrophobic cavity close to the NOS catalytic site. The kcat value for NOS II-catalyzed oxidation of N-parafluorophenyl N′-hydroxyguanidine was 80% of that found for NOHA, and its kcat/Km value was only 9-fold lower than that of NOHA. Interestingly, the Km value found for NOS II-catalyzed oxidation of N-(3-thienyl) N′-hydroxyguanidine was 25 μM, almost identical to that of NOHA. Recombinant NOS I and NOS III also oxidize several N-aryl N′-hydroxyguanidines with the formation of NO, with a clearly different substrate specificity. The best substrates of the studied series for NOS I and NOS III were N-(para-hydroxyphenyl) and N-(meta-aminophenyl) N′-hydroxyguanidine, respectively. Among the studied compounds, the para-chlorophenyl and paramethylphenyl derivatives were selective substrates of NOS II. These results open the way toward a new class of selective NO donors after in situ oxidation by each NOS family.