52245-57-5

Upstream product

• 622-77-5 N-benzylcyanamide 
• 100-46-9 benzylamine 

Relevant academic research and scientific papers

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

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.

Novel substrates for nitric oxide synthases.

Enzymatic generation of nitric oxide (NO) by nitric oxide synthase (NOS) consists of two oxidation steps. The first step converts L-arginine to N(G)-hydroxy-L-arginine (NOHA), a key intermediate, and the second step converts NOHA to NO and L-citrulline. To fully probe the substrate specificity of the second enzymatic step, an extensive structural screening was carried out using a series of N-alkyl (and N-aryl) substituted-N'-hydroxyguanidines (1-14). Among the eleven N-alkyl-N'-hydroxyguanidines evaluated, N-n-propyl (2), N-iso-propyl (3), N-n-butyl (4), N-s-butyl (5), N-iso-butyl (6), N-pentyl (8) and N-iso-pentyl (9) derivatives were efficiently oxidized by the three isoenzymes of NOS (nNOS, iNOS and eNOS) to generate NO. N-Butyl-N'-hydroxyguanidine (4) was the best substrate for iNOS (K(m)=33 microM) and N-iso-propyl-N'-hydroxyguanidine (3) was the best substrate for nNOS (K(m)=56 microM). When the alkyl substituents were too small (such as ethyl 1) or too large (such as hexyl 10 and cyclohexyl 11), the activity decreased significantly. This suggests that the van der Waals interaction between the alkyl group and the hydrophobic cavity in the NOS active site contributes significantly to the relative reactivity of compounds 3-11. Moreover, five N-aryl-N'-hydroxyguanidines were found to be good substrates for iNOS, but not substrates for eNOS and nNOS. N-phenyl-N'-hydroxyguanidine was the best substrate among them (K(m)=243 microM). This work demonstrates that N-alkyl substituted hydroxyguanidine compounds are novel NOS substrates which 'short-circuit' the first oxidation step of NOS, and N-aryl substituted hydroxyguanidine compounds are isoform selective NOS substrate.