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, CH(3), OH, OCH(3), and NH(2), led to NO formation rates between 8 and 41% of that of NO formation from the natural NOS substrate, N(omega)-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 O(2) 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 N(omega)-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(triple bond)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 k(cat) value for NOS II-catalyzed oxidation of N-para-fluorophenyl N'-hydroxyguanidine was 80% of that found for NOHA, and its k(cat)/K(m) value was only 9-fold lower than that of NOHA. Interestingly, the K(m) value found for NOS II-catalyzed oxidation of N-(3-thienyl) N'-hydroxyguanidine was 25 microM, 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 para-methylphenyl 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.

Role of Arginine Guanidinium Moiety in Nitric-oxide Synthase Mechanism of Oxygen Activation

“…In contrast, the loss of such a strong H-bond would decrease the stability of the Fe II -O 2 complex; this is observed with guanidines of families 2 and 3 for which the autoxidation rates increase by 100-fold (Table 2). Thus, the decrease of the guanidinium pK a value can be related to the decrease in NO production by the uncoupling of electron transfer and the associated release of high amounts of ROS (Table 2) (40,42,86).…”

“…This strongly supports the involvement of two protonation events leading to the formation of a Compound I as the oxidative species for L-Arg hydroxylation (Scheme 4, step 1). However, whereas low pK a arylguanidines failed to become hydroxylated, the corresponding N-hydroxyguanidines still led to significant production of NO (Table 2) (61,86,89). This suggests that the Fe III -peroxo complex might be sufficient to achieve NOHA oxidation (29); without the participation of an additional water molecule, low pK a substrates, such as NOHA, will stabilize the Fe III -peroxo complex and favor its direct reaction on the guanidinium moiety (Scheme 4, step 2).…”

Involvement of NO in the Endothelium-Independent Relaxing Effects ofNω-Hydroxy-l-arginine and Other Compounds Bearing a C=NOH Function in the Rat Aorta

“…L-NOHA was obtained from Alexis Corporation (Lä ufelfingen, Switzerland) and N -hydroxy-D-arginine from GlaxoSmithKline (Uxbridge, Middlesex, UK). N-Hydroxyguanidines, N-(4-chlorophenyl)-NЈ-hydroxyguanidine, N,NЈ-dicyclohexyl-NЉ-hydroxyguanidine, N-hydroxydebrisoquine, benzamidoxime, ClBZA, 4-nitrobenzamidoxime (NO 2 BZA), 4-n-(hexyloxy)benzamidoxime (HXBZA), 4-(methoxy)benzamidoxime (MXBZA), and 4-chloroacetophenone-oxime (ClBK) were synthesized following previously described procedures (Jousserandot et al, 1998;Renodon-Cornière et al, 2002).…”

“…4 and Table 1). The structural determinants required for the relaxant effect are also entirely different from those required for being an NO synthase substrate (Dijols et al, 2001;Renodon-Cornière et al, 2002): the substituted N-hydroxyguanidine, a poorly relaxant compound, is a selective substrate for the inducible NO synthase; by contrast, the identically substituted amidoxime ClBZA and ketoxime ClBK are potent relaxant compounds, whereas they are not substrate for any of the three NO synthase isoforms. Thus, structure activity relationships together with the above-mentioned failure of P450 and NO synthase inhibitors to blunt relaxation lend no support to the hypothesis of the involvement of a P450 or NO synthase in the relaxant effect of compounds with a CϭNOH function in the rat aorta.…”

“…The second step of the reaction consists in oxidative cleavage of the CϭNOH bond of the N-hydroxyguanidine function, with formation of the free radical NO and L-citrulline. Recently, it has been reported that non-␣-amino acid N-aryl-N-hydroxyguanidines are also oxidized by NO synthase with formation of NO (Renodon-Cornière et al, 2002). It has been known for several years, however, that oxidative cleavage of the CϭN bond of CϭNOH functions of various compounds, including L-NOHA, can be catalyzed also by hemoproteins like horse radish peroxidase, rat liver microsomal cytochrome P450 (P450), hemoglobin, and catalase.…”