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J. Med. Chem. 2002, 45, 944-954
N-Aryl N′-Hydroxyguanidines, A New Class of NO-Donors after Selective Oxidation by Nitric Oxide Synthases: Structure-Activity Relationship Axelle Renodon-Cornie`re,† Sylvie Dijols,† Ce´line Perollier,† David Lefevre-Groboillot,† Jean-Luc Boucher,† Roger Attias,† Marie-Agnes Sari,† Dennis Stuehr,‡ and Daniel Mansuy*,† Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, UMR 8601 CNRS, Universite´ Paris V, 45 Rue des Saints Pe` res, 75270 Paris Cedex 06, France, and Department of Immunology, Lerner Research Foundation, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, Ohio 44195 Received July 31, 2001; Revised Manuscript Received November 8, 2001
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 CdNOH 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. Introduction Nitric oxide (NO) is a key inter- and intracellular messenger molecule involved in the maintenance of vascular tone, neuronal signaling, and host response to infection.1,2 In mammals, the synthesis of NO is catalyzed by constitutively expressed neuronal and endothelial nitric oxide synthases (NOS I and NOS III, respectively), which are controlled by the Ca2+-dependent binding of the regulatory protein calmodulin (CaM). The inducible NOS (NOS II) is expressed in macrophages following induction by inflammatory mediators and binds CaM at basal levels of Ca2+.3,4 All three NOSs produce NO and L-citrulline from the oxidation of L-arginine (L-Arg), in the presence of reduced nicotinamide adenine dinucleotide phosphate (NADPH) and molecular oxygen, with intermediate formation of Nωhydroxy-L-arginine (NOHA) (eq 1).5-7 * To whom correspondence should be addressed. Tel: 33(0)1 42 86 21 87. Fax: 33(0)1 42 86 83 87. E-mail: Daniel.Mansuy@biomedicale. univ-paris5.fr. † Universite ´ Paris V. ‡ The Cleveland Clinic Foundation.
All NOSs are constituted by two domains, an NH2terminal oxygenase domain and a CO2H-terminal reductase domain, which are separated by the CaM binding region. Heme (iron protoporphyrin IX) and (6R)5,6,7,8-tetrahydro-L-biopterin (BH4) are the essential prosthetic groups found in the oxygenase domain, which also contains the substrate binding site. Flavin adenine dinucleotide and flavin mononucleotide are found in the reductase domain, which also bears an NADPH binding site.8-10 NOSs display high substrate specificity, and only very few compounds have been clearly shown to act as NOS substrates with the formation of NO. The
10.1021/jm011006h CCC: $22.00 © 2002 American Chemical Society Published on Web 12/27/2001
Hydroxyguanidines as NOS Substrates
analogues of L-Arg and NOHA with a longer side chain, homo-L-Arg and homo-NOHA, are less efficiently transformed than their parent compounds, whereas shorter side chain analogues, nor-L-Arg and nor-NOHA, are not transformed.11-13 The introduction of a CdC double bond in the L-Arg side chain gives E-dehydro-L-Arg, which is a poor substrate for NOSs.14 In a similar manner, rigidification of the L-Arg side chain by introduction of a phenyl ring abolishes NO formation.15,16 Modifications of the R-amino acid function of L-Arg give compounds such as NR-acetyl-L-Arg, L-Arg methyl ester, and D-Arg that are not substrates of NOS.11 The introduction of substituents on the guanidino function of L-Arg leads to potent inhibitors such as Nω-nitro-Larginine (NO2-L-Arg) and Nω-amino-L-arginine1,9,11,17 whereas Nω-methyl-L-arginine, which is also a widely used inhibitor of NOSs, is oxidized by NOSs with a very limited formation of NO.18,19 Replacement of the guanidino function of L-Arg with an acetamidino function results in Nδ-iminoethyl-L-ornithine, a potent NOS inhibitor.17,20 L-Indospicine and Nω-hydroxyindospicine are compounds in which the δ-NH group of L-Arg and NOHA has been replaced with a CH2 group. Both compounds fail to act as substrates for NOSs and do not interact with the NOS active site.21,22 The very limited number of substrates known so far for NOSs suggests that highly specific structural features are required for NO generation by these enzymes. However, some simple guanidines and alkyl-iso-thioureas, such as aminoguanidine, S-ethyl-iso-thiourea (SEITU), and N-phenyl S-methyl-iso-thiourea are strong inhibitors of NOSs.17,23-26 Spectroscopic studies have demonstrated that some of these simple molecules are bound in the active site of NOS, in close proximity to the heme.27-30 These results suggest that many compounds bearing a positively charged group, but not the R-amino acid function of L-Arg, can interact with the NOSs' active site. We thus started with the idea that some simple guanidines or N-hydroxyguanidines could be oxidized by NOSs with the formation of NO. In fact, we have reported preliminary results showing that N-(4chlorophenyl) N′-hydroxyguanidine (eq 2, X ) para-Cl) is efficiently oxidized by NOS II with concomitant formation of NO and the corresponding N-aryl urea.31
This new reaction of NOS II required NADPH and the presence of BH4 and was inhibited by the addition of L-Arg analogues (NO2-L-Arg and L-thiocitrulline (TC)) and by the heme ligand imidazole but was not inhibited by superoxide dismutase (SOD). This reaction thus clearly differed from the P450 and BH4-free NOS IIcatalyzed oxidation of N-hydroxyguanidines,32,33 which is dependent on O2°- derived from the oxidase function of NOS34-36 and strongly inhibited by SOD.32,33 These results clearly indicated that this new reaction proceeded at the active site of NOS II,31,33 in a manner similar to what is occurring in the second step of NO
Journal of Medicinal Chemistry, 2002, Vol. 45, No. 4 945
synthesis catalyzed by NOSs, the oxidation of NOHA to L-citrulline, and NO. In an effort to find new substrates of NOSs that lead to NO formation after selective oxidation by these enzymes and to determine the structural factors required for a compound to act as a substrate of NOSs, we have synthesized a series of N-aryl N′-hydroxyguanidines and studied their oxidation by purified recombinant NOS II. Furthermore, related compounds bearing an amidoxime, ketoxime, or aldoxime function instead of the N-hydroxyguanidine function were also synthesized and tested as potential NOS II substrates. Finally, these compounds were tested as substrates of NOS I, NOS II, and NOS III in a comparative manner. Our results show the importance of a monosubstituted N-hydroxyguanidine function for NO formation. They also show that several N-aryl N′-hydroxyguanidines are efficiently oxidized by NOS I, II, and III with the formation of NO and suggest that new, selective substrates should be found for each NOS and could constitute a new class of NO donors of pharmacological interest. Results Chemistry. A series of N-aryl N′-hydroxyguanidines, 1-23, that involve either a phenyl ring bearing various substituents or an heterocyclic aryl ring were prepared by a classical procedure starting from substituted anilines with the intermediate formation of the corresponding arylcyanamides (eq 3).
This two-step procedure allowed us to prepare compounds 1-11, 14-17, and 23 from commercially available anilines or from 3-aminothiophene, which was obtained by a previously reported method that is mentioned in the Experimental Section. In the case of N-aryl N′-hydroxyguanidines bearing an NH2 function, 12, 13, and 18, the starting compound was the corresponding nitroarene (Figure 1-1). After protection of its NH2 function by the formation of a tertbutyloxycarbonyl (Boc) derivative, the nitroarene was reduced (H2, Pd/C) to the corresponding aniline. A reaction with BrCN, then with NH2OH, and deprotection of the NH2 function by HCl in dioxane eventually led to the expected N-aryl N′-hydroxyguanidines 12, 13, and 18. The heterocyclic N′-hydroxyguanidine 22 was prepared by following the same strategy from 6-nitroindazole after similar protection of its NH function (Figure 1-2). Three N-hydroxyguanidines bearing a COOH function were also used in this study. Two of them, 19 and 21, were obtained by previously reported methods that are mentioned in the Experimental Section. The third one, 20, was synthesized by following a strategy similar to that of eq 3 applied to the tert-butyl ester obtained by the reaction of 4-nitrophenol with tert-butyl bromoacetate (Figure 1-3). For the sake of comparison, two N-alkyl N′-hydroxyguanidines, 24 and 25, were also prepared by using the procedure of eq 3 (with alkyl instead of aryl). Moreover, to study the influence of another N-alkyl or N-aryl
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Renodon-Cornie` re et al.
Figure 1. Synthetic routes followed for the preparation of N-aryl N′-hydroxyguanidines bearing NH2 (1 and 2) or COOH (3) functions. Key: (a) (t-BuOCO)2O; (b) H2, Pd/C; (c) BrCN; (d) NH2OH, HCl; (e) HCl in anhydrous dioxane.
substituent on the hydroxyguanidine function, two disubstituted N-hydroxyguanidines bearing their substituents either on the same nitrogen, 26, or on two different nitrogen atoms, 27, were also synthesized. Finally, several aryl compounds bearing a CdNOH function, such as amidoximes, ketoximes, and aldoximes, 28-37, were prepared in order to determine the structural factors necessary for a compound to be oxidized by NOS with NO formation. A detailed report of the synthesis and characteristics of the new Nhydroxyguanidines is included in the Experimental Section. NOS II-Catalyzed Oxidation of Various N-Aryl N′-Hydroxyguanidines, 1-23, by NADPH and O2. The formation of NO during oxidation of N-hydroxyguanidines by NADPH and O2 in the presence of purified, recombinant NOS II was measured by the classical spectrophotometric assay following the conversion of oxyhemoglobin to methemoglobin.37,38 The assay was always done in the presence of SOD and catalase; thus, the observed formation of methemoglobin was not due to any possible oxidation of the N-hydroxyguanidines by O2°- or H2O2 derived from the oxidase function of NOS II.33-35 NOS II oxidized not only N-(4-chlorophenyl) N′hydroxyguanidine, 5, as previously reported,31 but also several N-aryl N′-hydroxyguanidines, with a rate of NO formation that may be up to 41% of that found for the endogenous substrate, NOHA (Table 1). In fact, only five compounds among the 23 N-aryl N′-hydroxyguanidines that have been tested led to NO formation with rates higher than 10% of that found for NOHA. These compounds are N-phenyl N′-hydroxyguanidine itself, 1, and four of its para-substituted derivatives, 2, 5, 7, and 9, that bear F, Cl, CH3, and OH para substituents, respectively. In a more general manner, the introduction of a given substituent in the para position of the phenyl ring of 1 led to better activities of NO formation than
the introduction of the same substituent in ortho or meta positions. Table 1 shows that this is true for the Cl (compare 3-5), CH3O (see 10 and 11), and NH2 (see 12 and 13) substituents. If one now compares the activities observed for various para-substituted compounds, it clearly appears that both electronic and steric factors are important. NO formation rates are highly dependent on the size of the para substituent of the phenyl ring. The largest rates were observed with the smallest possible substituents H and F (20 and 41% as compared to NOHA); they were smaller with medium size substituents such as Cl, CH3, OH, and NH2 (between 8 and 17%) and close to zero with bigger substituents such as C(CH3)3, CH2CO2H, or OCH2CO2H. When comparing the rates observed in the case of substituents of similar size, it appears that electrondonating substituents led to higher rates than electronwithdrawing substituents. Thus, compounds 14 and 16 bearing 4-NO2 and 4-CF3 substituents led to very low rates of NO formation, close to the detection limit, whereas compounds 7 and 13 bearing 4-CH3 and 4-NH2 substituents led to activities 17 and 8% of those found for NOHA. Table 1 also shows that N-hydroxyguanidines bearing a heterocyclic aryl substituent, 22 and 23, are also oxidized by NOS II with the formation of NO, and the activity observed with the 3-thienyl derivative is 9% of that found with NOHA. Insertion of one or two CH2 groups between the phenyl ring and the N-hydroxyguanidine function of 1 led to compound 24, which acts as a substrate of NOS almost as well as 1, and to compound 25, which is a poorer substrate (Table 2). However, changes at the level of the N-hydroxyguanidine function itself led to compounds that did not act as substrates of NOS II with NO formation. This is the case of N-hydroxyguanidines bearing two substituents, either on two different nitrogen atoms, such as 27, or on the same nitrogen atom, such as 26. This is also the
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Journal of Medicinal Chemistry, 2002, Vol. 45, No. 4 947
Table 1. NO Formation from the Oxidation of Monosubstituted N-Aryl N′-Hydroxyguanidines RsNHsC(dNsOH)sNH2 Catalyzed by NOS II compd 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
R C6H54-F-C6H4 2-Cl-C6H43-Cl-C6H44-Cl-C6H44-Br-C6H44-CH3-C6H44-tert-butyl-C6H44-OH-C6H42-CH3O-C6H44-CH3O-C6H43-NH2-C6H44-NH2-C6H44-NO2-C6H43-CF3-C6H44-CF3-C6H43-CH2OH-C6H43-CH2NH2-C6H44-(CH2CO2H)-C6H44-(O-CH2-CO2H)-C6H44-[CH-(CH3)-CO2H]-C6H46-indazolyl 3-thienyl
activitya (% NOHA)b 20 ( 3 41 ( 6 5(1 5(1 13 ( 3 7.5 ( 2 17.5 ( 4
