Imidazole derivatives as antioxidants and selective inhibitors of nNOS

Nitric Oxide 14 (2006) 45–50 www.elsevier.com/locate/yniox

Imidazole derivatives as antioxidants and selective inhibitors of nNOS V. Sorrenti a,*, L. Salerno b, C. Di Giacomo a, R. Acquaviva a, M.A. Siracusa b, A. Vanella a a

Department of Biochemistry, Medical Chemistry and Molecular Biology, University of Catania, v.le A. Doria 6, 95125 Catania, Italy b Department of Pharmaceutical Sciences, University of Catania, v.le A. Doria 6, 95125 Catania, Italy Received 6 July 2005 Available online 7 November 2005

Abstract The reperfusion of ischemic tissue often delays its physiological and functional recovery; this paradoxical effect is ascribed to increased release of free radicals including O2
and NO. For these reasons, scavenging reactive oxygen species or inhibition the NO synthesis has been shown to result in an enhanced neuronal survival after cerebral ischemia. Many authors believe that therapy for stroke patients would be a cocktail of drugs with various mechanisms of action. Combination therapy is a difficult and complicated avenue for drug development because of the possibility of drug–drug interactions. An alternative approach would be to combine multiple activities within the same compound. In consideration of the free-radical scavenging and inhibitory effect on NOS of various natural and synthetic compounds, the aim of this study was to analyze the antioxidant properties of some imidazole derivatives previously synthesized in our laboratory. Results obtained in the present study provide evidence that tested compounds exhibit interesting antioxidant properties, expressed either by their capacity to scavenge free radicals or their ability to reduce lipid peroxidation. In particular, compounds A and B represent chemical structures which can be easily modified to improve the observed antioxidant properties and to provide new therapeutic strategies focused on multiple downstream events. Ó 2005 Elsevier Inc. All rights reserved. Keywords: Free radicals; Nitric oxide; Antioxidant; NOS inhibitors

Nitric oxide (NO) is synthesized from L-arginine by NO synthase (NOS). Three isoforms have been identified: neuronal NOS (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS). The constitutive isoforms (nNOS and eNOS) are regulated by Ca2+/calmodulin, whereas the inducible isoform (iNOS) is only expressed after induction and is Ca2+/calmodulin independent. Neurological disorders, such as cerebral ischemia, involve an enhanced formation of free radicals, e.g., reactive oxygen and nitrogen species in brain tissue [1,2]. In fact, the reperfusion of ischemic tissue often delays its physiological and functional recovery; this paradoxical effect is ascribed to increased release of free radicals including O2
and to activation of calcium/calmodulin-dependent nNOS with consequent overproduction of NO; this last by reaction with O2
produces peroxynitrite anion *

Corresponding author. E-mail address: [email protected] (V. Sorrenti).

1089-8603/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.niox.2005.09.005

with harmful effects on neuronal cells. For these reasons, scavenging reactive oxygen species or inhibition of NO synthesis has been shown to result in an enhanced neuronal survival after cerebral ischemia [3]. In previous research, we have shown that pre-treatment with antioxidants reduced percentage of mortality induced by partial cerebral post-ischemic reperfusion [4,5]. Moreover, increased nNOS activity was observed under the same experimental conditions and treatment with the selective NOS inhibitor 7-nitroindazole (7-NI) induced a significant neuroprotective effect [6]. It has been reported [1] that therapy for stroke patients will be a cocktail of drugs having various mechanisms of action. Combination therapy is a difficult and complicated avenue for drug development because of the possibility of drug–drug interactions. An alternative approach would be to combine multiple activities within the same compound. From this view point, Chabrier et al. [7] produced dual action compounds such as BN 80933 that provided

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Table 1 Inhibitory activities of compounds A–H against neuronal, inducible, and endothelial nitric oxide synthase (see Refs. [9–12])

R R' N

N

nX

Compound

n

X

R

R0

% of inhibition of nNOS at 500 lM (KilM)

% of inhibition of eNOS at 500 lM (KilM)

% of inhibition of iNOS at 500 lM (KilM)

A B C D E F G H

1 1 1 1 1 1 4 1

CO CO CO CO CO CO O CH2

H CH3 CH(CH3)2 H H H H H

NO2 NO2 NO2 CF3 CH3 C6H5 Br NO2

100 (22.5) 100 (74) 100 (22.5) 36.7 40 45a 100 (32) 50

0 (>5000) 0 (>5000) 52 (550) 0 0 30a 40 (625) 0

31.62 32 (1050) 25 (1300) 27.35 13 48a 21 (1550) 0

a

% of inhibition is expressed at 50 lM owing to the very low solubility at higher concentrations.

promising neuroprotection, as demonstrated in a rat model of transient cerebral ischemia [8]. We recently described the synthesis and the structure– activity relationships of a series of N-phenacyl, N-phenethyl, and N-phenylhydroxyethyl-imidazoles and their inhibitory effects on the various NOS isoforms [9–12]. Some of the compounds tested were considered selective nNOS inhibitors because of their much greater effect against nNOS with respect to eNOS [11]. Since many authors described free-radical scavenging and inhibitory effects on NOS of various natural and synthetic compounds [13–21] and since from the free-radical chemistry point of view the structure of some imidazole derivatives previously synthesized in our laboratory might justify possible their antioxidant properties, the aim of this study was to analyze the antioxidant activity of these compounds. For this purpose, among the numerous imidazole derivatives previously synthesized, we selected the following compounds: [N-(4-nitrophenacyl)imidazole (A); N-(4-nitrophenacyl)-2-methyl-imidazole (B); N-(4-nitrophenacyl)-2-isopropyl-imidazole (C); N-(4-trifluoromethylphenacyl)imidazole (D); N-(4-methylphenacyl)imidazole (E); N-(4-phenylphenacyl)imidazole (F); 1-[4-(4-bromophenoxy)butyl]-1H-imidazole (G); N-(4-nitrophenethyl)imidazole (H). Compounds A–F were selected as representative of most active and selective of N-phenacyl imidazoles, whereas G and H as most active and selective of N-phenethyl and N-phenylhydroxyethyl-imidazoles, respectively (Table 1). Experimental procedures Chemicals Nicotinamide-adenine dinucleotide, reduced form disodium salt, 1-diphenyl-2-picrylhydrazyl radical (DPPH), xylenol orange were obtained from Sigma Aldrich (St. Louis, MO, USA). All other chemicals were from Merck (Frankfurt, Germany).

Methods All the imidazole derivatives analyzed show a major absorption in the 220–260 nm range. To assay antioxidant activity of imidazole derivatives, their ability to quench a stable radical, to scavenge superoxide anion and to inhibit in vitro lipid peroxidation, was tested. Quenching of DPPH The free-radical scavenging capacity of different concentrations of A–H was tested by their ability to bleach the stable 1,1-diphenyl-2-picrylhydrazyl radical (DPPH). The reaction mixture contained 86 lM DPPH and different concentrations of A–H (50–100–250–500 lM) in 1 ml of ethanol. After 10 min at room temperature the absorbance was recorded at k = 517 nm. Trolox (50 lM) was used as reference compound. Scavenger effect on superoxide anion Superoxide anion was generated in vitro as described by Russo et al. [22]. A total volume of 1 ml of the assay mixture contained: 100 mM triethanolamine–diethanolamine buffer, pH 7.4, 3 mM NADH, 25 mM/12.5 mM EDTA/ MnCl2, 10 mM b-mercapto-ethanol, and different concentrations of compounds A–H (50–100–250–500 lM). After 20 min incubation at 25 °C, the decrease in absorbance at k = 340 nm was measured. SOD (80 mU) was used as reference compound. Determination of lipid hydroperoxide levels in the plasma of a healthy donor Plasmatic lipid hydroperoxide levels were evaluated by oxidation of Fe2+ to Fe3+ in the presence of xylenol orange (FOX assay) at k = 560 nm [23]. Heparinized venous blood was collected after overnight fasting; plasma was separated by centrifugation at 800g for 20 min. Plasma aliquots

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47

Fig. 1. Scavenger effect of A–H and Trolox expressed as capacity to bleach the stable 1,1-diphenyl-2-picrylhydrazyl radical (DPPH). Results are expressed as percentage decrease in absorbance at k = 517 nm with respect to control. Each value represents means ± SD of five experiments. *Significance of compounds A–D vs. E–H p < 0.001; **significance of compounds B and C vs. A and D p < 0.001.

(500 ll) were diluted 1:1 with oxygenated PBS and incubated at 37 °C for 2 h with or without different concentrations (0.5–5–50 lM) of A–D, in a total volume of 1 ml. Since compounds E–H failed to exert radical scavenging activity, their ability to inhibit lipid peroxidation was not evaluated. Results are expressed as percentage of inhibition respect to control (plasma incubated in absence of test compounds). Partition coefficient and solubility evaluation Since the ability to inhibit lipid peroxidation may be related to lipophilicity of tested compound, we calculated log P values of the most active imidazole derivatives (A– C) using an Advanced Chemistry Development (ACD/ Labs) Software Solaris (V. 4.67). Statistical Analysis One-way analysis of variance (ANOVA) followed by BonferroniÕs t test was performed in order to estimate significant differences among groups. Data were reported as mean values ± SD and differences between groups were considered to be highly significant at p < 0.001. Results This study tested free-radical scavenging activity of substances by their ability to bleach the stable DPPH radical [24]. This assay provides information on the reactivity of test compounds with a stable free radical. Because of its odd electron, DPPH gives a strong absorption band at k = 517 nm in visible spectroscopy (deep violet color). As this electron becomes paired off in the presence of a freeradical scavenger, the absorption vanishes, and the resulting decolorization is stoichiometric with respect to the number of electrons taken up. In this assay, all the tested compounds showed a DPPH quenching dose-dependent

capacity but substances characterized by a keto group (A–F) and in particular substances A–D, were more efficient than G and H (Fig. 1). Among the imidazole derivatives in which a keto group is present, compounds B and C, characterized by a methyl or isopropyl group in imidazole ring and by a nitro group in the phenyl ring, were the most active. In addition, at 500 lM concentration the action of B was equivalent to 30 lM of Trolox (reference compound) (Fig. 1). To investigate the superoxide anion scavenging capacity of imidazole derivatives, we used a method which excludes the Fenton-type reaction and the xanthine/xanthine oxidase system. Compounds A–D inhibited superoxide anion formation in a dose-dependent manner; compounds A–C, all characterized by a nitro group in the benzenic ring, showed a more potent capacity than compound D (Fig. 2). The same concentrations of compounds E–H showed no scavenging capacity. The effect of A–D on lipid hydroperoxide levels was also evaluated using human plasma, incubated with or without the synthesized compounds for 2 h at 37 °C. Compounds A and B were more active than C, while compound D was not able to inhibit lipid peroxidation (Fig. 3). Compounds A–C showed a moderate lipophilicity as reported in Table 2. Results obtained demonstrated that compound C was characterized by higher log P value respect to compounds A and B. Discussion Given that in acute or chronic neurological disorders the mechanisms of neurotoxicity involve numerous processes, targeting a single event may be insufficient to provide effective neuroprotection. Both reactive oxygen and nitrogen species are involved in neurotoxic processes and contribute to neuronal death [25–30]. These species can act independently, but can also interact directly to form higher reactive oxidant peroxynitrite [31]. Although the formation of NO

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Fig. 2. Scavenger effect of A–H and superoxide dismutase (SOD) activity on O2
expressed as percentage of inhibition of NADH oxidation. Rate of O2
production was 4 nmoles/min. Each value represents means ± SD of five experiments. *Significance of compounds A–D vs. E–H p < 0.001; **significance of compounds A–C vs. D p < 0.001.

Fig. 3. Effect of A–D on lipid hydroperoxide levels in human plasma incubated at 37 °C for 2 h. Results are expressed as percentage of inhibition respect to control. Each value represents means ± SD of 5 experiments. *Significance of compounds A and B vs. D p < 0.001; **significance of compounds C vs. D p < 0.01. ***Significance of compounds A and B vs. C p < 0.001. Table 2 Calculated log P values for imidazole derivatives A–C Compound

Log P

A B C

1.024 1.113 1.98

is increased after ischemia, controversial data have been reported regarding the administration of non selective inhibitors such as L-NA and L-NAME [32–35], whereas significant neuroprotective effects have been reported after treatment with selective nNOS inhibitors [6,36,37]. Moreover, the pre-treatment with antioxidants reduced percentage of mortality induced by cerebral post-ischemic reperfusion [4,5]. The possibility of acting at several sites in the neurotoxic cascade may be more effective, as suggested by several studies in which different treatments were associated [3,38–40].

Thus, a novel strategy for the treatment of acute or chronic neurological disorders would be the administration of a drug possessing both antioxidant and selective nNOS inhibitory activities. For these reasons, novel inhibitors of neuronal NOS with potent antioxidant properties have been synthesized [7,41]. Moreover, it has been demonstrated that various natural drugs such as curcumin, epigallocatechin gallate, resveratrol, and quercetin [16–21] possess antioxidant and specific iNOS-nNOS inhibitory properties. On the other hand, unselective NOS inhibitor L-NAME was demonstrated to reduce lipid peroxidation in N-nitrosodiethylamine-treated rats [13]. In the present study the free-radical scavenging activity, investigated by DPPH test, demonstrated that compounds characterized by a keto group (A–F), showed a higher quenching capacity than G and H. The scavenging activity of these compounds may be reasonably related with their structures; in fact the presence of a keto group with the adjacent unsaturated carbon–carbon double bond of phenyl ring can contribute to the stability of the radical ‘‘via resonance’’ and by providing attachment sites for free radicals. Among tested compounds, the most active ones were characterized by an electron withdrawing group (–NO2 or –CF3) in the benzenic ring (A–D); in particular, the typic electron withdrawing nitro group present in A–C may give additional resonance structures by interaction with free radicals, therefore increasing their free-radical scavenging activity with respect to E and F; in fact, the presence of methyl and phenyl groups in benzenic ring reduced the activity of compounds E and F. Furthermore, the presence of 2-methyl or 2-isopropyl group in the imidazole ring of compounds B and C, slightly

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increased their free-radical scavenging capacity respect to unsubstituted imidazole compound (A). Since the antioxidant effects of imidazole derivatives could be mainly due both to free-radical scavenging and chelating activities, in this study the direct superoxide anion scavenging capacity of these compounds was investigated using a method which excludes the Fenton-type reaction and xanthine/xanthine oxidase system [22]. Compounds A–D inhibited superoxide anion generation in a dose-dependent manner. In this test, compounds A–C, characterized by keto and nitro groups, showed greater scavenger efficiency than D. In addition, the inhibitor effects of A–C at 500 lM concentration were similar to the action of 80 mU/ml SOD. Compounds E–H which lack a nitro or a keto group showed no antioxidant activity. To study antioxidant activity in an ‘‘ex vivo cell-free system,’’ we evaluated antilipoperoxidative capacity in human plasma. The antilipoperoxidative activity is possible only for compounds with the typic electron withdrawing nitro group (A–C); in particular compounds A and B exhibited higher activity than C at the same concentrations. Reduced antilipoperoxidative capacity of compound C characterized by higher lipophilicity, may be due to steric bulkiness of isopropyl group which might reduce its interaction with lipohydroperoxides. Compound D, characterized by a trifluoromethyl group instead of a nitro group, showed no antilipoperoxidative capacity. This might be due to an insufficient electronic delocalization that disadvantages interaction with lipohydroperoxides. In conclusion, results obtained in the present study, allow us to affirm that A–C are nNOS inhibitors endowed with antioxidant properties which remain, however, lower than reference compounds. In consideration of these results, compounds A and B represent chemical structures which can be easily modified to improve the observed antioxidant properties and to provide new therapeutic strategies focused on multiple downstream events. References [1] U. Dirnagl, C. Iadecola, M.A. Moskowitz, Pathobiology of ischemic stroke: an integrated view, Trends Neurosci. 22 (1999) 391–397. [2] S.A. Lipton, P.A. Rosemberg, Excitatory aminoacid as a final common pathway for neurologic disorder, N. Engl. J. Med. 330 (1994) 613–622. [3] B. Spinnewyn, S. Cornet, M. Auguet, P.E. Chabrier, Synergistic protective effects of antioxidant and nitric oxide synthase inhibitor in transient focal ischemia, J. Cereb. Blood Flow Metab. 19 (1999) 139– 143. [4] A. Vanella, V. Sorrenti, C. Castorina, A. Campisi, C. Di Giacomo, A. Russo, J.R. Perez-Polo, Lipid peroxidation in rat cerebral cortex during post-ischemic reperfusion: effect of exogenous antioxidants and Ca2+-antagonist drugs, Int. J. Dev. Neurosci. 10 (1992) 75–80. [5] V. Sorrenti, C. Di Giacomo, M. Renis, A. Russo, C. La Delfa, J.R. Perez-Polo, A. Vanella, Lipid peroxidation in rat cerebral cortex during post-ischemic reperfusion: effect of drugs with different molecular mechanisms, Drugs Exp. Clin. Res. 20 (1994) 185–189. [6] V. Sorrenti, C. Di Giacomo, A. Campisi, J.R. Perez-Polo, A. Vanella, Nitric oxide synthetase activity in cerebral post-ischemic reperfusion

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