Synthesis and Antioxidant Activity of N-Benzyl-2-[4-(aryl)-1H-1,2,3-triazol-1-yl]ethan-1-imine Oxides

The synthesis, antioxidant capacity, and anti-inflammatory activity of four novel N-benzyl-2-[4-(aryl)-1H-1,2,3-triazol-1-yl]ethan-1-imine oxides 10a–d are reported herein. The nitrones 10a–d were tested for their antioxidant properties and their ability to inhibit soybean lipoxygenase (LOX). Four diverse antioxidant tests were used for in vitro antioxidant assays, namely, interaction with the stable free radical DPPH (1,1-diphenyl-2-picrylhydrazyl radical) as well as with the water-soluble azo compound AAPH (2,2′-azobis(2-amidinopropane) dihydrochloride), competition with DMSO for hydroxyl radicals, and the scavenging of cationic radical ABTS•+ (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) radical cation). Nitrones 10b, 10c, and 10d, having the 4-fluorophenyl, 2,4-difluorophenyl, and 4-fluoro-3-methylphenyl motif, respectively, exhibited high interaction with DPPH (64.5–81% after 20 min; 79–96% after 60 min), whereas nitrone 10a with unfunctionalized phenyl group showed the lowest inhibitory potency (57% after 20 min, 78% after 60 min). Nitrones 10a and 10d, decorated with phenyl and 4-fluoro-3-methylphenyl motif, respectively, appeared the most potent inhibitors of lipid peroxidation. The results obtained from radical cation ABTS•+ were not significant, since all tested compounds 10a–d showed negligible activity (8–46%), much lower than Trolox (91%). Nitrone 10c, bearing the 2,4-difluorophenyl motif, was found to be the most potent LOX inhibitor (IC50 = 10 μM).


Introduction
Oxidative stress (OS) is a state of imbalance between the production and accumulation of free oxygen radicals in cells and tissues and the ability of the antioxidant system to remove them [1].Antioxidants are chemical compounds that effectively neutralize the formation of free radicals.Their task is primarily to protect the body against free radicals, the excess of which may increase the risk of inflammation, arteriosclerosis, heart attacks, stroke, as well as neurodegenerative diseases (e.g., Parkinson's and Alzheimer's), among others [2][3][4].The action of antioxidants is multidirectional, however, and the individual antioxidants differ in their mode of action.They may act either by multiple mechanisms or by a predominant mechanism [5][6][7].Moreover, the biological importance of antioxidants is closely related to understanding the mechanisms of their action, which in turn determines the possibility of their practical use.

Chemistry
Nitrones 10a-d were prepared following the reactions shown in Scheme 1 as previously described [16], by reacting aldehydes 11a-b with N-benzylhydroxylamine.The progress of the reaction was monitored by TLC and the full conversion of the aldehydes 11ab into respective nitrones 10 was achieved within 15 min, at room temperature (rt).All In continuation to our studies to identify new nitrones for the therapy for pathological inflammation and oxidative stress (OS), the nitrones 10a-d (Figure 3), N-benzyl analogs of the previously reported compounds 8 and 9 [16] (Figure 2), have been synthesized with the intention of testing their antioxidant potency.The newly prepared series of compounds contains unfunctionalized nitrone 10a as well as derivatives substituted at the C4 of 1,2,3-triazole moiety with 4-fluorophenyl (10b), 2,4-difluorophenyl (10c), and 4fluoro-3-methylphenyl (10d), which were selected from the previously synthesized nitrones of series 8 and 9 [16] based on their observed antioxidant activity.

Chemistry
Nitrones 10a-d were prepared following the reactions shown in Scheme 1 as previously described [16], by reacting aldehydes 11a-b with N-benzylhydroxylamine.The progress of the reaction was monitored by TLC and the full conversion of the aldehydes 11ab into respective nitrones 10 was achieved within 15 min, at room temperature (rt).All

Chemistry
Nitrones 10a-d were prepared following the reactions shown in Scheme 1 as previously described [16], by reacting aldehydes 11a-b with N-benzylhydroxylamine.The progress of the reaction was monitored by TLC and the full conversion of the aldehydes 11a-b into respective nitrones 10 was achieved within 15 min, at room temperature (rt).All final products were purified by crystallization and their structure and purities were established by 1 H, 13 C, and 19 F NMR (Supplementary Materials, Figures S1-S11), and IR techniques and by elemental analysis (Section 3).In particular, nitrones 10a-d were isolated as pure Zstereoisomers at the double bond (CH=N), as determined and confirmed by the presence of the single sets of the diagnostic signals of the respective protons CH=N (δ = 7.12-7.15ppm) and CH 2 Ph (δ = 4.97-5.00ppm) in the 1 H NMR spectra of 10a-d.Based on a comparison of the literature data for other acyclic nitrones [17], Z-configuration was assigned for (Z)-10ad; however, the corresponding signals for the E-isomeric nitrones necessary to provide full correlation were not observed.
final products were purified by crystallization and their structure and purities were established by 1 H, 13 C, and 19 F NMR (Supplementary Materials, Figures S1-S11), and IR techniques and by elemental analysis (Section 3).In particular, nitrones 10a-d were isolated as pure Z-stereoisomers at the double bond (CH=N), as determined and confirmed by the presence of the single sets of the diagnostic signals of the respective protons CH=N (δ = 7.12-7.15ppm) and CH2Ph (δ = 4.97-5.00ppm) in the 1 H NMR spectra of 10a-d.Based on a comparison of the literature data for other acyclic nitrones [17], Z-configuration was assigned for (Z)-10a-d; however, the corresponding signals for the E-isomeric nitrones necessary to provide full correlation were not observed.Scheme 1. Synthesis of nitrones 10a-d.Reagents and conditions: a. details for preparation of aldehydes 11a-d given in [16]; b.BnNHOH × HCl, CH3CO2Na, rt, 15 min.

In Vitro Antioxidant and Anti-Inflammatory Activity
Herein, we have investigated in vitro the antioxidant evaluation of nitrones 10a-d with regard to their antioxidant ability as well as to their ability to inhibit soybean LOX on several diverse antioxidant tests and in comparison to nordihydroguaiaretic acid (NDGA) and Trolox as standards.All aerobic organisms produce free radicals that can a ack and damage lipids and DNA, inducing neurodegenerative diseases, cancer, and stroke.Since OS and inflammation present a complex character, we decided to evaluate the in vitro antioxidant activity of the synthesized molecules using four different antioxidant assays: All are spectrophotometric measurements which are simple, rapid, and convenient.DPPH is a stable free radical, advantageous for testing compounds in an ethanolic solution, which in its oxidized form presents a maximum absorbance at about 517 nm.The DPPH method is independent of the molecule's polarity.The reducing activity (RA) of the examined compounds with the stable free radical DPPH is given in Table 1.This interaction shows their radical scavenging ability in an iron-free system.Nitrones 10b, 10c, and 10d highly interact with DPPH (64.5-81%) after 20 min, whereas 10a presents a lower value.In general, the insertion of a substituent increases the reducing activity (RA).Thus, starting from 10a, all the other compounds in which one or two fluorine atoms or a fluorine atom and a methyl group are present, exhibit higher activities.It seems that an acceptor, such as a fluorine atom, with small molar refractivity (MR) in the para position offers antioxidant ability.The presence of a second fluorine atom as a substituent, 10c does not influence the interaction values, whereas a methyl group in meta position acting as a donor lowers activity (10b > 10d).RA is not influenced by lipophilicity within this nitrones group.The interaction values are increased for all after 60 min (78-81%), showing time

In Vitro Antioxidant and Anti-Inflammatory Activity
Herein, we have investigated in vitro the antioxidant evaluation of nitrones 10a-d with regard to their antioxidant ability as well as to their ability to inhibit soybean LOX on several diverse antioxidant tests and in comparison to nordihydroguaiaretic acid (NDGA) and Trolox as standards.All aerobic organisms produce free radicals that can attack and damage lipids and DNA, inducing neurodegenerative diseases, cancer, and stroke.Since OS and inflammation present a complex character, we decided to evaluate the in vitro antioxidant activity of the synthesized molecules using four different antioxidant assays: All are spectrophotometric measurements which are simple, rapid, and convenient.DPPH is a stable free radical, advantageous for testing compounds in an ethanolic solution, which in its oxidized form presents a maximum absorbance at about 517 nm.The DPPH method is independent of the molecule's polarity.The reducing activity (RA) of the examined compounds with the stable free radical DPPH is given in Table 1.This interaction shows their radical scavenging ability in an iron-free system.Nitrones 10b, 10c, and 10d highly interact with DPPH (64.5-81%) after 20 min, whereas 10a presents a lower value.In general, the insertion of a substituent increases the reducing activity (RA).Thus, starting from 10a, all the other compounds in which one or two fluorine atoms or a fluorine atom and a methyl group are present, exhibit higher activities.It seems that an acceptor, such as a fluorine atom, with small molar refractivity (MR) in the para position offers antioxidant ability.The presence of a second fluorine atom as a substituent, 10c does not influence the interaction values, whereas a methyl group in meta position acting as a donor lowers activity (10b > 10d).RA is not influenced by lipophilicity within this nitrones group.The interaction values are increased for all after 60 min (78-81%), showing time dependency.For the sake of comparison, NDGA was used as a standard reference compound.In our studies, the water-soluble azo AAPH was used as a thermal free radical initiator to induce the oxidative changes of linoleic acid to conjugated diene hydroperoxide.All nitrones except for 10c presented inhibition values (75-87%) lower than the common standard Trolox (93%) (Table 1).The compounds 10a and 10d are the most potent.Lipophilicity seems to play a significant role related to a positive result since the inserted methyl group in compound 10b (75%) increases the lipophilicity of compound 10d driving it to higher inhibition (87%).Nitrone 10c having two fluorine atoms and a clogP value of 2.24 exhibits the lowest anti-lipid peroxidation activity.
Hydroxyl ( • OH) free radical is counted as the most toxic.As a result, it reacts with important biological molecules such as DNA, lipids, or carbohydrates.We found it interesting to test the scavenging activity of the compounds in competition with DMSO.As shown in Table 1, all the compounds do not exhibit any activity compared to the standard compound Trolox.
In the ABTS •+ decolorization assay, the tested nitrones showed very low activity, except for nitrone 10b which is a mono-substituted fluor derivative.The compounds 10c and 10d exhibit equipotent results, lower activity than 10b (almost the half), and higher lipophilicity values.
We evaluated the synthesized nitrones for their ability to inhibit soybean LOX by the UV absorbance-based enzyme protocol, as shown in Table 1 [18].The appropriate stimulation of neutrophils cleaves arachidonic acid (AA) from membrane phospholipids, producing leukotrienes through lipoxygenase.Leukotriene B4 (LTB4) is a potent mediator of inflammation, considered to be important in the pathogenesis of neutrophil-mediated inflammatory diseases with a marked relation to the severity of cardiovascular diseases, stroke, and cancer [19].The enzyme lipoxygenase catalyzes the first two steps in the metabolism of AA, which is cleaved from membrane phospholipids to leukotrienes (LTB4).LTB4 generation is important in the pathogenesis of neutrophil-mediated inflammatory diseases.NDGA, a known inhibitor of soybean LOX, has been used as a reference compound with IC50 0.45µM.A perusal of the IC50′s inhibition values (Table 1) shows that the most potent inhibitors are the compounds 10c (IC50 10µM), 10b (IC50 62.5 µM), and 10a (IC50 85 µM).Compound 10d presents a lower activity of 45% at 100 µM.The structural moiety that significantly influences the inhibition in compounds 10b and 10c is the fluorine atom.In both compounds, this electronegative substituent is present.The most potent nitrone 10c possesses two fluorine atoms whereas 10b has one.The loss of the second  In our studies, the water-soluble azo AAPH was used as a thermal free radical initiator to induce the oxidative changes of linoleic acid to conjugated diene hydroperoxide.All nitrones except for 10c presented inhibition values (75-87%) lower than the common standard Trolox (93%) (Table 1).The compounds 10a and 10d are the most potent.Lipophilicity seems to play a significant role related to a positive result since the inserted methyl group in compound 10b (75%) increases the lipophilicity of compound 10d driving it to higher inhibition (87%).Nitrone 10c having two fluorine atoms and a clogP value of 2.24 exhibits the lowest anti-lipid peroxidation activity.
Hydroxyl ( • OH) free radical is counted as the most toxic.As a result, it reacts with important biological molecules such as DNA, lipids, or carbohydrates.We found it interesting to test the scavenging activity of the compounds in competition with DMSO.As shown in Table 1, all the compounds do not exhibit any activity compared to the standard compound Trolox.
In the ABTS •+ decolorization assay, the tested nitrones showed very low activity, except for nitrone 10b which is a mono-substituted fluor derivative.The compounds 10c and 10d exhibit equipotent results, lower activity than 10b (almost the half), and higher lipophilicity values.
We evaluated the synthesized nitrones for their ability to inhibit soybean LOX by the UV absorbance-based enzyme protocol, as shown in Table 1 [18].The appropriate stimulation of neutrophils cleaves arachidonic acid (AA) from membrane phospholipids, producing leukotrienes through lipoxygenase.Leukotriene B4 (LTB4) is a potent mediator of inflammation, considered to be important in the pathogenesis of neutrophil-mediated inflammatory diseases with a marked relation to the severity of cardiovascular diseases, stroke, and cancer [19].The enzyme lipoxygenase catalyzes the first two steps in the metabolism of AA, which is cleaved from membrane phospholipids to leukotrienes (LTB4).LTB4 generation is important in the pathogenesis of neutrophil-mediated inflammatory diseases.NDGA, a known inhibitor of soybean LOX, has been used as a reference compound with IC 50 0.45 µM.A perusal of the IC 50 ′ s inhibition values (Table 1) shows that the most potent inhibitors are the compounds 10c (IC 50 10 µM), 10b (IC 50 62.5 µM), and 10a (IC 50 85 µM).Compound 10d presents a lower activity of 45% at 100 µM.The structural moiety that significantly influences the inhibition in compounds 10b and 10c is the fluorine atom.In both compounds, this electronegative substituent is present.The most potent nitrone 10c possesses two fluorine atoms whereas 10b has one.The loss of the second fluorine atom (10b) lowers the activity as well as the absence of nitrone 10a.Substituents with low bulk, such as fluorine, and lipophilic contribution as π values increase the inhibitory activity.The strong inhibition of 10c could be therapeutically useful in stroke or neurodegeneration in combination with the high RA (%).It is worth mentioning that the high efficacy of the fluorinated derivatives of PBN (α-phenyl-N-tert-butyl nitrone), namely 4-F-PBN and 4-CF 3 -PBN, for spin-trapping experiments when compared to PBN has been recently described by Durand and co-workers [20].

Chemistry
General information-The 1 H, 13 C, and NMR spectra were taken in CDCl 3 on the Bruker Avance III spectrometers (600 MHz, Bruker Instruments, Karlsruhe, Germany) with TMS as the internal standard at 600 and 151 MHz, respectively.The 19 F NMR spectra were recorded in CDCl 3 on the Bruker AvanceNEO (Bruker Instruments, Karlsruhe, Germany) at 565 MHz.The IR spectra were measured on an Infinity MI-60 FT-IR spectrometer (Bruker Optik GmbH, Ettlingen, Germany).The melting points were determined on a Boetius apparatus and are uncorrected.The elemental analyses were performed by the Microanalytical Laboratory of this Faculty on the Perkin-Elmer PE 2400 CHNS analyzer (Perkin Elmer Corp., Norwalk, CT, USA).The following adsorbents were used: column chromatography, Merck silica gel 60 (70-230 mesh); analytical TLC, Merck TLC plastic sheets silica gel 60 F 254 (Merck KGaA, Darmstadt, Germany).
The 1 H-, 13 C-, and 19 F-NMR spectra of all the newly synthesized compounds are provided in Supplementary Materials.

General Procedure for the Preparation of Nitrones 10a-d
The respective aldehydes 11a-d, obtained directly from corresponding diethyl acetal according to the previously described procedure [13], were dissolved in ethanol (2 mL), and CH 3 CO 2 Na (1.3 mmol) was added followed by N-benzylhydoxylamine hydrochloride (1.1 mmol).The reaction mixture was stirred until the disappearance of the starting aldehyde was noticed on TLC.After that, 10% NaHCO 3 was added (5 mL) and the product was extracted with methylene chloride (3 × 5 mL).The organic extracts were combined, dried (MgSO 4 ), concentrated, and crystallized from diethyl ether to give the pure nitrone 10a-d.

In Vitro Assays
General biological assays: NDGA, Trolox, AAPH, and DPPH soybean LOX linoleic acid sodium salt were purchased from the Aldrich Chemical Co., Milwaukee, WI, USA.The phosphate buffer (0.1 M and pH 7.4) was prepared by mixing an aqueous KH 2 PO 4 solution (50 mL, 0.2 M), and an aqueous NaOH solution (78 mL, 0.1 M); the pH (7.4) was adjusted by adding a solution of KH 2 PO 4 or NaOH.For the in vitro tests, a Lambda 20 (Perkin-Elmer-PharmaSpec 1700, Perkin-Elmer Corporation Ltd., Lan Beaconsfield, Bucks, UK) UV-Vis double beam spectrophotometer was used.Each in vitro experiment was performed at least in triplicate and the standard deviation of absorbance was less than 10% of the mean.[21] To an ethanolic solution of DPPH (0.05 mM) in absolute ethanol, 10 µL from a stock solution (10 mM) in the DMSO of the compounds was added.The mixture was shaken vigorously and allowed to stand for 20 min or 60 min; absorbance at 517 nm was determined spectrophotometrically and the percentage of activity was calculated.All tests were undertaken on three or four replicates and the results were averaged (Table 1).NDGA was used as a reference compound.

Soybean LOX Inhibition Study
In Vitro [22] The tested compounds dissolved in DMSO were incubated at rt with sodium linoleate (0.1 mL) and 0.2 mL of the enzyme solution (1/9 × 10 −4 w/v in saline) in buffer Tris pH 9. The conversion of sodium linoleate to 13-hydroperoxylinoleic acid at 234 nm was recorded and compared with NDGA, the appropriate standard inhibitor (Table 1).[16] Ten microliters of the 16 mM linoleic acid sodium salt solution were added to the UV cuvette containing 0.93 mL of the 0.05 M phosphate buffer, pH 7.4 prethermostated at 37 • C. The oxidation reaction was initiated at 37 • C under air by the addition of 50 µL of the 40 mM AAPH solution.Oxidation was carried out in the presence of 10 µL of the compounds' stock 10 mM solution in DMSO, in the assay.Lipid oxidation was measured in the presence of the same level of DMSO.The rate of oxidation at 37 • C was recorded as the absorption values at 234 nm and compared to Trolox (Table 1).

Figure 2 .
Figure 2. Structures of the compounds 8 and 9 encountered in the previous work [16].

Figure 3 .
Figure 3. Structures of the nitrones 10a-d reported in the present work.

Figure 2 .
Figure 2. Structures of the compounds 8 and 9 encountered in the previous work [16].

Figure 2 .
Figure 2. Structures of the compounds 8 and 9 encountered in the previous work [16].

Figure 3 .
Figure 3. Structures of the nitrones 10a-d reported in the present work.

Figure 3 .
Figure 3. Structures of the nitrones 10a-d reported in the present work.

Table 1 .
Antioxidant activity of nitrones 10a-d a , and standards Trolox and NDGA.
Int. J. Mol.Sci.2024, 25, x FOR PEER REVIEW 5 of 10 dependency.For the sake of comparison, NDGA was used as a standard reference compound.

Table 1 .
Antioxidant activity of nitrones 10a-d a , and standards Trolox and NDGA.
a nitrones tested at 100 µM.Values are the means of three or four different determinations.no = no activity under the experimental conditions.Means within each column differ significantly (p < 0.05).b BioByte Corporation, C-QSAR database, 201 W Fourth Str., Suite # 204, Claremont CA 91711-4707, USA.nd, not determined.
Values are the means of three or four different determinations.no = no activity under the experimental conditions.Means within each column differ significantly (p < 0.05).b BioByte Corporation, C-QSAR database, 201 W Fourth Str., Suite # 204, Claremont CA 91711-4707, USA.nd, not determined.