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Evaluation of a new benzothiazole derivative with antioxidant activity in the initial phase of acetaminophen toxicity
⁎Corresponding author. Tel.: +52 57296000x62829. marcrh2002@yahoo.com.mx (Martha C. Rosales-Hernández)
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Received: ,
Accepted: ,
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.
Peer review under responsibility of King Saud University.
Abstract
Increasing evidence indicates that benzothiazoles and thioureas have the ability to inactivate reactive chemical species through their antioxidant activity. In this context, we designed and synthesized two benzothiazole-isothiourea derivatives, (E)-5-((benzo[d]thiazol-2-ylimino)(methylthio)methylamino)-2-hydroxybenzoic acid (compound 1) and (S,E)-2-((benzo[d]thiazol-2-ylimino)(methylthio)methylamino)-3-(4-hydroxyphenyl) propanoic acid (compound 2). The 2,2′-diphenyl-1-picrylhydrazyl radical reduction and Fenton reaction were used to evaluate the free radical scavenging activity of both compounds in vitro. The results indicated that compound 1 exhibited the highest scavenging activity. Hence, it was evaluated ex vivo using the initial phase of the acetaminophen-induced hepatotoxicity model. In particular, we demonstrated that compound 1 increased the reduced glutathione content and decreased the malondialdehyde levels. In addition, it was capable of inhibiting cytochrome P450 and producing a protective effect against the reactive intermediary N-acetyl-p-benzoquinoneimine.
Keywords
Benzothiazole derivatives
Isothiourea derivatives
Antioxidants
Acetaminophen
Cytochrome P-450
Hepatotoxicity
1 Introduction
Acetaminophen (APAP) is one of the most widely used drugs with analgesic and antipyretic properties. Although APAP is safe at therapeutic doses, it is well known that it causes acute hepatic centrilobular necrosis when consumed in large doses (Hazai et al., 2002). APAP overdose toxicity occurs in two phases. In the initial phase (0–2 h), this drug is bio-activated by cytochrome P-450 (CYP450) to form N-acetyl-p-benzoquinoneimine (NAPQI), a highly reactive electrophilic intermediary that is capable of depleting reduced glutathione (GSH) and increasing the formation of adducts with several proteins through covalent interactions. In the second phase (3–5 h), the APAP-dependent toxicity is determined by the mitochondrial permeability transition (MPT) and the release of alanine transaminase (ALT) (Bhattacharyya et al., 2013; Burke et al., 2010; Ji and Schüürmann, 2015; Zhang et al., 2015). It has been postulated that the production of NAPQI is involved with the development of oxidative stress because this reactive intermediary increases the production of reactive oxygen species (ROS), such as superoxide anion (O2•−) and hydrogen peroxide (H2O2), during the oxide-reduced CYP450 cycle. Then, these ROS may be converted to the hydroxyl radical (•OH) via the Fenton reaction in the presence of trace amounts of metal ions (James et al., 2003a; Namazi, 2009; Southorn and Powis, 1988), contributing to hepatocellular damage (Randle et al., 2008). Therefore, the •OH radical is capable of attacking the lipids in cellular membranes, favoring the formation of a carbon-centered radical (L•) that rapidly reacts with oxygen to form a lipid peroxy radical (LOO•); this reaction mechanism leads to the formation of aldehydes (malondialdehyde (MDA)) as by-products of lipid peroxidation (Kalyanaraman, 2013), with concomitant changes in the catalytic functions of several antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) (Singh et al., 2009).
N-acetylcysteine (NAC) (Fig. 1A) can be used to counteract the effects of the ROS generated during APAP metabolism because it promotes the intracellular synthesis of GSH and eliminates the electrophiles through conjugation and reduction reactions (Woodhead et al., 2012; Zembron et al., 2009). Nevertheless, NAC is not very effective because it depends on the APAP dose consumed (Carvalho et al., 2013). In this sense, it has been suggested that the use of antioxidants, such as polyphenols (Fig. 1B), carotenoids and vitamins (Chen et al., 2009; Uttara et al., 2009), could be a good alternative therapy to counteract the ROS-mediated APAP-induced toxicity. However, the antioxidants that have been used have several limitations, as they must be administered at high doses due to their low absorption and bioavailability (Thilakarathna and Rupasinghe, 2013).
Chemical structures of the antioxidant compounds derived from the thiol (A), polyphenol (B), and benzothiazole groups (C).
In this context, thioureas (Lima da Silva et al., 2015; Sudhamani et al., 2015) and several benzothiazoles (Keri et al., 2015) have been reported to be antioxidant agents. It has been demonstrated that benzothiazole derivatives have potential applications in several fields, including those in which high chemical and photophysical stabilities are required (Hrobárik et al., 2014, 2011, 2010), as well as those in which nitrogen and sulfur-containing heterocycles play an important role (Keri et al., 2015). It has been postulated that the good chemical properties of benzothiazoles are related to the electron-withdrawing ability of the thiazole ring, which acts as the edge substituent (Hrobárik et al., 2014, 2011, 2010). The benzothiazoles have also shown favorable results in radiotherapy due to their capability to inhibit the •OH radical (Fig. 1C) (Durgamma et al., 2014; Prouillac et al., 2009; Temiz et al., 2006). In addition, these heterocyclic compounds have inhibitory effects on enzymes, such as protoporphyrinogen oxidase, which can produce free radicals that result in concomitant toxicological effects on plants and animals (Jiang et al., 2011, 2010; Wu et al., 2014; Yang et al., 2013).
Considering that benzothiazoles, isothioureas and phenols exhibit multiple pharmacological properties, including antioxidant effects, in this work, we report the design and synthesis of two S-methylisothiourea-benzothiazole derivatives (compounds 1 and 2) with the aim of investigating whether the presence of these three groups in the same molecule produces an increased antioxidant effect. We used 5-aminosalicylic acid (5-ASA) and l-tyrosine (l-tyr) as phenols due to their ability to scavenge free radicals (Kanski et al., 2001; Molnár et al., 2015). The free radical scavenging activity of compounds 1 and 2 was evaluated in vitro using the 2,2′-diphenyl-1-picryl hydrazil (DPPH) reduction method and the Fenton reaction. Subsequently, the effect of compound 1 on ROS production during oxidative stress in the APAP overdose model was determined by quantifying the changes in the levels of MDA and GSH, as well as the changes in GPx activity. Additionally, we determined whether compound 1 exerts some effect on CYP450 activity. Finally, a possible mechanism of action of compound 1 was suggested.
2 Materials and methods
2.1 Synthesis
5-ASA (95%), l-tyr (99%) and DMSO (99%) were purchased from commercial sources (Sigma–Aldrich), and dimethyl N- dimethyl benzothiazol-2-ylcarbonimidodithioate (DBT) was prepared using the previously reported procedures (Merchán et al., 1982).
The melting points were measured on an Electrothermal IA 91000 apparatus (Electrothermal, Bibby Scientific, Staffordshire, ST15 OSA, UK) and were uncorrected. The 1H and 13C Nuclear Magnetic Resonance spectra (NMR) were obtained on a Varian Mercury 300 MHz spectrometer (Varian, Inc., Palo Alto, California 94304, USA) in deuterated dimethylsulfoxide (DMSO) solutions. The infrared (IR) spectra were recorded digitally on computer disks using an FT-IR Spectrum PerkinElmer GX SYSTEM (PerkinElmer, Shelton, Connecticut 06484-4794, USA) and further analyzed in the Origin program, version 6.0. Electrospray Ionization (ESI) High Resolution Mass Spectrometry was performed with a Bruker micrOTOF-Q II instrument (Bruker Daltonik GmbH, Bremen, Germany). The antioxidant capacity was analyzed with an ultraviolet–visible spectrophotometer (UV–vis) PerkinElmer LAMBDA 25 (Waltham, Massachusetts 02451, USA) and a Bruker BioSpin E-SCAN Electron Paramagnetic Resonance (EPR) Spectrometer (Bruker Biospin GmbH, D-76287 Rheinstetten/Karlsruhe, Germany). The ALT and MDA levels, the GSH and protein contents, and the GPx and GR catalytic activities were determined by UV–vis spectrophotometry in a Bio-Rad ELECTRA device (Bio-Rad Laboratories, Inc., Philadelphia, Pennsylvania 19103, USA).
2.1.1 Synthesis of (E)-5-((benzo[d]thiazol-2-ylimino)(methylthio)methylamino)-2-hydroxybenzoic acid (compound 1)
Sodium hydroxide (NaOH) (308.4 mg; 7.71 mmol) was dissolved in DMSO (30 mL), and 5-ASA (1180.6 mg; 7.71 mmol) was added. The reaction mixture was incubated at 95 ± 5 °C for 3 h, and DBT (2000 mg; 7.71 mmol) was incorporated. The reaction was monitored by TLC, and after 24 h, the reaction mixture was cooled to room temperature (RT). Then, 30 mL of distilled water was added and concentrated hydrochloric acid (35%) was added drop-wise until a brown solid appeared which was re-dissolved in excess HCl. The obtained solid was filtered and washed with distilled water. Purity 99%, yield 87%, red solid, mp: 121–122 °C. IR (ATR, ZnSe) νmax/cm−1: 3206, 3065, 2958 (XH, X = O, N), 1619 (m, C⚌O), 1597 (m, C⚌N), 1544 (s, C⚌N, Ar). 1H NMR (300 MHz, DMSO-d6): δ 11.4 (br, 1H, NH), 7.84 (d, 1H, J = 7.9 Hz, H-7), 7.68 (br, 2H, H-14, H-4), 7.54 (br, 1H, H-18), 7.36 (t, 1H, 3J = 7.6 Hz, H-5), 7.23 (t, 1H, 3J = 7.7, 7.3 Hz, H-6), 6.96 (d, 1H, J = 8.5 Hz, H-17), 2.50 (s, 3H, SCH3). The signals for COOH and OH were not observed (Fig. 2A). 13C NMR (75 MHz, DMSO-d6): δ 172.0 (COOH), 171 (br, C-11), 161 (br, C-2), 151 (br, C-9), 134 (br, C-18), 132 (br, C-8), 128 (br, C-13), 126.7 (C-5), 124.1 (br, C-6), 122.3 (br, C-7), 121 (br, C-14), 118.3 (C-17), 114.3 (br, C-4), 14.8 (SCH3). C-15 and C-16 were not observed. m/z: 360.0503 [M−H]+.
Numbers of the atoms in compound 1 (A) and compound 2 (B).
2.1.2 Synthesis of (S,E)-2-((benzo[d]thiazol-2-ylimino)(methylthio)methylamino)-3-(4-hydroxypheyl)-propanoic acid (compound 2)
NaOH (308.4 mg; 7.71 mmol) was suspended in 30 mL of ethanol, and then l-tyrosine (210.6 mg; 7.71 mmol) was added and the mixture was refluxed for 2 h. After cooling to room temperature, DBT (2000.0 mg; 7.71 mmol) was incorporated, and the reaction was heated to reflux temperature over 24 h and then filtered at RT. The solvent was evaporated from the solution, and the resulting sticky yellow solid was washed with chloroform until the solution was free from DBT, as determined by TLC. The remaining vitreous pale yellow solid was dried at RT. Purity 98%, yield 87%, mp: 100-102 °C. IR (ATR, ZnSe) νmax/cm−1: 3600, 3400, 3200 (XH, X = O, N); 1591 (m, C⚌O), 1546 (s, C⚌N, Ar), 1512 (m, C⚌N). 1H NMR (300 MHz, DMSO-d6): δ 10.70 (d, 1H, 3J = 8.2, NH), 9.4 (br, 1H, COOH), 8.33 (s, 1H, OH), 7.74 (d, 1H, 3J = 7.9 Hz, H-7), 7.55 (d, 1H, 3J = 8.2 Hz, H-4), 7.35 (t, 1H, 3J = 7.3 Hz, H-5), 7.20 (t, 1H, 3J = 7.9, 7.3, H-6), 7.04 (d, 2H, J = 8.2 Hz, H-16), 6.61 (d, 2H, J = 8.5 Hz, H-17), 4.03 (dt, 1H, 3J = 7.0 Hz, CH), 3.02 (AA’, 2H, CH2), 2.50 (s, 3H, SCH3) (Fig. 2B). 13C NMR (75 MHz, DMSO-d6): δ 171.5 (COOH), 170.8 (C-11), 163.8 (C-2), 155.7 (C-18), 151.1 (C-9), 131.1 (C-15), 130.2 (2C, C-16), 128.1 (C-8), 125.5 (C4), 122.8 (C-7), 121.0 (C-5), 119.8 (C-6), 114.7 (2C, C-17), 79.1 (C-13), 60.9 (C-14), 13.5 (SCH3). m/z: 388.0615 [M−H]+.
2.2 In vitro evaluation of the free radical scavenger capacity
2.2.1 Reduction of the radical 2,2′-diphenyl-1-picrylhydrazil (DPPH)
The DPPH radical-scavenging activity was performed according to the method described by Balaji et al. (2005). Initially, a 5-ASA stock solution (0.408 mM) was sequentially diluted with ethanol six times at 1:2 each. Then, an aliquot of each dilution (2 mL) was mixed with a DPPH solution in DMSO (2 mL, 30 μM). The mixtures were vigorously shaken and incubated for 60 min at RT in the dark. At the same time, a control containing each dilution of 5-ASA (2 mL) was mixed with DMSO. The absorbance was measured at 517 nm against DMSO. The percentage of DPPH radical scavenging (%DPPH-RSA) was determined according to the following equation:%DPPH-RSA = [1 − (Ai − Aj)/Ac] ∗ 100, where Ai is the absorbance of the mixture of 5-ASA or the other compounds and DPPH solutions, Aj is the absorbance of the 5-ASA solution with DMSO, and Ac is the absorbance of the DPPH solution with DMSO. Equimolar amounts of compounds 1 and 2 were evaluated using 5-ASA as a reference compound.
2.2.2 Fenton reaction assay
The formation of the adduct between N-tert-butyl-α-phenylnitrone (PBN; spin trapping) and the •OH radical was evaluated by Electron Paramagnetic Resonance (EPR). The Fenton reaction was performed to generate •OH in aqueous solutions in a final volume of 2500 μL. Solutions of FeCl2 in dichloromethane (0.32 mM) and PBN in DMSO (3 mM) were used. The reaction was initiated by the addition of H2O2 (0.5 M) and immediately transferred to capillary tubes (0.8–1.1 mm) (Méndez-Garrido et al., 2014). The EPR spectra were obtained using a Bruker E-SCAN-EPR spectrometer using a microwave frequency of 9.74 GHz (Band X9) at room temperature with the following EPR spectrometer settings: 3475.5 G, field center; potency, 0.872 mW; modulation, 1.10 G; 2 × 102, gain; 5.24 s, scan time; 10.24 ms, conversion; and 20 accumulations. The paramagnetic species was quantified by measuring the area under the curve (AUC) using the WINEPR program and the gyroscopic (g) factor (near g = 2.0032), which indicates the presence of free radicals. The •OH radical-scavenging activities of compounds 1 and 2 were determined in equimolar concentrations compared to 5-ASA (0.102 mM).
2.3 Ex vivo evaluation
2.3.1 Animals
Four-to-five-week-old male ICR mice were used. The animals were housed in polypropylene cages under controlled temperature and humidity conditions (22 °C ± 3 °C, 50% humidity) with free access to water and food (Purina rat chow). The animal procedures were conducted in accordance with the Mexican Official Standard NOM-062-ZOO-1999, the Technical Specifications for the Production, and the Care and Use of Laboratory Animals. The animal protocol was approved by the Research Committee for the Care and Use of Laboratory Animals (CICUAL) of the Escuela Superior de Medicina-Instituto Politécnico Nacional.
2.3.2 Drug administration protocols and tissue collection
After one week of adaptation, the mice were divided into four groups (n = 3 members for group). Group 1 was designated as the control group and was administered the vehicle (saline) through an intraperitoneal (i.p.) injection. The mice in Group 2 received a single APAP dose (500 mg/kg in 100 μL of saline, pH 9, i.p) (Ruepp et al., 2002). Group 3 received a single dose of NAC (1200 mg/kg in 100 μL of saline solution, i.p.) (James et al., 2003b) 0.5 h after APAP administration (500 mg/kg in 100 μL of saline, pH 9, i.p). The animals in Group 4 received an equimolar dose of compound 1 with respect to NAC 0.5 h after APAP administration (500 mg/kg in 100 μL of saline, pH 9, i.p). All animals were anesthetized using sodium pentobarbital (60 mg/kg, i.p.). The animals in Groups 1 and 2 were sacrificed 1 h after the administration, while the animals in Groups 3 and 4 were sacrificed 1.5 h after their corresponding treatments. The blood samples were collected by heart puncture, and the plasma was separated by centrifugation. Subsequently, the liver was perfused with PBS solution, pH 7.4, and it was dissected in various portions, which were washed with cold saline and were stored in separate Eppendorf vials at −80 °C for the subsequent development of the different biochemical assays.
2.3.3 Determination of hepatic lipid peroxidation
Lipid peroxidation was determined by quantifying the thiobarbituric acid-reactive substances (TBARS) (Ohkawa et al., 1979). One aliquot (75 μL) of homogenized tissue (0.1 ± 0.005 g per mL of water) was diluted with Tris-HCl buffer, pH 7.4 (175 μL; 0.15 M). After 0.5 h of incubation at 37 °C, 500 μL of thiobarbituric acid (0.3% (w/v)) dissolved in trichloroacetic acid (15% (w/v)) was added. Then, the samples were boiled for 1 h and centrifuged at 6000 rpm for 15 min. The level of lipid peroxidation is expressed as μmol/mg of protein of MDA at 540 nm.
2.3.4 Determination of the hepatic reduced glutathione (GSH) levels
The intracellular GSH levels were quantified by the reduction of the 5,5′-dithiobis-2-nitrobenzoic acid (DTNB) to 2-nitro-5-thiobenzoic acid (TNB), with some modifications (Ellman, 1959; Owens and Belcher, 1965). Portions of the liver tissue (0.05 ± 0.005 g) were homogenized in a metaphosphoric acid (MPA) solution (2.5 mL; 3% (w/v)). Then, the samples were centrifuged at 4500 rpm for 15 min, and a supernatant aliquot (167 μL) was diluted in phosphate buffer, pH 8 (500 μl; 0.1 M). Finally, DTNB (10 μL) was added, and the GSH levels were measured at 415 nm. The GSH content is expressed as μmol/mg of protein.
2.3.5 Determination of the catalytic activity of hepatic glutathione peroxidase (GPx) and glutathione reductase (GR)
The GPx activity was quantified by NADPH oxidation at 412 nm in the presence of cumene hydroxide using the Cayman Chemical kit (Item number: 703102). The GR and GPx activities are expressed as nmol/min/mg of protein. The GR activity was measured directly in hepatic homogenates (0.200 ± 0.005 g per mL) by the decrease in absorbance at 340 nm after NADPH oxidation using a kit from Cayman Chemical Co. (Item number: 703202).
2.3.6 Measurement of the serum alanine aminotransferase (ALT) levels
The ALT levels were quantified by NADPH oxidation at 340 nm in a coupled reaction with lactate dehydrogenase, according to the ALT1268 manual (Randox Laboratories). The ALT activity is expressed in U/L.
2.3.7 Preparation of hepatic microsomes
The hepatic microsomes were obtained as previously described (Kamath et al., 1971; Manno et al., 1991; Sampson and Karler, 1963). The liver sections were homogenized with 2–3 volumes of sucrose (0.25 M). The homogenate was centrifuged at 8500 rpm for 16 min at 4 °C, and the sediment was washed and mixed with CaCl2 (80 mM) in a 1:10 ratio and centrifuged at 13,500 rpm for 28 min at 4 °C. The pellet (sediment) was washed with KCl (0.15 M) and centrifuged again at 13,500 rpm for 10 min at 4 °C. The liver microsomes were resuspended in phosphate buffer (0.1 M, pH 7.4) containing glycerol (20% (v/v)) and were stored at −80 °C.
2.3.8 Determination of the presence of CYP450
The presence of CYP450 in the hepatic microsomes was determined using the method described by Omura and Sato (1964). A diluted sample of microsomes (1:10) in phosphate buffer, pH 7.4 (0.1 mM), containing glycerol (20% (v/v)) was mixed with 90 mg of sodium hydrosulfite (Na2S2O4). The samples were bubbled with high purity carbon monoxide for approximately 10 s. Finally, the presence of CYP450 was determined by its characteristic absorption peak at 450 nm on a UV–vis spectrophotometer that had previously been calibrated.
2.3.9 Quantification of the total protein content
The total protein content was quantified at 595 nm using the Bradford method and a Protein Assay kit from Cayman Chemical Co. (Item number: 704002).
2.4 Statistics
All data were expressed as the means ± standard error of the mean (SEM). For the normally distributed data, statistical significance was evaluated by one-way analysis of variance (ANOVA), followed by the Holm Sidak test. A value of P < 0.05 was considered to be a significant difference between groups.
3 Results
3.1 Chemistry
Compounds 1 and 2 were synthesized by nucleophilic substitution of the amine group from the sodium salts of 5-ASA and l-tyr to DBT, with the displacement of one molecule of MeSH as the leaving group. The reaction scheme is shown in Fig. 3. The amino groups of both 5-ASA and l-tyr were deprotonated by sodium hydroxide to obtain the corresponding sodium carboxylate salts; the reaction solvent was selected based on their solubility properties. In the last step, HCl was added to obtain the final protonated compound 1, whereas in the case of compound 2, acid-base equilibria were presumably established with the water present in the ethanolic media (95%). Thus, the additional acid was not required.![Scheme of the synthesis of (E)-5-((benzo[d]thiazol-2-ylimino)(methylthio)methylamino)-2-hydroxybenzoic acid (compound 1) and (S,E)-2-((benzo[d]thiazol-2-ylimino)(methylthio)methylamino)-3-(4-hydroxypheyl)propanoic acid (compound 2).](/content/184/2019/12/8/img/10.1016_j.arabjc.2016.02.004-fig3.png)
Scheme of the synthesis of (E)-5-((benzo[d]thiazol-2-ylimino)(methylthio)methylamino)-2-hydroxybenzoic acid (compound 1) and (S,E)-2-((benzo[d]thiazol-2-ylimino)(methylthio)methylamino)-3-(4-hydroxypheyl)propanoic acid (compound 2).
In previous reports with similar compounds, we have found that this reaction is stereospecific. The formation of an intramolecular hydrogen bond N—H⋯N (amine proton as the donor and heterocyclic nitrogen atom as the acceptor) leads to the formation of the E-isomer (Cruz et al., 2008, 2012, 2014). The structures of compounds 1 and 2 were elucidated by IR, 1H and 13C NMR, and MS. In general, the IR spectra of compounds 1 and 2 showed XH (X = N, O) absorption bands in the range of 3206–2958 and 3600–3200 cm−1, respectively, whereas the typical stretching of the C⚌O and C⚌N bands is observed in the corresponding ranges of 1619–1591 cm−1 and 1544–1546 cm−1, respectively. The frequency for C⚌N is in the expected range for similar compounds (Cruz et al., 2012, 2008). In the 1H NMR spectra, the amine proton signals of compounds 1 and 2 appear at δ 11.4 and 10.7, respectively, whereas the imine carbons are at approximately δ 171 in the 13C NMR spectra of both compounds. The 13C chemical shift (δ) for the imine carbon atom is in the expected range for similar compounds (Cruz et al., 2012, 2008). The signal for the remaining -SCH3 group appears at δ 2.50 as a singlet that integrates three protons in the 1H spectra of both compounds, and at δ 14.8 for compound 1 and 13.5 for compound 2 in the 13C spectra. Furthermore, the aliphatic proton signals in compound 2 appear at δ 3.02 (CH2) and 4.03 (CH), whose corresponding 13C absorptions are at δ 60.9 and 79.1, respectively. The mass spectrometric data ([M−H]− = 360.0503 m/z, 1; 388.0615 m/z, 2) are in agreement with the proposed structures.
Some specific spectroscopic details are in agreement with the E-configuration around the exocyclic C⚌N bond and the intramolecular hydrogen-bonded structures proposed in Fig. 2. In both the 1H and 13C NMR spectra of compound 1, many signals appear to be broad, mainly those corresponding to C-2, C-11 and to the aminosalicylic ring. This result suggests that the rotation of the N2-C13 bond is restricted, which is presumably caused by N12—H⋯N3 hydrogen bonding. The low field shift of the NH resonance, which appears as a broad signal at δ 11.4, is in agreement with this proposal and with the presence of a tautomeric equilibrium typical of azole heterocycles (Fig. 4). It is worth mentioning that the absence of the COOH and OH signals in 1H NMR spectrum is in agreement with an exchange of these protons due to the small water content in DMSO-d6.
Tautomeric equilibrium of compound 2.
In contrast, the XH (X = N, O) signals in compound 2 are very well differentiated. The NH is observed as a low field doublet at δ 10.70, the COOH at δ 9.4 is a broad signal and the OH at δ 8.33 is a sharp singlet. These results suggest a limited exchange among them and support the hypothesis that the NH is involved in the formation of a three-centered hydrogen bond, N3⋯H12⋯O (Gómez-Castro et al., 2014), with the heterocyclic N and carbonyl O atoms, as depicted in Fig. 2.
3.2 Free radical scavenging activity in vitro
3.2.1 Reduction of the radical 2,2′-diphenyl-1-picrylhydrazil (DPPH)
Table 1 shows that the %DPPH-RSA values are concentration-dependent. The scavenging activity of compound 1 was 87% at 0.051 mM, while NAC, l-tyr, DBT and compound 2 exhibit a decreased ability to reduce DPPH. However, at 0.102 mM, 5-ASA has a higher %DPPH-RSA against NAC. Additionally, l-tyr, DBT and compound 2 were not able to scavenge the DPPH radical at any concentration. In contrast, compound 1 showed a greater capacity to reduce the DPPH radical at all concentrations compared to 5-ASA and NAC.
Compound (mM)
0.013
0.026
0.051
0.102
0.204
0.408
%DPPH-RSA, 1 h
NAC
35
43
49
57
65
70
5-ASA
27
32
49
71
79
78
L-tyr
1
2
1
2
2
5
DBT
10
10
11
10
8
9
Compound 1
37
53
87
92
94
96
Compound 2
2
2
6
7
8
15
3.2.2 Fenton reaction
When the Fenton reaction was complete, the PBN-•OH adduct signal (Fig. 5A) was obtained, as shown in Fig. 5B. This signal corresponds to a hyperfine structure of a triplet of doublets (Méndez-Garrido et al., 2014), with gyroscopic factor values of g = 2.00802, g = 1.9994, and g = 1.99192 and hyperfine coupling constant values (mT) of aN = 1.3900, aH = 0.2376 and aN/aH = 5.8505. The triplet corresponds to the interaction between an unpaired electron and the nitrogen nuclear spin (Spin = 1, I = 1; 2I + 1), whereas the doublet corresponds to the interaction between an unpaired electron and the hydrogen nuclear spin (Spin = ½, I = ½; 2I + 1). Fig. 5C shows a similar spectral pattern for the PBN-•OH adduct. However, the adduct intensity and the AUC (undetermined) decrease in the presence of 5-ASA, l-tyr and DBT compared to the Fenton reaction alone and the Fenton reaction with NAC (Fig. 5D). These results suggest that 5-ASA, l-tyr and DBT have better •OH scavenging activity than NAC. In addition, the EPR measurements of compounds 1 and 2 showed that both were better scavengers of the •OH radical than 5-ASA, l-tyr, DBT, and NAC because the corresponding EPR signals were not well defined and it was difficult to determine the AUC (Fig. 5D).
PBN-•OH adduct formation and its EPR spectra in the different samples. The interaction between the •OH radical and PBN to form the PNB-•OH adduct (A) and the EPR spectrum of the PNB-•OH adduct (B). EPR spectra of the Fenton reaction (1) and the Fenton reactions with NAC (2), 5-ASA (3), l-tyr (4), DBT (5), compound 1 (6), and compound 2 (7). All compounds were added to separate reactions (C). The AUC values obtained from the Fenton reaction alone, or in the presence of NAC, 5-ASA, l-tyr, DBT, and compounds 1 and 2 (D).
3.3 Ex vivo evaluation
3.3.1 Hepatic lipid peroxidation
The MDA levels were determined in the control group and in the groups treated with APAP (Group 2), APAP + NAC (Group 3) and APAP + compound 1 (Group 4). The MDA levels in the APAP group were significantly increased (3.34 μmol/mg protein ± 0.70) compared to the control group (0.70 μmol/mg protein ± 0.09) (Fig. 6A). According to our results, in the NAC-treated group, the MDA levels decreased after 50 min of treatment (2.14 μmol/mg protein ± 0.02) compared to the group treated with APAP alone. Furthermore, the group treated with compound 1 (1.60 μmol/mg of protein ± 0.10) showed a tendency to restore the MDA levels to those of the control group (0.70 μmol/mg of protein ± 0.09) (Fig. 6A).
Ex vivo effects on lipid peroxidation (LPO) (A), the reduced glutathione (GSH) levels (B), and glutathione peroxidase (GPx) (C) and glutathione reductase (GR) activities (D). The MDA and GSH levels are expressed as μmol/mg of protein. The GPx and GR activities are expressed as nmol/min/mg of protein. The differences in the means of the groups of animals were expressed as follows: MDA ∗ p < 0.05 (APAP vs APAP + NAC and control) and GSH ∗ p < 0.05 (APAP + compound 1 vs control, APAP and APAP + NAC); ∗∗ p < 0.05 (APAP + NAC vs APAP) ∗∗∗ p < 0.05 (control vs APAP).
3.3.2 Determination of the hepatic reduced glutathione (GSH)
The GSH levels were determined in the control group and the groups treated with APAP, APAP + NAC and APAP + compound 1. As depicted in Fig. 5B, there was a significant decrease in the GSH content in the APAP-treated group (1.67 μmol mg of protein ± 0.37) compared to the control group (6.60 μmol mg of protein ± 0.68). The GSH levels in the NAC-treated group were increased after the APAP administration (8.39 μmol/mg of protein ± 0.19). However, the group treated with compound 1 showed a significant increase in the GSH content (43.71 μmol/mg protein ± 1.51) compared to the NAC-treated group (Fig. 6B).
3.3.3 Catalytic activity of hepatic glutathione peroxidase (GPx) and glutathione reductase (GR)
Our results suggest that the GPx (27.08 nmol/min/mg of protein ± 7.82) and GR (1.61 nmol/min/mg of protein ± 0.45) activities in the APAP-treated group were increased compared to the control group; however, there were no significant differences between the groups (Fig. 6C and D). Furthermore, the same figures show that GPx (8.97 nmol/min/mg of protein ± 0.91) and GR (1.19 nmol/min/mg of protein ± 0.17) activities in the NAC-treated group were similar to the control group. In contrast, the GPx (70.94 nmol/min/mg of protein ± 33.48) and GR (2.66 nmol/min/mg of protein ± 1.10) activities of the group treated with compound 1 were higher than the groups treated with vehicle, NAC and APAP. However, there were no significant differences between groups.
3.3.4 Serum alanine aminotransferase (ALT) levels
The ALT activity in the APAP-treated group (24 ± 5.4 U/L) was not significantly different from the saline-treated group (24 ± 4.5 U/L) after 1 h of APAP action. In the APAP-NAC-treated group, the ALT levels were significantly increased 50 min after the NAC treatment (82 ± 29.0 U/L). Meanwhile, the compound 1-treated group exhibited significantly reduced ALT levels (14 ± 12 U/L).
3.3.5 Presence of CYP450
Fig. 7 shows the UV–vis spectrum corresponding to the complex between CYP450 and carbon monoxide in the hepatic microsomes of the control group, which presents a maximum absorbance (Soret peak) at 450 nm. In addition, in the same figure, it can be observed that the characteristic peak, which is indicative of the CYP450 activity, is missing in the groups treated with compound 1 and NAC, suggesting that this hemoprotein may have been inhibited by both compounds. The loss of this peak is associated with reduced CYP450 activity and a conversion to a form with a maximum wavelength at 420 nm, as is observed for the samples belonging to the groups treated with compound 1 and NAC. However, this was not observed in the group treated with APAP alone.
UV–vis spectra of the CYP450-CO complex depicting the presence of CYP450 at a wavelength of 450 nm.
4 Discussion
Different studies in humans and animals show that the APAP toxicity during the first 2 h (initial phase) is determined by a decrease in the GSH levels and the production of adducts between cysteine residues and NAPQI ((cystein-S-yl)-NAPQI-protein), but the ALT levels are not changed (Dai et al., 2006; Li et al., 2013).
In this study, the administration of a single dose of APAP (500 mg/kg) causes a significant decrease in the GSH content after 1 h, but there are no apparent changes in the ALT levels compared to the control group. The NAPQI-induced decrease in the GSH content produced oxidized glutathione (GSSG) (Agarwal et al., 2012) and increased the GPx and GR activities, which are similar to reported values (Ferret et al., 2001) with a concomitant loss of its cytoprotective effect because these enzymes play an important role in the regulation of the GSH/GSSG levels (Aoyama and Nakaki, 2015; Brigelius and Flohé, 2003; Wu et al., 2004). Although these enzymes are responsible for maintaining the GSH levels when ROS are overproduced, the use of exogenous antioxidants is necessary to counteract the oxidative stress produced by an APAP overdose.
NAC is the most effective treatment against APAP overdose because it stimulates GSH synthesis (Acharya and Lau-Cam, 2010; Saito et al., 2010) and inhibits CYP450 similar to other thiol molecules (Conaway et al., 2001). However, high doses cause an imbalance in cellular homeostasis as a result of the excess production of ROS, including O2•−, H2O2 and •OH (Silva et al., 2008), cell lysis (Hallé et al., 2011) and the concomitant release of MDA and ALT. According to our results, NAC administration (1200 mg/kg) after the APAP overdose does not modify the hepatic GSH levels and GPx and GR activities compared to the control group. However, it increases the MDA and ALT levels, suggesting that the liver was damaged by oxidative stress. Therefore, the effectiveness of NAC is dose- and time-dependent, as previously reported (Gómez et al., 2013; Zhang et al., 2013).
Hence, due to the complications that can be presented with NAC treatment, the identification of new molecules is justified. In this study, we propose to combine benzothiazole, thiourea and phenols in the same molecule using S-methylisothiourea-benzothiazole as a core and 5-ASA and l-tyr as phenols due to their ability to scavenge free radicals (Kanski et al., 2001; Molnár et al., 2015). Thus, compounds (E)-5-((benzo[d]thiazol-2-ylimino)(methylthio)methylamino)-2-hydroxybenzoic acid (1) and (S,E)-2-((benzo[d]thiazol-2-ylimino)(methylthio)methylamino)-3-(4-hydroxypheyl)-propanoic acid (2) were synthesized, and their structures were confirmed by IR, 1H and 13C NMR spectroscopy, as well as mass spectrometry.
The results showed that equimolar doses of compound 1 and NAC significantly decreased the MDA levels after APAP administration compared to those of NAC, suggesting that compound 1 is capable of inactivating the ROS generated during APAP metabolism. The protective effect of compound 1 may be due to its ability to donate an electron. Fig. 7A shows the possible stabilization mechanism of the new phenoxy radical generated by compound 1 during the scavenging of ROS. The free radical is initially stabilized by resonance into the aromatic ring of 5-ASA (Ramírez-Durán et al., 2013); this is followed by tautomeric equilibrium, which involves the migration of a hydrogen atom between the isothiourea and the five-membered ring of the benzothiazole, which are present in the chemical structure of compound 1. In contrast, this effect was not evident for compound 2 because the new phenoxyl radical is only stabilized in the aromatic ring of l-tyr due to the interruption of the resonance caused by the presence of a saturated chain in this compound (Fig. 8B).
Possible mechanisms of stabilization of the new radicals obtained from compounds 1 (A) and 2 (B).
The EPR results from the Fenton reaction showed that compound 1 decreases the signal of the PBN-•OH adduct compared to compound 2. Additionally, it was shown that compound 1 is a better antioxidant than 5-ASA and NAC because it stabilizes the DPPH radical by 86% at 0.051 mM (18 μg/mL), suggesting that it is a good scavenger of ROS and is even better than other compounds, such as benzothiazole ester (40%), blackberry extracts (28%) and vitamin C (86%). Higher concentrations (100 μg/mL) of these substances are required to stabilize the DPPH radical to the same level as compound 1 (Bhat and Belagali, 2014; Gawron et al., 2012). Hence, the antioxidant effects of compound 1 are attributed to its chemical properties, as mentioned above, and these effects were not observed for compound 2, which was not administered in vivo.
On the other hand, the results showed that an equimolar dose of compound 1, in relation to NAC, after the APAP administration, significantly increased the GSH levels. Therefore, it is possible that compound 1 contributes to the overexpression of the gamma-glutamylcysteine synthase (GCS), an enzyme that participates in the GSH synthesis. The increase in the GCS levels would explain the significant increase in the levels of this tripeptide, which has also been observed following treatment with resveratrol and pterostilbene (Ghazali et al., 2012).
In addition to the antioxidant effect exhibited by compound 1, this study demonstrated its ability to inhibit CYP450 activity, as shown by the loss of the Soret peak at 450 nm and its conversion to a form with a maximum absorbance at 420 nm (Guengerich et al., 2009). As a result, NAPQI and ROS overproduction are prevented, consequently avoiding the propagation of lipid peroxidation.
5 Conclusions
In conclusion, the structures of compounds 1 and 2 were confirmed by spectroscopic and spectrometric methods. In addition, it was demonstrated that the nature of the phenol group determines the antioxidant effect of compounds 1 and 2. The antioxidant effect of compound 1, the 5-ASA derivative, is mainly due to resonance effects and tautomeric equilibrium, which are structurally limited in compound 2 (the l-tyr derivative). Therefore, it is possible that the protective effect of compound 1 against the toxicity produced by an overdose of APAP (in the first phase) is due to its ability to directly inactivate the reactive species and increase the levels of GSH. Additionally, compound 1 decreases the production of NAPQI by inhibiting CYP450. Thus, compound 1 shows better effects than NAC in the first phase of the APAP-induced toxicity, indicating that the former compound could be a good candidate to be evaluated in other diseases in which reactive species are produced. Therefore, it would also be interesting to assess the effect of compound 1 during the second phase of APAP-induced toxicity in this model.
Authorship contributions
Martha C. Rosales-Hernández, Itzia I. Padilla-Martínez participated in research design.
Laura C. Cabrera-Pérez, Jessica E. Mendieta-Wejebe conducted experiments.
Feliciano Tamay-Cach, Alejandro Cruz contributed to new reagents or analytical tools.
Laura C. Cabrera-Pérez, Martha C. Rosales-Hernández, Itzia I. Padilla-Martínez performed data analysis.
Laura C. Cabrera-Pérez, Martha C. Rosales-Hernández, Jessica E. Mendieta-Wejebe, Itzia I. Padilla-Martínez wrote or contributed to writing of the manuscript.
Acknowledgments
This research was financially supported by PRODUCTOS MEDIX, S.A. de C.V. [Empresa farmacéutica; Agreement: MEDIX SIP-2014-RE-014] and COFAA-SIP/IPN [Project: 20161374; 20161383; 20160675].
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