Synthesis and Antioxidant Activity of 2-Amino-5-R-1,3,4-Oxadiazoles with Hindered Phenol Fragments ^†

Abstract

Compounds with hindered phenolic moiety are known to be effective inhibitors of oxidative processes in different materials; moreover, a number of phenols show a wide spectrum of biological activity. At the same time, five-membered heterocycles exhibit unique properties, including antioxidant activity. One of the ways to create new effective antioxidants with a set of useful properties is to combine hindered phenol and a heterocyclic fragment in one molecule. In this work, new 1-acyl-4-R-thiosemicarbazides were obtained during a reaction between 3-(4-hydroxy-3,5-di-tert-butylphenyl)propanoic acid hydrazide and a number of isothiocyanates. 2-Amino-5-R-1,3,4-oxadiazoles were prepared in good yields by heterocyclization of 1-acyl-4-R-thiosemicarbazides in presence of iodoxybenzoic acid and triethylamine. The antioxidant activity of 1,3,4-oxadiazoles was studied in vitro and was found to be higher than that of 4-methyl-2,6-di-tert-butylphenol.

1. Introduction

All organic substances undergo oxidative degradation under the influence of reactive oxygen species (ROS) such as hydroxyl HO^•, superoxide O₂^•, peroxide ROO^•, alkoxyl RO^• and nitroxyl NO^• radicals [1]. ROS induce oxidative damage of cell membranes, lipids, proteins, and DNA repair system breakdown, which is connected with many degenerative diseases, such as cancer, atherosclerosis, and Alzheimer’s disease [2,3,4]. Antioxidants aim to scavenge free radicals and inhibit oxidative stress processes through their ability to inhibit the chain process of free radical oxidation, that allows them toplay crucial roles in conserving intricate cellular functions [5]. A large number of natural antioxidants as well as synthetic ones have been found. Natural antioxidants include ones that are endogenous enzymatic (glutathione peroxidase, superoxide dismutase and catalase) [6], nonenzymatic (uric acid, lipoic acid, bilirubin, glutathione, metatonin) [7] and exogenous (carotenoids, vitamin E, A and C, natural flavonoids) [8]. Synthetic antioxidants are represented by a wide range of classes of organic compounds, such as amines [9], benzotriazole derivatives [10], alkylaminothiadiazoles [11] and others, but the most common structures in synthetic antioxidants are flavonoids [12,13], coumarins [14,15], and hindered phenols [16,17]. Despite the fact that the mechanism of oxidation, as well as the mechanism of action of antioxidants, has been studied for several decades, there is no universal approach to the creation of new antioxidants. The most rational way to obtain new antioxidants is to combine a fragment known for its antioxidant properties in a structure—for example, hindered phenol and structures with functional groups of various natures or heterocycles [18]. Thus, it was shown that chalcon derivatives with di-tert-butylphenol fragments are effective antioxidants [14]. Additionally, an increase in the antioxidant properties of phenols under some conditions was achieved by introducing a sulfur atom into the structure of an oxidation inhibitor [19,20]. Antioxidants containing heterocyclic fragments in their structures are of considerable interest [21,22]. Such compounds are able to act by several mechanisms at once: inhibition of free radical processes, decomposition of hydroperoxides, and chelating metals [23].

In continuation of our previous studies [24,25], in this work we combined in one structure a fragment of 2,6-di-tert-butylphenol and 2-alkyl/arylamino-1,3,4-oxadiazole and studied the antioxidant properties of the synthesized structures.

2. Results and Discussions

2.1. Synthesis

Preparation of the target compounds is outlined in Scheme 1. Initially, 1-acylthiosemicarbazides 1a–c were obtained by the reaction between 3-(4-hydroxy-3,5-di-tert-butylphenyl)propanoic acid hydrazide with a number of isothiocyanates. The reaction was carried out by boiling of the starting reagents in isopropanol for 5 h. The yields of thiosemicarbazides were 77–95%. The ¹H NMR spectra of the obtained compounds show peaks in the region of 9.5–10 ppm, corresponding to the protons of the NH-NH fragment.

In accordance with the convenient method of [26], the reaction was carried out in an alcoholic alkali solution with the presence of iodine at 0 °C. N-substituted 2-amino-1,3,4-oxadiazoles were obtained in 48–58% yield with a strong resinification of the reaction mixture, which made it difficult to isolate the products. It was also not possible to achieve complete conversion of the starting 1-acylthiosemicarbazides even by boiling of the reaction mixture. In this regard, the method of applied cyclization proposed by the author of [27] was applied: the reaction was carried out in chloroform in the presence of 2-iodoxybenzoic acid and triethylamine at room temperature. The yields of the target compounds increased to 74–80% (

Table 1. The yields of oxadiazoles 2a–c.

Methods	2a	2b	2c
Method A	58%	56%	48%
Method B	74%	80%	75%

).

In the ¹H NMR spectra of compounds 2a–c, peaks corresponding to phenol fragment were observed: the singlet peak near 6.71 ppm is attributed to the O–H of the hindered phenol; peaks at 7.42–7.54 ppm with the integration of two protons were assigned to the two symmetrical aromatic ring protons. In addition, singlet peaks in the range of 9.23–10.36 ppm, corresponding to the protons of the secondary amino group, were observed.

2.2. Antioxidant Properties

The antioxidant properties of the obtained 2a–c compounds were tested in a model reaction of oleic acid oxidation. The oxidation of oleic acid was investigated at 65 °C using a thermostated apparatus upon bubbling of air into a cell for the oxidation at a constant rate of 2–4 mL min^–1. The level of lipid peroxidation was estimated via the concentration of primary oxidation products, hydroperoxides (LOOH) and secondary carbonyl products, which form complexes with thiobarbituric acid (TBARS, thiobarbituric acid reactive species) [14]. Butylated hydroxytoluene was used as a standard. The results of antioxidation ability tests are shown in Figure 1.

In the presence of compounds 2a–c, the concentrations of both hydroperoxides and secondary oxidation products decreases. All compounds have a significant effect on the concentration of hydroperoxides, according to this parameter, the activity of the synthesized substances significantly exceeds activity of BHT. In the presence of 2c and 2c, the concentration of TBARS also significantly decreases; compound 2a exhibits an efficiency comparable to that of BHT.

3. Materials and Methods

3.1. General Information

NMR ¹H and ¹³C spectra of solutions in DMSO-d₆ were recorded on a Bruker AM-300 spectrometer. All experiments were performed according to the standard methods of Bruker. Chemical shifts were reported relative to Me₄Si. The values of SSCCs are given in Hz. The mass spectra were recorded on an MS-30 Kratos device (Eu, 70 eV). A peak of the molecular ion M⁺ was observed for all synthesized compounds. The melting points of the obtained compounds were determined in an open capillary. Elemental analysis was carried out using an Elemental analyzer Vario micro cube. The course of reactions and purity of the compounds obtained was monitored by TLC on silica gel plates in a 10:1 toluene-ethanol solvent system.

3.2. Synthesis and Analytical Data of Preparated Compounds

3.2.1. Synthesis of Compounds 1a–c

A mixture of 20 mmol of hydrazide and 20 mmol of isothiocyanate in 70 mL of propanol-2 was stirred and refluxed for 5 h. The reaction mixture was cooled to room temperature; the formed precipitate was filtered off and recrystallized using a suitable solvent.

1-[2-(4-hydroxy-3,5-di-tert-butylphenyl)ethyl]-4-phenylthiosemicarbazide 1a: Yield—85%. M.p = 165–167 °C (ethanol:water—70:30). ¹H NMR (DMSO-d₆, δ, ppm): 1.36 s (18H, t-Bu), 2.34–2.62 m (4H, CH₂CH₂), 6.68 s (1H, HO), 7.05 s (2H, Har), 7.15–7.44 m (5H, Har), 9.56 br.c. (2H, NH), 10.02 s (1H, NH). MS: (M⁺) m/z 427. Calculated, %: C—67.41; H—7.78; N—9.83; S—7.50; found, %: C—67.26; H—7.98; N—9.68; S—7.35.

1-[2-(4-hydroxy-3,5-di-tert-butylphenyl)ethyl]-4-(4-methylphenyl)thiosemicarbazide 1b: Yield—77%. Mp = 203–204 °C (isopropanol:water—1:1). ¹H NMR (DMSO-d6, δ, ppm): 1.35 s (18H, t-Bu), 2.31 s (3H, CH₃), 2.44 t (J = 7.3, 2H, CH₂), 2.75 t (J = 7.2, 2H, CH₂), 6.71 s (1H, OH), 6.97 s (2H, Nar), 7.14 d (2H, Nar), 7.30 d (2H, Nar), 9.48 br.s (2H, NH), 9.86 s (1H, NH). MS: (M⁺), m/z 444. Calculated, %: C—67.99; H—7.99; N—9.51; S—7.26; found, %: C—67.81; H—7.85; N—9.68; S—7.36.

1-[2-(4-Hydroxy-3,5-di-tert-butylphenyl) ethyl]-4-(3-ethoxyphenyl)thiosemicarbazide 1c: Yield—80%. Mp = 165–167 °C (ethanol:water—1:1). ¹H NMR (DMSO-d6, δ, ppm): 1.35 s (18H, t-Bu), 2.31 t (3H, CH₃), 2.45 t (2H, CH₂), 2.75 t (2H, CH₂), 3.99 m (2H, CH₂), 6.71 s (1H, OH), 6.89 d (2H, Nar.), 6.97 s (2H, Nar), 7.24 d (2H, Nar), 9.45 br.s (2H, NH), 9.88 s (1H, NH). MS: (M⁺) m/z 471. Calculated, %: C—66.21; H—7.91; N—8.91; S—6.80; found, %: C—66.02; H—7.91; N—8.91; S—6.80.

3.2.2. Synthesis of Compounds 2a–c

Method A: To a suspension of 14 mmol of 1-acylthiosemicarbazide in ethanol, 0.7 mL of 4N NaOH solution was added. The reaction mixture was cooled and I₂ in an aqueous solution of KI was added dropwise at the temperature of 0–5 °C until a nonfading color of iodine appeard. The reaction mixture was heated to boiling point, then cooled and poured into 200 mL of ice water; the precipitate was filtered off and the product was crystallized using a suitable solvent.

Method B: To 113 mmol of 1-acylthiosemicarbazide in chloroform, 113 mmol of triethylamine and 23 mmol of iodoxybenzoic acid (IBX) were added. The reaction mixture was stirred at room temperature for 2.5 h. A solution of K₂CO₃ was added to the reaction mixture, the target substance was extracted from the aqueous layer with chloroform, and then the resulting extracts were dried over calcined Na₂SO₄. The solvent was evaporated, threated with cold water and the product was filtered off and crystallized using a suitable solvent.

2-N-(phenyl)amino-5-[2-(4-hydroxy-3,5-di-tert-butylphenyl) ethyl]-1,3,4-oxadiazole 2a: Yield—58% (Method A), 74% (Method B), Mp. = 186–188 °C, (ethanol:water—5:1). ¹H NMR (DMSO-d6, δ, ppm): 10.36 s (1H, NH), 7.54 d (J = 8.2 Hz, 2H, Har), 7.34 t (J = 7.6 Hz, 1H, Har), 6.91 s (2H, Har), 6.76 s (1H, OH), 3.01 t (J = 7.0 Hz, 2H, CH₂-CH₂), 2.90 t (J = 7.3 Hz, 2H, CH₂-CH₂), 1.35 s (18H, t-Bu). MS: (M⁺) m/z 393. Calculated, %: C—73.25; H—7.94; N—10.68; found, %: C—73.11; H—8.13; N—10.52.

2-N-(4-methylphenyl)amino-5-[2-(4-hydroxy-3,5-di-tert-butylphenyl) ethyl]-1,3,4-oxadiazole 2b: Yield—56% (Method A), 80% (Method B), Mp. = 172–174 °C, (ethanol:water—3:1). ¹H NMR (DMSO-d₆, δ, ppm): 10.23 s (1H. NH), 7.42 (d, J = 8.1 Hz, 2H, Har), 7.14 (d, J = 8.2 Hz, 2H, Nar), 6.91 s (2H, Har), 6.76 s (1H, OH), 2.89 (t, J = 7.0, 2H, CH₂-CH₂), 2.99 t (J = 6.8, 2H, CH₂-CH₂), 1.35 s (18H, t-Bu). MS: (M⁺) m/z 437. Calculated, %: C—73.68; H—8.16; N—10.31; found, %: C—73.79; H—8.33; N—10.15.

2-N-(4-ethoxyphenyl)amino-5-[2-(4-hydroxy-3,5-di-tert-butylphenyl)ethyl]-1,3,4-oxadiazole 2c: Yield—48% (Method A), 75% (Method B), Mp. = 179–181 °C, (ethanol:water—3:1). ¹H NMR (DMSO-d₆, δ, ppm): 9.33 s 1H, NH, 7.42 (s 2H, Har), 6.90 s (2H, Har), 6.51 s (1H, OH), 3.97, br. s (2H, CH₂), 2.8–3.07 m (2H, CH₂-CH₂), 1.35 br s (18H, t-Bu). MS: (M⁺) m/z 438. Calculated, %: C—71.37; H—8.06; N—9.60; found, %: C—71.25; H—8.18; N—9.80.

3.3. Antioxidant Properties of Preparated Compounds

Determination of the concentration of LOOH in oleic acid.

The studied compounds (1 mmol L^–1) were added to oleic acid and thermostated at 65 °C for 6 h. 0.5 mL of oleic acid, 9 mL of glacial acetic acid, 6 mL of chloroform, and 0.5 mL of a saturated freshly prepared KI solution were poured into a flask. The flask was shaken for 2 min, then 50 mL of distilled water and 0.5 mL of 1% starch solution were poured into it. Thereafter, they were immediately titrated with 0.01 N Na₂S₂O₃ solution. The LOOH concentration was calculated according to the following formula:

[LOOH] = [(V − V₀)•0.001269K•100]/m

V is the volume of 0.01 N Na₂S₂O₃ solution, consumed during the titration of working sample, mL; V₀ is the volume of 0.01 N Na₂S₂O₃ solution, consumed during the titration of control sample, mL; K is the conversion factor to the exactly 0.01 N Na₂S₂O₃ solution; m is the mass of studied oleic acid; 0.001269 is the amount of I₂ expressed in g, equivalent to 1 mL of 0.01 N Na₂S₂O₃ solution. The [LOOH] content equal to 1% corresponds to 78.7 mM of active O₂ per 1 L of lipids (mmol L^–1).

Determination of the concentration of TBARS in oleic acid.

The studied compounds (1 mmol L^–1) were added to oleic acid and thermostated at 65 °C for 6 h. After cooling, samples (0.01 mL) of oleic acid were taken from the thermostat and put into a test tube. A mixture of phosphate buffer (0.8 mL), distilled water (1.2 mL), and freshly prepared thiobarbituric acid solution (0.8%, 1 mL) were added; the tube was heated for 10 min in a boiling water bath, and after cooling the absorption of the samples was measured in comparison with that of control at λ = 532 nm. The concentration of carbonyl compounds was calculated according to the formula:

[TBARS] = (E•3)/0.156

(1)

[TBARS] is the content of carbonyl compounds, nmol L^–1; E is the absorbance of a sample relative to the control (mixture without oleic acid); 3 is the sample volume, mL; and 0.156 is the extinction of malondialdehyde (1 nmol) dissolved in 1 mL at λ = 532 nm [14].

4. Conclusions

In this study, the synthesis and antioxidant activity of three 2,6-di-tert-buthylphenol derivatives linked to 2-amino-5-R-1,3,4-oxadiazoles are described. Carrying out the cyclization of 1-acylthiosemicarbazides by the action of iodoxybenzoic acid made it possible to increase the yields of the target compounds in comparison with the previously mentioned convenient method. All investigated substances exhibited antioxidant capacity superior or comparable to that of BHT. 2-N-(4-ethoxyphenyl)amino-5-[2-(4-hydroxy-3,5-di-tert-butylphenyl)ethyl]-1,3,4-oxadiazole 2c possesses the best antioxidant properties among the studied oxadiazoles.