RU2706692C1 - New sulfur derivatives of 2-ethyl-6-methyl-hydroxypyridine - Google Patents

Abstract

FIELD: medicine; pharmacology.

SUBSTANCE: present invention refers to compounds of formulas

and

applicable in pharmacology and medicine.

EFFECT: novel antioxidants are disclosed.

3 cl, 4 tbl, 5 ex

Description

       The invention relates to the field of pharmacology and medicine, in particular, to new antioxidants based on sulfo derivatives of 2-ethyl-6-methyl-3-hydroxypyridine.

       Antioxidants based on 2-ethyl-6-methyl-3-hydroxypyridine and its salts (hydrochloride and succinate) are widely used in medical practice for the treatment of diseases accompanied by oxidative stress and the formation of reactive oxygen species, including ischemia due to acute cerebrovascular accident , atherosclerosis, and other diseases associated with tissue hypoxia. Voronina T.A. Antioxidant Mexidol. Psychopharmacol. Biol. Narcol. 2001. Vol. 1. No. 1. P. 2–12. The main disadvantage of 2-ethyl-6-methyl-3-hydroxypyridine is its instability to oxidation by atmospheric oxygen and the need to use preservatives in the composition of drugs based on it to prevent oxidative degradation. Thus, there is the problem of creating water-soluble antioxidants based on 2-ethyl-6-methyl-3-hydroxypyridine, which retain effectiveness in removing active oxygen species, but are much more resistant to atmospheric oxygen.

       The technical result of the present invention is to increase the resistance of 2-ethyl-6-methyl-3-hydroxypyridine derivatives to the action of atmospheric oxygen while maintaining biological antioxidant activity. The technical result is achieved by obtaining new sulfonic derivatives of 2-ethyl-6-methyl-3-hydroxypyridine.

       The present invention provides a new compound of general formula (I)

(I),

(I)

where R1 is H or SO ₃ H; R2 is H or SO ₃ H.

       Embodiments of the invention are a compound of formula (1)

(1)

(one)

and a compound of formula (2)

(2)

(2)

       Compounds (1) and (2) can be used as antioxidants in the manufacture of drugs.

       The term “medicinal product” is used in the present invention in the sense established by the Federal Law dated 12.04.2010 N 61-FZ (as amended on 11.28.2018) “On the circulation of medicines”, article 4.

        The term “production” of a medicinal product means the production of a medicinal product in the sense established by the Federal Law dated 12.04.2010 N 61-ФЗ (as amended on 11.28.2018) “On the Circulation of Medicines”, Article 4, namely, as activities for the production of medicines organizations producing drugs at one stage, several or all stages of the technological process, as well as the storage and sale of manufactured drugs.

       The drug of the present invention can be produced in various dosage forms that correspond to the methods of its administration and use and provide the desired therapeutic effect, for example, using methods and pharmaceutical procedures well known in the art and described in the Remington Pharmaceutical Scientific Reference. Remington's Pharmaceutical Sciences, seventeenth edition, ed. Alfonso R. Gennaro, Mack Publishing Company, Easton, Pa., Eighteenth edition, 1990. Examples of dosage forms of the present invention include, but are not limited to, any and all forms suitable for injection, intranasal, transdermal, glass, sublingual, and oral administration.

       Preferably, the compounds (1) and (2) will be used in the composition of liquid dosage forms containing from 1.0 to 50.0 wt. %

       Dosage forms may contain excipients.

       The term "excipient" refers to substances of inorganic or organic origin used in the production process, manufacture of drugs to give them the necessary physico-chemical properties.

Such excipients are solvents and fillers, preservatives, surface-active substances (surfactants), film formers, pH regulators, and flavoring agents.

       The following examples demonstrate the invention. The examples are illustrative and are not intended to limit the scope of the invention.

Example 1

       An example illustrates the preparation of compounds 1 and 2 of the present invention.

       In 250 ml of an aqueous solution of 2-ethyl-6-methyl-3-hydroxypyridine hydrochloride (17.4 g; 0.1 mol), sodium metabisulfite (19.0 g; 0.1 mol), and 1 mg of iron sulfate at pH 4.9 and with stirring, a stream of air (oxygen content 21%) was passed at 50 ° C for 72 hours. the temperature of the solution was adjusted to pH 1-2 with a solution of 1 N. sulfuric acid and purged with air until complete removal of sulfur dioxide. Next, the solution was adjusted to pH 7-7.5 with a NaOH solution, evaporated to dryness in vacuo, the residue was extracted with acetone to remove the unreacted starting pyridine base, then dissolved in a minimum of water, adjusted to pH 4-5 with a 1N solution. sulfuric acid and evaporated in vacuo to dryness. The dry residue was extracted for a long time with methanol. A mixture of compounds 1 and 2 was obtained by evaporation from a methanol solution, which was separated by high pressure preparative high performance liquid chromatography (HPLC) on 21 mm diameter columns filled with C18 reverse phase in gradient elution mode, the mobile phase is acetonitrile / 0.1% formic acid (A / B)

Compound (1). Yield 14% based on the starting 2-ethyl-6-methyl-3-hydroxypyridine. NMR ¹ H (DMSO-d6), δ-scale: 1.16 (3H, d); 2.35 (3H, s); 2.72 (2H, d); 7.13 (1H, s); 10.38 (1H, s). Mass spectrum, m / z: 217.04 (100%). IR spectrum, cm ^-1 : 1176-1264 (S = O).

Compound (2). Yield 8% based on the starting 2-ethyl-6-methyl-3-hydroxypyridine. NMR ¹ H (DMSO-d6), δ-scale: 1.25 (3H, d); 2.53 (3H, s); 3.09 (2H, d); 7.69 (1H, s); 11.48 (1H, s). Mass spectrum, m / z: 217.04 (100%). IR spectrum, cm ^-1 : 1176-1264 (S = O).

Example 2

            An example illustrates the antioxidant activity of the compounds of the present invention in an oxidative stress model.

Oxidative stress-induced hepatotoxicity was simulated on a HepG2 liver cell culture using hydrogen peroxide (H ₂ O ₂ ) and the effectiveness of the compounds as antioxidants was evaluated using a spectrophotometric MTT test based on the principle that living cells convert MTT (3- tetrazolium dye (4, 5-dimethylthiazol-2-yl) -2,5-diphenyl-tetrazolium bromide) into insoluble formazan. Siddiqui et al., Front. Pharmacol 2018, 9: 797 . Jiang et al., Oxid. Med. Cell Longev. 2014, 2014: 310504 . HepG2 liver cells were pre-incubated at a density of 1x10 ⁴ cells / cell in culture medium without or with the addition of 200 μM compound 1, compound 2, and a reference substance (2-ethyl-6-methyl-3-hydroxypyridine succinate or 3-GPS) for 20 min, then H ₂ O _{2 was} added at a cytotoxic concentration of 150 μM and the cells were incubated for 6 hours, after which the medium was replaced, 50 μg MTT was added, incubated for another 4 hours, the medium was separated from formazan crystals, 200 μl DMSO was added to each well to dissolve the crystals and absorbance was measured at 550 nm. In the control, cells were not exposed to compounds and H ₂ O ₂ . Survival results are presented in Table 1 as a percentage of control as the mean ± the average error in each group (n = 10). Statistical differences between groups were evaluated using Student t-test. Differences were considered significant at a significance level of p <0.05.

Table 1. The effect of compounds 1 and 2 on the survival of HepG2 cells

* Significant difference from "N ₂ About ₂ " (p <0,05).

Table 1 shows that H ₂ O ₂ significantly causes cell death compared with the control, while compounds 1, 2 and the reference substance significantly increase the survival of cells exposed to H ₂ O ₂ , which indicates the antioxidant properties of these compounds.

Example 3

This example shows that the compounds of the present invention protect the liver under oxidative stress caused by CCl ₄ .

A model of oxidative stress using carbon tetrachloride (CCl ₄ ) was used to evaluate the hepatoprotective effect of compounds 1 and 2. CCl ₄ belongs to the class of hepatotoxins that act after metabolic activation. CCl _{4 is} metabolized with the participation of cytochrome p450 (CYP2E1), forming the reactive free radical CCl ₃ •, which reacts with oxygen to form the trichloromethyl peroxyl radical CCl ₃ OO •, which causes lipid peroxidation and acute toxic hepatitis. Weber LW et al., Hepatotoxicity and mechanism of action of haloalkanes: carbon tetrachloride as a toxicological model. Crit. Rev. Toxicol. 2003, 33 (2): 105-36 . Male Wistar rats received 48 h, 24 h, and 2 h before CCl ₄ administration. injections physical solution (control, n = 6), 50 mg / kg of compound 1 (n = 6), 50 mg / kg of compound 2 (n = 6), or 50 mg / kg of 2-ethyl-6-methyl-3- hydroxypyridine succinate (3-GPS, reference substance, n = 6) and then received a single b.p. injection of CCl ₄ at a dose of 0.4 mg / kg, which caused acute toxic hepatitis. The effectiveness of the tested compounds was evaluated by the activity of the enzymes alanine transaminase (ALT) and aspartate transaminase (AST) in serum 48 hours after the introduction of CCl ₄ . The results are presented in Table 2 as the average value ± the average error in the activity of ALT or AST enzymes in serum in each group. For comparison, the results of analyzes in the group of intact rats (n = 10) are given. Statistical differences between groups were evaluated using Student t-test. Differences were considered significant at a significance level of p <0.05.

Table 2. The effect of compounds 1 and 2 on the activity of ALT and AST.

* Significant difference from “Phys. p + CCl ₄ "(p <0.05).

Table 2 shows that the injection of CCl ₄ causes a significant increase in the activity of the enzymes ALT and AST in the blood serum, which indicates the development of acute toxic hepatitis. Compounds 1 and 2 exhibit a significant protective antioxidant effect, comparable to the effect of the reference substance, preventing the development of hepatotoxicity under conditions of oxidative stress caused by the introduction of CCl ₄ .

Example 4

       This example shows that compounds 1 and 2 are more resistant to atmospheric oxygen than the starting 2-ethyl-6-methyl-3-hydroxypyridine succinate.

       A comparative assessment of the resistance to air oxygen of compounds 1 and 2 of the present invention and the comparison substance 2-ethyl-6-methyl-3-hydroxypyridine succinate (3-GPS) was carried out in 5% aqueous solutions of these compounds at pH 4.9 under free access to oxygen at 60 ° C for 2 weeks. Analysis of samples of solutions of compounds 1 (n = 4) and 2 (n = 4) was carried out by HPLC as described in Example 1. Analysis of samples of solutions of the reference substance (n = 4) was performed by HPLC on a Luna 5 u C18 100A, 250x4.6 column mm in gradient elution mode, the mobile phase is 0.07% phosphoric acid-water / 0.07% phosphoric acid-acetonitrile (A / B). The change in the substance content in the samples after two weeks of the experiment was calculated as a percentage according to the formula ΔС = 100% ∙ (Ct-C0) / C0, where Ct is the concentration of the substance in the solution after two weeks of experiment, C0 is the initial concentration of the substance. The results are shown in Table 3 as the average value ± the average error of the change in the concentration of the substance (ΔС) from four experiments. Statistical differences between groups were evaluated using Student t-test. Differences were considered significant at a significance level of p <0.05.

Table 3. The change in the content of compounds in the samples after two weeks of experience.

 * Significant difference from “3-GPS” (p <0.05).

       Table 3 shows that under conditions of free access to air oxygen, compounds 1 and 2 are significantly more stable than the reference substance, and the content of the main substance in the experimental conditions decreases by 0.3 and 0.7% for compounds 1 and 2, respectively, against 6.3% reduction for the reference substance.

Example 5

       Preparation of ampouled solutions of compounds 1 and 2.

       Compounds 1 or 2 were mixed with water for injection in the amounts shown in Table 4 under aseptic conditions to prepare a sterile solution, without the addition of preservatives. The resulting solution was filled in ampoules with a volume of 2, 5, or 10 ml.

Table 4. Ampouled solutions of compounds 1 and 2.

Claims (7)

1. The compound of General formula (I)

(I) where R1 is H or SO ₃ H; R2 is H or SO ₃ H, provided that R1 and R2 are not both H. 2. The compound according to claim 1, where R1 = H and R2 = SO ₃ H of the formula (2)

(2). 3. The compound according to claim 1, where R1 = SO ₃ H and R2 = H of the formula (3)

(3).