RU2244546C1 - Agent eliciting antioxidant and antiviral activities - Google Patents

Abstract

FIELD: organic chemistry, medicine, pharmacy.

SUBSTANCE: invention relates to biologically active compounds. Agent represents 3,6-dioxocyclohexa-1,4-diene-1,2,4,5-tetrasulfonate sodium. The new agent elicits antioxidant properties and therefore it can be used in food industry, in pharmaceutical compositions and cosmetic products. Also, the new agent elicits antiviral activity owing to it can be used as both the independent medicinal agent and in compositions with other preparations used for treatment of viral infections.

EFFECT: expanded assortment of medicinal agents and antioxidants, realization of indicated prescription.

1 tbl, 8 dwg

Description

The invention relates to biologically active compounds used in medicine and the pharmaceutical industry, applicable in the food industry in cosmetics. The invention is based on the preparation of a synthetic analogue of ubiquinone, which is close in biological action to a natural compound.

Synthesized 3,6-dioxocyclohexa-1,4-diene-1,2,4,5-tetrasulfonate sodium with antioxidant and antiviral activities. The compound is not toxic. The presence of biological activity allows us to recommend it to expand the arsenal of antiviral agents and anti-oxidative agents that occur both in the body and in pharmaceutical, food, cosmetic and other compositions.

The resulting compound was tested in vitro and in vivo. It can be used for therapeutic and prophylactic purposes, as in biochemical processes, it behaves like a natural ubiquinone. The compound is water-soluble, due to which it can be widely used in the biotechnological, chemical, and pharmaceutical industries [.

Antioxidants are known: vitamin E, glutathione, N-acetylcysteine, vitamin C (ascorbic acid), some essential amino acids, coenzyme Q (Mashkovsky MD Medicines. M. 2000. V.2. S.182-188).

The most important functions of this group of drugs include their interaction with active oxygen radicals.

Their action is aimed at the following physiological and pathological processes in the body.

1. Participation in basic metabolic processes and, in particular, redox reactions that determine the intensity of catalytic processes in cells.

2. Interaction with reactive oxygen species (free radicals) that form during pro-inflammatory activation of macrophages and tissue hypoxia.

3. Participation in the processes of electron transport and oxidative phosphorylation in mitochondria.

4. Interaction with vitamins (redox transition in B vitamins), with amino acids (sulfur-containing), with proteins.

5. Interaction with lipid peroxides of cell membranes and lipoproteins.

Vitamin E and vitamin C used in medicine are often used in the food industry and in pharmaceutical compositions as antioxidants.

The known drug olifene (J. High-molecular compounds. 1975. T.17 (A), No. 3. P.557-563. Mashkovsky MD Medicines, Moscow, “New Wave”, 2000, T.2, C. 187), which is

where n is from 1 to 5.

The drug has antioxidant activity, but consists of a mixture of polymer products, which vary from the synthesis conditions, because of which there is a large variation in units of activity (Mashkovsky MD Medicines. Moscow. “New Wave”, 2000. V.2. S.187).

The known drug ubiquinone (Mashkovsky MD Medicines. Moscow. “New Wave”, 2000. V.2. P.187), which represents the following structure:

6-decaprenyl-2,3-dimethoxy-5-methyl-1,4-benzoquinone, i.e. quinoid structure with a long terpenoid tail. Ubiquinone is a fat-soluble compound, but it is poorly soluble in water, which limits its use.

A highly active analogue of ubiquinone is idebenone (J. Molecules, 2000. No. 5, pp. 1439-1460), which is 2,3-dimethoxy-5-methyl-6- (10-hydroxydecyl) -1,4-benzoquinone:

This drug is new and not widely available in Russia. Not all of its properties are known, which makes its widespread use difficult.

In a number of antiviral drugs, rimantadine has recently become widespread:

α-methyl-1-adamantylmethylamine hydrochloride.

(Mashkovsky M. D. Medicines. Moscow. “New Wave”, 2000. V.2. S.325).

This drug gives good results, but it is a drug with a narrow spectrum of action, and therefore are constantly looking for new antiviral agents.

The aim of this invention is the creation of an affordable drug with antioxidant properties, a drug with an established structure, with antiviral activity.

This tool is necessary to increase the arsenal of drugs (drugs with biological activity), and to increase the arsenal of antioxidants used in compositions for various purposes.

The problem was solved by synthesis of a hydroquinone derivative, which is sodium 3,6-dioxocyclohexa-1,4-diene-1,2,4,5-tetrasulfonate. The compound was obtained by the reaction of parabenzoquinone and sodium thiosulfate. The resulting salt is water-soluble and fat-soluble, has low toxicity. The similarity of the chemical and biological properties of the synthesized compound with natural ubiquinone, the high bioavailability of the compound make it possible to use it as an antioxidant. Suddenly, when checking, its antiviral activity was revealed.

The authors chose the quinoid structure of the new compound close to natural as the goal of the synthesis. Based on the fact that substituents of the SO ₂ or SO _{3 type} are often used to increase the bioavailability of pharmacological substances (Khavinson V.Kh. et al. Free radical oxidation and aging. St. Petersburg, Nauka, 2003), the compound obtained is as follows structure:

which, due to the formation of resonance structures, including O = S = O, and due to the creation of a single π-electronic structure of the molecule (moreover, a strongly delocalized π-system), acquired donor-acceptor properties and the ability to interact with reactive oxygen species. The combination of the aromatic nucleus with sulfate groups led to the reactivity of the compound to interact with free radicals.

The synthesis of 3,6-dioxocyclohexa-1,4-diene-1,2,4,5-tetrasulfonate was carried out with polar aprotic solvents from parabenzoquinone with an excess of sodium thiosulfate. The synthesis was carried out in the presence of sulfuric acid at a temperature of 30-70 ° C. The molar ratio of sodium thiosulfate to parabenzoquinone was 6: 1; in some experiments, the amount of sodium thiosulfate was more than 6 moles per 1 mol of parabenzoquinone. The yield of the final product is from 70% to 80%. The reaction product is a black solid, powdery substance, soluble in polar solvents.

The decomposition temperature is 394 ° C. The toxicity of the drug = LD ₅₀ = 5100 ± 135 mg / kg. The compound does not have the ability to cumulate, does not cause irritation to the skin and allergic reactions.

Based on the data obtained, we can conclude about the reaction mechanism. The reaction proceeds by the mechanism of electrophilic substitution of hydrogen in the nucleus of parabenzoquinone. The reaction product can be sulfonated until complete replacement of hydrogen in the nucleus of parabenzoquinone.

1. The formation of sulfonating agent

SO ₃ : Na ₂ S ₂ O ₂ → SO ₃ + Na ₂ S

2Ar-SO ₃ H + Na ₂ S → 2Ar-SO ₃ Na + H ₂ S ↑

2. π-complex

3. σ-complex

4. The formation of sodium tetrasulfonate

The compound is capable of polymerizing when heated in solvents (with an increase in the number of units, the solubility decreases, which leads to the formation of precipitation). In solutions forms aggregates. Slowly decomposes in the light. It enters into redox reactions like benzoquinones. Capable of forming salts and chelate complexes with metals and organic amines. It exhibits antioxidant properties. To analyze the obtained compound, 18 mass spectrometric analyzes were carried out after synthesis (Na ₂ S ₂ O ₃ + C ₆ H ₄ O ₂ ). The ion spectra of both charges were investigated. The results are presented in figures 1 and 2.

Measurement conditions:

Device: Voyager DE time-of-flight laser ionization mass spectrometer.

Sample weight 1 μg. The sample volume is 1 μl.

Matrices: synapinic and alpha-cyanocinnamic acids in a solution of 70% acetonitrile, 29.9% bidistilled water, 0.1% trifluoroacetic acid. Laser intensity 60-80% maximum.

The measurement accuracy was 0.2-1%.

In all the presented samples, a set of peaks with a step of 22 amu starting from 445 manifests itself in the synapinic acid network, which confirms the presence of 3,6-dioxocyclohexa-1,4-diene-1,2,4,5-tetrasulfonate sodium (with a different amount of sodium ions) (see figure 1). This is indirectly evidenced by the spectrum of negatively charged ions, in which a step of 23 amu is visible, assuming that doubly charged ions are formed.

Synthesis of sodium 3,6-dioxocyclohexa-1,4-diene-1,2,4,5-tetrasulfonate.

The synthesis was carried out in a water-acetone solution, where the water content in the water-acetone mixture was varied in the range from 1:40 to 1:10. This ratio affects the speed and controllability of the reaction. With increasing water content, the reaction rate decreases. The amount of sulfuric acid in the reaction mixture is 0.05% by volume.

0.2 L of a saturated aqueous solution of sodium thiosulfate and 10 ml of concentrated sulfuric acid were added to 1.8 L of a 2% solution of parabenzoquinone in acetone. The molar ratio of parabenzoquinone to sodium thiosulfate is 1: 6. The addition of sulfuric acid to the reaction mixture increases the yield of the final product by approximately 15%.

The reaction mixture is stirred, it is initially heated to a temperature of + 40 ° C, after which the reaction proceeds with the release of heat (enzothermally). The reaction temperature is maintained within + 45-65 ° C, periodically cooling the reactor.

The end of the reaction is determined by the final cessation of heat and cooling the reaction mixture.

12 hours after the end of the reaction, the reaction mixture was filtered through a sterile calico filter to remove traces of acetone, and the precipitate was dried at room temperature for 24 hours. The crushed precipitate is placed in a soxhlet and washed with diethyl ether for 24 hours. The product is dried in vacuo. Yield: 70% of the taken parabenzoquinone.

Chromatography of the products of synthesis of 3,6-dioxocyclohexa-1,4-diene-1,2,4,5-tetrasulfonate was carried out by reverse phase liquid chromatography on a Dynamax 60A column using a liquid chromatograph (Gilson, France). The chromatogram (figure 3) shows the main peak of the material with residual impurities, probably a dimer (compounds 1 and 2, respectively). The total content of impurities from experiment to experiment was no more than 14%, in some experiments it was below 8%. Thus, sodium 3,6-dioxocyclohexa-1,4-diene-1,2,4,5-tetrasulfonate obtained at a temperature of 50-55 ° C is characterized by a rather high degree of homogeneity.

An example of the synthesis of 3,6-dioxocyclohexa-1,4-diene-1,2,4,5-tetrasulfonate sodium.

To a solution of 7.5 kg of parabenzoquinone in a mixture of acetone and water (20 l of acetone and 1.2 l of water) was added 1 l of concentrated sulfuric acid and a saturated aqueous solution of sodium thiosulfate (66 kg of sodium thiosulfate).

The solution was heated to 40 ° C, then the heating was turned off and the reaction was carried out, cooling the solution to + 50-55 ° C (the reaction proceeds with the release of heat). The end of the process was recorded by the cessation of heat. The reaction mixture was kept after termination of the process (the absence of heating indicates that all parabenzoquinone reacted) for 12 hours without treatment. Then the resulting mass was filtered through a coarse calico filter. The precipitate was dried for 24 hours in an oven. The mass of sediment was crushed to granules of about 4 mm in size and placed in filter cartridges, which were installed in soxhlets, where they were filtered with diethyl ether for 24 hours. Then the precipitate was dried in an oven and crushed. The product yield of 33.9 kg, which is 70% (parabenzoquinone). The decomposition temperature is + 394 °. Analysis by mass spectrometry confirms the receipt of 95% pure 3,6-dioxocyclohexa-1,4-diene-1,2,4,5-tetrasulfonate sodium.

C ₆ O ₄ S ₄ Na ₄ Calculated Found C 14.00% O 43.38% C 13.80% O 43.01% LD ₅₀ = 5200 mg / kg

The effect of sodium 3,6-dioxocyclohexa-1,4-diene-1,2,4,5-tetrasulfonate on oxygen free radicals.

The antioxidant properties of mono-, di- and oligocyclohexasulfonates were investigated using the method described by Mohanti et al. (Mohanti JG, Jaffe JS, Schulman ES, Raible DG A highly sensitive flujrescent micro-assay of H ₂ O ₂ releasse from activated human keucocytes using a dihydroxyphenoxazine derivative. J. Of Immunological methodds. 1997. Vol.202. P.133- 1411).

The generation of oxygen and hydrogen peroxide free radicals is a natural function of activated macrophages. White blood cells were obtained from donated blood by centrifugation in 6% dextran in the presence of EDTA. If necessary to obtain an enriched fraction of neutrophils, the Percoll gradient was used. Human albumin was used as a cell stabilizer. The experiments were carried out in 96-well plates using an Appkied Biosystems Cytofluorimeter. Registration was performed for scoproletin and dihydroxyphenoxazine at wavelengths of 360/460 and 590/645, respectively. Macrophage activation was performed with phorbol myristyl acetate according to the method described by Bozeman (Bozeman P. M., Learn D. B., Thomas E. I. Assay of a human leukocyte enzymes myelooperoxidase and eosinophil peroxidase. J. Immunol. Methods, 1990. Vol. 126. P. 125-132).

Determination of antioxidant activity was carried out in 96-well plates, in a volume of 0.2 ml in a culture medium containing bovine serum albumin. The cell content was 10 × 10 ⁵ ml. Dihydroxyphenoxazine was dissolved in dimethyl sulfoxide and added at a concentration of 1 × 10 ^-4 M. Fluorescence analysis was performed on a 96-well Cytofluorimeter with data of 360/460. The results were taken into account by quenching the fluorescence.

The results of the study of the antioxidant activity of the proposed compounds are presented in figure 4.

The abscissa is the concentration of drugs in μ M

Y-axis -% luminescence quenching

Curve 1 - control.

Curve 2 - 3,6-dioxocyclohexa-1,4-diene-1,2,4,5-tetrasulfonate sodium.

As can be seen from the data presented in figure 4, the proposed compound has a high activity on the action of oxygen free radicals, i.e. 83-84%.

The effect of sodium 3,6-dioxocyclohexa-1,4-diene-1,2,4,5-tetrasulfonate on the protection of cells from oxidative stress caused by the suppression of de novo glutathione synthesis.

It is known that the resistance of animal and human cells is largely determined by the synthesis of de novo glutathione. If glutathione synthesis is impaired, cell viability is significantly reduced, as they become sensitive to endogenous oxidative stress caused by endogenous production of oxygen free radicals. These processes were studied in detail in tests for the sensitivity of mutant fibroblasts isolated from patients with Ataxia Friedreich. A feature of this pathology is that the cells of these patients are highly sensitive to the effects of free oxygen radicals, which is associated with a mutation in the mitochondrial protein Frataxin encoded by nuclear genes (Badckock M., de Silva M., Oakss R., et al. Regulation ofmitochoondrial iron accumulation by Yfh Ip, a putative homolog of frataxin. S cience, 1997. Vol.276. P. 1709-1912). The blockade of the synthesis of glutathione is usually carried out using an inhibitor of L - butionine (S, R) - sulfoximide. In the presence of this inhibitor, cells actively respond to any effect that can cause oxidative stress. This MDCK cell line approach has been used in tests with the compound of the invention. To enhance the generation of free radicals, xanthine was added to the culture medium, which resulted in the formation of singlet oxygen due to the induction of cellular xanthine oxidase. In these experiments, it was necessary to find out whether cell protection under these conditions is possible using sodium 3,6-dioxocyclohexa-1,4-diene-1,2,4,5-tetrasulfonate. The results of these studies are presented in figure 5.

The abscissa shows the concentration of drugs in mcg

Ordinate - extinction at 550 nm

Curve 1 - control.

Curve 2 - catalase.

Curve 3 - 3,6-dioxocyclohexa-1,4-diene-1,2,4,5-tetrasulfonate sodium.

As can be seen from the obtained data, the claimed compound possesses the most pronounced antioxidant activity under conditions of suppression of glutathione synthesis. Its activity in protecting cells from oxidative stress was even higher than ubiquinone and approached an analogue of ubiquinone known as Idebenone (Lenaz G., Bovina C., D'Aurelio M., et al. Role of mitochondria in oxidative stress and aging, Ann . Of New York Acad. Of Sci, 2002. Vol. 599. P. 199-213 Walckock et al., 1997. Jauslin ML, WirthTh., Meier Th., Schoumacher F. A cellular model for Friedreich Ataxia reveals small-molecule glutathione peroxidase mimetics as a novel treatment strategy. Human Mol. Gen., 2002. Vol. 11. P.3055-3063).

As can be seen from the data presented (figure 5), 3,6-dioxocyclohexa-1,4-diene-1,2,4,5-tetrasulfonate has a pronounced ability to protect cells from both endogenous oxidative stress and free radicals formed in the culture medium, that is, outside the cells. Thus, the proposed compound provides protection of cells from oxidative stress even in the absence of de novo glutathione synthesis, which indicates a more complex mechanism of the action of this drug on cellular metabolism under conditions of oxidative stress. A likely point of application of the antioxidant effect of the compound is participation in FAD oxidation-reduction reactions, similar to ubiquinone. Moreover, these processes can occur both in blood plasma and on the surface of the plasma membranes of cells or in the cytosol (Morre DJ, Pogue R., Morre DM A multifunctional hydroquinone oxidase of the external cell surface and sera. Biofactors. 1999. Vol. 9. P. 179-187).

The effect of sodium 3,6-dioxocyclohexa-1,4-diene-1,2,4,5-tetrasulfonate on the stability of the reduced form of riboflavin.

To obtain data on the participation of the proposed compound in cell metabolism, of particular interest were studies of their relationship with the metabolism of vital vitamins, which include vitamins of group B2, which are part of enzymes as cofactors involved in electron transport in the mitochondrial chain of oxidative phosphorylation at the boundary of reducing donors equivalents and electrons. The key places in this chain are occupied by FAD (flavin adenine dinucleotide) and NADP (nicotinamide adenine dinucleotide phosphate) —dependent enzymes and ubiquinone. The latter, in turn, is an electron donor to cytochrome b, the first in the chain of mitochondrial cytochromes (Scheme 1). Therefore, spectrophotometric studies were carried out to study the oxidation and reduction of riboflavin and the effect of sodium 3,6-dioxocyclohexa-1,4-diene-1,2,4,5-tetrasulfonate on these processes. As a result of this work, the mechanism of action of the drug at the level of riboflavin and its stabilization in a restored state was studied.

Riboflavin is a weak base capable of undergoing reversible redox reactions with sodium 3,6-dioxocyclohexa-1,4-diene-1,2,4,5-tetrasulfonate, which is a weak acid. Moreover, the fact is known that the property of the pyridine cycles underlying riboflavin contributes to the formation of complex compounds of significant stability with groups such as SO ₃ .

R = N is the oxidized form of riboflavin.

R = NH is the reduced form of riboflavin.

R = O is the oxidized form of sodium 3,6-dioxocyclohexa-1,4-diene-1,2,4,5-tetrasulfonate.

R-OH is a reduced form of sodium 3,6-dioxocyclohexa-1,4-diene-1,2,4,5-tetrasulfonate.

A kind of redox capacity is created. Free radicals and other strong oxidizing agents convert riboflavin to the form R = N, and sodium 3,6-dioxocyclohexa-1,4-diene-1,2,4,5-tetrasulfonate not only absorbs them, but also has the ability to restore its original form .

The recovery of riboflavin is significantly accelerated in an acidic environment under conditions of excess H +. Specific oxidation-reduction mechanisms can be described within the framework of the scheme below.

Scheme 1. The redox transition of riboflavin in the presence of compounds such as dihydroquinones.

Sodium 3,6-dioxocyclohexa-1,4-diene-1,2,4,5-tetrasulfonate (shown in the form as a model dihydroquinone nucleus, left) restores riboflavin through an intermediate complex, which, as already mentioned, is more actively formed in acidic environment. Catalysts or carrier intermediaries are possible in this process (Baum E.Z., Ding W. D., Siegel M.M., et al. Flavins inhibit cytomegalovirus UL 80 protease via disulfide bond formation. Biochemistry, 1996. Vol.35. P.5847-5855).

The proposed compound is a functional stabilizer of flavin-containing enzymes and vitamins.

In addition, it is known that with most pathological processes and inflammation in conditions of cellular respiration, acidosis develops. Pharmacological preparations with the properties of 3,6-dioxocyclohexa sodium under these conditions are able to actively restore the cofactors of cellular and mitochondrial enzymes, thereby ensuring the maintenance of their activity during the development of pathological processes.

Based on the antioxidant properties of sodium 3,6-dioxocyclohexa-1,4-diene-1,2,4,5-tetrasulfonate, it has been used to stabilize vitamins in foods, as long-term storage of foods, soft drinks, juices or syrups is observed both a decrease in buffer capacity and a drop in pH.

Food additives 3,6-dioxocyclohexane-1,4-diene-1,2,4,5-tetrasulfonate were tested in an amount of 0.025-50%. They achieved stabilization of vitamins without fear of toxicity (low LD ₅₀ ), without fear of causing allergies. The organoleptic properties of the products did not change. Additives of large quantities of the claimed compounds in addition to stabilizing vitamins gave antioxidant properties to food compositions (as ascorbic acid).

Redox reactions underlie the majority of pathological processes. With them and with inflammation in conditions of impaired cellular respiration, acidosis develops.

Figure 6 presents the energy curve characterizing the potentials of the electron transport chain of mitochondria, which indicates the position of the ubiquinone and FAD-prosthetic group of succinate dehydrogenase having a riboflavin nucleus in the active center. These data allow us to comprehend the mechanism of ongoing processes.

UH - ubiquinone

FMN - flavin mononucleotide

c, C ₁ , a ₂ , and ₃ - cytochromes

O ₂ - oxygen

The composition of flavin dehydrogenases includes flavin nucleotides, which are tightly bound to the apoenzyme and cannot be cleaved from it at any stage of the catalytic cycle. The redox properties of flavoproteins are due to the ability of the isoalloxazine ring of riboflavin to reversibly transition from the oxidized state to the reduced state, in which two electrons in the composition of hydrogen atoms are attached to the ring (Scheme 1). In studying respiratory chains, two flavoproteins associated with the membrane are of particular interest: succinate dehydrogenase, which catalyzes the oxidation of succinate and NAD (F) -H ₂ dehydrogenase, which catalyzes the reduction of its flavin prosthetic group, coupled with the oxidation of NAD (F) × H ₂ .

Proteins containing iron-sulfur centers take part in the electronic transport in the respiratory chain (the coordination relationship of iron atoms with sulfhydryl groups of cysteine is implied). They are part of some flavoproteins, for example, succinate and NAD (F) -H ₂ dehydrogenases, or they serve as the only prosthetic protein groups. Respiratory chains contain a large number of FeS centers. In the mitochondrial electron transport chain, probably about ten of these proteins function, and the key enzymes of the oxidative phosphorylation chain are indicated in FIG. 6. Depending on the structure, FeS centers can carry out simultaneous transfer of 1 or 2 electrons (e), which is associated with a change in the valence of iron atoms.

As already indicated, quinones are fat-soluble compounds having a long terpenoid tail connected to the quinoid nucleus capable of reversible oxidation-reduction by the addition of two hydrogen atoms. The most common is ubiquinone, which functions in the respiratory chain between the flavoproteins and cytochromes.

Unlike other electron carriers, quinones are not bound to specific proteins. A small pool of ubiquinone is dissolved in the lipid phase of the membranes, and therefore its turnover under conditions of impaired redox processes plays a critical role in impaired tissue respiration (Severin E.S. et al., Biochemistry, M., Medicine, 2000, p. 78).

The cytochromes participating in the final stage of the electron transfer chain are a group of proteins containing iron-porphyrin prosthetic groups (gems). Using cytochromes, electron transfer is carried out, during which the valence of iron changes:

Fe ²⁺ << Fe ³⁺ + e ^-

In mitochondria, the electron transport and oxidative phosphorylation chain includes five cytochromes (b, c, c ₁ , a, a ₃ ), which differ in absorption spectra and redox potentials. The differences in these parameters are due to the protein components of cytochromes, the nature of the side chains of their porphyrins, and the method of heme attachment to proteins. Terminal cytochromes (a + a ₃ ) transfer electrons to molecular oxygen, representing the actual cytochrome oxidase, in the reaction center of which, in addition to two gems, two copper atoms are contained. The formation of water occurs when four electrons are transferred to an oxygen molecule. Cytochrome oxidases carry out transfer _of only two electrons to O ₂ , which results in the occurrence of hydrogen peroxide. Hydrogen peroxide is further destroyed by catalase or peroxidase, which is reflected in the structure and functioning of the electron transport chain of mitochondria of animal and human cells (Fig.6).

FAD - the prosthetic group of succinate dehydrogenase; UH - ubiquinone; b, c, c ₁ , a, and ₃ are cytochromes.

Solid lines indicate the processes occurring in the membrane; intermittent - in the cytosol of the cell; the zigzag line shows the action sites of electron transport inhibitors.

Thus, the respiratory chain of electron transfer in mitochondria consists of a large number of intermediate carriers - cytochromes, which carry out electron transport from organic substrates to O ₂ , and ubiquinone is localized at the very beginning of this chain.

The key compound 3,6-dioxocyclohexa-1,4-diene-1,2,4,5-tetrasulfonate is close to natural quinones, and in particular to ubiquinone in structure. 7 shows the structural formula of 3,6-dioxocyclohexa-1,4-diene-1,2,4,5-tetrasulfonate and its model obtained using the Alchemie program. The ball model of the compound shows that oxygen atoms located around the circumference of the molecule are easily accessible to interact with water molecules, cations, and redox enzymes.

7:

The black balls in the center are carbon atoms (benzene rings). (1)

Gray are sulfur atoms. (2)

Black are oxygen atoms. (3)

Dark gray - hydrogen atoms. (4)

A view along a long axis is presented.

This drug exhibits high activity as a “scavenger” of free radicals and, like coenzyme Q ₁₀ , provides an increase in the efficiency of electron transport in the respiratory chain and inactivation of free radicals in cases of impaired cellular respiration and hypoxia. This function of sodium 3,6-dioxocyclohexa-1,4-diene-1,2,4,5-tetrasulfonate seems to be the most important for explaining its antioxidant properties.

The effect of sodium 3,6-dioxocyclohexa-1,4-diene-1,2,4,5-tetrasulfonate on the stability of recombinant interferon alpha-2

One of the problems with recombinant protein preparations is their low stability. On the one hand, recombinant proteins are rapidly oxidized by atmospheric oxygen, and, on the other hand, in a partially denatured state they quickly aggregate. Both oxidation and aggregation lead to their inactivation.

At the same time, many recombinant proteins are currently used in the composition of the so-called external dosage forms: suppositories, ointments, creams, gels. This leads to the fact that these drugs directly interact with atmospheric oxygen and are quickly inactivated during storage. Protein oxidation is mainly carried out by sulfhydryl groups of cysteine and methionine. Oxidation of cysteine residues leads to partial protein denaturation, which, in turn, makes them more sensitive to proteolysis. In this regard, to stabilize recombinant interferon-alpha-2, vitamin E is used as a suppository. Previously, the composition of vaginal suppositories with the addition of ε-aminocaproic acid and riboflavin (Ailamazyan E.K., Kiselev V.I., Yuriev M.Z. ., Kiselev O.I. Patent of the Russian Federation No. 2180593). These components have a dual purpose. On the one hand, aminocaproic acid is a protease inhibitor, and, on the other hand, has weak antioxidant properties. Both of these properties are aimed at protecting and stabilizing the main active component of suppositories - interferon-alpha-2.

However, the activity of additional components is equally aimed at inhibiting viral and cellular proteases and inactivating free radicals generated in the focus of inflammation.

However, with respect to the stabilization of recombinant preparations, the antioxidant properties of sodium 3,6-dioxocyclohexa-1,4-diene-1,2,4,5-tetrasulfonate are particularly attractive. The compound was tested for its effect on the stabilization of recombinant proteins. Given the low toxicity of the compound, such a compound is compatible with both external dosage forms and injectable.

Studies of the stabilization of recombinant α -2-interferon with a new compound have shown good results.

On Fig presents data on the study of the effect of 3,6-dioxocyclohexa-1,4-diene-1,2,4,5-tetrasulfonate sodium on the stability of a preparation containing recombinant 1-2-interferon for 2 weeks at a temperature + 5 ° C. Curve 1 - storage of the drug with the addition of 3,6-dioxocyclohexa-1,4-diene-1,2,4,5-tetrasulfonate sodium. Curve 2 - storage of the drug without any stabilizers. The abscissa axis is days, the ordinate axis is biological activity in ME.

As can be seen in Fig. 8, compound 1 has significant stabilizing ability for the preparation of recombinant α -2-interferon. This allows us to recommend it for the stabilization of recombinant preparations of human proteins and, in particular, α-2-interferon.

An example of the manifestation of the antioxidant properties of sodium 3,6-dioxocyclohexa-1,4-diene-1,2,4,5-tetrasulfonate (use in a food composition).

Dry fortified beverage - a composition which includes riboflavin (vitamin B _2), were tested for retention of activity when stored riboflavin (the effect of atmospheric oxygen, ultraviolet radiation, natural light, air temperature A composition without the additive was 3.6 dioksotsiklogeksa-1. , 4-diene-1,2,4,5-tetrasulfonate sodium (control samples); another composition contained a substance according to the invention.

The amount of riboflavin in the composition is 0.025%

The amount of 3,6-dioxocyclohexa-1,4-diene-1,2,4,5-tetrasulfonate in the composition is 0.025%.

Storage conditions for the composition: accelerated aging - 7 days, long aging - 180 days, storage temperature - + 38 ° C, UV irradiation and exposure to open sunlight were used.

The quantitative content of riboflavin in the images of drinks was determined according to GOST 7047-55. Organoleptic tests according to GOST 66875-86.

As a result of the studies, it was found that when storing samples of drinks with riboflavin and 3,6-dioxocyclohexa-1,4-diene-1,2,4,5-tetrasulfonate, the content of riboflavin decreased by 1.65 mg in the control samples not containing the claimed compound, by 2.45 mg, which is 16% more than in samples with the proposed invention according to the invention tool (table 1).

The content of riboflavin in the sample, mg Storage mode when: Name of the test sample Sample weight, g Storage time, day Starting sample B2 mg temperature 38 ° С ultraviolet radiation exposure to sunlight (daylight hours) Dry fortified drink with riboflavin and 3,6-dioxocyclohexa-1,4-diene-1,2,4,5-tetrasulfonate 20,0 7 5,0 4.35 3.90 4,55 Dry fortified drink with riboflavin without 3,6-dioxocyclohexa-1,4-diene-1,2,4,5-tetrasulfonate 20,0 7 5,0 3,55 3.85 4,55

Storage of samples of drinks with riboflavin for more than 90 days led to partial changes in organoleptic characteristics: color and taste (when stored without the means proposed according to the invention).

The storage of beverage samples with the addition of 3,6-dioxocyclohexa-1,4-diene-1,2,4,5-tetrasulfonate did not lead to changes both quantitatively in terms of riboflavin and organoleptic indicators at the indicated times.

Example. To the dry components of fruit juices for the preparation of drinks was added dry 3,6-dioxocyclohexa-1,4-diene-1,2,4,5-tetrasulfonate in the ratio (in weight):

dry fruit extracts: the claimed tool is 1: 0.5.

Checking the storage according to GOST requirements showed that the organoleptic characteristics (color, taste) have not changed. Sodium 3,6-dioxocyclohexa-1,4-diene-1,2,4,5-tetrasulfonate remained practically unchanged during storage.

From tests it was unequivocally established that the new compound 3,6-dioxocyclohexa-1,4-diene-1,2,4,5-tetrasulfonate sodium is an antioxidant (antioxidant), acceptable due to low toxicity in the food industry.

Antiviral activity of sodium 3,6-dioxocyclohexa-1,4-diene-1,2,4,5-tetrasulfonate.

The antiviral activity of the claimed compounds was investigated on a model of influenza infection of mice. Mice were infected with a 1-fold lethal dose of the virus. The drug was administered orally at a dose of 30 mg / kg. Data on the determination of antiviral activity are summarized in table 2.

table 2 The activity of 3,6-dioxocyclohexa-1,4-diene-1,2,4,5-tetrasulfonate in relation to lethal influenza infection of mice caused by influenza virus A drug Scheme Dose mg / kg single total Accounting for the death of animals 79% > 70% % of the fallen protection index % of the fallen protection index 3,6-dioxo-cyclohexa- Preventative 30/60 63.1 ± 11.4 25 84.2 ± 8.6 10
- 1,4-diene-1,2,4,5- Medical 30/120 65.0 ± 10.9 23 85.0 ± 8.2 10
- sodium tetrasulfonate Standard
5 days 30/150 25.0 ± 9.9 70
+++ 80.0 ± 9.2 16 Standard
5 days 30/150 25.0 ± 9.9 67
+++ 75.0 ± 9.9 eleven Standard
7 days 30/210 25.0 ± 9.9 64
+++ 60.0 ± 10.9 33
+ Standard
5 days 60/300 5.0 ± 5.0 98
++++ 35.0 ± 10.9 63
+++

Thus, antiviral activity is an additional property of 3,6-dioxocyclohexa-1,4-diene-1,2,4,5-tetrasulfonate.

Antiviral activity can be attributed to the influence on the initial stages of the replicative cycle of influenza viruses and, in particular, on the interaction with cellular receptors and (or) on the lysosomal stage of virion decapsidation. It can be assumed that the formation of multidimensional structures takes place in solution. Then aqueous solutions will be more active against viral infection.

The uniqueness of this compound is primarily manifested in its exceptional antioxidant properties. Due to the unique structure of π-bond systems, this compound is distinguished by the fact that it exhibits higher antioxidant activity compared to ubiquinone; the advantage of the synthesized compound is its water solubility. The obtained new antioxidant has high bioavailability and an active effect on the enzymes of the cytoplasm of cells and intercellular fluid.

The antiviral activity of the claimed drug allows you to recommend it for protection against viral influenza infection.

Claims (1)

An agent exhibiting antioxidant and antiviral activity, which is sodium 3,6-dioxocyclohexa-1,4-diene-1,2,4,5-tetrasulfonate.

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