RU2430105C1 - Potassium salt of carboxymethyl ester of 3-oxy-ursan-12-ene-28 acids exhibiting hepatoprotective, antioxidant and anti-inflammatory activity - Google Patents

Abstract

FIELD: medicine. ^ SUBSTANCE: invention refers to potassium salt of carboxymethyl ester of 3-oxy-ursan-12-ene-28 acid of formula I: A method for preparing is based on transformations of ursolic acid produced by received from juice production waste. ^ EFFECT: compound exhibits expressed hepatoprotectory, antioxidant activity and anti-inflammatory characteristics, and also higher solubility as compared with ursolic acid that will allow to use it in medicine as a complex hepatoprotector. ^ 1 cl, 5 ex, 4 tbl, 1 dwg

Description

The invention relates to the pharmaceutical industry, specifically to a new chemical compound - potassium salt of carboxymethyl ether 3-hydroxy-ursan-12-ene-28th acid (according to IUPAC nomenclature - potassium salt 2 - ((1S, 2R, 4aS, 6aS 6bR, 10S, 12aR, 12bR, 14bS) -10-hydroxy-1,2,6a, 6b, 9,9,12a-heptamethyl-1,2,3,4,4a, 5,6,6a, 6b, 7,8,8a, 9,10,11,12,12a, 12b, 13,14b-eicosahydropicene-4a-carbonyloxy) acetic acid) of the formula I

possessing hepatoprotective, antioxidant and anti-inflammatory activity, which will make it possible to use it in medicine as a pharmaceutical.

Most modern hepatoprotective drugs are made from plant materials. Dosage forms are represented either by total water-alcohol and dry extracts of the corresponding plants, or by extracts of extract components similar in properties. Examples are legalon, silibor, catergen, valiliv, flaming, flacumin. The active principle of drugs of this group are mainly flavonoids, such as rutin, quercetin [M.D. Mashkovsky. Medicines In two volumes. Ed. “New Wave”, Moscow, 1996, v. 1, p. 603. Medicines approved for use in the USSR. Ed. M.A. Klyueva, E.A. Babayana. M., "Medicine", 1979, pp. 61-65], dihydroquercetin [M.B. Plotnikov, N. A. Tyukavkina, T. M. Plotnikova. Medicines based on dikvertin. Tomsk Ed. Tomsk University. 2005, 228 p., V.V. Kugach, N.I. Nikulshina, V.I. Ishchenko. Dosage forms of flavanoids. (Overview). Chemical Pharmaceutical Journal. 1988, No. 8, pp. 1018-1025]. The main side effect of drugs based on flavonoids is associated with their effect on the gastrointestinal tract, which manifests itself mainly in the form of nausea, heartburn, dyspepsia, and sometimes vomiting [M.D. Mashkovsky. Medicines In two volumes. Ed. “New Wave”, Moscow, 1996, v. 1, p. 603. Medicines approved for use in the USSR. Ed. M.A. Klyueva, E.A. Babayana. M., "Medicine", 1979, S. 61-65].

Dihydroquercetin is the main bioflavonoid (content of 90% and higher) of the drug dicvertin, which is made from waste products from larch wood [patent RU No. 2088256. "Means for the complex therapy of dicvertin diseases and a method for its production" Publ. 12/02/1996, Discoveries. Inventions 1997, No. 24]. Dihydroquercetin has antioxidant, anti-inflammatory, capillary-protective, gastro- and hepatoprotective properties [V.K.Kolkhir, N.A. Tyukavkina, V.A. Bykov. New antioxidant remedy "Dikvertin", Practical herbal medicine. 1997, No. 1. S.12-16]. The indicated effects provide this compound with the status of a basic “substance for the creation on its basis of combined preparations that expand the spectrum of its biological activity [MB Plotnikov, N. A. Tyukavkina, T. M. Plotnikova. Medicines based on dikvertin. Tomsk. Ed. Tomsk University. 2005, 228 p., V.V. Kugach, N.I. Nikulshina, V.I. Ishchenko. Dosage Forms of Flavonoids. (Review). Chemical-Pharmaceutical Journal. 1988, No. 8, pp. 1018-1025 ].

Another potentially valuable compound for medicine isolated from plant materials is ursolic acid. It has been established that it exhibits hepatoprotective, anti-inflammatory, antioxidant and antitumor activity [Liu Jie. Pharmacology of oleanolic acid and ursolic acid, J. of Ethnopharmacology. 1995. V. 49, Issues 2-1, pp. 57-68., R. Saravanam, P. Viswanathan, R. Viswanathan Pugalendi. Protective effect of ursolic acid on ethanol-mediated experimental liver damage in rats, Life Sciences. 2006. V.78. Issue 7, pp. 713-719].

The indicated properties testify to its high potential as a base product for the preparation of derivatives with high biological activity.

Ursolic acid belongs to plant metabolites and is found in a variety of plant sources. Ursolic acid of high purity can be easily obtained from available domestic raw materials — waste products from juices — cranberry meal [patent RU No. 181636, S. A. Shevtsov, V. A. Raldugin, G. I. Shchukin. Method for producing ursolic acid. Publ. 09/27/1995] and chokeberry [patent RU No. 2329048, L. P. Kozlova, E. V. Malykhin, S. M. Obut, S. A. Popov, O. P. Sheremet. A method of producing ursolic acid. Publ. 07/20/2008].

The disadvantage of this compound, limiting its use in medicines, is the low solubility in polar and non-polar solvents. This property can manifest itself in the form of low bioavailability, which usually requires an increase in dosages.

The objective of the invention is the creation on the basis of ursolic acid hepatoprotector with antioxidant and anti-inflammatory activity, characterized by a higher solubility in water compared with ursolic acid and obtained from renewable domestic raw materials.

The solution of this problem is achieved by creating a compound of formula I, which is a potassium salt of carboxymethyl ether 3-hydroxy-ursan-12-en-28-oic acid, which has increased solubility in water and exhibits hepatoprotective, antioxidant and anti-inflammatory activity. Currently, drugs based on this compound are not on the market.

The advantage of the hydroxyacetic acid derivatives to which compound I belongs is that they are stronger acids than ursolic and form stable salts that do not hydrolyze in aqueous solutions. Due to this property, compound I has a higher solubility compared to ursolic acid and its salts. It has been shown that salts of ursolic acid, which is a weak acid, are unstable and easily hydrolyzed in an aqueous medium, and free ursolic acid precipitates from solutions [N.M. Sell, R.E. Kremers, Metallic Salts of Ursolic Acid, bid. Eng. Chem. Anal. Ed., 1935, 7 (2), p.105-106].

An analogue of the claimed compounds in antioxidant and hepatoprotective action is dihydroquercetin [(2R, 3R) -3,5,7,3 ', 4'-pentahydroxyflavanone] of the formula (II)

The prototype and analogue of the claimed compounds in structure and biological properties is a pentacyclic triterpene compound - ursolic acid III

Comparison compound for anti-inflammatory properties is the non-steroidal anti-inflammatory drug indomethacin (substance "Fluka" BioChemika).

Compound I is prepared as follows: ursolic acid III is alkylated with ethyl chloroacetate in dimethylformamide in the presence of potassium carbonate, ethyl ether IV is obtained, which is separated by filtration after the reaction mixture is poured into water. Ethyl ether IV without drying is treated with a solution of an alcoholic solution of KOH under boiling, and the carboxyethyl fragment is selectively hydrolyzed, after which the potassium salt I is separated by crystallization (see drawing).

The biological activity of compound I was studied by determining hepatoprotective, antioxidant and anti-inflammatory properties in outbred mice. The antioxidant dihydroquercetin (II) (99.9% purity) and the non-steroidal anti-inflammatory drug indomethacin (substance Fluka BioChemika) were used as comparison preparations. Ursolic acid was a structural analogue of the studied compound.

To study the hepatoprotective and antioxidant effects, a standard experimental model of toxic CCl ₄ hepatitis in mice was used. The model was reproduced according to the methodological recommendations [Guide to the experimental (preclinical) study of new pharmacological substances. Moscow. Publishing House of Medicine. 2005, 240 pp.]. Toxic hepatitis was modeled by intragastric administration to mice of a 25% solution of CCl ₄ in vegetable oil. Compound I was injected into the stomach at a dose of 20 mg / kg in the form of a water-twin suspension 1 hour before hepatotoxin. The reference compound, dihydroquercetin (DHQ), was administered in a similar manner at a dose of 50 mg / kg. The hepatoprotective effect was determined by a decrease in serum markers of cytolysis and cholestasis - alanine aminotransferase (ALT), aspartate aminotransferase (ACT) and alkaline phosphatase (ALP), antioxidant properties were evaluated by a decrease in the concentration of peroxidation products (TBCRC), determined by the reaction with thiobarbit BC Handbook of clinical and chemical laboratory diagnostics, Minsk: Belarus, 2000, T. 2, p.207].

Serum samples were taken one day after the introduction of agents. The analysis of biochemical parameters was carried out using standard sets of reagents (Olveks Diagnosticum, Germany) on a biochemical analyzer (Photometer 5010, Germany).

It was found that compound I, when administered intragastrically at a dose of 20 mg / kg, has a pronounced hepatoprotective effect, reducing the severity of cytolytic processes by 2.5-3.5 times and the severity of cholestasis by 1.6 times against toxic hepatitis. It was shown that, in terms of the anticytolytic and anticholestasis effect, compound I is superior to its structural analogue and reference preparation (Table 1).

It was shown that compound I has a significant antioxidant effect, reducing the concentration of TBKRS in the blood by 1.4 times relative to the control, while the claimed compound is not inferior to the reference agents in terms of the antioxidant effect (Table 2).

The anti-inflammatory activity of the ursolic acid derivative was investigated on a standard model of histamine-induced inflammation [Guide to the experimental (preclinical) study of new pharmacological substances. Moscow. Publishing House of Medicine. 2005, 240 pp.]. Compound I and its structural analogue were administered intragastrically at a dose of 20 mg / kg 1 hour prior to subplant implantation of phlogogen. The reference drug indomethacin was administered in the same manner at an effective dose of 20 mg / kg. The anti-inflammatory effect was evaluated by a decrease in comparison with the control of the edema index, which was calculated as a percentage as the ratio of the difference between a healthy and inflamed paw to a healthy mass. It was shown that compound I reduces the severity of swelling of the paws of mice by 1.8 times. In terms of anti-inflammatory effect, the claimed compound was not inferior to ursolic acid, but was 1.2 times less effective than the reference preparation indomethacin (Table 3).

Thus, the new compound - the potassium salt of carboxymethyl ether 3-hydroxy-ursan-12-ene-28-one acid (I) - has the following advantages, namely:

- a higher hepatoprotective effect compared with ursolic acid and dihydroquercetin;

- antioxidant activity similar to dihydroquercetin;

- anti-inflammatory activity, not inferior to that of ursolic acid;

- higher solubility in water than ursolic acid;

- the use in the synthesis of available ursolic acid obtained from waste from the food industry.

The invention is illustrated by the following examples.

Example 1. Obtaining potassium salt of carboxymethyl ester of 3-hydroxy-ursan-12-en-28-oic acid (I)

To a suspension of ursolic acid (0.92 g, 2 mmol) in DMF (10 ml), crushed K ₂ CO ₃ (0.45 g, 3.3 mmol) and ethyl 2-chloroacetate (0.25 g, 2 mmol) are added. The mixture was stirred at room temperature for 4 hours. The reaction mixture was poured into water 30 ml and filtered.

The crude precipitate without drying is placed in a solution of KOH (0.4 g, 7.2 mmol) in ethanol (10 ml). The mixture is boiled for 1 hour and cooled to room temperature, the precipitated crystals are separated by filtration and washed with chilled ethyl alcohol (+ 5 ° C, 5 ml), dried in air, and potassium salt I of 0.97 g (94%) is obtained as a needle crystals. Mp> 300 ° C (decomp.)

NMR spectra are taken for free acid V. To obtain acid V, potassium salt I is treated with dilute sulfuric acid, washed with water and dried in air:

H ¹ NMR spectrum ^of compound V (Py-d ₅ , 300 MHz, δ _C ppm): 0.83 s (3H), 0.90 s (3H), 0.91 s (3H), 0.92 m (3H), 1.00 s ( 3H), 1.15 s (3H), 1.21 s (3H), 2.49 d (1H, J = l 1.0 Hz), 3.41 m (1H), 4.92 d (1H, J = 15.5 Hz), 5.03 d (1 H, J = 15.5 Hz), 5.3 m (1 H).

¹³ C NMR spectrum of compound V (Py-d ₅ , 75 MHz, δ _C ppm): 174.78, 168.87, 136.47, 123.90, 76.00, 59.26, 53.67, 51.14, 46.24, 45.86, 40.25, 37.82, 37.22, 37.19 , 37.02, 36.99, 35.12, 34.87, 31.33, 28.77, 26.67, 26.31, 25.92, 22.54, 21.68, 21.49, 19.16, 16.63, 15.24, 15.20, 14.44, 13.60.

Example 2. The study of the solubility of potassium salt I and acid V in comparison with the solubility of ursolic acid III and its potassium salt

The solubility of acids and their salts in ethanol is studied by the gravimetric method for saturated solutions. The solubility of acids and their salts in water is studied using UV absorption spectra for saturated solutions.

Table 1 Solubility of ursolic acid, carboxymethyl ether 3-hydroxy-ursan-12-en-28-oic acid and their potassium salts Solvent Ursolic Acid III K ursolic acid salt Acid V K salt (I) ethanol 10 ± 1 g / l - 65 ± 10 g / l 35 ± 5 g / l water 6 ± 2 mg / l 70 ± 15 mg / l 90 ± 15 mg / l 120 ± 20 mg / l

From the data presented in table. 1, it is seen that the solubility of acid V and its salt I in ethanol significantly exceeds that for ursolic acid. The solubility of acid V and its salt I in water also exceeds the corresponding values for ursolic acid and its potassium salt.

The higher solubility of K salt I in water compared with free acid V may possibly increase the bioavailability of compound I.

Example 3. The study of hepatoprotective properties in a model of acute toxic hepatitis

Acute toxic hepatitis was caused in outbred male mice weighing 20-25 g by a single intragastric administration of a 25% solution of CCl ₄ in sunflower oil at the rate of 0.1 ml per 10 g of body weight [Guide to experimental (preclinical) study of new pharmacological substances. Moscow. Publishing House of Medicine. 2005, 240 pp.]. Compound I and ursolic acid were administered 1 hour before the reproduction of hepatitis intragastrically as a suspension in distilled water with the addition of Tween-80 emulsifier at a dose of 20 mg / kg (in a volume of 0.2 ml per 10 g of animal weight). Dihydroquercetin was administered in the same manner at an effective dose of 50 mg / kg. Control animals were injected with water-twin suspension in an equivalent volume. In each group there were 9-10 individuals.

After one day, the animals were killed by decapitation under light ether anesthesia and the activity of ALT, ACT, and alkaline phosphatase was determined in the blood serum of mice using standard reagent kits (Olveks Diagnostic).

The results were statistically processed using the STATISTIKA 6 software package.

It was found that compound I under conditions of toxic hepatitis has a significant anti-cytolytic effect, reducing the activity of ALT and ACT in the blood by 3.6 and 2.4 times, respectively, compared with the control. Similar effects in reference agents were less pronounced: ursolic acid decreased the activity of ALT and ACT by 2.8 and 1.7 times against the control, and dihydroquercetin - by 2.9 and 1.5 times, respectively. It was shown that compound I showed a significant anticholestasis effect, decreasing the level of alkaline phosphatase in the blood by 1.6 times. Ursolic acid reduced this indicator by 1.2 times compared with the control. Dihydroquercetin did not show anticholestasis, significantly increasing the level of alkaline phosphatase relative to the control. Thus, compound I has a pronounced hepatoprotective effect, surpassing its structural analogue and reference preparation in anticytolytic and anticholestasis (Table 1).

table 2 The effect of compound I on the biochemical parameters of the blood serum of mice with induced CCl ₄ hepatitis The control Compound I UK DKV ALT, Unit / L 159.3 ± 20.4 43.8 ± 11.1 *** 57.1 ± 0.2 *** 54.3 ± 10.9 *** ACT, U / L 121.5 ± 11.1 51.3 ± 11.3 ** 71.1 ± 13.5 * 80.9 ± 11.1 * Alkaline phosphatase, units / l 530.3 ± 47.4 326.8 ± 25.7 ** 442.6 ± 55.8 735.9 ± 66.7 * * P <0.05; ** P <0.01; *** P <0.001 - differences with control are significant K - control, CC - ursolic acid, DHQ - dihydroquercetin, ALT, ACT - alanine, aspartate aminotransferase, alkaline phosphatase - alkaline phosphatase.

Thus, it was shown that compound I, when administered intragastrically at a dose of 20 mg / kg, has a pronounced hepatoprotective effect, reducing the severity of cytolytic processes by 2.5-3.5 times and the severity of cholestasis by 1.6 times against toxic hepatitis.

Example 4. The study of antioxidant properties in a model of acute toxic hepatitis

The antioxidant effect of compound I was determined on the model of toxic hepatitis CCl ₄ described above. The influence of agents on the severity of lipid peroxidation processes was evaluated by the level of TBA-reactive compounds (TBARS) in blood serum, which are secondary products of peroxidation. Compound I and comparison agents were administered 1 hour before hepatitis was reproduced intragastrically as a suspension in distilled water with the addition of Tween-80 emulsifier at a dose of 20 mg / kg (in a volume of 0.2 ml per 10 g of animal weight). The doses administered were: for ursolic acid and the compound 1-20 mg / kg; for dihydroquercetin - 50 mg / kg. Control animals were injected with water-twin suspension in an equivalent volume. In each group there were 9-10 individuals. After a day, the animals were killed by decapitation under light ether anesthesia and the concentration of TBARS in the blood serum of mice was determined by the conventional method [Kamyshnikov BC Handbook of clinical and chemical laboratory diagnostics, Minsk: Belarus, 2000, T.2, p.207].

The results were statistically processed using the STATISTIKA 6 software package. Differences were considered significant with a probability of p <0.05.

It was found that compound I had a significant antioxidant effect, reducing the concentration of TBKRS in the blood by 1.4 times relative to the control, ursolic acid and dihydroquercetin almost equally (1.3 times) reduced this indicator, but the effect of ursolic acid was not significant (table. 2). Thus, compound I was not inferior to the reference agents in antioxidant properties.

Table 3 The effect of compound I on the level of TBA-reactive compounds in blood serum Group The control Compound I UK DKV TBKRS, μmol / L 4.9 ± 0.5 3.6 ± 0.2 * 3.8 ± 0.3 3.7 ± 0.3 * * P <0.05; ** P <0.01 - differences with control are significant ^# P <0.05 differences with DQV significant K - control, CC - ursolic acid, DHA - dihydroquercetin, TBKRS - TBA reactive compounds.

Example 5. The study of the anti-inflammatory properties of compound I on a model of histamine inflammation.

The experiments were carried out on outbred mice males weighing 20-25 g. Animals were divided into groups of 8 individuals. Compound I was administered intragastrically as a suspension in distilled water with the addition of Tween-80 emulsifier at a dose of 20 mg / kg (in a volume of 0.2 ml per 10 g of animal weight). Separate groups of mice were similarly injected with a structural analogue of ursolic acid at a dose of 20 mg / kg, as well as a reference anti-inflammatory drug indomethacin at an effective dose of 20 mg / kg. Control animals received an equivalent amount of a water-twin emulsion. 1 hour after the administration of agents, all mice were subplanarly injected with a 0.5% histamine solution of 0.05 ml / mouse in the hind paw. 5 hours after phlogogen administration, mice were sacrificed by cervical dislocation of the spine, both hind legs were cut off, and the mass of each was determined. The anti-inflammatory effect was evaluated by the value of the inflammation index, which was determined as the ratio of the mass difference between the inflamed and intact paws to the intact mass, expressed as a percentage. The results were statistically processed using the STATISTIKA 6 software package. Differences were considered significant with a probability of p <0.05.

The data of the experiment are given in table. 3. It was shown that compound I reduced the severity of edema of the paw of mice as well as ursolic acid (1.8 times), while indomethacin reduced edema by 2.1 times. Thus, under the conditions of inflammation caused by histamine, compound I was not inferior in anti-inflammatory effect to its structural analogue, but was 1.2 times less effective than the reference drug indomethacin.

Table 4 The effect of agents on the value of the histamine edema index Group The control Compound I UK Indomethacin Edema index,% 29.57 ± 3.11 # 16.94 ± 1.06 ** 16.82 ± 3.00 * 13.83 ± 1.55 ** * p <0.05; ** p <0,01 - differences with control are significant; ^# p <0.05 differences with indomethacin are significant.

Thus, it was found that compound I is not inferior to ursolic acid in anti-inflammatory activity.

Claims (1)

Potassium salt of carboxymethyl ether 3-hydroxy-ursan-12-en-28-oic acid of the formula I

possessing hepatoprotective, antioxidant and anti-inflammatory activity.

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