RU2364599C2 - Antioxidant, possessing hepatoprotective and hemostimulating activity - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to novel compound, namely to (1'S,5'S,9'S,10'R)-methyl-5-[2-(3-benzyl-6-carboxy-4-oxo-10-oxa-3-azatricyclo[5.2.1.0^1.5]deca-8-ene-9-yl)ethyl]-1,10-dimethyl-6-methylene-decahydronaphtylene-1-carboxylate of formula (I)

.

EFFECT: possesses anti-oxidant, hepatoprotective and hemostimulating activity, has low toxicity, and can be applied in medicine.

1 cl, 4 tbl, 4 ex

Description

The invention relates to a new chemical compound, specifically to (1'S, 5'S, 9'S, 10'R) -methyl-5- [2- (3-benzyl-6-carboxy-4-oxo-10-oxa-3-azatricyclo [5.2 .1.0 ^1,5 ] dec-8-en-9-yl) ethyl] -1,10-dimethyl-6-methylene-decahydronaphthalene-1-carboxylate of the formula (I),

possessing antioxidant, hepatoprotective and hemostimulating activity.

This property suggests the possibility of using the compound in medicine as a pharmaceutical preparation.

In modern medical practice, antioxidants of synthetic and natural (plant) origin are used. Among plant-based antioxidants, flavonoids, for example, rutin, quercetin [1, 2], and dihydroquercetin [3], have found application. An analogue in the pharmacological properties of the claimed compound is dihydroquercetin [(2R, 3R) -3,5,7,3 ', 4'-pentahydroxyflavanone] of the formula (II).

Dihydroquercetin is the main bioflavonoid (90% and higher) of the drug diquertin, the production of which from larch wood has been established in recent years. Dihydroquercetin has antiradical and antioxidant activity, anti-inflammatory, capillaroprotective, gastro- and hepatoprotective properties [3]. 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. The main disadvantage of drugs based on flavonoids is a possible side effect on the gastrointestinal tract, which manifests itself mainly in the form of nausea, heartburn [1, 2].

The problem to which the invention is directed, is to create, on the basis of domestic plant materials, an antioxidant of a new structural type with high hepatoprotective activity. An important element of the task is to obtain an agent with improved pharmacological properties aimed at correcting systemic side effects that occur when using highly toxic drugs, such as cytostatics. The drugs of this group, widely used in antitumor therapy, cause severe disorders in the form of myelo- and hemodepression, immunological disorders, functional and morphological damage to various organs, etc. [4, 5]. An analysis of literature data shows that the synthesis of new compounds from plant materials in order to expand the range of non-toxic antioxidants with additional (in addition to antioxidant activity) protective properties is an urgent task.

The problem is solved by a new chemical compound - (1'S, 5'S, 9'S, 10'R) -methyl-5- [2- (3-benzyl-6-carboxy-4-oxo-10-oxa-3-azatricyclo [5.2.1.0 ^1,5 ] dec-8-en-9-yl) ethyl] -1,10-dimethyl-6-methylene-decahydronaphthalene-1-carboxylate of the formula (I) having hepatoprotective and hemostimulating activity, including against the background of the introduction of the body of cytostatic drugs.

The structural analogue of these compounds is methyl 5- [2- (4-benzyl-3,5-dioxo-10-oxa-4-azatricyclo [5.2.1.0 ^2,6 ] dec-8-en-9-yl) ethyl ] -1,10-dimethyl-6-methylene-decahydronaphthalene-1-carboxylate of the formula (III) having antidepressant activity [6].

The method of obtaining compound (I) containing a labdanoid backbone in the structure connected to a 10-oxa-3-azatricyclo [5.2.1.0 ^1,5 ] dec-8-ene fragment is carried out according to the above scheme 1. Acylation 16 - [(N- benzyl) aminomethyl] methyl amberthianate (IV) obtained by aminomethylation of methyl amberthianate according to the procedure in [7], maleic anhydride proceeds under mild conditions (chloroform, 20 ° С) and leads to the formation of substituted 10-oxa-3-azatricyclo [5.2.1.0 ^1,5 ] -6-carboxy-dec-8-en-4-one (I). The compound is formed as a mixture of (1R, 5S, 6R, 7R) - and (1S, 5R, 6S, 7S) -diastereoisomers (1: 1) (according to ¹ H NMR spectrum) (84% yield).

Scheme 1

The biological activity of compound (I) was studied by determining the antioxidant and hepatoprotective activity in a model of toxic CCl ₄ hepatitis in mice, as well as in rat damage caused by the introduction of cyclophosphamide. The antioxidant dihydroquercetin (II) was used as a comparison drug.

Previously, in an experiment on outbred mice weighing 18-23 g, acute toxicity was determined with a single intragastric route of administration. It was found that the LD _{50 of} compound (I) exceeds the maximum possible dose for a single administration of 1000 mg / kg.

To study the antioxidant and hepatoprotective effects, a standard experimental model of toxic hepatitis CCl ₄ in mice was used. The model was reproduced according to the guidelines [8]. A solution of CCl ₄ in vegetable oil (25%) was administered intragastrically to male mice. Compound (I) was injected into the stomach at a dose of 100 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. A day later, the activity of alanine aminotransferase (ALT), aspartate aminotransferase (ACT), alkaline phosphatase (ALP) and the concentration of malondialdehyde (MDA) were determined by standard methods in mice [9]. It was established that compound (I) under the conditions of toxic hepatitis has a significant anti-cytolytic effect, reducing the activity of ALT and ACT in the blood, respectively, by 1.5 and 1.9 times compared with the control. Agent (I) is not inferior in its effect on the ALT level, but 1.6 times more in terms of its effect on ACT. According to the severity of the anticholestasis effect, compound (I) is not inferior to DHQ (Table 1).

A study of the protective effect of compound (I) under conditions of cyclophosphamide (CF) damage was studied in Wistar female rats. CF was administered once intraperitoneally at a dose of 125 mg / kg in a solution of 0.9% NaCl to all animals. Compound (I) in the form of a water-twin suspension was administered to a group of rats (12 pcs.) In the stomach at a dose of 50 mg / kg for three days after administration of CF. The reference compound, dihydroquercetin (DHQ), was administered at the same dose in the same way to a separate group of rats (10 pcs.). The control group was administered only cyclophosphamide (10 pcs.). At the end of the experiment, the composition of the peripheral blood and leukocyte formula were determined. The activity of alanine aminotransferase (ALT), aspartate aminotransferase (ACT), alkaline phosphatase (ALP), the concentration of total protein, and glucose were studied in blood serum using standard reagent kits. The concentration of malondialdehyde (MDA) was studied by the conventional method [9].

The results of the study of biological activity are given in table.2-4. It was found that compound (I), when administered intragastrically at a dose of 50 mg / kg against the background of cyclophosphamide lesion, is inferior to DHQ in anticytolytic properties, but surpasses it in anticholestic and antioxidant effects. It was found that compound (I) tends to decrease cytopenia caused by cyclophosphamide, increasing the number of leukocytes and platelets relative to the control, respectively, 1.4 and 1.2 times, the number of neutrophils - 1.8, and monocytes - 2.5 times . It was shown that the hemostatic effect of compound (I) is superior to that for DKV.

Thus, the present invention has the following advantages.

- Compound (I) has a high antioxidant and hepatoprotective activity, as well as a hemostimulating effect against the background of the introduction of the cytostatic cyclophosphamide.

- Use for its synthesis of the original obtained from available raw materials - needles or gum resin of Siberian cedar Pinus sibirica R. Mayr.

The invention is illustrated by the following examples.

Example 1. Synthesis of (1R, 5S, 6R, 7R) - and (1S, 5R, 6S, 7S) - (1'S, 5'S, 9'S, 10'R) -Metyl-5- [2- (3-benzyl-6 -carboxy-4-oxo-10-oxa-3-azatricyclo [5.2.1.0 ^1,5 ] deca-8-en-9-yl) ethyl] -1,10-dimethyl-6-methylene-decahydronaphthalene-1-carboxylates (I).

To a stirred solution of 0.87 g (1.96 mmol) of furfurylamine (IV) [7] in 30 ml of benzene was added 0.19 g (1.96 mmol) of maleic anhydride. The reaction mixture was stirred at 20 ° C for 48 h. The solvent was evaporated in vacuo, the residue was chromatographed on silica gel (eluent was chloroform). Received 0.90 g (84%) of a mixture of (1R, 5S, 6R, 7R) - and (1S, 5R, 6S, 7S) -diastereoisomers of 10-oxa-3-azatricyclo [5.2.1.0 ^1,5 ] deca-8-ene (I) as a colorless oil. UV spectrum, λ _max. , nm (log ε) 258 (1.71), 264 (1.67). IR, cm ^-1 : 692 (C = C); 1132, 1229, 1723 (C = O). ¹ H NMR (δ, ppm, J, Hz): 0.45 (6H, C ^{16 _'3} H), 0.97 m (2H, H ^4'), 1.00 m (2H, H ^{2 '),} 1.14 s (6H, C ^{15 'H} _3), 1.24 m (2H, H ^9'), 1.45 m (2H, H ^{3 ', 11),} 1.52 m (4H, H ^{5', 11),} 1.73 m ( 4H, H ^{4 ', 8'} ), 1.81 m (6H, H ^{3 ', 7', 12 '} ), 1.95 m (2H, H ⁸ ), 2.14 d.m. (2H, H ^{2 '} , J _heme 13.1), 2.22 m (2H, H ^12' ),

2.35 m (2H, H ^{7 '} ), 2.83 br. s (2H, H ⁶ ), 2.87 br. s (2H, H ⁵ ), 3.50 d, 3.53 d (2H, H ² , J 13), 3.58 s (6H, OCH ₃ ), 3.66 d, 3.68 d (2H, H ² , J 13), 4.33 s, 4.36 s (2H, H ^{13 '} ), 4.40 d, 4.60 d

(4H, CH ₂ Ph, J 12), 4.78 s (2H, H ^{13 '} ), 5.14 br. s (2H, H ⁷ ), 5.94 d (1H, H ⁸ , J 1.3), 5.96 d (1H, H ⁸ , J 1.4), 7.21 m (4H, H ^{2``, 6 ''} ), 7.26 m ( 2H, H ^{4 ''),} 7.30 m (2H, H ^{3 ', 5''). 13} C NMR spectrum, δ, ppm: 12.31 k, 12.38 k (C ^{16 '} ), 19.70 t (C ^3' ), 20.79 t, 21.10 t (C ^{11 '} ), 25.54 t (C ^12' ), 25.99 t (C ^{8 '} ), 28.56 k, 28.58 k (C ^15' ), 37.92 t (C ^{2 '} ), 38.41 t (C ^7' ), 38.98 t (C ^{4 '} ), 40.10 s, 40.15 s (C ^{1 '} ), 44.07 s (C ^10' ), 46.81 t, 46.82 t (CH ₂ ), 46.93 t, 47.00 t (C ² ), 47.19 d (C ⁶ ), 50.66 d, 50.72 d (C ⁵ ), 50.98 k (OCH ₃ ), 55.28 d, 55.59 d (C ^{5 '} ), 56.01 d (C ^9' ), 81.66 d, 81.70 d (C ⁷ ), 89.60 s, 89.68 s (C ¹ ), 106.20 t, 106.24 t (C ^{13 '} ), 127.58

g (C ^{4 '),} 127.77 d, 127.82 d (C ^2'',6''), 127.99 d, 128.29 d (C ^8), 128.69 d (C ^3'',5''), 135.16 s, 135.18 s (C ^{1 ''} ), 147.42 s, 147.63 s (C ^{6 '} ), 149.60 s, 149.81 s (C ⁹ ), 172.02 s, 172.09 s (C ⁴ ), 173.57 s (CO ₂ H), 177.42 s (C ¹⁴ ). Found,%: C 72.79; H, 7.40; N, 2.55. C ₃₃ H ₄₆ NO ₆ . Calculated,%: C 72.53; H 7.33; N, 2.56.

Example 2. The study of hepatoprotective properties in a model of acute toxic hepatitis.

Acute toxic hepatitis was caused in outbred male mice by a single intragastric administration of a 25% solution of CCl ₄ in sunflower oil at a rate of 0.1 ml per 10 g of body weight. Compound (I) was administered intragastrically at a dose of 100 mg / kg in the form of a water-twin suspension 1 hour before the reproduction of hepatitis. The control animals were similarly injected with water-twin suspension in an equivalent volume, the comparison group was dihydroquercetin at a dose of 100 mg / kg. A day later, in the blood serum of mice, the activity of alanine aminotransferase (ALT), aspartate aminotransferase (ACT) and alkaline phosphatase (ALP) was determined using standard reagent kits (Biocon, Olvex Diagnosticum). The results were statistically processed using the STATISTIKA 6 software package.

It was found that compound (I) under the conditions of toxic hepatitis has a significant anti-cytolytic effect, reducing the activity of ALT and ACT in the blood by 1.5 and 1.9 times, respectively, compared with the control. Agent (I) is not inferior in its effect on the ALT level, but 1.6 times more in terms of its effect on ACT. It was found that under the action of compound (I), the level of alkaline phosphatase significantly decreases (1.5 times compared with the control), which indicates the anticholestasis effect of the agent. According to the severity of the anticholestasis effect, compound (I) is not inferior to DHQ. Under the conditions of this experiment, both agents showed no effect on the intensity of peroxidation: the concentration of MDA in the corresponding groups did not differ significantly from the control (Table 1).

Table 1 The effect of compound (I) on the biochemical parameters of the blood serum of mice with induced CCl ₄ hepatitis Group Biochemical parameters ALT, mkkat / l ACT, mkkat / l Alkaline phosphatase, mkkat / l MDA, μmol / L The control 880.80 ± 37.12 554.00 ± 48.34 2.91 ± 0.26 3.56 ± 0.35 (I) 592.47 ± 40.76 *** 301.46 ± 29.36 ** 1.97 ± 0.27 * 3.89 ± 0.22 ^# DKV 577.24 ± 25.81 *** 480.47 ± 41.14 2.18 ± 0.14 * 3.28 ± 0.11 * P <0.05; ** P <0.01; *** P <0.001 - differences with control are significant;
^# P <0.05 differences with DQV significant

Thus, it was shown that compound (I), when administered intragastrically at a dose of 100 mg / kg, has a hepatoprotective effect, reducing the severity of cytolytic and cholestatic processes against toxic hepatitis.

Example 3. The study of hepatoprotective and antioxidant properties against toxic damage to rats with cyclophosphamide

The experiment was performed on female Wistar rats, which were injected once intraperitoneally with cyclophosphamide at a dose of 125 mg / kg (in a solution of 0.9% NaCl). Compound (I) was injected into the stomach of 12 rats at a dose of 50 mg / kg for three days after administration of CF (in the form of a water-twin suspension). The reference compound, dihydroquercetin (DHQ), was administered at the same dose in the same way to a separate group of rats (10 pcs.). The control was animals with the introduction of only CF (10 pcs.). At the end of the experiment, the activity of alanine aminotransferase (ALT), aspartate aminotransferase (ACT), alkaline phosphatase (ALP), the concentration of total protein and glucose were studied in blood serum using standard reagent kits (Biocon, Olvex Diagnosticum). The concentration of malondialdehyde (MDA) was determined by the conventional method [9].

The results are presented in table.2. It was found that under these conditions, both compound (I) and the reference drug did not significantly affect ALT activity. With respect to ACT, compound (I) showed only a tendency toward a decrease in activity (1.3 times as compared with the control), while DHQ caused a small but significant decrease in the activity of this enzyme in the blood (1.4 times). Compound (I) showed a pronounced tendency to anticholestasis: the activity of alkaline phosphatase under the influence of compound (I) decreased relative to control and DHQ 1.3 and 1.4 times, respectively. The anticholestatic effect was not found in the reference agent. The concentration of MDA in animals with the introduction of agent (I) was 1.3 times lower than in the group with the introduction of DHQ, which under the conditions of this experiment increased the intensity of peroxidation. The influence of both agents on indicators of total metabolism (protein, glucose) was not noted.

The data in the table show that compound (I), when administered intragastrically at a dose of 50 mg / kg against a background of cyclophosphamide lesion, is inferior to DHQ in anticytolytic properties, but surpasses it in anticholestatic and antioxidant effects.

table 2 The effect of compound (I) on the average values of biochemical parameters of rat blood serum on the background of cyclophosphamide intoxication Group ALT, mcat / g protein ACT, mkat / g protein Alkaline phosphatase, mcat / g protein MDA, μmol / L Total protein, g / l Glucose, mol / L the control 54.3 ± 6.8 73.9 ± 7.9 129.0 ± 9.5 1.93 ± 0.09 63.1 ± 2.34 16.43 ± 1.02 (I) 58.4 ± 7.2 58.4 ± 5.5 102.6 ± 15.8 1.93 ± 0.08 65.27 ± 4.12 14.29 ± 0.91 DKV 55.2 ± 5.2 53.7 ± 3.9 * 146.3 ± 13.6 2.57 ± 0.09 * 63.14 ± 1.52 14.19 ± 0.70 * P <0.05 - differences with control are significant

Example 4. The study of hemostatic effect against toxic damage to rats by cyclophosphamide

The experiment was performed on female Wistar rats, which were injected once intraperitoneally with cyclophosphamide at a dose of 125 mg / kg (in a solution of 0.9% NaCl). Compound (I) was injected into the stomach of 12 rats at a dose of 50 mg / kg for three days after administration of CF (in the form of a water-twin suspension). The comparison drug, dihydroquercetin (DHQ), was administered in the same dose in the same way to a separate group of rats (10 pcs.). The control was animals with the introduction of only CF (10 pcs.). At the end of the experiment, the morphological composition of the peripheral blood was determined using a MEDONIC hemoanalyzer. The leukocyte formula was counted under a microscope in blood smears stained with hematoxylineosine.

The results are presented in table.3. It was found that compound (I) tends to decrease cytopenia caused by cyclophosphamide, increasing the number of leukocytes and platelets relative to the control, respectively, 1.4 and 1.2 times. The hemostatic effect of dihydroquercetin manifested itself as a tendency to a 1.6-fold increase in the number of leukocytes. Both compounds are characterized by a slight significant increase in the quantitative indicators of hemoglobin in red blood cells (MCH, MCHC). At the same time, under the action of DHQ, a slight significant decrease in the number of red blood cells and hematocrit was observed.

Table 3 The effect of compound (I) on the average values of peripheral blood rats on the background of intoxication with cyclophosphamide Group Rbc HCT Wbc HBV Plt Mcv RDW% MPV SIT ICSU the control 5.06 ± 0.21 28.0 ± 1.2 0.9 ± 0.1 15.5 ± 0.8 93.9 ± 8.4 55.3 ± 1.0 10.2 ± 0.4 8.8 ± 0.01 30.6 ± 0.6 55.5 ± 0.6 (I) 4.77 ± 0.19 26.2 ± 1.0 1.3 ± 0.1 15.6 ± 0.7 109.6 ± 11.9 54.9 ± 0.6 10.3 ± 0.2 8.3 ± 0.03 * 32.8 ± 0.5 * 59.7 ± 1.0 * DKV 4.19 ± 0.20 * 22.6 ± 0.9 * 1.4 ± 0.2 14.2 ± 0.6 88.3 ± 8.9 55.3 ± 0.8 10.2 ± 0.6 8.3 ± 0.1 * 33.9 ± 0.3 * 61.3 ± 0.8 * Norm 6.87 ± 0.14 38.7 ± 0.7 17.0 ± 0.9 19.7 ± 0.6 257.0 ± 21.2 57.1 ± 0.9 13.3 ± 0.7 9.0 ± 0.1 29.7 ± 0.5 52.0 ± 0.4 * P <0.05 - differences with control are significant RBC is the number of red blood cells, HCT is the hematocrit, WBC is the number of leukocytes, HBV is hemoglobin, PLT is platelet count, MCV is the average volume of red blood cells, RDW% is the percentage distribution on the absolute weight of red blood, MPV is the volume of platelets, MSN is the average hemoglobin content in erythrocyte, MCHC - the average concentration of hemoglobin.

Against the background of neutrophenia caused by cyclophosphamide, a tendency to an increase in the number of granulocyte cells was observed under the action of compound (I): the number of segmented neutrophils increased 1.8 times in comparison with the control, young neutrophils appeared. In the same group of animals, relative monocytosis was revealed, which was expressed in a 2.5-fold increase in the number of monocytes compared to the control. The marked tendency to stimulate leukocyte cells under the action of compound (I) was more pronounced than in DQW (Table 4).

Table 4 The effect of compound (I) on the average values of rat leukocyte counts on the background of intoxication with cyclophosphamide Group eosinophils p / nuclear s / nuclear Monocytes Lymphocytes the control 0 0 1.43 ± 0.48 2.43 ± 0.81 96.14 ± 0.51 (I) 0 0.14 ± 0.14 2.57 ± 0.84 6.0 ± 1.0 91.29 ± 1.02 DKV 0 0 0.71 ± 0.29 4.71 ± 0.47 94.57 ± 0.48 Norm 0.86 ± 0.46 0 19.29 ± 1.44 7.14 ± 1.06 72.61 ± 1.44

Thus, it was shown that compound (I), when administered intragastrically at a dose of 50 mg / kg, against the background of hemo-depression caused by cyclophosphamide administration, is superior to DHQ in terms of hemostimulating effect.

Information sources

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Claims (1)

(1'S, 5'S, 9'S, 10'R) -methyl-5- [2- (3-benzyl-6-carboxy-4-oxo-10-oxa-3-azatricyclo [5.2.1.0 ^1,5 ] deca-8 en-9-yl) ethyl] -1,10-dimethyl-6-methylene-decahydronaphthalene-1-carboxylate of the formula (I),

possessing antioxidant, hepatoprotective and hemostimulating activity.