RU2353620C1 - (z)-methyl-16-(5-oxo-2-phenyl-oxazol-4-ilidenmethyl)-15,16-epoxy-8(17),13(16),14-labdatrien-18-oate, possessing antioxidative, hepatoprotective and hemostimulating activity - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to (Z)-methyl-16-(5-oxo-2-phenyl-oxazol-4-ilidenmethyl)-15,16-epoxy-8(17),13(16),14-labdatrien-18-oate of formula (I)

(I). Compound (I) possesses high antioxidative, hepatoprotective and hemostimulatng activity and can be used for correction of side effects, arising with introduction of highly toxic medications used in anti-tumor therapy.

EFFECT: obtaining compound, which possesses high antioxidative, hepatoprotective and hemostimulatng activity.

1 cl, 4 tbl, 4 ex

Description

The invention relates to medicine, specifically to a new chemical compound Z-methyl-16- (5-oxo-2-phenyl-oxazol-4-ylidenemethyl) -15,16-epoxy-8 (17), 13 (16), 14- labdatriene-18-oatu 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 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 - Z-methyl-16- (5-oxo-2-phenyl-oxazol-4-ylidenomethyl) -15,16-epoxy-8 (17), 13 (16), 14-labdatriene-18 -atom of formula (I), which has a pronounced antioxidant, hepatoprotective and hemostatic activity, including against the background of the introduction of cytostatic drugs into the body.

A structural analogue of this compound is the 16-aminomethyl derivative of lambertianic acid of the formula (III) having nootropic activity [6].

The method of producing compound (I) having the structure of azlactone of the labdanic type is implemented according to the above scheme 1. Formylation of methyl ester of lambertanoic acid (IV) leads to the 16-formyl derivative (V) [7], which is isolated by crystallization. The interaction of aldehyde (V) with benzoylglycine (hippuric acid) leads to the formation of labdanoid 5 (4H) -oxazolone (I). Compound (I) is formed in up to 76% yield as an individual isomer with a (Z) double bond configuration. The advantages of the invention include a method for producing compound (I) by chemical modification of the available plant metabolite of Siberian cedar Pinus sibirica R. Mayr. - methyl ester of lambertanoic acid (IV). The latter is easily distinguished from a forest product - cedar resin or from cedar needles, which is a large-tonnage waste of the cutting area [8]. The physicochemical constants of the new, first-obtained compound (I) are shown in Example 1.

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 CCl ₄ hepatitis in mice was used. The model was reproduced according to the guidelines [9]. 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. After a day, the activity of alanine aminotransferase (ALT), aspartate aminotransferase (ACT), alkaline phosphatase (ALP) and the concentration of malondialdehyde (MDA) were determined by conventional methods [10]. It was found that compound (I) significantly reduces the activity of transaminases in the blood against the background of toxic hepatitis, not inferior to dihydroquercetin in anticytolytic and superior to its anticholestatic effect (table 1).

The 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 [10].

The results of the study of biological activity are given in table.2-4. It was found that compound (I) against the background of cyclophosphamide significantly reduces the activity of alkaline phosphatase. The marked anticholestasis effect is 1.7 times higher than the corresponding effect of DHQ. Compound (I) also reduces the concentration of MDA by 1.3 times compared with DHQ, which indicates its antioxidant activity. It was found that when cyclophosphamide is damaged, compound (I) exhibits a hemostimulating effect, significantly increasing the number of leukocytes in peripheral blood (1.7 times relative to the control), and also increases the number of red blood cells, platelets and hemoglobin by 1.2-1.3 times compared to DKV.

Thus, the new compound is Z-methyl-16- (5-oxo-2-phenyl-oxazol-4-ylidenomethyl) -15,16-epoxy-8 (17), 13 (16), 14-labdatriene-18- Oatu formula (I) - has the following advantages, namely:

- It has a high antioxidant and hepatoprotective activity, as well as a hemostatic effect against the background of the introduction of the cytostatic cyclophosphamide.

- Use for the synthesis of compound (I) of the original obtained from available plant materials - needles or gum resin of Siberian cedar Pinus sibirica R. Mayr.

The invention is illustrated by the following examples.

Example 1. Z-methyl-16- (5-oxo-2-phenyl-oxazole-4-ylidenomethyl) -15,16-epoxy-8 (17), 13 (16), 14-labdatriene-18-ooat (I )

To a solution of 1.00 g (2.79 mmol) of 16-formylmethyl amberthianate (V) [7] in 15 ml of acetic anhydride, 0.50 g (2.79 mmol) of hippuric acid and 0.38 g (2.79 mmol) of potassium carbonate were added with stirring. The reaction mixture was stirred for 5 hours and left overnight. The precipitate was filtered off, washed with water, dried under vacuum, and recrystallized from a mixture of petroleum ether: sulfuric ether = 2: 1. Received 1.06 g (yield 76%) of compound (I). _Mp 112-115 ° C. [α] ²⁰ _D 1.16 (c 7.68, CHCl ₃ ). IR spectrum, cm ^-1 : 702, 780, 883 (Ph); 883 (C = C); 983, 1551 (furan); 1173, 1380, 1720 (CO ₂ Me); 1645, 1759, 1789 (azlactone). UV spectrum, λ _max. , nm (log ε) 232 (3.41), 266 (3.75), 392 (4.18), 409 (4.17). ¹ H NMR spectrum (δ, ppm, J, Hz): 0.43 s (3H, C ^{20 '} H ₃ ), 0.82 etc. (1H, H ^{1 '} , J 12, 3), 0.90 etc. (1H, H ^{3 '} , J 12, 3), 1.06 s (3H, C ^19' H ₃ ), 1.13 dd. (1H, H ^{5 '} , J 11, 2.4), 1.38 dm. (1H, H ^{2 '} , J 12), 1.48 d (1H, H ^9' , J 9), 1.60-1.80 m (6H, H ^{7 ', 2', 6 ', 11', 11 ', 1'} ) , 1.89 dm (1H, H ^{6 '} J 12), 2.05 dm. (1H, H ^{3 '} , J 11), 2.36 etc. (1H, H ^{7 '} , J 11, 3), 2.51 m (1H, H ^12' ), 2.66 m (1H, H ^{12 '} ), 3.51 s (3H, OCH ₃ ), 4.53 s, 4.92 s (2H, H ^{17 ', 17'} ), 6.40 d (1H, H ^{14 '} , J 1.8), 6.92 s (1H, H ⁶ ), 7.41 t (2H, H 3`` <sup>, 5''</sup> , J 7), 7.48 t .t. (1H, H <sup>4 '',</sup> J 7, 1), 7.68 d (1H, H <sup>15,</sup> J 1.8), 8.07 dd (2H, H <sup>2``, 6 ''</sup> , J 7, 1). C ¹³ NMR spectrum (CDCl ₃ , δ _C ppm): 12.36 k (C ^{20 '} ), 19.54 t (C ²¹ ), 23.49 t (C ^12' ), 23.89 t (C ^{11 '} ), 25.90 t ( C ^{6 '} ), 28.39 t (C ^19' ), 37.74 t (C ^{3 '} ), 38.19 t (C ^7' ), 38.65 t (C ^{1 '} ), 39.78 s

(C ^{4 '} ), 43.87 s (C ^10' ), 50.81 k (OCH ₃ ), 54.17 d (C ^{9 '} ), 55.77 d (C ^5' ), 106.58 t (C ^{17 '} ), 113.67 d (C ^{14 '} ), 115.00 d (C ⁶ ), 125.32 s (C1 ^'' ), 127.86 d (C ^2'',6'' ), 128.18 s (C ⁴ ), 128.49 d (C ^3'',5'' ) , 132.64 d (C ^{4 ''} ), 137.80 s (C ^{13 '} ), 146.80 s (C ^16' ), 147.14 s (C ^{8 '} ), 147.71 d (C ^15' ), 162.14 s (C ² ), 167.53 s (C ⁵ ), 177.27 s (C ^{18 '} ). Found: C 74.03, H 7.11, N 2.7. C ₃₁ O ₅ NH ₃₅ . Calculated: C 74.25, H 6.99, N 2.79.

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 level of malondialdehyde (MDA) was determined by the conventional method [10]. The results were statistically processed using the STATISTIKA 6 software package.

It was found that compound (I) under the conditions of toxic hepatitis has an anticytolytic effect, significantly reducing the activity of transaminases in the blood (1.3-1.5 times in comparison with the control). Agent (I) is not inferior to dihydroquercetin in its anticytolytic effect, as evidenced by the absence of statistically significant differences in indices between the corresponding groups. It was found that under the action of compound (I), the level of alkaline phosphatase significantly decreases (1.9 times compared with the control), which indicates the anticholestasis effect of the agent. In terms of severity of the anticholestasis effect, compound (I) is 1.4 times superior 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) 701.50 ± 61.29 * 373.16 ± 49.00 * 1.57 ± 0.21 ** 4,55 ± 0,46 ^# 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 carried out on rats of Wistar females, 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 results are presented in table.2. It was found that compound (I) significantly reduces the activity of alkaline phosphatase relative to the control and reference compounds. The marked anticholestasis effect is 1.7 times higher than that of DKV. Compound (I) also significantly reduced (1.3 times) the concentration of MDA relative to DHQ, which under the conditions of this experiment increased the intensity of peroxidation. Against the background of CF, compound (I) did not affect the activity of transaminases, while DHQ significantly reduced the activity of one of them - ACT. The effects of both compounds on the indicators of total metabolism (protein, glucose) were not observed.

From the data of table 2 it can be seen that compound (I), when administered intragastrically at a dose of 100 mg / kg against a background of cyclophosphamide damage, is superior to DHQ in its anticholestic and antioxidant effect.

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. mcat / g protein Alkaline phosphatase, mcat / g protein MDA, μmol / L Total protein, g / l Glucose, mmol / 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) 62.6 ± 2.9 81.4 ± 7.1 ## 87.1 ± 14.2 * 1.92 ± 0.07 # 60.26 ± 4.17 14.93 ± 0.64 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;
^# P <0.05 differences with DHQ are significant.

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

The experiment was carried out on rats of Wistar females, 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 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 hematoxylin-eosin.

The results are presented in table.3. It was found that compound (I) exhibits a hemostimulating effect, significantly increasing the number of leukocytes in peripheral blood (1.7 times relative to the control). The corresponding effect of DHQ was less significant (1.5 times relative to the control). In relation to other blood indices, there is a tendency to normalize them under the action of compound (I), the positive effect of which exceeded the effect of DHQ. So, under the action of agent (I), the number of red blood cells and platelets increased by 1.3 and 1.2 times, respectively; hematocrit and hemoglobin increased by 1.3-1.2 times compared with DHQ (although the average content and concentration of hemoglobin in the red blood cell were significantly higher than in the control).

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 Hgb 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) 5.34 ± 0.20 30.2 ± 1.0 1.5 ± 0.1 * 16.6 ± 0.7 107.9 ± 9.6 55.0 ± 0.5 10.8 ± 0.6 8.7 ± 0.04 31.3 ± 0.3 56.4 ± 0.6 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, HGB is hemoglobin, PLT is platelet count, MCV is the average volume of red blood cells, RDW% is the percentage distribution of the absolute weight of red blood, MPV is the volume of platelets.

Against the background of cyclophosphamide-induced neutropenia, moderate lymphocytosis was observed in the groups. Under the action of compound (I) and DHQ, animals developed relative (statistically unreliable) monocytosis, which was more pronounced in the reference group.

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 1.14 ± 0.51 3.71 ± 0.42 95.14 ± 0.40 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 100 mg / kg, against the background of hemodepression caused by cyclophosphamide administration, is superior to DHQ in terms of hemostimulating effect.

Information sources

1. M.D. Mashkovsky. Medicines, in two volumes, ed. “Torsing”, Kharkov, 1998, v.2, 55.

2. Medicines approved for use in the USSR. Ed. M.A. Klyueva, E.A. Babayana. M .: Medicine, 1979, p. 61-65.

3. M.B. Plotnikov, N.A. Tyukavkina, T.M. Plotnikova. Medicines based on dikvertin. Tomsk Tomsk University Publishing House. 2005, 228 p.

4. M.L. Gershanovich, V.A. Filov, M.A. Akimov, A.A. Akimov. Introduction to the pharmacotherapy of malignant tumors. SPb. Satis Publishing House, 1999, 152 pp.

5. V.A. Tutelian, M.M. Gapparov, L.Yu. Telegin, V.M. Devichensky, L.A. Pevnitsky, Bulletin of experimental biology and medicine. 2003.V. 136. No. 12. S.604-607.

6. T.G. Tolstikova, I.V. Sorokina, T.V. Voevoda, S.V. Chernov, E.E. Schulz, G.A. Tolstikov. Reports of the Academy of Sciences. 2001.V. 376. No. 1. S.271-273.

7. Klok D.A., Shakirov M.M., Grishko V.V., Raldugin V.A. Izvestia AN. Chemical series. 1995. No. 11. S.2514-2517.

8. T.G. Tolstikova, I.V. Sorokina, M.P. Dolgikh, Yu.V. Kharitonov, S.V. Chernov, E.E. Schulz, G.A. Tolstikov. Chemical Pharmaceutical Journal. 2004.V. 38. No. 10. S.13-15.

9. Guidance on the experimental (preclinical) study of new pharmacological substances. M .: Medicine, 2005, 832 p.

10. Kamyshnikov B.C. Handbook of clinical and chemical laboratory diagnostics. Minsk: Belarus, 2000.V.2, p.207.

Claims (1)

(Z) -Metyl-16- (5-oxo-2-phenyl-oxazol-4-ylidenomethyl) -15,16-epoxy-8 (17), 13 (16), 14-labdatriene-18-oat of the formula (I ),

possessing antioxidant, hepatoprotective and hemostimulating activity.