RU2346940C1 - 16-{2-benzoylamino-2-[(3,5-di-tret-buthyl-4-hydroxuphenyl)alkyl-carbamoyl]vinyl}-labdatriens with antioxidant, hepatoprotection and hemostimulating effect - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention concerns new compounds, particularly 16-{2-benzoylamino-2- [(3,5-di-tet-buthyl-4-hydroxyphenyl)-alkylcarbamoyl]vinyl}-labdatriens of the formula (I)

with antioxidant, hepatoprotection and hemostimulating effect, allowing for application under toxic hepatitis and in correction of side effects of cytostatic medicines (e.g. cyclophosphane). Compounds of the formula 1 are obtained from raw plant material, Siberian cedar needles or oleo-resin.

EFFECT: obtaining compounds with antioxidant, hepatoprotection and hemostimulating effect.

4 tbl, 5 ex

Description

The invention relates to new chemical compounds, specifically to 16- {2-benzoylamino-2 - [(3,5-di-tert-butyl-4-hydroxyphenyl) alkylcarbamoyl] vinyl} -labdatrienes of the formula (I)

possessing antioxidant, hepatoprotective and hemostimulating activity.

These properties suggest the possibility of using compounds in medicine as pharmaceuticals.

In medical practice, antioxidants of the group of screened phenols are used. For example, the drug dibunol (2,6-di-tert-butyl-4-methylphenol) is used to treat cancer and papillomatosis of the bladder, cystitis, burns, trophic and radiation ulcers [1].

Probucol [4,4 '- (isopropylidendithio) bis- (2,6-di-tert-butyl) phenol] is prescribed as a lipid-lowering agent for hypercholesterolemia with a risk of coronary heart disease [2]. Thus, derivatives of 2,6-di-tert-butylphenol can serve as drugs of various orientations, which makes the synthesis and pharmacological study of substances including the named structural fragment promising.

The problem to which the invention is directed, is the development of phenolic antioxidants with additional properties based on the available plant labdanoid lambertianic acid. An important element of the task is to obtain an antioxidant of a new structural type that can reduce the toxic side effects of cytostatic drugs, which usually include hemo- and myelodepression, immunological disorders, functional and morphological damage to various organs, etc.

Interest in the development of such agents based on plant materials has increased significantly in the new century due to the need for their inclusion in the complex chemotherapy of tumors. The range of such chemotherapeutic corrector drugs is currently not wide enough. Mostly used drugs in the form of complex mixtures of extractive substances [3], which have certain disadvantages associated with individual intolerance and allergic effects.

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 new chemical compounds 16- {2-benzoylamino-2 - [(3,5-di-tert-butyl-4-hydroxyphenyl) alkylcarbamoyl] vinyl} -labdatrienes of the formula (I), namely, (Z) - methyl-16- {2-benzoylamino-2- [2- (3,5-di-tert-butyl-4-hydroxyphenyl) ethylcarbamoyl] vinyl} -15,16-epoxy-8 (17), 13 (16 ), 14-labdatriene-18-oatom (Ia) or (Z) -methyl-16- {2-benzoylamino-2- [3- (3,5-di-tert-butyl-4-hydroxyphenyl) propylcarbamoyl] - vinyl} -15,16-epoxy-8 (17), 13 (16), 14-labdatriene-18-oatom (Ib), which exhibit pronounced antioxidant, hepatoprotective and hemostimulating properties and are suitable for correcting ii toxic effects of cytostatic chemotherapy.

An analogue in the properties of the claimed compounds 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 [4]. The indicated effects provide this compound with the status of a basic substance for the creation on its basis of agents possessing new pharmacological properties. The main disadvantages of complex preparations, which include dihydroquercetin, are the development of side effects in the gastrointestinal tract, such as nausea, heartburn [5].

An analogue in the structure of these compounds is the 16-aminomethyl derivative of lambertianic acid of the formula (III) with nootropic activity [6].

The method of obtaining compounds (I) containing a labdanoid backbone in the structure, connected with fragments of spatially hindered phenols, is implemented according to Scheme 1. Formylation of the methyl ester of lambertianic 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 (VI). Compound (VI) is formed in up to 76% yield as an individual isomer with a (Z) double bond configuration. The interaction of azlactone (VI) with 3,5-di-tert-butyl-4-hydroxyphenylethyl (propyl) -amines (VIIa, b) smoothly leads to the corresponding 4-substituted carbamoyl vinylbenzamides (Ia, b) (yield 76-91%). An advantage of the invention is a method for producing compounds (Ia, b) by chemical modification of an 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]. Physicochemical constants of new, first-obtained compounds are given in examples 1, 2.

The biological activity of compounds (Ia, b) was studied by determining toxicity, hepatoprotective properties in a model of toxic hepatitis, antioxidant and hemostimulating activity in a model of toxic damage to rats caused by cyclophosphamide. The antioxidant dihydroquercetin (II) was used as a comparison drug.

Acute toxicity was determined on outbred mice weighing 18-23 g with a single intragastric route of administration. The toxicity parameters were calculated according to the Kerber method. It was found that the LD _{50 of} compounds (Ia, b) exceeds the maximum possible dose for a single injection 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. Compounds (Ia, b) were 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 compounds (Ia) and (Ib), when administered intragastrically at a dose of 100 mg / kg, have a hepatoprotective effect, reducing the severity of cytolytic and cholestatic processes against toxic hepatitis. Agent (Ia) reduces the activity of transaminases in the blood by 1.3, agent (Ib) - by 1.7-1.9 times in comparison with the control. Both agents are not inferior to dihydroquercetin in terms of their effect on ALT levels, while agent (Ib) exceeds the reference compound by 1.7 times in terms of its effect on ACT activity. Compound (Ia) is not inferior in magnitude of the anticholestasis effect, and compound (Ib) is 1.5 times higher than DHQ.

The study of the hepatoprotective, antioxidant, and hemostimulating effects of compounds (Ia, b) 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. Compounds (Ia, b) were administered in the form of a water-twin suspension to two groups of rats (10 each) in a 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 method of [10].

The results of the study of biological activity are given in table.2-4. It was established that against the background of intoxication with cyclophosphamide, the anticytolytic effect of compounds (Ia) and (Ib) is lower, and the antioxidant effect is 1.3-1.4 times higher than that of DKV. It was shown that compound (IB) against the background of hemodepression caused by the introduction of cyclophosphamide, reduces leukopenia and exceeds DKV in stimulating effect on the white blood cell. Agent (Ia) under the same conditions does not exhibit a significant hemostimulating effect.

Thus, the present invention has the following advantages, namely:

- High hepatoprotective activity of compounds (Ia) and (Ib) in toxic hepatitis.

- Antioxidant and hemostimulating effects of compound (Ib) against the background of the introduction of cyclophosphamide cytostatic.

- Use for the synthesis of compounds (Ia, b) 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. Preparation of (Z) -Metyl-16- {2-benzoylamino-2- [2- (3,5-di-tert-butyl-4-hydroxyphenyl) ethylcarbamoyl] vinyl} -15,16-epoxy- 8 (17), 13 (16), 14-labdatriene-18-ooate (Ia).

1.16 (с 7.68, CHCl₃). ИК спектр, см^-1: 702, 780, 883 (Ph); 883 (C=C); 983, 1551 (фуран); 1173, 1380, 1720 (СО₂Ме); 1645, 1759, 1789 (азлактон). УФ спектр, λ_макс, нм (lg ε) 232 (3.41), 266 (3.75), 392 (4.18), 409 (4.17). Спектр ЯМР ¹H (δ, м.д., J, Гц): 0.43 с (3Н, С^20,H₃), 0.82 т.д. (1Н, Н^1', J 12, 3), 0.90 т.д. (1H, Н³', J 12, 3), 1.06 с (3Н, С^19, Н₃), 1.13 д.д. (1Н, Н⁵', J 11, 2.4), 1.38 д.м. (1Н, Н²', J 12), 1.48 д (1Н, Н^9', J 9), 1.60-1.80 м (6Н, н^{7',2',6',11',11',1'}), 1.89 д.м (1Н, Н^6' J 12), 2.05 д.м. (1Н, Н^3', J 11), 2.36 т.д. (1Н, Н^7', J 11, 3), 2.51 м (1Н, Н^12'), 2.66 м (1Н, Н^12'), 3.51 с (3Н, ОСН₃), 4.53 с, 4.92 с (2Н, Н^17',17'), 6.40 д (1Н, Н^14', J 1.8), 6.92 с (1Н, Н⁶), 7.41 т (2Н, Н^3",5", J 7), 7.48 т.т. (1Н, Н^4", J 7, 1), 7.68 д (1Н, Н¹⁵, J 1.8), 8.07 д.д. (2Н, Н^2",6", J 7, 1). Спектр ЯМР С¹³ (CDCl₃, δ_с м.д.): 12.36 к (С^20'), 19.54 т (С^2'), 23.49 т (С^12'), 23.89 т (С^11'), 25.90 т (С^6'), 28.39 к (С^19'), 37.74 т (С^3'), 38.19 т (С^7'), 38.65 т (С^1'), 39.78 с (С^4'), 43.87 с (С^10'), 50.81 к (ОСН₃), 54.17 д (С^9'), 55.77 д (С^5'), 106.58 т (С^17'), 113.67 д (С^14'), 115.00 д (С⁶), 125.32 с (С^1"), 127.86 д (С^2",6"), 128.18 с (С⁴), 128.49 д (С^3",5"), 132.64 д (С^4"), 137.80 с (С^13'), 146.80 с (С^16'), 147.14 с (С^8'), 147.71 д (С^15'), 162.14 с (С²), 167.53 с (С⁵), 177.27 с (С^18'). Найдено: С 74.03, Н 7.11, N 2.7. С₃₁O₅МН₃₅ Вычислено: С 74.25, Н 6.99, N 2.79.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 formed was filtered off, washed with water, dried under vacuum, and recrystallized from a mixture of petroleum ether: sulfuric ether = 2: 1. Obtained 1.06 g (yield 76%) of Z- (1S, 4aR, 5S) -methyl-1.4a-dimethyl-6-methylene-5- {2- [2- (5-oxo-2-phenyl-4-oxazolylidenemethyl) fur-3-yl-ethyl} -decahydronaphthalene-1-carboxylate (VI). _Mp 112-115 ° C.

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 ", 5"} , J 7), 7.48 t . (1H, H ^{4 "} , J 7, 1), 7.68 d (1H, H ¹⁵ , J 1.8), 8.07 dd (2H, H ^{2", 6 ",} J 7, 1). C ¹³ NMR spectrum (CDCl ₃ , δ _s ppm): 12.36 k (C ^{20 '} ), 19.54 t (C ^2' ), 23.49 t (C ^{12 '} ), 23.89 t (C ^11' ), 25.90 t (C ^{6 '} ), 28.39 k (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 (C ^{1 "),} 127.86 g (C ^{2", 6 "),} 128.18 s (C ^4), 128.49 d (C ^{3", 5 "),} 132.64 g (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 MH ₃₅ Calculated: C 74.25, H 6.99, N 2.79.

(2Н, NHCH₂), 4.56 с, 4.91 с (2Н, H^17,17), 5.07 с (1Н, ОН), 6.32 д (1Н, Н¹⁴, J 1.8), 6.54 т (1Н, CONH, J 6), 6.83 с (1Н, Н^1'), 7.00 с (2Н, Н^2",6"), 7.34 д (1Н, Н¹⁵, J 1.8), 7.46 т (2Н, H^7',9', J 7), 7.54 т (1Н, Н^8', J 7), 7.91 д (2Н, H^6',10',J 8), 8.55 ш.с. (1Н, Н^3'). Спектр ЯМР С¹³ (CDCl₃, δ_с м.д.): 12.44 к (С²⁰), 19.69 т (С²), 23.21 т (С¹²), 24.12 т (С¹¹), 26.07 т (С⁶), 28.56 к (С¹⁹), 30.09 к

34.06 с

35.58 т

37.93 т (С³), 38.43 т (С⁷), 38.86 т (С¹), 39.98 т (CH₂N), 41.56 с (С⁴), 44.07 с (С¹⁰), 50.96 к (ОСН₃), 54.70 д (С⁹), 55.90 д (С⁵), 106.48 т (С¹⁷), 110.72 д (С^1'), 112.78 д (С¹⁴), 125.11 д (С^2",6"), 126.31 с (С^2'), 127.32 д (С^6',10'), 128.58 д (С^7',9'), 129.33* с (С¹³), 129.81* с (С^5'), 132.05 д (С^8'), 133.19 с (С^1"), 135.76 с (С^3",5"), 143.14 д (С¹⁵), 146.24 с (С¹⁶), 147.50 с (С⁸), 152.10 с (С^4"), 164.80 с (CONH), 166.22 с (С^4'), 177.58 с (С¹⁸). Найдено: С 74.61, N 3.68, Н 8.73. C₄₇O₆N₂H₆₂ Вычислено: С 75.20, N 3.73, Н 8.28.To a solution of 0.50 g (1.0 mmol) of azlactone (VI) in 7 ml of benzene was added 0.27 g (1.1 mmol) of 3,5-ditretbutyl-4-hydroxyphenylethylamine, the reaction mass was heated (70 ° C) for 6 hours. The solvent was removed in vacuo. , the residue was chromatographed on silica gel (eluent: chloroform). Crystallization from diethyl ether gave 0.57 g (76% yield) of compound (Ia). _Mp 91-93 ° C. [α] ₅₈₀ 9.40 ° (c 1.90, CHCl _3). IR spectrum, cm ^-1 : 713, 750, 769 (Ph); 890 (furan); 1122, 1188, 1722 (CO ₂ Me); 1516, 1665 (CONH); 3426 (OH). UV spectrum, λ _max , nm (log ε) 316 (3.26). ¹ H NMR spectrum (δ, ppm, J, Hz): 0.48 s (3H, C ²⁰ H ₃ ), 0.94 etc. (1H, H ¹ , J 12, 3), 0.99 etc. (1H, H ³ , J 12, 3), 1.14 s (3H, C ¹⁹ H ₃ ), 1.25 dd. (1H, H ⁵ , J 11, 3), 1.37 s (18H, 2C (CH ₃ ) ₃ ), 1.48 d (1H, H ² , J 12), 1.56 s (2H, H ^9.11 ), 1.68- 1.80 m (4H, H ¹ ' ⁶ ' ^2.11 ), 1.87 etc. (1H, H ⁷ , J 10, 3), 1.96 dm. (1H, H ⁶ , J 12), 2.12 d.m. (1H, H ³ , J 11), 2.33 m (2H, H ^12.7 ), 2.58 m (1H, H ¹² ), 2.79 t (2H, CH ₂ Ar, J 6), 3.58 s (3H, OCH ₃ ), 3.61 m

(2H, NHCH ₂ ), 4.56 s, 4.91 s (2H, H ^17.17 ), 5.07 s (1H, OH), 6.32 d (1H, H ¹⁴ , J 1.8), 6.54 t (1H, CONH, J 6 ), 6.83 s (1H, H ^{1 '} ), 7.00 s (2H, H ^{2 ", 6"} ), 7.34 d (1H, H ¹⁵ , J 1.8), 7.46 t (2H, H ^{7', 9 '} , J 7), 7.54 t (1H, H ^{8 '} , J 7), 7.91 d (2H, H ^{6', 10 '} , J 8), 8.55 br.s. (1H, H ^{3 '} ). C ¹³ NMR spectrum (CDCl ₃ , δ _s ppm): 12.44 k (C ²⁰ ), 19.69 t (C ² ), 23.21 t (C ¹² ), 24.12 t (C ¹¹ ), 26.07 t (C ⁶ ) , 28.56 k (C ¹⁹ ), 30.09 k

34.06 s

35.58 t

37.93 t (C ³ ), 38.43 t (C ⁷ ), 38.86 t (C ¹ ), 39.98 t (CH ₂ N), 41.56 s (C ⁴ ), 44.07 s (C ¹⁰ ), 50.96 k (OCH ₃ ), 54.70 d (C ⁹ ), 55.90 d (C ⁵ ), 106.48 t (C ¹⁷ ), 110.72 d (C ^{1 '} ), 112.78 d (C ¹⁴ ), 125.11 d (C ^{2 ", 6"} ), 126.31 s ( C ^{2 '} ), 127.32 d (C ^{6', 10 '} ), 128.58 d (C ^{7', 9 '} ), 129.33 * s (C ¹³ ), 129.81 * s (C ^5' ), 132.05 d (C ^{8 '} ), 133.19 s (C ^{1 "} ), 135.76 s (C ^{3", 5 "} ), 143.14 d (C ¹⁵ ), 146.24 s (C ¹⁶ ), 147.50 s (C ⁸ ), 152.10 s (C ^4" ), 164.80 s (CONH), 166.22 s (C ^{4 '} ), 177.58 s (C ¹⁸ ). Found: C 74.61, N 3.68, H 8.73. C ₄₇ O ₆ N ₂ H ₆₂ Calculated: C 75.20, N 3.73, H 8.28.

Example 2. Obtaining (Z) -Metyl-16- {2-benzoylamino-2- [3- (3,5-di-tert-butyl-4-hydroxyphenyl) propylcarbamoyl] vinyl} -15,16-epoxy- 8 (17), 13 (16), 14-labdatriene-18-ooate (IB).

1.96 д.м. (1Н, Н⁶ J 12), 2.12 д.м. (1Н, Н³, J 11), 2.38 м (2Н, H^12,7), 2.58 т (3Н, СН₂Ar, Н¹², J 6), 3.41 д.д.

3.58 с (3Н, ОСН₃), 4.56 с, 4.92 с (2Н, H^17,17), 5.03 с (1Н, ОН), 6.33 д (1Н, Н¹⁴, J 1.8), 6.52 т (1Н, CONH, J 6), 6.81 с (1Н, H^1'), 6.97 с (2Н, H^2",6"), 7.35 д (1Н, Н¹⁵, J 1.8), 7.47 т (2Н, Н^7',9', J 7), 7.55 т (1Н, Н^8', J 7), 7.93 д (2Н, Н^6',10'; J 8), 8.59 ш.с. (1Н, H^3'). Спектр ЯМР С¹³ (CDCl₃, δ_с м.д.): 12.50 к (С²⁰), 19.76 т (С²), 23.27 т (С¹²), 24.21 т (С¹¹), 26.13 т (С⁶), 28.61 к (С¹⁹), 30.21 к (С(СН₃)₃), 31.59 т

35.09 т

34.14 с

37.99 т (С³), 38.51 т (С⁷), 38.92 т (С¹), 39.69 т (CH₂N), 40.04 с (С⁴), 44.13 с (С¹⁰), 51.03 к (ОСН₃), 54.74 д (С⁹), 55.95 д (С⁵), 106.55 т (С¹⁷), 110.60 д (С^1'), 112.87 д (С¹⁴), 124.76 д (С^2",6"), 126.53 с (С^2'), 127.38 д (С^6''^10'), 128.65 д (С^7',9'), 129.79 с (С¹³), 132.03 с (С^5'), 132.14 д (С^8'), 133.25 с (С^1”), 135.62 с (С^3",5"), 143.17 д (С¹⁵), 146.36 с (С¹⁶), 147.57 с (С⁸), 151.74 с (С⁴"), 164.88 с (CONH), 166.22 с (С^4'), 177.65 с (С¹⁸). Найдено: С 75.32, N 3.62, Н 8.63. C₄₈O₆N₂H₆₄ Вычислено: С 75.39, N 3.66, Н 8.38.To a solution of 0.50 g (1.0 mmol) of azlactone (VI) obtained in Example 1, 0.27 g (1.1 mmol) of 3,5-di-tert-butyl-4-hydroxyphenylpropylamine was added in 7 ml of benzene, the reaction mass was heated (70 ° C) ) for 6 hours. The solvent was removed in vacuo, the residue was chromatographed on silica gel (eluent: chloroform). Crystallization from diethyl ether gave 0.69 g (91% yield) of compound (IB). _Mp 85-87 ° C. [α] _580, 11.06 ° (c 3.23, CHCl ₃ ). IR spectrum, cm ^-1 : 713, 747, 769 (Ph); 890 (furan); 1155, 1231, 1723 (CO ₂ Me); 1514, 1665 (CONH). 3325 (OH). UV spectrum, λ _max , nm (log ε) 315 (3.24). ¹ H NMR spectrum (δ, ppm, J, Hz): 0.48 s (3H, C ²⁰ H ₃ ), 0.93 etc. (1H, H ¹ , J 12, 3), 0.98 etc. (1H, H ³ , J 12, 3), 1.14 s (3H, C ¹⁹ H ₃ ), 1.25 dd. (1H, H ⁵ , J 11, 3), 1.40 s (18H, 2C (CH ₃ ) ₃ , 1.45 m (1H, H ² ), 1.55 s (2H, H ^9.11 ), 1.66-1.80 m (4H , H ¹ ' ⁷ - ² ' ¹¹ ), 1.86 m

1.96 dm (1H, H ⁶ J 12), 2.12 d.m. (1H, H ³ , J 11), 2.38 m (2H, H ^12.7 ), 2.58 t (3H, CH ₂ Ar, H ¹² , J 6), 3.41 dd

3.58 s (3H, OCH ₃ ), 4.56 s, 4.92 s (2H, H ^17.17 ), 5.03 s (1H, OH), 6.33 d (1H, H ¹⁴ , J 1.8), 6.52 t (1H, CONH, J 6), 6.81 s (1H, H ^{1 '} ), 6.97 s (2H, H ^{2 ", 6"} ), 7.35 d (1H, H ¹⁵ , J 1.8), 7.47 t (2H, H ^{7', 9 '} , J 7), 7.55 t (1H, H ^{8 '} , J 7), 7.93 d (2H, H ^{6', 10 '} ; J 8), 8.59 br.s. (1H, H ^{3 '} ). C ¹³ NMR spectrum (CDCl ₃ , δ _s ppm): 12.50 k (C ²⁰ ), 19.76 t (C ² ), 23.27 t (C ¹² ), 24.21 t (C ¹¹ ), 26.13 t (C ⁶ ) , 28.61 k (C ¹⁹ ), 30.21 k (C (CH ₃ ) ₃ ), 31.59 t

35.09 t

34.14 s

37.99 t (C ³ ), 38.51 t (C ⁷ ), 38.92 t (C ¹ ), 39.69 t (CH ₂ N), 40.04 s (C ⁴ ), 44.13 s (C ¹⁰ ), 51.03 k (OCH ₃ ), 54.74 d (C ⁹ ), 55.95 d (C ⁵ ), 106.55 t (C ¹⁷ ), 110.60 d (C ^{1 '} ), 112.87 d (C ¹⁴ ), 124.76 d (C ^{2 ", 6"} ), 126.53 s ( C ^{2 '} ), 127.38 d (C ^6' '^10' ), 128.65 d (C ^{7 ', 9'} ), 129.79 s (C ¹³ ), 132.03 s (C ^{5 '} ), 132.14 d (C ^8' ), 133.25 s (C ^{1 ”} ), 135.62 s (C ^{3", 5 "} ), 143.17 d (C ¹⁵ ), 146.36 s (C ¹⁶ ), 147.57 s (C ⁸ ), 151.74 s (C ⁴ "), 164.88 s (CONH), 166.22 s (C ^{4 '} ), 177.65 s (C ¹⁸ ). Found: C 75.32, N 3.62, H 8.63. C ₄₈ O ₆ N ₂ H ₆₄ Calculated: C 75.39, N 3.66, H 8.38.

Example 3. 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 (0.1 ml per 10 g of body weight). Compounds (Ia) or (Ib) were administered intragastrically to mice (9 pcs. In each group) at a dose of 100 mg / kg in the form of a water-twin suspension 1 hour before the reproduction of hepatitis. Control animals (9 pcs.) Were similarly injected with water-twin suspension in an equivalent volume, the comparison group (9 pcs.) - 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 method of [10]. The results were statistically processed using the STATISTIKA 6 software package.

The results of the experiment are presented in table 1. It was found that compounds (Ia) and (Ib) show a significant anticytolytic effect. Agent (Ia) reduces the activity of transaminases in the blood by 1.3, agent (Ib) - by 1.7-1.9 times in comparison with the control. Both agents are not inferior to dihydroquercetin in terms of their effect on ALT levels (the differences between the groups are not significant), and agent (IB) exceeds the reference compound by 1.7 times in terms of its effect on ACT activity. Under the action of compounds (Ia) and (Ib), the activity of alkaline phosphatase significantly decreases, which indicates their anticholestasis effect. Agent (Ia) reduces the activity of alkaline phosphatase in the blood by 1.5, and agent (Ib) - by 2 times compared with the control. In terms of severity of the anticholestasis effect, compound (Ia) is not inferior, and compound (Ib) significantly exceeds DQW by 1.5 times. Both agents, as well as the reference compound, in the conditions of this experiment did not affect the intensity of peroxidation processes: the concentration of MDA in the corresponding groups did not differ significantly from the control (Table 1).

Table 1
The effect of compounds (Ia) and (Ib) 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 (Ia) 655.80 ± 55.46 ** 411.07 ± 43.58 * 1.99 ± 0.12 * 3.75 ± 0.12 " (Ib) 515.58 ± 60.41 *** 290.14 ± 46.17 ** 1.41 ± 0.16 ** 3.07 ± 0.24 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 DHQ are significant.

Thus, it was shown that compounds (Ia) and (Ib), when administered intragastrically at a dose of 100 mg / kg, have a hepatoprotective effect, reducing the severity of cytolytic and cholestatic processes against toxic hepatitis. Compound (Ia) is not inferior in magnitude to the effect, and compound (Ib) is superior to DHQ.

Example 4. The study of hepatoprotective and antioxidant properties 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). Compounds (Ia) and (Ib) were injected into the stomach of rats (10 each in a group) in the form of a water-twin suspension 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 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 established that against the background of cyclophosphamide, compounds (Ia) and (Ib) did not show an anticytolytic effect: the activity of both transaminases had no statistical differences with the control. The corresponding effect of DHQ was manifested only in relation to ACT, the activity of which in the reference group decreased by 1.4 times relative to the control. Both agents and DHQ in this experiment did not exert anticholestasis. Compounds (Ia) and (Ib) did not significantly affect the concentration of MDA in the blood, unlike DHQ, which significantly increased the intensity of peroxidation relative to the control. The differences between DHQ and compounds (Ia) and (Ib) in the concentration of MDA were 1.3 and 1.4 times, respectively. No significant difference between the agents and the reference compound in terms of the effect on the total metabolic rate (protein, glucose) was found.

table 2
The effect of compounds (Ia) and (Ib) on the average values of rat serum biochemical parameters against the background of cyclophosphamide intoxication Group ALT. mcat / g protein ACT. mcat / g protein Bf. 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 (Ia) 67.3 ± 3.8 69.5 ± 4.7 ^# 122.8 ± 15.1 1.98 ± 0.05 59.89 ± 2.32 13.53 ± 0.55 * (Ib) 65.3 ± 4.3 86.4 ± 9.2 ^# 146.2 ± 16.7 1.86 ± 0.17 58.73 ± 2.92 14.64 ± 0.36 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.

Thus, it was shown that when administered intragastrically at a dose of 50 mg / kg against the background of intoxication with cyclophosphamide, the anticytolytic effect of compounds (Ia) and (Ib) is lower, and the antioxidant is higher than that of DHQ.

Example 5. 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 established that against the background of hemophilia caused by cyclophosphamide, compound (Ib) causes shifts in peripheral blood indices unidirectional with DQF. Compound (IB) significantly reduces leukopenia, increasing the number of leukocytes in the blood by 1.9 times relative to the control, which exceeds the similar effect of DQF (1.6 times). Agent (IB) showed a small (1.2-fold) decrease in the number of red blood cells compared with the control, which correlates with the same decrease in hematocrit. The relative decrease in erythrocyte mass in animals of this group is compensated by a small significant increase in the quantitative indicators of hemoglobin in erythrocytes (MCH, MCHS). Compound (Ia) did not cause significant changes in peripheral blood counts and, thus, did not exhibit a hemostimulating effect.

Table 3
The effect of compounds (Ia) and (Ib) on the average values of rat peripheral blood parameters against the background of cyclophosphamide intoxication 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 (Ia) 5.25 ± 0.31 28.4 ± 1.2 1.1 ± 0.2 16.3 ± 0.3 97.4 ± 8.8 53.9 ± 0.8 10.7 ± 0.6 8.6 ± 0.05 * 31.1 ± 0.6 57.7 ± 0.5 * (Ib) 4.33 ± 0.24 * 23.7 ± 1.4 * 1.7 ± 0.3 * 14.2 ± 0.6 101.1 ± 17.2 58.5 ± 1.9 10.6 ± 0.3 8.5 ± 0.1 * 33.0 ± 0.8 * 57.4 ± 0.3 * 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 - the number of red blood cells, HCT - hematocrit, WBC - the number of leukocytes, NOV - hemoglobin, PLT - platelets, MCV - the average volume of red blood cells, RDW% - the percentage distribution of the absolute weight of red blood, MPV - the volume of platelets, MSN - the average content of hemoglobin in erythrocyte, MCHC - the average concentration of hemoglobin.

Against the background of neutrophenia caused by cyclophosphamide, there was a tendency to an increase in the number of granulocyte cells under the action of compounds (Ia) and (Ib). In the same groups, relative monocytosis was revealed in the blood of animals. The noted tendency to stimulate leukocyte cells was more pronounced in compound (IB) than in DQW (Table 4).

Table 4
The effect of compounds (Ia) and (Ib) on the average values of rat blood leukocyte counts on the background of cyclophosphamide intoxication Group eosinophils p / nuclear s / nuclear monocytes lymphocytes the control 0 0 1.43 ± 0.48 2.43 ± 0.81 96.14 ± 0.51 (Ia) 0.29 ± 0.18 0 3.43 ± 0.84 4.57 ± 0.57 91.86 ± 0.99 (Ib) 0.43 ± 0.20 0 1.29 ± 0.68 5.71 ± 1.04 92.57 ± 1.64 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 (Ib), when administered intragastrically at a dose of 50 mg / kg against the background of hemosuppression caused by the introduction of cyclophosphamide, reduces leukopenia and is superior to DQW in stimulating effect on blood leukocyte growth. Agent (Ia) under the same conditions does not exhibit a significant hemostimulating effect.

Information sources

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3. EG Goldberg, Zueva EP, Preparations from plants in the complex therapy of malignant neoplasms. Tomsk Tomsk University Publishing House. 2000, 128 p.

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

5. Medicines approved for use in the USSR./ Ed. M.A. Klyueva, E.A. Babayana. M., Medicine, 1979, S. 61-65.

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. 2. 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.

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Claims (1)

16- {2-Benzoylamino-2 - [(3,5-di-tert-butyl-4-hydroxyphenyl) alkylcarbamoyl] vinyl} -labdatrienes of the formula (I)

possessing antioxidant, hepatoprotective and hemostimulating properties.