RU2174982C2 - Betulinol 3,28-di-o-nicotinate eliciting hepatoprotective and anti-hiv activity - Google Patents

Abstract

FIELD: organic chemistry. SUBSTANCE: invention describes a novel compound, namely, betulinol 3,28- -di-O-nicotinate of the formula (I)

Description

проявляющему гепатопротекторную и анти-ВИЧ активность.The invention relates to a new biologically active chemical compound, specifically to betulin 3.28-di-O-nicotinate (1), of the formula

showing hepatoprotective and anti-HIV activity.

 Compound (1) and its properties are not described in the literature.

 The state registration number of the compound (1) is N 10640198.

 In recent years, there has been an intensive search for highly effective drugs based on available natural substances that have a complex of valuable pharmacological properties. In this regard, the synthesis of low-toxic modified analogues of natural compounds combining various types of biological activity is of particular interest.

белоствольных берез вида Betula pendula достигает 35- 40%. Известно, что бетулин (2), наряду с гиполипидемическим действием, оказывает желчегонное и гепатопротекторное действия [1] . Бетулин (2) обладает также анти- ВИЧ активностью [2] . В связи с этим поиск новых соединений в ряду лупановых тритерпеноидов, в частности на основе бетулина (2), обладающих более высокой гепатопротекторной и анти-ВИЧ активностью, является актуальным.The closest in structure and properties to the proposed compound is the triterpenoid of the lupane group - betulin (2), the content of which in the cortex

White-stemmed birch species Betula pendula reaches 35-40%. It is known that betulin (2), along with a lipid-lowering effect, has choleretic and hepatoprotective effects [1]. Betulin (2) also has anti-HIV activity [2]. In this regard, the search for new compounds in the series of lupane triterpenoids, in particular based on betulin (2), which have higher hepatoprotective and anti-HIV activity, is relevant.

 In the claimed technical solution, a new low-toxic betulin derivative is synthesized, namely, betulin 3.28-di-O-nicotinate (1), which exhibits hepatoprotective and anti-HIV activity.

 In the study of the hepatoprotective activity of compound (1), the known drugs Karsil and Silibor were used as reference preparations [3]. However, it should be noted that achieving a good effect with the use of these drugs is possible only with prolonged use in significant doses (at least 50 mg / kg). In addition, karsil, being an imported drug (Bulgaria), is now inaccessible to a large mass of patients. Thus, a very urgent task is to find a more effective, low toxic and cheap hepatoprotective drug.

 As a comparison drug in the study of anti-HIV activity of compound (1), azidothymidine was used.

Acute toxicity (LD ₅₀ ) and effective dose (ED ₅₀ ) of compound (1) were determined on white outbred mice weighing 20-25 g when introduced into the stomach. The toxicity parameters and units of action were calculated according to Litchfield and Wilcoxon. Based on the Decree of the State Committee for Standards of the Council of Ministers of the USSR of March 10, 1976 N 579, this compound belongs to class IV low-hazard substances (LD ₅₀ = 6.5 mg / kg). The ED _{50 of} compound (1) was 11.7 mg / kg.

 The hepatoprotective effect of compound (1) was evaluated by the bile secretory and biliary excretory functions of the liver in intact rats and in animals with experimental hepatitis caused by carbon tetrachloride. As a comparison drug with compound (1), a structural analogue, betulin (2), and the well-known pharmacological hepatoprotective preparations of the flavonoid structure, silibor and carlsil, were used. The results of the experiments are given in table. 1 and 2.

It was found that compound (1) at a dose of 50 mg / kg increases the secretion of bile by rat hepatocytes at all observation times 1.5–2 times more effective than the reference drugs betulin and silibor. In addition, compound (1) likewise enhances the bile formation function of rat hepatocytes (Table 1). In the table. Figure 2 shows the effect of compound (1) on the functional state of the liver with CCl ₄ hepatitis in rats. With hepatitis, the biliary and biliary excretory functions of the liver are sharply inhibited in comparison with intact animals. As can be seen from the table. 2, compound (1) turned out to be more active even in a lower (5 times) dose (10 mg / kg) than the comparison drug - carlsil. Compound (1) increases the functional activity of hepatocytes throughout the duration of the experiments, while Karsil - only in the 2nd and 3rd hours. In addition, compound (1) increases bile formation (total bile weight) in animals with experimental CCl ₄ hepatitis more than 1.5 times more active than carzil.

 The anti-HIV activity of compound (1) was investigated on a traditional model of the highly sensitive to HIV MT-4 cell line - transplantable human lymphocytes primarily infected with human immunodeficiency virus type 1 - HIV-1 / EVK strain. A known viral reverse transcriptase inhibitor, azidothymidine (AZT), which is widely used in the practice of treating HIV infection, but with high toxicity, low efficiency in blocking HIV reproduction in monocyte / macrophage cells, etc., was used as a pharmacological analogue [4].

The effectiveness of the antiviral effect of the compound (1) was evaluated using a set of methods that allow to obtain sufficiently complete information about the features of its anti-HIV action:
1) determination of the cytotoxicity of the drug;
2) determining the degree of protection of infected HIV cells from death due to infection by determining their viability by trypan blue exclusion;
3) quantitative determination of the accumulation of p24 viral protein by the enzyme immunoassay.

It was found that compound (1) effectively inhibits the accumulation of the virus-specific protein p24 (ID ₅₀ = 0.02 μg / ml), without exerting a protective effect, and is not toxic to MT-4 cells in the concentration range 0.001 - 20.0 μg / ml (IC ₅₀ > 20.0 μg / ml). (tab. 3). The IS index of compound (1)> 1000 (suppression of accumulation of antigen p24> 1000), which indicates its pronounced anti-HIV effect. Compound (1) in all respects: ID ₅₀ (effective dose), IC ₅₀ (toxic dose) and IS (therapeutic index) has significant advantages compared to betulin, in which ID ₅₀ = 23, IC ₅₀ = 45, IS = 1.9 [2].

 The synthesis of compound (1) was carried out by the interaction of betulin with nicotinic acid chloride in the environment of pyridine - tributylamine with a yield of 93.6%. An analytically pure product was obtained by extraction with benzene.

 Thus, a new low-toxic derivative was proposed among the triterpenoids of the lupane group-3.28-di-O-nicotinate of betulin (1). Compound (1) has low toxicity, has a pronounced hepatoprotective activity, exceeding the effect of the known hepatoprotectors silibor and carcil in a lower dose, combined with pronounced anti-HIV activity.

 The invention is demonstrated by the following examples.

 Example 1. Obtaining dinulinum betulin (1).

To a solution of 1 mmol (0.44 g) betulin in a mixture of anhydrous 5 ml of pyridine and 5 ml of tributylamine, 3 mmol (0.43 g) of nicotinic acid hydrochloride freshly prepared according to the procedure of [5] was added with stirring and cooling (0-5 ^° C). The temperature was brought to room temperature and stirred for another 4 hours. The reaction mass was poured into 50 ml of cold water, acidified with HCl and extracted with benzene. The extract was washed with water, 5% HCl, again with water, dried with Na ₂ SO ₄ and evaporated in vacuo. Yield 0.59 g (93.6%). R _f = 0.55 (eluent chloroform - methanol = 20: 1). _Mp mp = 116-118 ^o C. Found.%: C-77.54, H-8.21, N-4.05. C ₄₂ H ₅₆ O ₄ N ₂ . Calculated,%: С-77.26, Н-8.64, N-4.29. UV spectrum (EtOH), λ _max / HM: 263 (Ig ε 4.18). IR spectrum (υ, cm-1, liquid paraffin): 1730 (С = O), 1650 (CH-CH ₂ ), 1600 (Ar), 1470, 1390, 1330, 1300, 1260, 1220, 1150, 1050, 990, 910 (CH = CH ₂ ), 870. ¹ H NMR spectrum (δ, ppm, J / Hz): 0.82, 0.84, 0.91, 0.94, 1.00 (5c, 15H, 5 CH ₃ ), 1.00- 2.00 (m, CH ₂ , CH), 1.63 (s, 3H, CH ₃ ), 2.48 (ddd, 1H, H19, J = 5.7, 10.6, 11.6), 4.05 (d, 1H, H28, J = 11 ), 4.45-4.53 (m, 2H, H28, H29), 4.60-4.67 (m, 2H, H3, H29), 7.25-7.40, 8.18-8.26, 8.67-8.73, 9.12-9.20 (all m, 8H, H aroma.). Spectrum ¹³ C NMR (δ ppm): 38.3 (C1), 23.7 (C2), 82.2 (C3), 37.6 (C4), 55.6 (C5), 18.1 (C6), 34.6 (C7), 40.9 (C8 ), 50.2 (C9), 37.0 (C 10), 20.8 (C11), 25.1 (C12), 38.1 (C13), 42.7 (C14), 27.0 (C15), 29.5 (C16), 47.7 (C17), 46.6 ( C18), 48.9 (C19), 149.8 (C20), 29.8 (C21), 34.0 (C22), 28.0 (C23), 16.0 (C24), 16.7 (C25), 16.1 (C26), 14.7 (C27), 63.7 ( C28), 110.0 (C29), 19.1 (C30), 164.8, 165.8 (-OC = O), 123.2, 123.2, 126.2, 126.6, 137.0, 137.0, 149.8, 150.8, 153.1, 153.3 (С aroma,),
Example 2. The study of the hepatoprotective activity of the compound (1).

The experiments were carried out on 120 rats weighing 180-200 g. The unit of action of ED ₅₀ = 11.7 mg / kg and a toxic dose of LD ₅₀ = 6.5 mg / kg was determined by the method [6]. The effect of compound (1) on the functional state of the liver was evaluated by the bile secretory and biliary excretory function of the liver according to the procedure [7] in intact rats and in animals with experimental CCl ₄ hepatitis. Bile secretion was expressed in mg / min per 100 g of animal mass, and bile secretion was expressed in the total amount of bile excreted for each hour and in total for 4 hours of observation per 100 g of rat mass. Lupane group triterpenoid - betulin (2) and known hepatoprotectors - silibor and karsil were used as comparison preparations.

 In the first series of experiments, the effect of compound (1) on the functional state of the liver in intact animals was studied. Lupane group triterpenoid Betulin (2) and silibor in equal doses of 50 mg / kg, usually used in practice in the treatment of silibor, were used as comparison preparations. The results of the experiments are given in table. 1.

In the next series of experiments, we studied the effect of compound (1) on the functional activity of rat liver in hepatitis caused by CCl ₄ by the method [8]. Compound (1) was taken at a dose of 10 mg / kg after determining a therapeutic dose of ED ₅₀ = 11.7 mg / kg, and the carlsel comparison drug at a dose of 50 mg / kg commonly used in practice [9]. The results of the experiments are given in table. 2.

 Thus, the compound (1) has a pronounced hepatoprotective activity, in a lower dose exceeding the effect of the known hepatoprotectors - silibor and carcil.

 Example 3. The study of anti-HIV activity of the compound (1).

The anti-HIV properties of the present compound (1) were studied in a traditional model of primary HIV-infected MT-4 lymphoid cells. A strain of HIV-1 / EVK was used in the work. The cytotoxicity of the compound was evaluated in a culture of transplantable human MT-4 lymphocytes. The preparation was dissolved in ethanol and added to the wells of 96-well culture plates (three wells for each dilution) in the appropriate dilutions during cell sieving. The inoculum concentration was 5 • 10 ⁵ cells / ml. The cells were further cultured on growth medium (RPMI-1640 medium supplemented with 10% bovine serum, 0.06% L-glutamine, 100 μg / ml gentamicin and 60 μg / ml lincomycin) at 37 ^° C and 5% carbon dioxide for 4 days. At the end of the incubation, the fraction of viable cells in the Goryaev chamber was counted after staining with trypan blue. A dose-dependent curve was constructed and the concentration of compounds causing the death of 50% of the cells — CD ₅₀ — was determined. To evaluate the anti-HIV activity of the compound, MT-4 cells were infected with a strain of HIV-1 / EVK with a multiplicity of infection of 0.2-0.5 infectious units per cell. After the virus was adsorbed for 1 h at 37 ^° C, infected and control cells were diluted with growth medium to an inoculum concentration of 5 × 10 ⁵ cells / ml and introduced into the wells of 96-well culture plates. Then, a solution of the proposed compound (1) was added (three wells for each dilution) and then cultivated as described above. The concentration of the compound was from 0.001 to 20 μg / ml. The inhibitory effect of compound (1) was evaluated on the 4th day of cultivation by measuring the amount of p24 viral antigen by enzyme immunoassay. In addition, anti-HIV activity was determined based on the degree of protection of infected cells from death due to viral infection, by counting the proportion of viable cells in the Goryaev chamber after staining with trypan blue. Based on the results obtained, dose-dependent curves were constructed and quantitative characteristics of inhibition of HIV reproduction were determined: ID ₅₀ — concentration of the compound, 50% suppressing virus production or providing 50% cell protection; IS - selectivity index: ratio of toxic dose of compound IC ₅₀ to its effective dose ID ₅₀ . The results of the experiments are given in table. 3.

List of references
1. Vasilenko Yu.K., Semenchenko V.F., Frolova L.M., Konopleva G.E., Parfentieva E.P., Skulte I.V. Pharmacological properties of birch bark triterpenoids // Exper. and wedge. pharmacol. 1993.V. 56. N 4.P. 53-55.

 2. Fujioka T., Kashiwada Y., Kilkuskie R. E., Cosentino L. M., Ballas L. M., Jiang J. B., Janzen W. P., Chen I.-S., Lee K.-H. Anti-AIDs agents, II. Betulinic acid and Platanic acid as anti-HIV principles from Syzigium Claviflorum, and the anti-HIV activity of structurally related triterpenoids // J. Nat. Prod. 1994. V. 57. P. 243-247.

 3. Mashkovsky M. D. Drugs. M.: Medicine, 1993.T.P. S. 612.

 4. Plyasunova OA, Egoricheva I.N., Fedyuk N.V., Pokrovsky A.G., Baltina L.A., Murinov Yu.I., Tolstikov G.A. The study of anti-HIV activity of β-glycyrrhizic acid // Questions of Virology, 1992, N 5-6, S. 235-238.

 5. Naumova BS, Chekmareva IB, Zhdanovich ES, Preobrazhensky N.A. Preparation of nicotinic acid derivatives // Chem. Pharm. Journal, 1969.V.3. N 5. S. 11-12.

 6. Belenky M.L. Elements of a quantitative assessment of the pharmacological effect. L.-1963. 186 p.

 7. Skakun N.P., Oleinik L.N. Comparative effect of atropine and metacin on exocrine liver function // Pharmacol Journal. and toxyl. 1967. N 3. S. 334-337.

 8. Nikolaev S.M. Herbal medicines for damage to the hepatobiliary system. Novosibirsk 1992.155s.

 9. Nasyrov X.M., Chepurina L.S., Kireeva R.M. The study of hepatoprotective and choleretic action of derivatives of glycyrrhizic acid // Experiment. and clinic. Pharmacology. 1995. N 6. S. 60-63.

Claims (1)

1. 3.28-di-O-nicotinate betulin,

showing hepatoprotective and anti-HIV activity.

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