3,28-di-o-nicotinate betulin, showing hepatoprotective and anti-hiv activity

 

(57) Abstract:

Describes a new chemical compound, namely 3,28-di-O-nicotinate betulin formula I

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showing hepatoprotective and anti-HIV activity. The connection is a low-toxic substance, has a pronounced hepatoprotective activity, which exceeds the effect of betulin and celebra, combined with anti-HIV activity. 3 table.

The invention relates to new biologically active chemical compound, specifically to 3,28-di-O-nicotinate betulin (1), formula

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showing hepatoprotective and anti-HIV activity.

The compound (1) and its properties are not described in literature.

The State registration number of the compounds (1) - N 10640198.

In recent years, an intensive search of highly effective drugs based on the available natural substances, possessing valuable pharmacological properties. In this regard, of particular interest is the synthesis of low modified analogues of natural compounds, combining different types of biological activity.

The closest in structure and properties to the proposed connection is triterpen tiget 35 - 40%. It is known that betulin (2), along with the hypolipidemic effect, has choleretic and hepatoprotective activity [1] . Betulin (2) also has anti - HIV activity [2] . In this regard, the search for new compounds in the series lipanovich the triterpenoids, in particular on the basis of betulin (2), with a higher hepatoprotective and anti-HIV activity, is relevant.

In the claimed technical solution synthesized a new low-toxic derivative of betulin, namely 3,28-di-O-nicotinate betulin (1), showing hepatoprotective and anti-HIV activity.

In the study of hepatoprotective activity of compound (1) as the reference drugs used well-known drugs Carsil and zelibor [3]. However, it should be noted that achieving a good effect in the use of these drugs is only possible with long-term use in large doses (at least 50 mg/kg). Additionally, Kars, being imported drug (Bulgaria), now became available to a large mass of patients. Thus, a very important task is the search for more effective, non-lethal and cheap hepatoprotective drug.

As the comparison drug under study, the effective dose (ED50) of the compound (1) was determined on outbred mice weighing 20-25 g at the introduction into the stomach. The toxicity parameters and unit actions were calculated by Litchfield and Wilcoxon signed. On the basis of the Decision of the state Committee of standards of the USSR on 10 March 1976 579 N this connection belongs to class IV hazardous substance (LD50=6.5 mg/kg). ED50compound (1) was 11.7 mg/kg

Hepatoprotective effect of compound (1) was estimated by zhelchesekretornoyj and the excretory functions of the liver of intact rats and in animals with experimental hepatitis induced by carbon tetrachloride. As Comparators with compound (1) used structural analog - betulin (2) and the known pharmacological hepatoprotective drugs flavonoid patterns - zelibor and Kars. The results of the experiments are given in table. 1 and 2.

It is established that the compound (1) dose of 50 mg/kg increases the secretion of bile by hepatocytes of rats during all periods of observation in 1.5-2 times more effective than Comparators - betulin and celebra. In addition, the compound (1) similarly increases zheleobrazovatel function of rat hepatocytes (table. 1). In table. 2 shows the effect of compound (1) on the functional SOS is sustained fashion the liver as compared with intact animals. As can be seen from the table. 2, the compound (1) was more active even at lower (5 times) dose (10 mg/kg) than the comparison drug - Kars. The compound (1) increases the functional activity of hepatocytes during all periods of the experiments, while the Kars - only in the 2nd and 3rd hours. In addition, the compound (1) increases the bile production (total weight of bile) in animals with experimental CCl4-hepatitis more Kars more than 1.5 times.

Anti-HIV activity of compound (1) was investigated on the traditional model highly sensitive to HIV cell line MT-4 - transplantable human lymphocytes, initially infected with human immunodeficiency virus type 1 strain HIV-1/coding gain. As a pharmacological analogue used a known inhibitor of viral reverse transcriptase - azidothymidine (AZT), which is widely used in practice, the treatment of HIV infection, but with high toxicity, low efficacy in blocking the reproduction of HIV in cells monocytes/macrophages and other [4].

The effectiveness of antiviral activity of a compound (1) was estimated using complex methods, allowing to obtain sufficient information about the characteristics of its anti-HIV actions:

1) opredeleniya infection by determining their viability by exclusion Trifanova blue;

3) quantification of the accumulation of viral protein p24 by ELISA.

It is established that the compound (1) effectively suppresses the accumulation virousspecificakih protein p24 (ID50=0.02 µg/ml), without protective actions, and in the concentration range 0.001 - 20.0 µg/ml is not toxic to cells MT-4 (IC50> 20.0 µg/ml). (PL. 3). The index IS compound (1) > 1000 (suppressing the accumulation of p24 antigen > 1000), indicating that his pronounced anti-HIV action. Connection (1) all parameters: ID50(effective dose), IC50(toxic dose) and IS (therapeutic index) has significant advantages compared with betulin, which ID50=23, IC50=45, IS=1.9[2].

Synthesis of compound (1) was performed by the interaction of betulin with the acid chloride of nicotinic acid in the environment pyridine - tributylamine with the release of 93.6%. Analytically pure product was obtained by extraction with benzene.

Thus, the proposed new low-toxic derivative among the triterpenoids lobanovo group-3,28-di-O-nicotinate betulin (1). Connection (1) allatoxin, has a pronounced hepatoprotective activity, exceeding the effect of known hepatoprotectors of celebra the Oia is demonstrated by the following examples.

Example 1. Getting dinicotinate betulin (1).

To a solution of 1 mmol (0.44 g) in a mixture of betulin 5 ml of anhydrous pyridine and 5 ml of tributylamine under stirring and cooling (0-5oC) was added 3 mmol (0.43 g), freshly prepared by the method of [5] the hydrochloride of the acid chloride of nicotinic acid. The temperature was brought to room temperature and stirred for another 4 h, the Reaction mass was poured into 50 ml of cold water, acidified with HCl and was extracted with benzene. The extract was washed with water, 5% HCl solution, again with water, dried Na2SO4and evaporated in vacuum. Yield 0.59 g (93.6%). Rf=0.55 (eluent chloroform - methanol = 20:1). TPLpl.=116-118oC. Found,%: C-77.54, N-8.21, N-4.05. C42H56O4N2. Calculated,%: C-77.26, N-8.64, N-4.29. UV spectrum (EtOH),max/HM: 263 (Ig 4.18). IR spectrum (cm-1, vaseline oil): 1730(C= O), 1650(CH-CH2), 1600 (Ar), 1470, 1390, 1330, 1300, 1260, 1220, 1150, 1050, 990, 910 (CH=CH2), 870. Range1H NMR (, memorial plaques, J/Hz): 0.82, 0.84, 0.91, 0.94, 1.00 (5c, 15H, 5 CH3), 1.00-2.00 (m, CH2CH), 1.63 (s, 3H, CH3), 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 aromatic.). Range13With NMR ( M. D.): 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 (10), 20.8 (C11), 25.1 (C12), 38.1 (C13), 42.7 (C14), 27.0 (C15), 29.5 (C16), 47, 26.2, 126.6, 137.0, 137.0, 149.8, 150.8, 153.1, 153.3 (With the aroma),

Example 2. The study of hepatoprotective activity of compound (1).

Experiments were performed on 120 rats weighing 180-200 g Unit steps U50= 11.7 mg/kg toxic dose LD50= 6.5 mg/kg was determined by the method of [6]. Effect of compound (1) on the functional state of the liver was assessed by zhelchesekretornoyj and the excretory functions of the liver by the method of [7] in intact rats and in animals with experimental CCl4-hepatitis. Bile secretion was expressed in mg/min per 100 g weight of the animal, and biliary function by the total number of selected bile for each hour and the total amount of 4 h of observation per 100 g weight of rats. As Comparators used triterpenoid lobanovo group - betulin (2) and known hepatoprotectors - zelibor and Kars.

In the first series of experiments studied the effect of compound (1) on the functional state of the liver in the intact animals. As Comparators used triterpenoid lobanovo group - betulin (2) and silimar in equal doses of 50 mg/kg, commonly used in practice when treating siliborum. The results of the experiments are given in table. 1.

In the next series of experiments studied the effect of compounds (1) the dose of 10 mg/kg after determining therapeutic dose ED50=11.7 mg/kg, and drug comparisons Kars in commonly used in practice [9] the dose of 50 mg/kg the results of the experiments are given in table. 2.

Thus, the compound (1) has a pronounced hepatoprotective activity at lower dose exceeding the effect of known hepatic - celebra and Kars.

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

Anti-HIV properties represented by compounds (1) were studied on the traditional model of primary HIV-positive lymphoid cells MT-4. In this work, we used a strain of HIV-1/coding gain. The cytotoxicity of the compounds was assessed in the culture of human T lymphocytes man line MT-4. The drug was dissolved in ethanol and appropriate dilutions were made in wells of 96-well culture plates (three wells for each dilution) for the screening of cells. The seeding concentration was 5105cells/ml the cells were cultured in growth medium (medium RPMI-1640 with the addition of 10% serum fetal cow, 0,06% L-glutamine, 100 µg/ml gentamicin and 60 μg/ml of lincomycin) at 37oC and 5% carbon dioxide for 4 days. At the end of incubation was estimated percentage of viable cells in the cell Goryaeva after akrasian the cells CD50. To assess anti-HIV activity of the compounds of cells MT-4 were infected with HIV-1 strain/coding gain with a multiplicity of infection of 0.2-0.5 infectious units per cell. After adsorption of virus for 1 h at 37oC infected and control cells were diluted with growth medium before seeding concentration 5105cells/ml and added into wells of 96-well culture plates. Then made the solution proposed connection (1) (three wells for each dilution) and then cultured as described above. The concentration ranged 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 viral p24 antigen by ELISA. In addition, it was determined anti-HIV activity, based on the degree of protection of infected cells from death due to virus infection, calculating the percentage of viable cells in the cell Goryaeva after dyeing Trifanova blue. On the basis of the results obtained built a dose-dependent curves and determine the characteristics of inhibition of reproduction of HIV: ID50- the concentration, 50% inhibitory products of a virus or providing 50% protection of the cells; IS the index of selectivity: on the table. 3.

References

1. Vasilenko Y. K., Semenchenko C. F., Frolova L. M, Konopleva, E., parfentiev E. P. , Schulte I. C. Pharmacological properties of triterpenoids of birch bark //Experimental. and the wedge. Pharmacol. 1993. So 56. # 4. S. 53-55.

2. Fujioka T., Kashiwada y, Kilkuskie, R. E., L. M. Cosentino, L. M. Ballas , 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. Medicines. M.:Medicine, 1993. So P.F. 612.

4. Plyasunov O. A., Egorychev I. N., Fedyk N. In., Pokrovsky A. G., Baltina L. A., Murinov Y. I., Tolstikov, A. Study of anti-HIV activity, glycyrrhizic acid //problems of Virology, 1992, N 5-6, S. 235-238.

5. Naumova B. S., Chekmareva I. B., Zhdanovich E. C., N. Preobrazhenskaya.A. Obtaining derivatives of nicotinic acid //Chem.-Pharm. Magazine, 1969. So 3. N 5. C. 11-12.

6. Belenky, M. L. elements of a quantitative evaluation of the pharmacological effect. HP-1963. 186 C.

7. The Roller N. P., Oleinik L. N. Comparative effects of atropine and metacin on the exocrine function of the liver /Journal of Pharmacol. and tonsil. 1967. N 3. C. 334-337.

8. Nikolaev, S. M. Herbal drugs damage heat otecting and choleretic actions derived glycyrrhizic acid //Experimental. and clinically. Pharmacology. 1995. N 6. S. 60-63.

1. 3,28-di-O-nicotinate betulin,

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showing hepatoprotective and anti-HIV activity.

 

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