Derivatives of betulin as inhibitors of complement

 

(57) Abstract:

Describes betulin derivatives of General formula I

where R1, R2the same or different and independently or together.

- SO2(OH) and their salts

- P(O)(OH)2and their salts

- CH2COOH and their salts

where R3- (CH3)(=CH2)

- CH(CH3)2

as inhibitors of the complement. I exhibit higher activity, get them from the available triterpene betulin allocated from birch bark. 5 table.

The proposed invention relates to the field of medicine, specifically to immunology.

The complement system is part of the immune system, which is activated when ingested alien bacteria and antigens. After the destruction of foreign bodies activation of complement is terminated. However, a wide range of immune, autoimmune and immunodeficiency diseases, accompanied by the defeat of their own body tissues, is associated with excessive and (or) late activation of the complement system. To the group of acute conditions associated with activation of the complement system, which may include respiratory delesne, wound disease, asthma, recurrent (post-surgery) vascular stenosis, the syndrome of multiple organ failure, hemorrhage, Guillain-Barre syndrome. In a group of chronic conditions include paroxysmal nocturnal hemoglobinuria, glomerulonephritis, systemic lupus erythematosus, rheumatoid arthritis, disease Alsheimer, rejection of organs transplant, myasthenia gravis, multiple sclerosis. Often the activation of the complement system, leading to complications that arise due to incomplete biocompatibility of the materials in devices for hemodialysis, artificial hearts, and others In this regard, the establishment of effective modulators of the complement system, is able to prevent its destructive effect on the body, is one of the topical problems of modern molecular immunology

Known macromolecular therapeutic agents based on natural and recombinant forms of the natural inhibitors of the complement system (C1-ING and sCRl) [Asghar S. S. Pharmacological manipulation of the complement system in human diseases. //Frontiers in Bioscience. 1996. March 1. V. 1 P. 15-25]. The reason for the attractiveness of recombinant proteins as therapeutic drugs, is already incorporated in them biological properties, However, the high cost recombinase low molecular weight inhibitors of the complement system.

Today found a lot of strong low-molecular-weight inhibitors of the activation of complement in vitro. Among them, antitumor, protivovospalitelnye, antifibrinolytic agents. Rosmarinic acid (1) inhibits activation of the complement with IC50180 µm [Sahu A., Rawal N. Inhibition of

complement by covalent attachment of rosmarinic acid to activated C3b //Biochem. Pharmacol. 1999. V. 57, P. 1439-1446]. This natural connection is difficult, moreover, insufficiently active.

The most similar structure and the result achieved by the proposed subject of the invention are synthetic analogues of Cleanaway acid (2). From table.1 shows that the most active compound in this series inhibits activation of the complement with IC5031.4 µm [H. Assefa, Nimrod A. Synthesis and evaluation of potential complement inhibitory semisynthetic analogs of oleanolic acid. //Bioorg.Med.Chem.Lett. 1999. V. 9. P. 1889-1894].

The obvious disadvantages of such an inhibitor should include enough high activity, low availability of the source Cleanaway acid, the multistage obtain its derivatives, has the required property.

The technical purpose of this invention is the synthesis of more active inhibitor of complement.

50to 7 μm. They are derived from allocated from the bark of birch triterpene betulin.

Inhibitors of complement General formula

where R1, R2the same or different and independently or together represent:

- SO2(OH) and their salts (A)

- P(O)(OH)2and their salts (In)

- CH2COOH and its salts (C)

where R3- (CH3)(=CH2) (I)

- CH(CH3); (II)

Example 1

3,28-Disulfate betulin and its derivatives (IA-VIA). The sulfation of betulin (1, R1=R2=H) and its derivatives (II-VI, Rl=R2=H) is conducted according to the following scheme. To a mixture of 9.5 mmol of sulfuric acid and 9.5 mmol of acetic anhydride in 10 ml of pyridine at a temperature of from 18 to 90°C was added 1.6 mmol of the corresponding derivative of betulin and stirred for 30-60 minutes. The mixture is then neutralized precipitate the inorganic salts filtered off, the solution evaporated to dryness. The resulting crystals are washed twice with 20 ml water and dried. The yields of products 90-97%.

Disulfate betulin (IA):1H-NMR (CDCl3-CD3D, 1:1, , M. D.): 0.68-1.99 (M, CN, (CH2)n, m), 3.65 (1H,=CHOS, etc), 4.0 (2H, CH2OS, D. D.). 4.55, 4.70 (2H, CH2=C, 2 S.).

Example 2

3,28-Diphosphate betulin and its derivatives (IB-VIB of trichloride phosphorus under mechanical stirring at 90-95°C for 24 h, blowing with argon, then add another 10.7 mmol of phosphorus oxychloride and heated under the same conditions for another 76 hours the Excess phosphorus oxychloride is distilled off under vacuum. The remaining oily liquid is crystallized at a temperature of -4° C with 10 ml of hexane. The precipitate was separated by decantation, poured in 32 ml of heptane, 20 min refluxed separates nerastvorim precipitate by decantation and the solution placed in a refrigerator (-4° C) for crystallization. The precipitated crystals filtered off, dissolved in 5 ml of water and dried over P2O5and CON. Output 68-75%.

Diphosphate betulin (IB):1H-NMR (CDCl3-CD3D, 1:1, , MD): 0.68-1.99 (M, CN, (CH2)n. m), 3.61 (1H,=CHOP, etc), 3.90 (2H, SNOW, D. D.), 4.55, 4.70 (2H, CH2=C, 2C.).

Example 3

3,28-Di-O-carboxymethylation and its derivatives (IC-VIC) are obtained by the following method. To 3.2 mmol of sodium hydride in 20 ml of tetrahydrofuran, add 1.6 mmol betulin derivative, and then 3.2 mmol of ethyl ether bromoxynil acid. The reaction mixture is boiled under stirring for 30 minutes, then evaporated. Add 10 ml of water and 2 ml of concentrated hydrochloric acid and evaporated to 3 ml After cooling, the precipitated crystals filtered off, washed with 5 ml of water and dried. Outputs cont(1H,= ONSOON, so), 3.95 (2H, ONSOON, D. D.), 4.49 (4H, SNAN, s), 4.55, 4.70 (2H, CH2=C, 2 S.).

Example 4

20,29-Dihydrovitamin (II, R1=R2=H) produced by the catalytic shriramana betulin in tetrahydrofurane at 5% Ohm palladium on coal at room temperature and atmospheric pressure. The quantitative output.

Example 5

20,29-Dihydro-20,29-dichlorodibenzofuran (V, R1=R2=H) and 20.29-dihydro-20,29-dibromoethylene (IV, R1=R2=H) are obtained in heterophase mixture of betulin (I, R1=R2=H), methylene chloride and chloroform (or bromoform) with 50% aqueous solution of sodium hydroxide in the presence of a phase transfer catalyst for 20 hours the Mixture was evaporated and extracted with a final matter, tetrahydrofuran, recrystallized from ethyl acetate. Outputs 85 and 60%, respectively.

20,29-Dihydro-20,29-dichlorodibenzofuran (V, R1=R2=H): so pl. 201-202,50C.1H-NMR (CDCl3): 3.72 (1H, d, J 10.7, 28-SNON), 3.22 (1H, d, J 10.7, 28-CH2IT). 3.18 (1H. m, 3H). 2.30 (1H, m, N). 1.22 (3H, s, CH3), 0.99 (3H, s, CH3), 0.98 (3H, s, CH3), 0.96 (3H, s, CH3), 0.81 (3H, s, CH3), 0.74 (3H, s, CH3). Found, %: C 70.84, N 9.59. WITH31H50Cl2O2. Calculated, %: C 69.39, N 9.65.

20,29--CH2IT), 3.22 (1H, d, J 10.2, 28-CH2IT). 3.18 (1H, m, 3H), 2.40 (1H, m, N), 1.66 (3H, s, CH3), 1.59 (3H, s, CH3), 1.36 (3H, s, CH3), 1.00 (3H, s, CH3), 0.94 (3H, s, CH3), 0.81 (3H, s, CH3). Found, %: C 60.59, H 8.20. C31H50Br2O2. Calculated, %: C 60.82, N 8.83.

Example 6

20,29-Dihydro-20,29-methylisatin (III, R1=R2=H) is obtained from 2 mmol betulin (I, R1=R2=H) and 3 mmol diiodomethane in ether in the presence of metallic copper activated zinc dust during the day. The solution is filtered, evaporated, and recrystallized from ethyl acetate. An output of 60%. so pl. 233-234,5° C,1H-NMR (CDCl3): 3.72 (1H, d, J 10.7, 28-SNON), 3.22 (1H, d, J 10.7, 28-SNON), 3.18 (1H, m, 3-H), 2.30 (1H, m, 19-H), 1.22 (3H, s, CH3), 0.99 (3H, s, CH3), 0.98 (3H, s, CH3), 0.96 (3H, s, CH3), 0.81 (3H, s, CH3), 0.74 (3H, s, CH3), 0.24 (4H, m, CH2-CH2); EI-MS m/z 457 M+.

Found, %: C 81.52, N, 11.47. SNO. Calculated, %: C 78.06, N 11.52.

Example 7

30-Bromethalin (VI) receive bromirovanii 1.5 mmol betulin (I) of 1.6 mmol of N-bromosuccinimide in 20 ml of carbon tetrachloride within 24 hours After evaporation and washing with 10 ml of hot water get crystalline powder. Yield 91%.1H-NMR (CDCl3): 5.00, 5.11 (2H, s, 29-CH2=), 3.95 (2H, s, 30-Snug), 3.72 (1H, d, J 10.7, 28-SNON), 3.22 (1H, d, J 10.7, 28-SNON), 3,18 (1H, m, 3-

Definition antihemolytic activity (AA) is carried out in a standard hemolytic system of the following composition: sensitized rabbit antibodies to sheep erythrocytes, the sample analyte, the complement of the Guinea pig, peronally buffer (pH 7.4) containing ions of CA2+and Mg2+(VBS++) [Kozlov L. C., Rostovtseva L. I., The Poseur Ie, Sutovsky N. S., Sorokina, I. B., Barkov T-I. // Bioorganic. Chemistry, 1985, T. 11, S. 1510-1518].

Preparation of the buffer. Under stirring and heated dissolve 3 g (0.0145 mol) of medinal and 5.75 g (0.0312 mol) of veronal in 1.5 l of distilled water. Dissolve 85 g (1.45 mol) of NaCl and bring the volume to 2 L. Add anhydrous NaOH (0.4-0-5 g) to pH 7.4. Then add 5 ml of 1 M solution of magnesium chloride and 1.5 ml of 1 M solution of calcium chloride.

Preparation of erythrocytes. Red blood cells are stored at 4°C in sterile test tubes in a solution of Olivera under anaerobic conditions. In experiments using erythrocytes in VBS++. In all experiments use only cold buffer solution (to reduce spontaneous lysis of erythrocytes). For the experiment, sterile taken 2-3 ml of the suspension of erythrocytes, add 20 ml of buffer, gently shaken until homogeneous sostoaniy sediment. After that add the buffer to achieve the previous level. The described operation is repeated up until the supernatant is not bleached. The resulting suspension of erythrocytes further standardizes.

Preparation of sensitized erythrocytes. To 10 ml of a suspension of erythrocytes (1· 109cells/ml) was added 10 ml of a solution of rabbit antibodies obtained by dilution buffer (VBS++) in the ratio of 1:400, mixed and incubated for 30 min at 37° C, shaking occasionally. Suspension is centrifuged at 3000g 5 minutes Then the supernatant is carefully poured, add a little VBS++and resuspended residue.

Standardization of sensitized erythrocytes. Erythrocyte suspension was adjusted to a concentration of 1.5· 108cells/ml to 1900 μl of distilled water was added 100 μl of the suspension of sensitized erythrocytes and shake up until the solution became transparent. You must ensure that the optical density of the solution was in the range of 1.000-1.050. For this purpose, if necessary, dilute the suspension of erythrocytes buffer and repeat the operation preparation lysed solution and optical density measurement. Further use standardized erythrocytes.

After incubation in tubes make additional 3 ml of cold buffer. All the tubes centrifuged 5 min at 3000g, measure the optical density of the supernatant (A414ex and a414 p/l AVG). Further calculated according to the formula I values lysis build a graph of the dependence of lysis concentration of complement.

L=AEX/A414 p/l AVG·100% (I)

ANDEX- the optical density of the supernatant samples 5-16;

A414 p/l AVG. the average optical density of the supernatant samples 1-3.

You also need to pay attention to the background lysis of erythrocytes ANDCE(lysis in pure buffer). It is desirable that it does not exceed the value of optical density of 0.1. If it is larger than 0.2, it is necessary to take a new batch of red blood cells.

For further experiments use the concentration of complement in which there is 75-80% lysis of erythrocytes.

To determine antihemolytic activity of substances in each tube is ntrol for complete lysis is performed by adding distilled water instead of buffer. Control 75-80% of the lysis is carried out in samples 4-6.

The tubes are thoroughly shaken and incubated at 37° C for 25 minutes After that, the contents of the tubes is cooled to 4° C, diluted with buffer 4 times and centrifuged at 3000g for 5 minutes then the supernatant is separated, the amount of released hemoglobin in the sample is measured spectrophotometrically at 414 nm.

Derivatives of betulin General formula

where R1, R2the same or different and independently or together represent

- SO2(OH) and their salts,

- P(O)(OH)2and their salts,

- CH2COOH and their salts,

R3- - (CH3)(=CH2),

- CH(CH3)2

as inhibitors of the complement.



 

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