RU2463041C1 - Method of obtaining betulinic acid - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to pharmaceutical industry, in particular to method of obtaining betulinic acid. Method of obtaining betulinic acid, including betulin dispersing in donor coordinating solution, oxidation of betulin by chrome (IV) compound in presence of aluminium sulfate under specified conditions, purification of obtained betulonic acid and its reduction by sodium borohydride in isopropyl alcohol to betulinic acid.

EFFECT: method makes it possible to increase target product output.

5 cl, 1 tbl, 4 ex

Description

The proposed method relates to the field of organic chemistry, namely to the production of natural biological active substances, and can be used to produce betulinic acid.

Betulinic acid is of interest for medicine and pharmacy as an anti-HIV-1 and antitumor active substance, as well as the fact that its derivatives have immunostimulating, antioxidant, hepatoprotective and antiulcer effects (1). Betulinic acid is a natural biological active substance found in the bark and leaves of many plants, but its isolation from them is unprofitable due to its low content (about 0.1% by weight of dry raw materials).

Currently known methods for producing betulinic acid from betulin - the main product of processing the outer layer of the bark of Betula birch (birch bark) and which is a triterpene alcohol of natural origin. Moreover, most of the known methods are quite complex, include the use of a large number of different types of organic solvents, are characterized by the duration of individual stages and the relatively low yield of betulinic acid relative to betulin. A known two-stage method for producing betulinic acid from betulin, including the first stage of the oxidation of betulin to betulonic acid with chromic anhydride in acetic acid at 15-20 ° C for 10 minutes, purification of the obtained betulonic acid - the main product of the oxidation reaction, from by-products, namely extraction with diethyl ether, washing the extract with 10% sodium chloride (NaCl) solution, treating the extract sequentially with sodium hydroxide to form betulonic acid sodium salt and hydrochloric acid to obtain Ia betulonic acid, solvent removal and recrystallization betulonic acid, followed by reduction of betulonic acid to betulinic acid (2).

As a result, the yield of betulinic acid is less than 50% (based on the initial betulin).

The disadvantages of this method are the low yield of the target product, as well as the complexity and duration of the process, because each stage includes a large set of different types of processing and the use of a large number of organic solvents (acetic acid, diethyl ether, methyl alcohol, tetrahydrofuran).

Closest to the proposed method for the combination of essential features and the achieved technical result is a known method for producing betulinic acid, selected by the authors as a prototype.

This method is carried out by oxidizing betulin, previously dissolved in glacial acetic acid, chromium oxide (VI) in a solution of acetic acid to betulonic acid, purifying betulonic acid through sodium salt by diluting the reaction mixture with an aqueous NaCl solution, double extracting the reaction mixture with diethyl ether, washing with ether extract with an aqueous NaCl solution, treating the extract with a solution containing sodium hydroxide (NaOH), converting the sodium salt of betulonic acid to free betulonic acid, then would be formed betulonic acid sodium salt in diethyl ether solution is converted into a free betulonic acid and recrystallized, betulonic acid and then reduced with sodium borohydride in diethyl ether to a solution of betulinic acid (3).

In this case, the yield of the target product is ~ 65% with respect to the initial betulin.

The disadvantage of this method is the low yield of the target product, multi-stage and duration of the process, as well as the use of a large number of organic solvents at all stages of the process.

The objective of the invention is to develop a method for producing betulinic acid, which allows to achieve a high yield of the target product, simple in execution and not requiring the use of a large number of organic solvents.

The problem is solved by the proposed method for producing betulinic acid, including the oxidation of pre-prepared betulin with a chromium compound (VI), in particular chromium oxide (VI), in solution, purification of the obtained betulonic acid and its reduction with sodium borohydride in an organic solvent to betulinic acid, according to the invention before the oxidation of betulin is dispersed in a donor coordinating solvent, the oxidation of betulin is carried out at a temperature of 15-35 ° C for 60-180 minutes in the presence of sulfate aluminum (Al ₂ (SO ₄ ) ₃ ) when it is contained in the reaction mixture of 0.4 · 10 ^-4 -0.4 · 10 · ² mol / l, for purification of the obtained betulonic acid, the liquid phase containing betulonic acid is separated from the precipitate and dilute it with water until a dispersion forms, then the resulting dispersion is filtered, the precipitate, which is betulonic acid, is washed with water, after which betulonic acid is reduced with sodium borohydride in isopropyl alcohol to betulinic acid.

It is preferable that as a donor coordinating solvent take a solvent that is not subjected to oxidation, namely acetone, pyridine, dimethylformamide, diethyl ether.

Preferably, the dispersion of betulin in a donor coordinating solvent is carried out under the influence of ultrasound at a frequency of 20-50 kHz for 0.5-5 minutes.

, или хлорпиридиновый хромат

.Preferably, as the chromium compound (VI), along with chromium oxide (CrO ₃ ), either potassium dichromate (K ₂ Cr ₂ O ₇ ), or potassium chromate (K ₂ CrO ₄ ), or pyridine dichromate are used

or chloropyridine chromate

.

Preferably, the liquid phase is diluted with water 5-10 times at room temperature.

A new technical result of the proposed method is to increase the yield of the target product to 90-95% compared to 65% of the prototype, reducing the number of operations, which simplifies the production method and leads to a reduction in the time for its implementation, as well as a reduction in the use of organic solvents and, as a result reducing environmental pollution.

This technical result is due to the fact that before oxidation, betulin is dispersed in a donor coordinating solvent, the oxidation of betulin is carried out at a temperature of 15-35 ° C for 60-180 minutes in the presence of aluminum sulfate (Al ₂ (SO ₄ ) ₃ ) when it is contained in the reaction mixtures of 0.4 · 10 ^-4 -0.4 · 10 ^-2 mol / l, for purification of the obtained betulonic acid, the liquid phase containing betulonic acid is separated from the precipitate and diluted with water until a dispersion is formed, then the resulting dispersion is filtered, the precipitate, representing a betulonovy ki slot, washed with water, after which betulonic acid is reduced with sodium borohydride in isopropyl alcohol to betulinic acid.

The proposed method is as follows.

, или хлорпиридиновый хромат

, окисление бетулина осуществляют при температуре 15-35°С в течение 60-180 мин и по окончании реакции окисления полученную бетулоновую кислоту очищают путем отделения жидкой фазы, содержащей бетулоновую кислоту, от осадка и разбавления ее водой до образования дисперсии, после чего полученную дисперсию фильтруют, осадок, представляющий собой бетулоновую кислоту, промывают водой, после чего бетулоновую кислоту восстанавливают борогидридом натрия в изопропиловом спирте до бетулиновой кислоты.Prior to the betulin oxidation reaction, it is dispersed in a donor coordinating solvent, namely acetone, or pyridine, or dimethylformamide, or diethyl ether, preferably when exposed to ultrasound at a frequency of 20-50 kHz for 0.5-5 minutes, and then to the solution betulin add an aqueous solution of aluminum sulfate Al ₂ (SO ₄ ) ₃ when its content in the reaction mixture is from 0.4 · 10 ^-4 mol · l ^-1 to 0.4 · 10 ^-2 mol / l and a solution of the chromium compound (VI) namely chromium oxide (CrO ₃₎ or potassium dichromate (K ₂ Cr ₂ O ₇₎ or potassium chromate (K ₂ CrO ₄₎ or pi idinovy dichromate

or chloropyridine chromate

the betulin oxidation is carried out at a temperature of 15-35 ° C for 60-180 min and, upon completion of the oxidation reaction, the obtained betulonic acid is purified by separating the liquid phase containing betulonic acid from the precipitate and diluting it with water to form a dispersion, after which the resulting dispersion is filtered , the precipitate, which is betulonic acid, is washed with water, after which betulonic acid is reduced with sodium borohydride in isopropyl alcohol to betulinic acid.

In this case, the yield of the target product - betulinic acid - is 90-95%.

Examples of specific uses of the proposed method.

Example 1

Prior to the betulin oxidation reaction, 1 g of betulin was dispersed in 92 ml of acetone under ultrasound at a frequency of 20 kHz for 5 min, and then an aqueous solution of aluminum sulfate Al ₂ (SO ₄ ) _{3 was} added to the betulin solution, when it was contained in the reaction a mixture of 0.4 · 10 ^-2 mol / l, and a solution of a chromium compound (VI), namely chromium oxide (CrO ₃ ), betulin was oxidized at a temperature of 15 ° C for 180 min, and upon completion of the oxidation reaction, the resulting betulonic acid was purified by separating the liquid phase containing betul acid, from the precipitate and diluting it with water 5 times at room temperature to form a dispersion, after which the resulting dispersion was filtered, and the precipitate, which was betulonic acid, was washed with water and the betulonic acid was reduced with sodium borohydride in isopropyl alcohol to betulinic acid.

At the same time, the yield of the target product, betulinic acid, was 93%. The selectivity, yield and characteristics of the target product - betulinic acid - are presented in the table.

Characteristics of the obtained betulinic acid:

IR spectrometer: 3419 (br.s.) νO-H; 3070 (narrow words) ν = CH; 2941 (compare int.), 2870 (narrow int.) ΝC-H _{as, s} carbon skeleton; 1697 (narrow int.) ΝC = O from COOH; 1641 (narrow cf.) νC = C; 1454 (narrow int.) ΔO-H; 1375 (narrow int.) ΔCH _3sy ; 1043 (narrow int); 1029, νCH2OH; 883 (narrow int.) ΔС = СН ₂ .

¹³ C NMR spectrum:

-CH ₃ : 27.21 (C23); 14.0 (C24); 14.2 (C25); 14.2 (C26); 12.7 (C27); 17.4 (C30);

-CH, -CH ₂ (carbon skeleton): 38.54 (C1); 26.71 (C2); 39.06 (C4); 55.45 (C5); 16.41 (C6); 33.9 (C7); 40.3 42.25 (C8); 49.99 (C9); 38.32 (C10); 18.99 (C11); 24.87 (C12); 37.63 (C13); 42.04 (C14); 25.5 (C15); 28.14 (C16); 54.96 (C17); 46.65 (C18); 48.59 (C19); 150.38 (C20); 30.15 (C21); 31.76 (C22);

-COOH: 177.2 (C28); C = O: 76.8 (C3); = CH ₂ : 109.2 (C29).

PMR spectrum:

0.80 (s.) 24 - CH3; 0.85 (s.) 23 - CH3; 0.98 (s.) 27 - CH3; 1.00 (s.) 25 - CH3; 1.02 (s.) 26 - CH3; 1.02-2.00 complex of CH, CH2; 1.68 (s.) 30 - CH3; 2.28 (m.) 19 - H; 3.00 (2 d.) 3α - H; 4.50 (s.) And 4.62 (s.) 29 - N.

t _PL = 302-303 ° C.

Example 2 was carried out analogously to example 1, while betulin was dispersed in dimethylformamide, under the influence of ultrasound at a frequency of 50 kHz for 0.5 min, and the oxidation was carried out with potassium dichromate (K ₂ Cr ₂ O ₇ ), at a temperature of 35 ° C for 60 min, when the content in the reaction mixture of aluminum sulfate Al ₂ (SO ₄ ) ₃ 0.4 · 10 ^-4 mol / l, and the liquid phase was diluted with water 10 times.

The yield and characteristics of the target product betulinic acid are presented in the table.

Example 3 was carried out analogously to example 1, while betulin was dispersed in pyridine without ultrasound, and the oxidation was carried out with potassium chromate (K ₂ CrO ₄ ) at a temperature of 35 ° C. The yield and characteristics of the target product - betulinic acid - are presented in the table.

), и концентрация сульфата алюминия Al₂(SO₄)₃ в реакционной смеси 0,4·10^-4 моль/л, а жидкую фазу разбавляли водой в 10 раз.Example 4 was carried out analogously to example 1, while betulin was dispersed in diethyl ether without ultrasound, the oxidation was carried out with potassium pyridine dichromate (

), and the concentration of aluminum sulfate Al ₂ (SO ₄ ) ₃ in the reaction mixture was 0.4 · 10 ^-4 mol / L, and the liquid phase was diluted with water 10 times.

The yield and characteristics of the target product betulinic acid are presented in the table.

Data on the selectivity and yield of the target product in examples 1-4 Example No. Characterization of the obtained betulinic acid Selectivity,% Exit, % t _pl , ° C ¹³ C NMR spectrum one 98 93 302-303 76.8 (C3); 150.38 (C20); 177.2 (C28); 109.6 (C29) 2 95 90 299-301 76.8 (C3); 150.3 (C20); 177.2 (C28); 109.6 (C29) 3 97 92 301-302 76.8 (C3); 150.3 (C20); 177.2 (C28); 109.6 (C29) four 98 95 304-306 76.8 (C3); 150.3 (C20); 177.2 (C28); 109.6 (C29)

As can be seen from the obtained results, the proposed method allows to achieve the yield of the target product - betulinic acid - 90-95% with respect to the initial betulin, with the simplicity of its execution and short implementation time, as well as with small amounts of use of organic solvents, and, as a result, the implementation of the proposed method practically does not lead to environmental pollution.

Information sources

1. G.A. Tolstikov, O.B. Flekhter, E.E. Schultz and others Betulin and its derivatives. Chemistry and biological activity. Chemistry for Sustainable Development, 2005, 13, p.1.

2. Le Bang Sean, A.P. Kaplun, A.A. Shpilevsky. Synthesis of betulinic acid from betulin and the study of its solubilization using liposomes. Bioorganic Chemistry, 1998, vol. 24, No. 10, pp. 787-793.

3. The prototype. RF patent No. 2269541, application No. 2004137442/04, dated December 21, 2004, for “A method for producing betulinic acid”.

Claims (5)

1. A method of producing betulinic acid, comprising oxidizing pre-prepared betulin with a chromium compound (VI), in particular chromium oxide (VI), in solution, purification of the obtained betulonic acid and its reduction with sodium borohydride in an organic solvent to betulinic acid, characterized in that before by oxidation, betulin is dispersed in a donor coordinating solvent, the oxidation of betulin is carried out at a temperature of 15-35 ° C for 60-180 min in the presence of aluminum sulfate (Al ₂ (SO ₄ ) ₃ ) when it is contained in the reaction the mixture of 0.4 · 10 ^-4 -0.4 · 10 ^-2 mol / l, for purification of the obtained betulonic acid, the liquid phase containing betulonic acid is separated from the precipitate and diluted with water until a dispersion is formed, then the resulting dispersion is filtered, the precipitate, which is betulonic acid, is washed with water, after which betulonic acid is reduced with sodium borohydride in an organic solvent to betulinic acid. 2. The method according to claim 1, characterized in that as a donor coordinating solvent take a solvent that is not subjected to oxidation, namely: acetone, pyridine, dimethylformamide, diethyl ether. 3. The method according to claim 1, characterized in that the dispersion of betulin in a donor coordinating solvent is carried out under the influence of ultrasound with a frequency of 20-50 kHz for 0.5-5 minutes 4. The method according to claim 1, characterized in that, as the chromium compound (VI), along with chromium oxide (CrO ₃ ), use either potassium dichromate (K ₂ Cr ₂ O ₇ ) or potassium chromate (K ₂ CrO ₄ ), or pyridine dichromate

or chloropyridine chromate

. 5. The method according to claim 1, characterized in that the liquid phase is diluted with water 5-10 times at room temperature.