RU2490273C1 - Method of producing methyl pheophorbide (a) - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to organic chemistry, particularly to chemistry of natural compounds and production of methyl pheophorbide (a), which is meant for synthesis of porphyrins and chlorins for photodynamic therapy purposes. The disclosed method of producing methyl pheophorbide (a) is realised as follows: extracting chlorophyll (a) from Spirulina Platensis microalgae and demetallisation thereof by treating with concentrated acetic acid while exposing the reaction mass to ultrasound at frequency 25-30 kHz for 2.0-2.5 hours at temperature of 45-50°C. The formed pheophytin (a) is then extracted by precipitation with water, after which it is methylated in a medium of tetrahydrofuran in the presence of sulphuric acid at 50-55°C for 1.0-1.5 hours; the end product is extracted by precipitation with water and purified by twofold precipitation from methylene chloride into ethanol.

EFFECT: invention significantly reduces physiological and environmental hazard of the process due to the 55-60-fold reduction of the amount of toxic methanol used, 4-5-fold reduction of the duration of the process and twofold increase in output of the product.

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Description

The invention relates to the field of organic chemistry, in particular to the chemistry of natural compounds, namely the production of methyl-pheophorbide (a), which is intended for the synthesis of porphyrins and chlorins based on it for the purposes of photodynamic therapy.

State of the art

Innovative medical technologies based on nanotechnologies and cellular technologies allow creating devices for recording intermolecular interactions based on optical and nanowire biosensors for the diagnosis and treatment of viral and cancer diseases. For these purposes, the method of photodynamic therapy is widely used [Photodynamic tumor therapy, 2 ^nd and 3 ^rd generation photosensitizers. Ed. JG Moser, Harwood Academic Publishers, 1998, Amsterdam]. This approach is based on the introduction of some special substances into the body, namely dyes (sensitizers), which cause the formation of reactive oxygen species in response to light exposure. Due to their spectral properties, the most effective sensitizers are porphyrins and chlorins, which are obtained from methylpheophorbides. So, arginyl-chlorin e ₆ and lysyl-chlorin p _{6 are} patented as photosensitizers for the photodynamic therapy of cancer [Pat. 5,378,835 (01/1995) USA / Nakazato M. Method of Prodacing Water-soluble Sodium Pheo-phorbide a // CA - 1993. - V.117. - 178303y]. Methylpheophorbide is synthesized by methanolysis of pheophytin, which is a metal-free analogue of chlorophyll. Chlorophyll is isolated from plant materials.

Thus, the development of new effective methods for obtaining individual chlorophyll derivatives from plant materials, in particular high purity methylpheophorbide (a), with a good yield and in large quantities, is an urgent task.

A known method of producing methylpheophorbide (a), including the extraction of chlorophyll derivatives from plant materials, namely from the lipophilic fraction of plants of the genus Serrotula coronate L, methylation of chlorophyll derivatives and chromatographic separation of compounds [Kuchin A.V., Karmanova L.P., Belykh D. AT. A method of producing methyl esters of pheophorbides. RF patent No. 99106221, publ. 03/20/2001].

A known method of producing methylpheophorbide (a) [Fischer N., Stern A. Die chemie des Pyrrols. - Bd.II, 1940. - S.74-75], including stirring the microalgae Spirulina Platensis in methanol in the presence of concentrated sulfuric acid at room temperature in a dark place for 2 days. The reaction mass is filtered on a glass porous filter, washed with a mixture of methanol-diluted sulfuric acid. The filtrate was partially evaporated, diluted with cold distilled water and neutralized with an aqueous solution of potassium hydroxide. The precipitate is filtered on a glass filter through a layer of Celite, washed repeatedly with boiling water, dried and washed with hexane. Raw methylphosphorbide is removed from the sorbent first with acetone, then with a mixture of acetone-chloroform (1: 1), after which the solution is evaporated. The product is rubbed in methanol, filtered on a glass filter, washed with methanol and hexane.

Crude methylphosphorbide is dried and purified by silica gel column chromatography (eluent is methylene chloride ether 95: 5). Then the purified methylphosphorbide (a) is recrystallized from a mixture of methylene chloride-methanol, the crystalline precipitate is filtered off on a glass filter, washed with methanol. 0.06 g of methylpheophorbide (a) is obtained from 20 g of algae.

A known method of producing methylpheophorbide (a) [Porphyrins: structure, properties, synthesis / K.A. Askarov, B.D. Berezin et al. - M .: Nauka, 1985. - 333 p.], Including the extraction of a mixture of pheophytins (a + b) from dried nettle leaves, the isolation of pheophytin (a) and its metabolism. To extract pheophytins (a + b), finely divided nettle dried in the dark nettle leaves are poured with ethanol. After a day, the chlorophyll solution (a + b) is filtered off, diluted hydrochloric acid is added to the filtrate. After 12 hours, the precipitated precipitate of pheophytins (a + b) is filtered off. From 300 g of nettle powder, 2.0-2.2 g of pheophytin are obtained (a + b). To separate pheophytin (a + b), a mixture of pheophytins is dissolved at reflux in chloroform with a solution of Girard T reagent in methanol and acetic acid, the solution is evaporated under vacuum, the solid residue is dissolved in methylene chloride and filtered through glass wool, the filtrate is chromatographed on alumina. The substance obtained after partial evaporation of methylene chloride is reprecipitated from methylene chloride to methanol and pure pheophytin (a) is obtained. Theophytin (a) is methylated with methanol in the presence of dilute sulfuric acid in the dark at room temperature for 15 hours. Then the mixture is diluted with chloroform, washed twice with water, dried, reprecipitated from methylene chloride to methanol. From 20 g of nettle, 0.1 g of methylpheophorbide (a) is obtained.

Closest to the essence of the invention, i.e. the prototype is a method of producing methylpheophorbide (a) [Water-soluble porphin rin derivatives for photodynamic therapy, their use and manufacture. US Patent 6777402, August 17, 2004]. The method is as follows. Mix the plant material, namely Spirulina pla-tensis, or Spirulina maxima, or Chlorella, or Powdered Dry Nettle Leaves, or Frozen Spinach Leaves, in methanol in the presence of concentrated sulfuric acid for 3 hours at room temperature. In this case, chlorophyll (a) is extracted from plant material, demetallization of the resulting chlorophyll (a) with sulfuric acid leads to the formation of pheophytin (a), and methylation of pheophytin (a) with methanol, resulting in the production of methylpheophorbide (a).

In this case, the reaction mixture contains methanol with the desired product methylpheophorbide dissolved in it (a), an excess of methanol, an excess of sulfuric acid, the remains of plant material without chlorophyll, plant material from which chlorophyll is not fully recovered, chlorophyll, which has not been completely demetallized, pheophytin (a), which is not fully methylated.

Then, the target product is isolated from the reaction mass. For this, the reaction mass is diluted with methanol and filtered through Celit, washing the precipitate repeatedly with methanol. The filtrate was extracted twice with hexane, diluted with chloroform, washed with an aqueous solution of potassium chloride, then with water and the solvents were distilled off.

To purify methylpheophorbide (a), the precipitate after solvent stripping is chromatographed on alumina (eluent hexane-chloroform), the solvents are distilled off, the product is recrystallized twice from methylene chloride-methanol.

The disadvantages of this method are:

1. Extremely high physiological and environmental hazards of the process due to the use of large amounts of methanol in the synthesis. So, to obtain 0.1 g of pure methylpheophorbide (a), destruction of the cell walls of plant materials, extraction of the contents of the cells, washing, chromatography, reprecipitation, recrystallization requires 1500-1700 ml of methanol. Methanol is a strong nervous and vascular poison. The special toxicity of methyl alcohol is usually associated with the formation of formaldehyde and formic acid from it in the body. Methanol poisoning is possible even by inhalation of its vapor, in contact with skin. The maximum permissible concentration of methanol in the air is 5 mg / m ³ . Methyl alcohol is especially dangerous when it enters the digestive system, as 30 ml can be fatal. All work with the use of methanol should be carried out using personal protective equipment, in a fume hood in the presence of well-functioning ventilation. Since in the synthesis of methylpheophorbide (a), in addition to methylation itself, methanol is used for washing, reprecipitation, recrystallization, etc., therefore, a large amount of methanol waste accumulates in the synthesis process and there is a problem of their disposal.

2. Significant duration and complexity of the process. The processes of isolation of chlorophyll (a) from plant materials, its demetallization into pheophytin (a) and methylation of pheophytin (a) proceed in one reaction mass for 3 hours. In this case, the reaction mixture contains methanol with the desired product methylpheophorbide dissolved in it (a), an excess of methanol, an excess of sulfuric acid, the remains of plant material without chlorophyll, plant material from which chlorophyll is not fully recovered, chlorophyll, which has not been completely demetallized, pheophytin (a), which is not fully methylated. This leads to the fact that the stages of separation (washing with methanol, extraction with hexane, washing with an aqueous solution of potassium chloride and then with water, distilling off the solvents) and purification (chromatography on a column of aluminum oxide, distillation of solvents, double recrystallization) take a lot of time (7- 8 days) and are rather laborious and require the installation of a plant for distillation of solvents, preparation of chromatographic columns, replacement of alumina in columns after chromatography of each portion of the product.

3. Low yield of the target product. 1.75 g of methylpheophorbide (a) are obtained from 1 kg of plant material.

SUMMARY OF THE INVENTION

The inventive task was to find a method for producing methylpheophorbide (a) from Spirulina Platensis microalgae by extracting chlorophyll (a) from it, demetallizing it to obtain pheophytin (a), methylating the latter with methanol in the presence of sulfuric acid, and subsequent isolation and purification of the target product, which allowed would significantly reduce the physiological and environmental hazards of the process, reduce its duration and complexity, and increase the yield of the target product.

This goal is achieved by the method of producing methylpheophorbide (a) from the microalga Spirulina Platensis by extracting chlorophyll (a) from it, demetallizing it to obtain pheophytin (a), methylating the latter with methanol in the presence of sulfuric acid and subsequent isolation and purification of the target product, characterized in that the selection of chlorophyll (a) and its demetallization is carried out in concentrated acetic acid when exposed to the reaction mass of ultrasound with a frequency of 25-30 kHz for 2.0-2.5 hours at a temperature of 45-50 ° C, then pheophytin ( ) is isolated by precipitation with water, after which it is subjected to methylation in tetrahydrofuran in the presence of sulfuric acid at 50-55 ° С for 1.0-1.5 hours, the target product is isolated by precipitation with water, and its purification is carried out by two reprecipitation from methylene chloride in ethanol.

The invention allows to obtain the following advantages:

- significantly reduce the physiological and environmental hazards of the process, as the amount of poisonous methanol used is reduced by a factor of 55-60, since it is used only for methylation of pheophytin (a);

- significantly (4-5 times) reduce the duration of the process;

- 2 times increase the yield of the product.

Information confirming the reproducibility of the invention

To implement the method using the following substances:

1. Microalga Spirulina Platensis

2. Acetic acid GOST 19814-74

3. Methanol GOST 2222-95

4. Tetrahydrofuran GOST 8313-88

5. Methylene chloride GOST 8728-88

6. Sulfuric acid GOST 2184-77

7. Ethanol GOST 5964-93

The implementation of the method is illustrated by the following example.

Example 1

Extraction of chlorophyll from plant materials and its demetallization into pheophytin (a). 1000 g of microalgae Spirulina Platensis is poured into 900 ml of 80-85% acetic acid and heated for 2.0-2.5 hours at 45-50 ° C when exposed to ultrasound at a frequency of 25-30 kHz. Under these conditions, acetic acid, fully extracting chlorophyll, simultaneously turns it into pheophytin (a). The reaction mixture is filtered, the filtrate is poured into water, the precipitate of pheophytin (a) is filtered off. From 1000 g of microalgae powder, 5 g of pheophytin (a) are obtained.

Obtaining methylpheophorbide (a). 5 g of pheophytin (a) and 45-50 ml of methanol containing 4-5% sulfuric acid to acidify the medium are heated with stirring in 90-100 ml of tetrahydrofuran at 50-55 ° C for 1.0-1.5 hours.

Isolation of methylpheophorbide (a). A mixture of bluish-green color is poured into 1.0-1.2 l of ice water, the precipitate is filtered off on a glass filter and dried.

Purification of methylpheophorbide (a). The product is purified by double reprecipitation from methylene chloride to ethanol. Obtain 3 g of pure methylpheophorbide (a). NMR ¹ H-spectrum: 9.41, 9.23, 8.56 (3H, all s, meso-H); 7.88 (1H, q, -CH.dbd.CH ₂ ), 6.25-6.10 (2H, -CH.dbd.CH ₂ ), 6.28 (1H, s, cyclopentanone-H), 4.50, 4.25 (2H, m, 7 -H, 8-H); 3.55 (2H, q, 4-CH ₂ CH ₃ ); 3.93, 3.70, 3.63, 3.39, 3.15 (15H, all s, 5.upsilon. -CH ₃ ); 2.75-2.20 (4H, m, -CH ₂ CH ₂ COOCH ₃ ); 1.85 (3H, d, 8-CH ₃ ); 1.71 (3H, m, 4-CH ₂ CH ₃ ); 0.55 and -1.68 ppm (2H, 2 broad s, 2.upsilon. -NH-).

The yields of the target product obtained by the claimed method for various parameters of its implementation are shown in the table.

Table The yield of the target product No. p / p Options Yield, g The frequency of ultrasound, kHz Ultrasound processing time, hour Temper. sonication, ° C Methylation time, hour Temper. methylation, ° С one 25 2.1 48 1.0 fifty 3.1 2 27 2.0 45 1.3 52 3.3 3 thirty 2.5 fifty 1.5 55 3.5

Claims (1)

A method of producing methylpheophorbide (a) from Spirulina Platensis microalgae by extracting chlorophyll (a) from it, demetallizing it to obtain pheophytin (a), methylating the latter with methanol in the presence of sulfuric acid and subsequent isolation and purification of the target product, characterized in that the isolation of chlorophyll ( a) and its demetallization is carried out in concentrated acetic acid under the influence of ultrasound on the reaction mass with a frequency of 25-30 kHz for 2.0-2.5 hours at a temperature of 45-50 ° C, then pheophytin (a) is isolated by precipitation with water, after cheg it is subjected to methylation in tetrahydrofuran medium in the presence of sulfuric acid at 50-55 ° C for 1.0-1.5 hours, the target product is isolated by precipitation with water, and its purification is carried out by two reprecipitation from methylene chloride to ethanol.