RU2330037C1 - METHOD OF CHLORINE e6 PRODUCTION - Google Patents

Abstract

FIELD: medicine; pharmacology.

SUBSTANCE: invention refers to pharmacology, namely to production of biologically active compounds, specifically to improved method of chlorine e6 production applied for production of photosensitisers for photodynamic cancer therapy. Method is performed by processing sulphuric acid concentrated spirulina methanolic suspensions followed by filtration, dilution with water, alkali neutralisation by, filtration of settled deposition through celite layer followed with washing in hot water and petroleum-ether, elution with acetone celite, silica gel based chromatography, recrystallisation of mixed chloride methylene- methanol, release of produced alkylpheophorbide and its dilution with acetone, alkali processing, hydrochloric acid neutralisation, a filtration through celite layer, washing in water, aqueous ammonia, lyophilisation, dilution with aqueous alkali, filtration through sephadex layer, neutralisation, centrifugation and lyophilisation.

EFFECT: production of pure product with reagents taken in small amounts.

1 cl, 6 ex

Description

The invention relates to pharmacology, in particular to the production of biologically active compounds, specifically to an improved method for producing chlorin e6, which can be used as an initial intermediate for the production of numerous photosensitizers for the photodynamic therapy of cancer and other neoplasms, a fluorescent diagnostic agent, and also pharmacological preparations with wide spectrum of action.

Several methods are known for producing mainly water-soluble salts of chlorin e6, in which chlorine e6 itself is formed as a result of successive chemical procedures and is used as an intermediate intermediate without special isolation and characteristics of its physicochemical and spectral properties, which greatly complicates the preparation of standardized drugs for it basis.

Thus, for example, a method is known for producing trisodium and tripotassium salts of chlorin e6, which consists in treating the biomass of cyanobacteria of the genus Spirulina with an aqueous-alcoholic solution of alkali, followed by the isolation of numerous chlorine derivatives from the extract, their combined alkaline hydrolysis in vacuum, neutralization, and the precipitation of precipitates containing Along with numerous chlorine derivatives, chlorin e6 is also obtained, from which, when NaOH or KOH are added, a water-soluble product is obtained (1). In this method, the composition and mutual content of water-soluble chlorins is not defined, which makes it difficult to obtain from it a medicinal product for medical use. The main and fundamental disadvantage of this method of obtaining water-soluble salts of chlorin e6 is the fact that due to the use of spirulina biomass as a feedstock, the resulting product cannot have a constant composition.

A known method for producing water-soluble chlorins (2), according to which chlorins containing three carboxylate residues (hydroxycarbonylethyl, hydroxycarbonylmethyl and nuclear carboxyl groups) form water-soluble salts with a linear or cyclic primary or secondary amine or with a basic amino acid. As an example, we used chlorin released by special processing of spirulina with a yield of about 50%. The disadvantages of this method include the fact that to obtain aqueous solutions of chlorins, a product was used containing a mixture of various chlorins, in which the content of chlorin e6 varied widely and amounted to 80-90%. The relatively low content of e6 chlorin is due to fundamental difficulties in the isolation of this substance from the spirulina biomass using the above technology.

Another known method for the preparation of water-soluble salts of chlorin e6 is the hydrolysis of the corresponding trimethyl ether in tetrahydrofuran in the presence of NaOH for 2 days at room temperature (3).

The main disadvantage of this method is the preliminary preparation of the corresponding trimethyl ether, the synthesis of which is a rather complicated synthetic task. In the process of alkaline saponification of three ester residues, there is the possibility of the presence in the reaction mixture of numerous products of incomplete saponification, i.e. mono- and dimethyl ethers, and for the complete hydrolysis of ester residues, a long time is necessary, which negatively affects the stability of chlorin e6 in alkaline media.

There is also known a method for producing a mixture of alkaline salts (sodium or potassium) of chlorins of variable composition, consisting of salts of chlorin e6, purpurin 5 and chlorin p6, according to which the biomass of spirulina is treated with acetone to extract chlorophyll a, followed by treatment of the acetone extract with hydrochloric acid to convert chlorophyll a into pheophytin a, its isolation by centrifugation after neutralization of the solution, hydrolysis of the ester residue in the hydrochloric acid - acetone-hexane system, isolation by filtration of the "raw" pheoforb ida, by its partial purification by reprecipitation from an acetone-water mixture, drying in air, initial alkaline hydrolysis in the acetone - 1% aqueous alkali system, repeated hydrolysis by adding a large amount of alkali when heated to 40-60 ° C for an indefinite time interval (20-90 minutes), neutralization of the product with hydrochloric acid, its separation by centrifugation, reprecipitation from acetone to separate from the degradation products and drying in a wide temperature range (40-100 ° С) from 1 hour to 30 days to (4). As a result of such a complex and ambiguous processing of spirulina microalgae, a product of indeterminate composition is obtained, the physicochemical and photophysical characteristics of which, as well as the biological properties, will depend on many factors. The use of such a non-standardized product as a pharmacological agent is not possible.

A known method of producing chlorin e6 (18-carboxy-20- (carboxymethyl) -8-ethenyl-13-ethyl2b3-dihydro-3,7,12,17-tetramethyl-21H, 23H-porphin-2-propionic acid) by extraction of chlorophyll and from spirulina biomass with an aqueous-alcoholic alkali solution, followed by neutralization with acid and obtaining a precipitate of pheophytin a, its purification by re-dissolution in acetone, alkaline hydrolysis of purified pheophytin a, followed by neutralization with acid to form a chlorin e6 precipitate (5). In this method, the composition and mutual content of chlorins is not determined, since pheophytin a precipitates together with hydrophobic lipids and carotenoids. In addition, at the last stage of the hydrolysis of pheophytin a to chlorin e6, phytol is cleaved. As a result, the final product may be contaminated with phytol impurities and chlorine e6-related compounds. In addition, the content of the main components - tetrapyrroles, in particular chlorophyll a, is not standardized in the raw spirulina biomass used; phycobiliproteins and phycobilicyanins; lipids and proteins, which also leads to a different content of impurities in the final product.

Closest to the described method, the technical essence is the method of producing chlorin e6, followed by conversion to its bis-N-methylglucamine salt by treating lyophilized spirulina in methyl alcohol in the presence of sulfuric acid, followed by filtration, evaporation of methanol, addition of hexane to the oily residue, separation hexane layer, adding methylene chloride to the lower layer, diluting the mixture with cold water, separating the organic layer, washing with water, evaporation, chromatographs by columnization with silica gel in the chloroform-ether system, evaporation of the fraction containing methylpheophorbide a, crystallization of methylpheophorbide a from a mixture of chloroform-methanol. With vigorous stirring in an inert gas stream, add an NaOH solution to the acetone solution of the isolated methylpheophorbide, and heat it up to 50 ° С, add an aqueous NaOH solution, keep it at 50-60 ° С, cool it, neutralize to pH 4.5-5.0. The precipitate obtained after centrifugation containing chlorin e6 is washed with water, suspended in pyrogen-free water, centrifuged 2-3 times and transferred to the N-methyl-D-glucamine salt (6). The method has a number of significant disadvantages that do not allow it to be carried out on an industrial scale. So, for example, obtaining methylfeoforbide a in amounts exceeding several grams is impossible, since a 2- or 3-fold extraction with hexane from a large volume (several liters) of a viscous filtrate containing methanol, sulfuric acid and numerous extractive products from spirulina is extremely difficult for the following reasons:

a) due to the difficulty of finding the interface between the two phases, since they are almost black in color and low transparency;

b) for complete extraction with hexane, intensive shaking is required, which is possible only when using dividing funnels of a small volume (not more than 0.5-1 l);

c) intensive shaking leads to a significant increase in the time of separation of the two phases (1-2 hours);

d) after separation of the hexane layer, the lower layer after adding methylene chloride and water and subsequent shaking is very difficult to delaminate due to the fact that protein products and other surface-active substances pass into the aqueous layer;

e) to completely separate the organic layer in methylene chloride from methanol and sulfuric acid, repeated washing with water is required, which significantly increases the time of the washing operation;

f) before evaporation to a minimum volume of the separated organic layer, it is necessary to dry it from water, since the presence of water is unacceptable during chromatographic purification on silica gel.

Chlorin e6 with the accompanying compounds is transferred into the corresponding N-methylglucamine salt in the form of a solution, which is used as a photosensitizer. The main disadvantage of this method is the preparation of exclusively aqueous solutions of the N-methylglucamine salt of chlorin e6, which does not allow the maximum use of the wide possibilities of using chlorin e6 for medical purposes, due to the insufficient stability of aqueous solutions during storage. In all of the above methods, as a result of sequential chemical operations, chlorin e6 is obtained only in the form of an aqueous solution. Chlorin e6 was not isolated in dry form and was characterized by physicochemical methods; its spectral properties were not described. At the same time, the results of special studies indicate that individual chlorin e6 has the maximum photosensitizing effect (7).

The aim of this invention is to develop a method for producing chlorine e6 of high purity, on the basis of which it is possible to obtain highly active dosage forms. To obtain highly purified, constant in composition and storage stable chlorin e6, a method for producing chlorin e6 is proposed based on a standardized starting material methylpheophorbide a, which is obtained from spirulina biomass using an improved technology that allows the production of methylpheophorbide a in large quantities and using fewer organic solvents .

In accordance with the invention, a method for producing chlorin e6 is described, characterized in that a suspension of lyophilized spirulina in methyl or absolute ethanol is treated with concentrated sulfuric acid in an amount of 5-10% of the solution volume for 10-12 hours with stirring, followed by filtration, the solution is diluted with water, neutralized with aqueous alkali to pH 4.5-5.0, the precipitated alkylfeoforbide precipitate is separated by filtration through a layer of celite, followed by washing with hot water and a light petroleum ether, the product was eluted with celite acetone, the solution was evaporated, methylene chloride-ether chromatographed on silica gel with a methylene chloride system, recrystallized from a mixture of methylene chloride-methanol and alkylpheophorbide and separated by filtration, dissolved in acetone with vigorous stirring in an inert gas stream, added an aqueous alkali solution and heated at 40-60 ° C for 1.5 hours, the solution is cooled, acidified with hydrochloric acid to pH 4.0-5.0, the precipitate formed is filtered off through a celite pad, washed with distilled water, the product is washed off with celite with an aqueous solution of ammonia, the solution is lyophilized, the residue is dissolved in an aqueous alkali solution, followed by filtration through a Sephadex G-25 layer, the fraction containing chlorin e6 is acidified with hydrochloric acid to pH 4.0-5.0, the chlorine e6 precipitate is separated by centrifugation, suspended in water and lyophilized.

The process scales easily without changing the processing time of the spirulina. Neutralization of the filtrate after separation of spirulina and dilution with cold water proceeds without a noticeable exothermic effect; precipitation occurs quickly, within a few minutes, and for its "ripening" to improve filtration through celite takes no more than 1-2 hours. The use of hot water for washing the precipitate filtered on celite allows obtaining a free-flowing product at the end of this operation, convenient for washing it with petroleum ether and practically completely separating it from lipids, phytol, and other products that impede chromatographic purification. Methylpheophorbide a is easily extracted from celite so washed by washing with acetone.

The invention is illustrated by the following examples.

Example 1. To a suspension of 200 g of lyophilized spirulina in 600 ml of methanol, 30 ml of concentrated sulfuric acid are poured with vigorous stirring, stirred for 10-12 hours, the resulting blue-green product consisting of spirulina, filtered through a No. 2 glass filter, diluted with 600 ml water, neutralized with an aqueous solution of alkali to a pH of 4.5-5.0, the precipitated loose precipitate is filtered off through a layer of celite 545, the celite is washed with hot water, then with light petroleum ether, the product is eluted with cellite with acetone, the solution is evaporated to a minimum of the volume, the residue is chromatographed on a column of silica gel G-25 in the system methylene chloride - ether (95: 5), the main fraction was evaporated and recrystallized from methylene chloride-methanol. 0.155 g of methylpheophorbide a is obtained.

UV spectrum in chloroform, λ _max nm (ε. 10 ^-3 ): UV-VIS λ _max (CHCl ₃ ) 668 nm (e 45.3), 610 (13.1), 536 (14.0), 506 (15.8), 412 (104.8). (yield on chl. about 1-1.5%).

Example 2. Similarly, from 200 g of freeze-dried spirulina in 600 ml of absolute ethanol, 0.120 g of ethylfeoforbide a is obtained.

PMR (400 MHz, CDCl ₃ ): 9.47 (1H, s, 10-H); 9.32 (1H, s, 5-H); 8.56 (1H, s, 20-H); 7.95 (1H, m, 3 ¹ -H); 6.25 (1H, dd, 3 ² -H); 6.17 (1H, dd, 3 ² -H); 3.67 (1H, q, 8 ¹ -H); 3.68 (3H, s, 12 ¹ ); 3.39 (3H, s, 2 ¹ ); 3.21 (3H, s, 7 ¹ ); 1.80 (d, H-18 ¹ ); 1.68 (t, H-8 ² ); 4.47 (q, H-18); 4.20 (dd, H-17); 2.62 (dt, H _a -17 ¹ ); 2.31 (dt, H _a -17 ¹ ); 2.50 (dt, H _b -17 ² ); 2.24 (t, H _{b ′} -17 ² ); 0.53 (1H broad s, NH); -1.65 (1H, s, NH).

Example 3. To a solution of 1 g of methylpheophorbide a in 100 ml of acetone with vigorous stirring in a stream of inert gas add 120 ml of a 10% aqueous solution of caustic potassium at a temperature of 50 ° C for 1.5 hours, controlling the formation of chlorin e6 by TLC (on Selica gel Merck (0.04-0.063 mm), in the system methylene chloride chloride 9: 1). The solution is cooled to 10-15 ° C, acidified with dilute hydrochloric acid (1: 3) to pH 4.0-5.0, the precipitated volumetric precipitate is filtered through a layer of celite 545, the celite is washed with distilled water until there are no chlorine and acetone ions in the mother liquor; chlorin e6 is washed off with 100 ml of water containing 1 ml of concentrated aqueous ammonia, the solution is lyophilized and 0.85 g (87%) of “raw” chlorin e6 is obtained in the form of a dark green air powder. The resulting chlorin e6 was dissolved in 20 ml of a 1% NaOH solution, purified on a Sephadex G-25 column, eluting with a 1% alkali solution, the main fraction was separated, acidified with diluted hydrochloric acid to pH 4.0-5.5, the precipitate was centrifuged, washed with distilled water to the absence of chlorine ions in the filtrate, the precipitate is suspended in 50 ml of distilled water and lyophilized. Obtain 0.76 g (77%) of chlorin e6 (C ₃₄ H ₃₆ N ₄ O ₆ m.m. 596.68) in the form of a dark green air powder with a content of the main substance of at least 98% (according to HPLC).

Absorption spectrum in water at pH 9.0 λ _max nm (ε 10 ^-3 ): 654 (36.750).

PMR spectrum for chlorin e6 trimethyl ether obtained by treating chlorin e6 with an ether solution of diazomethane

PMR 400 MHz, (CDCl ₃ ): 9.68, 9.55. 8.72 (1H, 1H, 1H, meso-H, all s); 8.05, 6.34, 6.13 (m, d, d, 1H, 1H, 1H, CH = CH ₂ ); 5.34 and 5.21 (1H, 1H, two d, CH ₂ COOCH ₃ ); 4.43-4.38 (m, 2H, 7.8-H); 4.24, 3.75, 3.62, 3.56, 3.46, 3.28 (all s, 3 × CH ₃ macrocycles and 3 × COOCH ₃ ); 3.78 q and 1.70 t, (5H, CH ₂ CH ₃ ); 2.53 and 2.18 (two m, 4H, CH ₂ CH ₂ COOCH ₃ ); 1.73 d (8-CH ₃ ); -1.31 and -1.48 (two broad s, 2NH).

Example 4. To a solution of 4 g of methylpheophorbide a in 750 ml of acetone with vigorous stirring in a stream of inert gas at a temperature of 40-50 ° C, add 100 ml of 10% NaOH, mix for 15 minutes, add another 350 ml of 10% NaOH, stirred for 45 minutes at 60 ° C, controlling the conversion of methylpheophorbide a to chlorin e6 by TLC. After completion of the process, the solution is cooled to 15-20 ° C, acidified by cooling with dilute hydrochloric acid (1: 3) to a pH of 4.0-5.0. The precipitate formed is separated by centrifugation, washed twice with distilled water, then suspended in distilled water and lyophilized. Obtain 3.4 g, (86%) of "raw" chlorin e6 in the form of a dark green air powder. Yield at least 95% of the main substance (according to HPLC).

Example 5. From 4 g of methylpheophorbide a and after alkaline treatment and acidification (example 4), the precipitated precipitate of chlorin e6 is filtered through a layer of celite 545, celite is washed first with distilled water to remove inorganic salts and acetone, then chlorine e6 adsorbed on celite is washed off with 200 ml of distilled water to which 3 ml of concentrated aqueous ammonia is added, chromatography on Sephadex G-25, eluting with a 1% alkali solution, the main fraction is collected, acidified with diluted formic acid to pH 4.5-5.5, lyophilized and half chayut 3.04 g (77%) chlorin e6 with the content of the basic substance is not less than 98%.

Received chlorin e6 in the form of a light air powder of dark green color is stored in tightly closed bottles, protecting from moisture, at a temperature of -15 ° C.

Example 6. Similarly receive chlorin e6 from ethylfeoforbida a.

BIBLIOGRAPHY

1. RU 2054476, C1, 02.20.1996.

2. RU 2144538, C1, 01.20.2000.

3. US 5002962, A, 03/26/1991.

4. RU 2183956, C1, 06/27/2002.

5. RU 2228750, C1, 05.20.2004.

6. RU 2276976, C2, 02/10/2006.

7. Yu.A. Bely, A.V. Tereshchenko, P.L. Volodin, M.A. Kaplan, G.V. Ponomarev. A comparative study of the photodynamic effects of chlorin-type photosensitizers on the intact retina of experimental animals. Refractive surgery and ophthalmology, 2006, 6, (No. 2), 55-59.

Claims (1)

The method of producing chlorin e6 formula 1 from spirulina, characterized

the fact that a suspension of freeze-dried spirulina in methyl or absolute ethanol is treated with concentrated sulfuric acid in an amount of 5-10% of the solution volume for 10-12 hours with stirring, followed by filtration, the resulting solution is diluted with water, neutralized with aqueous alkali to pH 4, 5-5.0, the precipitated alkylfeoforbide precipitate was separated by filtration through a celite pad followed by washing with hot water and light petroleum ether, the product was eluted with celite acetone, the solution was evaporated, the residue was chromatographed on si methylene chloride chloride system, the main fraction is evaporated, the residue is recrystallized from methylene chloride-methanol mixture, the alkylpheophorbide is filtered off, dissolved in acetone, the aqueous alkali solution is added and heated at 40-60 ° C, the solution is cooled, acidified with hydrochloric acid to pH 4 , 0-5.0, the precipitated precipitate is separated by filtration through a celite pad, washed with distilled water, the product is washed with celite with an aqueous ammonia solution, the solution is lyophilized, the residue is dissolved in an aqueous alkali solution, followed by a filter through a Sephadex layer, the fraction containing chlorin e6 is acidified to pH 4.0-5.5, the precipitate of chlorin e6 is separated by centrifugation, suspended in water and lyophilized.