The method of producing chlorin e6

 

(57) Abstract:

The invention relates to chemistry and medicine. The extraction is carried out chlorophyll and spirulina biomass with subsequent treatment of the extract with acid to obtain pheophytin and. Carry out alkaline hydrolysis and his selection of the target product. Moreover, when preparing pheophytin and to hydrolysis spend it cleaning by reconstitution of pheophytin and in acetone. Chlorin e6isolated from the reaction mixture after the hydrolysis, washed with acidified distilled water. The invention improves the yield and purity of the product. 1 C.p. f-crystals.

The present invention relates to organic chemistry and medicine, in particular to an improved method of producing chlorine 6(18-carboxy-20-(carboxymethyl)-8-ethynyl-13-ethyl-2,3-dihydro-3,7-12,17 tetramethyl-N, N porphin-2-propionic acid) or its salts.

Chlorin e6it belongs to the class of plant porphyrins obtained on the basis of chlorophyll a. It is known that vegetable porphyrins, including chlorophyll and its derivatives are pharmacologically active compounds and can be used in medical practice, including as photosensitizers for (PDT)[1]. In the lia.

In particular, the known method of producing chlorin e6from nettle leaves [2]. For this nettle leaves pour ethanol. The result: an extract containing a mixture of chlorophylls a and b. The extract is treated with acid, resulting from each form of chlorophyll contained in the extract, is formed corresponding to the shape of pheophytins a and b. Subsequent alkaline saponification of different forms of pheophytin leads to the formation in the reaction mass at least two products: chlorine e6and the birthplace of the g7. Along with these major products in the reaction mixture present their esters, as well as vitolinya remains.

The disadvantage of the described method of producing chlorin e6is the complexity of the technological process, a small yield and low purity of the target product (from 1 kg of dried nettle leaves receive 1 g of chlorin e6the purity of which does not exceed 60%).

The prototype is a method of producing chlorine her, described in the patent [3]. As a source of raw materials use biomass of cyanobacteria, for example the genus Spirulina grown in the light in controlled conditions. The biomass of cyanobacteria containing only one form of chlorophyll - chlorophyll a, process water spirtovodochnoj or hydrochloric acid, formed pheophytin is filtered off and washed with a nonpolar organic solvent. Then pheophytin dissolved in alcohol, the solution add CON and spend alkaline hydrolysis when heated in the absence of oxygen. The formation of the alkali salt of chlorin. After cooling, the reaction mixture chlorin e6precipitated with hydrochloric acid followed by filtration and washing. The yield of the target product is 3.5 g with a purity of 90%.

In the described prototype has significant drawbacks.

In the method of producing chlorin e6in particular in the process of hydrolysis, as well as at the final stage of selection of chlorin e6from the reaction mixture there are conditions conducive to the formation of unwanted impurities, which reduce the yield and purity of the final product.

The task of the invention was to improve the method of producing chlorin e6order to enhance the yield and purity of the final product.

This object is achieved in that at the stage of dissolution of pheophytin prior to the process of synthesis of chlorin, and also in the final stages of obtaining chlorin e6apply additional operaon the possibility of increasing the purity of the solution pheophytin, used in the synthesis of chlorin6. In the synthesis reaction of chlorine use alkaline hydrolysis of pheophytin dissolved in alcohol. As a result of low solubility of pheophytin in alcohol may be present undissolved particles pheophytin, which reduces the yield of the target product and the contamination of its impurities.

To address this deficiency has been developed for the operation of pre-dissolution teofilina in acetone, cleaned from impurities, formed, for example, during storage, with subsequent reconstitution it in acetone.

At the final stage of obtaining chlorin e6there was an additional operation of the washing of the final product, which also helped improve its purity.

It is established that the chlorin e6isolated from the reaction mixture contains impurities that reduce the degree of its purity. The removal of these impurities were removed by washing chlorin e6on the filter, acidified distilled water. Thus, by combining the cleansing of pheophytin before hydrolysis and leaching of chlorine after completion of the synthesis reaction in this way obtained konechno the second spirulina. In the method prototype, the final product is 3.5 g with the same degree of purity, i.e., increase the yield of the final product compared to the prototype is more than 40%.

Further objectives and advantages of the invention will become apparent from the additional detailed description of the method of producing chlorin e6in specific examples of implementation of this method.

Example 1.

1 kg of biomass of cyanobacteria pour 1.5 liters of alkaline ethanol solution, which is obtained by dissolving 40 ml of 1N. NaOH in 1 l of distilled water and subsequent mixing with ethanol in the ratio 1:2 respectively.

The biomass was washed with this solution for 3-5 minutes, filtered, and then extracted with ethanol for the extraction of chlorophyll a. In the extract obtained was added 1.2 ml of concentrated model HC1 per liter of extract. The resulting precipitate pheophytin and is filtered and washed on the filter with hexane. The output of pheophytin and is 10 g, the purity of 94.5%.

Received pheophytin pererastayut in acetone. In the reaction vessel with ethanol add solution teofilina in acetone. The reaction mixture is heated nitrogen purging in the course is on the nitrogen. After that the reaction mixture is cooled and neutralized with concentrated hydrochloric acid to the sludge of chlorin e6. Chlorine allocate on a filter, washed with acidified water with a pH of 1.8 and air-dried in the dark. Yield 5.0 g, purity of 92.5%.

Example 2.

Pheophytin and receive, as in example 1. The acetone solution pheophytin without purification by filtration is mixed with ethanol. Further operations are performed as described in example 1. Output and chlorine 4.8 g of 1 kg of dry biomass of spirulina at a purity of 90%.

Implementation of the proposed method in accordance with example 2 is reasonable only in the case of freshly prepared pheophytin and with a high degree of purity.

Example 3. The method in accordance with example 1 with the washing of chlorin e6on the filter with distilled water with a pH of 4.5. The output of chlorin 5.0 g, 1 kg of dry biomass of spirulina with the purity of 92.5%.

Sources of information

1. Sukhin, M., Meshcheryakov, A. L. et al. the Results of biological testing of a new type of photosensitizer on the basis of chlorine in the diagnosis and therapy of malignant tumors. International medical reviews. 1996, № 4, № 5.

2. Auth.mon. No. 4734016/04 the USSR who ICCA O. N. and others).

1. The method of producing chlorin e6(18-carboxy-20-(carboxymethyl)-8-ethinyl-13-ethyl-2,3-dihydro-3,7-12,17-tetramethyl-N, N porphin-2-propionic acid), including extraction of chlorophyll a from spirulina biomass with subsequent treatment of the acid extract with getting pheophytin and alkaline hydrolysis and his selection of the target product in the form of chlorin e6, characterized in that in the preparation of pheophytin and to hydrolysis spend it cleaning by reconstitution of pheophytin and in acetone, and the chlorin e6isolated from the reaction mixture after the hydrolysis, washed with acidified water.

2. The method according to p. 1, characterized in that the chlorin e6isolated from the reaction mixture, washed with distilled water with a pH of 1.8 to 4.5.

 

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