RU2416614C2 - Photosensitiser and preparation method thereof - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: method of preparing photosensitiser involves treatment of a suspension of lyophilically dried spirulina, for example, in methanol or a absolute alcohol until alkyl pheophorbide is obtained and with reaction of the latter with ethylenediamine for example, preferably in an organic solvent, extraction of the corresponding ether of a monoamide of chlorine e 6, its dissolution in alcohol and adding, for example, adipic acid in molar ratio 2:1. Apyrogenic water is added to the photosensitiser of structural formula

(1), such that content of chlorine salts is equal to 30-40 mg per 10 ml of the alcohol-water mixture (1:1).

EFFECT: replacing the negative charge on the periphery of the chlorine macrocycle with a positive charge by introducing into the peripheral substitute amine groups to obtain cation-charged chlorines in order to change distribution of the photosensitiser in the body in order to increase affinity to tumours, possibility of controlling distribution owing to combination of different alkyls and different organic acids with different basicity in the structural formula through a novel drug formulation, having good therapeutic properties for treating cancer and other diseases through photodynamic therapy methods for cancer and other neoplastic diseases of different origins compared to existing medicinal agents containing chlorine e 6.

5 cl, 6 ex, 9 dwg

Description

The invention relates to pharmacology, in particular to the production of biologically active compounds of the chlorin series (see review: ESNyman, PHHynninen, J. Photochem. Photobiol. B: Biology, Vol.73, pp. 1-28 (2004). Reserch advances in the of tetrapyrrolic photosensitizers for photodynamic therapy.). The invention can be used to obtain salts of chlorin e6 monoamide esters of general formula (1), which can be used as highly effective photosensitizers (hereinafter FS) for the photodynamic therapy of cancer and other neoplasms of various genesis, as well as fluorescence diagnostics of cancer cells.

For example, derivatives of chlorin e6 monoamide of general formula (1) can be obtained as adipates of chlorin e6 monoamide ester adipates (Fig. 1 — a particular case of general formula (1) for a dibasic adipic acid salt is shown).

Fig 1.

I, R = Me; Et

Thus, salts of monobasic acids, for example, acetic or pyruvic, dibasic, for example adipic or oxalic, or tribasic, for example citric or horsetail, can be obtained. These compounds can be used in the same cases in which known photosensitizers are used, drugs approved for use in Russia, the basis of which are chlorine e6 salts (Fig. 2),

Fig. 2.

C ₃₄ H ₃₆ N ₄ O ₆

MW 596.68

or NPE6 (aspartylchlorin e6) (Fig. 3),

Fig. 3.

C ₃₈ H ₄₁ N ₅ O ₉

MW 711.77

since they are all derivatives of the same macrocycle - chlorin e6 and therefore have similar spectral characteristics (the main absorption band at 662 nm in alcohol and other organic media and fluorescence spectra), and in other cases when it is necessary to fundamentally change the biodistribution of the drug in organism, since the FS offered by us belong to cationically charged chlorins. All the known and officially approved for use FS in the Russian Federation (Fig. 2) belong to the class of water-soluble salts (potassium, sodium or N-methylglucamine) of chlorin e6, which is a tricacid and, therefore, refers to anionically charged PS (see RU 2144538, RU 2183956, RU 2276976, RU 2276976, US 6969765, US 5330741, WO 2004/089409 A3).

Mono-L-Aspartylchlorin e6 (NPE6, TALAPORFIN or LASERPHYRIN) (see Fig. 3), which is an amide derivative of chlorin e6, is a tetra-acid, and its sodium salts are also perfectly soluble in water (see: Roberts, WG, et al ., In Vitro Characterization of Monoaspartyl Chlorin e6 and Diaspartyl Chlorin e6 for Photodynamic Therapy, J. Nat'l. Can. Inst., 80: 330-336 (1988).

A characteristic feature of chlorin e6 is the fact that, depending on the solvent, its spectral characteristics vary greatly. Thus, it is known that in aqueous media its absorption maximum shifts to the short-wavelength region from 662 nm in alcohol to 655 nm in an aqueous solution at pH 9.0 and higher.

It is known that in cancer foci, due to the rapid growth of cells, a slight change in pH occurs downward, therefore, the aim of the present invention was to create a PS on the one hand having the same photophysical characteristics as the known PSs in order to use the same licensed lasers for PDT, and on the other hand, replace the negative charge on the periphery of a chlorine macrocycle with a positive one by introducing an amino group in the peripheral substituent in order to change the distribution of PS in the body and to increase even more pnost FS to tumors. Moreover, the found structure additionally provides the ability to control the distribution of PS in the body due to the combination of various alkyls and various organic acids of different basicities.

Thus, the task was solved in the synthesis of derivatives of monoamide chlorin e ₆ General formula (1),

where R ₁ means a linear or branched alkyl having from 1 to 4 carbon atoms, R ₂ means salts of pharmaceutically acceptable organic acids.

This substance can be used as a photosensitizer containing derivatives of chlorin e6 monoamide, which have the above general formula (1).

This substance can be obtained in the following way: provide the interaction of alcohol containing R ₁ , and chlorin e6 to form the ester of pheophorbide chlorin e6, containing R ₁ , provide the interaction of the formed ester with ethylenediamine to form the monoamide of the above ester, provide the interaction of the formed monoamide with a pharmaceutically acceptable organic acid to form a salt of the above monoamide. Currently, as a raw material containing chlorin e6, it is advisable to use spirulina.

As examples of implementation, currently selected are some of the most economically feasible components in terms of cost and ease of preparation.

Spirulina is treated with methanol or ethanol containing 5-10% conc. sulfuric acid at room temperature for 12 hours, then the biomass is filtered off, methylpheophorbide a (II) (Fig. 4) or ethylpheophorbide a (III) (Fig. 5), which is in a solution of methylene chloride, chloroform or another suitable solvent, is isolated from the filtrate treated with 0.5 g · equiv of freshly distilled ethylenediamine for 3-4 hours at room temperature until the reaction is completed (spectral and chromatographic test), the solvent is removed in vacuo, the crystalline residue is suspended in dist. water, filtered off, washed with dist. water until there is no alkaline reaction in the filtrate due to the presence of unreacted ethylenediamine, dried under vacuum at room temperature and the corresponding amide methyl ester monoethylenediamine monoamide chlorin e6 (IV) is obtained (Fig. 6). Similarly, the corresponding ethyl ester (V) is obtained (Fig. 7) starting from ethylfeoforbide a (II) and ethylenediamine in quantitative yield.

Fig. 4

Fig. 5.

Fig.6

Fig. 7

Chlorinamidamines are readily soluble in organic solvents (methylene chloride, chloroform, dichloroethane), somewhat more difficult in acetone or alcohol. These compounds in a two-phase system (organic solvent - water) are extremely easily transferred to the aqueous phase when it is acidified due to the formation of the chlorinamide trication.

All known FSs based on chlorin e6 for intravenous use are either aqueous solutions (concentration ~ 30 mg of chlorin e6 in 10 ml of water, i.e. 0.35% solution for RADACHLORIN or 0.5% solution for PHOTODITAZINE followed by dilution to 100 ml), or a prepared solution of a powder of 100 mg of trisodium salt of chlorin e6 together with polyvinylpyrrolidone in 100 ml of water. Thus, the concentration of PS calculated by the content of chlorin e6 in the injection solution does not exceed 0.3-1.0 mg / ml.

To obtain PS (I, R = Me, Et), proceeding from compounds IV and V (Fig. 6) and (Fig. 7), proceed as follows: dissolve 30 mg of chlorin IV or V in 5 ml of ethanol rectified, add 5 ml of pyrogen-free water, filtered through 0.22 mmk millipores, adjusted the total injection volume to 100 ml with water and used as intended.

Among the numerous derivatives of chlorin e6 and mesochlorin e6 tested as potential PS for PDT, two monoamides with ethylenediamine are known (compounds VI and VII, Fig. 8 and Fig. 9), however, they did not differ in any noticeable positive properties from traditional chlorin e6 and therefore were not used in the future to develop a practically important drug based on them. The synthesis of compounds VI and VII was carried out starting from pheophorbide a (or mesofeophorbide a) or directly from chlorin e6 (mesochlorin e6). The presence of carboxyl groups in compounds VI and VII nullified the effect of the amino group, since the formation of the zwitterionic structure greatly complicated the isolation of these amides, due to their solubility in both acidic and alkaline aqueous media.

Fig. 8

Fig. 9

The advantages in obtaining chlorinamido amines IV and V compared with the known PS based on chlorin e6 salts are as follows:

1. Disclosure of the cyclopentane exocycle in alkylpheophorbide a to chlorine e6 derivatives with ethylene diamine occurs under mild conditions and with a quantitative yield, in comparison with the use of aqueous alkali for the reactions of the traditional conversion of pheophorbides a to chlorine e6;

2. In the process of obtaining amidochlorins, there are no processes of degradation of chlorins to a mixture of other derivatives (for example, purpurine, chlorin e4, etc. (see the link from http://www.sciencedirect.com/ search for chlorin e6 N.A. Isakau, TV Trukhacheva and PTPetrov Isolation and identification of impurities in chlorin e6 Journal of Pharmaceutical and Biomedical Analysis, In Press, Corrected Proof, Available online May 24, 2007, or patent RU 2183956).

3. The production of amidochlorins starting from alkylfeoforbide a can easily be scaled to several tens of grams in a conventional chemical laboratory without the use of special and expensive equipment compared to the synthesis of chlorin e6, the preparation of which requires a reaction under conditions that are most protected from the action of atmospheric oxygen, using relatively large volumes of aqueous solutions per 1 g of raw materials, the use of repeated washing with large volumes of pyrogen-free water of sediment of chlorin e6 from the accompanying impurities followed by centrifugation.

4. The stability of photosensitizers prepared from amidochlorins during storage is much higher than photosensitizers based on chlorine e6 salts, which is very important when creating lexforms.

5. To increase the solubility of amidochlorins in water-alcohol solutions when creating injectable forms of PS by interaction with organic acids, the corresponding salts are obtained which are stable in dry form or in water-alcohol solutions during storage.

Example 1. To a suspension of 100 g of lyophilized spirulina in 300 ml of methanol, 30 ml of conc. sulfuric acid for 5 minutes, stirred for 12 hours, the spirulina biomass is filtered on a No. 2 glass filter, the biomass on the filter is washed with 100 ml of methanol, the combined filtrate is evaporated to half volume in vacuo, diluted with dist. water to 500 ml, neutralized with cooling to 10-15 ° C with an aqueous 10% alkali solution (KOH or NaOH), the precipitate formed is filtered through a 2 cm layer of celite on a glass filter (D 9 cm), washed with hot water until inorganic salt filtrate, dried in air, celite washed with petroleum ether or hexane from fat-soluble impurities, and then the product is extracted with a mixture of methylene chloride-acetone (10: 1), evaporated to dryness, chromatographed on a silica gel column, the fraction containing methylpheophorbide and evaporated in vacuo dry, doba 1 ml of freshly distilled ethylenediamine in 20 ml of methylene chloride is added, stirred for 2 hours, 50 ml of water are added to the solution, shaken, the organic layer is separated, washed with water 2 more times until the unreacted ethylene diamine is completely removed, the organic layer is separated, evaporated, chromatographed on a column with silica gel in a methylene chloride-methanol system (95: 5), the main fraction was separated, evaporated to give 350 mg of 18-aminoethylcarbamoyl-20-methoxycarbonylmethyl-8-ethenyl-13-ethyl-2,3-dihydro-3,7,12, 17-tetramethyl-21H, 23H-2- (2-methoxycarbonyl ethyl) porphin (IV).

Example 2. Similarly, from 100 g of lyophilized dried spirulina and 300 ml of abs. alcohol receive 230 mg of 18-aminoethylcarbamoyl-20-methoxycarbonylmethyl-8-ethenyl-13-ethyl-2,3-dihydro-3,7,12,17-tetramethyl-21H, 23H-2- (2-ethoxycarbonylethyl) porphin (V )

Example 3. A mixture of 250 mg of methylpheophorbide a and 0.5 ml of ethylenediamine in 15 ml of methylene chloride was kept under stirring for 3 hours and 270 mg of chlorin IV was isolated.

Example 4. Similarly, from 250 mg of ethylfeoforbid a and 0.5 ml of ethylenediamine receive 220 mg of chlorin V.

Example 5. To a solution of 50 mg of chlorin IV in 10 ml of a mixture of methylene chloride-ethanol (1: 1) is added the calculated amount of 13 mg (1 g · -eq) adipic acid in 5 ml of ethanol, the solution is evaporated to dryness in vacuum and get salt - adipate IV (1, R = Me).

Example 6. Similarly, from chlorin V get salt-adipate V (1, R = Et).

The prior art:

RU 2144538 (from 2000.01.20) A method for producing water-soluble chlorins;

RU 2183956 (from 2002.06.27) Photosensitizer and method for its preparation;

US 6969765 (2005-11-29) Photosensitizer and method for production thereof;

US 5330741 (1994-07-19) Long-wavelength water soluble chlorin photosensitizers useful for photodynamic therapy and diagnosis of tumors;

RU 2276976 (from 2006.02.10) Photosensitizer and method for its preparation;

RU 2152790 (from 2000.07.20) Agent for the photodynamic diagnosis and treatment of cancer;

WO 2004/089409 A3 (2004.10.21) Agent for the photodynamic diagnosis and therapy of malignant tumors.

Claims (5)

1. Derivatives of monoamide chlorin e6 of the General formula (1),

where R ₁ means a linear or branched alkyl having from 1 to 4 carbon atoms,
R ₂ means a salt of pharmaceutically acceptable organic acids. 2. A photosensitizer containing derivatives of chlorin e6 monoamide, characterized in that these derivatives are indicated in paragraph 1. 3. The method of producing derivatives according to claim 1, characterized in that they provide the interaction of an alcohol containing R ₁ and chlorin e6 to form a chloroform e6 pheoforbide ester containing R ₁ ,
provide the interaction of the formed ester with ethylene diamine to form the monoamide of the above ester; provide the interaction of the formed monoamide with a pharmaceutically acceptable organic acid to form the salt of the above monoamide. 4. The method according to claim 3, characterized in that spirulina is used as the raw material containing chlorin e6. 5. The method according to any one of claims 3 and 4, characterized in that they provide the interaction of the formed ester with ethylenediamine in a medium containing alcohol.