The method of obtaining water-soluble chlorin

 

(57) Abstract:

The invention relates to the chemistry of biologically active compounds, particularly to a method of obtaining new water-soluble chlorin, which may find application as photosensitizers for photodynamic therapy of cancer. Order to obtain a stable water-soluble chlorine method, which consists in the interaction of the source of chlorine with a solution of the base. According to the invention as a source of chlorine use compounds of General formula (1)

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or (2)

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where R1= -CH(OAIk)CH3, -CH=CH2, -CHO, -C(O)CH3;

R2= H or lower alkyl;

R3= -CH3, -SNO,

and as the basis of linear or cyclic primary or secondary amine, amino acid or the salt of the amino acids. The original pre-chlorin periostat by gradually adding water to its acetone solution, and a stoichiometric amount of base is added directly to the wet precipitate of chlorine and subsequent sterowanie 1,5-2%-aqueous solution of the desired product with a concentrated solution of the appropriate base or concentrated Hcl to a pH of 7.5-8.5. This method p. the century 1 C.p. f-crystals, 4 PL.

The invention relates to the chemistry of biologically active compounds, particularly to a method of obtaining new water-soluble chlorin General formula (1) and (2).

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where R1= -CH(OAlk)CH3, -CH=CH2, -CHO, -C(O)CH3;

R2=H or lower alkyl;

R3=-CH3, -CHO;

L = volume of the ligand (linear or cyclic primary or secondary amine, amino acid or the salt of the amino acids), and n 1.

These compounds may find application as photosensitizers (PS) for photodynamic therapy (PDT) of cancer and fluorescent labels.

Known FS chlorophyll-type high trapnest against malignant tumors, such as chlorine in the form of sodium salt [Kostenich, G. A. , Zhuravkin I. N., Zhavrid E. A., Experimental Grounds for Using Chlorin E(6) in the Photodynamic Therapy of Malignant Tumors, J. Photochem. Photobiol. 1994, v.22, N 3, 211 - 217; Zorina I.e., Photodynamic activity of derivatives of chlorin molecular and cellular aspects. Diss. on saisc. academic Art. candles. Biol. Sciences. Minsk, 1992], Terentieva salt of mono-L-aspartyl chlorin [Boomer J. C., Burnham B. F. Tetrapyrrol Polyaminomonocarboxylic Acid Therapeutic Agents. U. S. Cl. 514/410. Pat. No. 4,977,177, 11/1990] , trinacria salt lysyl-chlorin [Leach, M. W., R. J. Higgins, J. E. Boggan, Lee S.-J., Autry, S., Smith K. M. Effectiveness of a Lysylchlo potential photosensitizers derived pheophorbide [Nakazato M. Method of Producing Water-soluble Sodium Pheophorbide U. S. Cl.540/145. Pat. N 5378835, 01/1995] and bacteriochlorin [Dougherty, T. J. Bacteriochlorophyll-a derivatives useful in photodynamic therapy. U. S. Cl. 514/410. Pat. N 5173504, 12/1992].

A method of obtaining trinational salt lysyl-chlorin which consists in the interaction of the methyl ester of purpurine-18 obtained by the reaction of allometrically of methylpheophorbide with an aqueous solution of lysine in methylene chloride in the presence of pyridine. The mixture is stirred for 12 hours at room temperature and remove the solvent in high vacuum. The resulting crude product was then purified using reverse-phase HPLC and the solvent removed lyophilic drying. With the aim of obtaining injectable solution for the PDT drug is dissolved in phosphate buffer, add 0.1 N NaOH solution, bring the solution to a physiological pH 7.35 with 0.1 N NCl and filtered through a microporous filter [Smith K. M., Lee S.-J. Long-wavelength Water Soluble Chlorin Photosensitizers Useful for Photodynamic Therapy and Diagnosis of Tumors. U. S. Cl. 424/9. Pat. N 5330741, 07/1994].

The disadvantages of this method include poor reproducibility, the complexity and the use of highly toxic reagents, making it unsuitable for pharmaceutical production. In addition, the obtained water-soluble target sustainable product in the ch M. W., Higgins R. J., J. E. Boggan, Lee S.-J., Autry, S., Smith K. M. Effectiveness of a Lysylchlorin / Chlorin mixture in Photodynamic Therapy of the Subcutaneous 9L Glioma in the Rat. Cancer Research 1992, V. 52, 1235 - 1239; Smith, K. M., Lee S.-J. Long-wavelength Water Soluble Chlorin Photosensitizers Useful for Photodynamic Therapy and Diagnosis of Tumors. U. S. Cl. 424/9. Pat. N 5330741, 07/1994].

A method of obtaining water-soluble sodium salt of pheophorbide According to this method pheophorbide dissolved in diethyl ether and to the solution slowly, dropwise, with stirring, add very dilute alkali solution in n-propanol, isopropanol or mixtures of these solvents. The reaction is carried out until complete precipitation of the salt of pheophorbide in the precipitate, which is separated by centrifugation and dried in vacuum. The product is dissolved in water, obtaining a solution with a concentration of 0.5% and pH 9.2 - 9.5, which is then diluted with phosphate buffer with pH 7.4 - 7.8 [Nakazato M. Method of Producing Water-soluble Sodium Pheophorbide U. S. Cl.540/145. Pat. N 5378835,01/1995].

The disadvantage of this method is that at physiological pH values, it is impossible to obtain a sufficiently concentrated (>1%) injecting a salt solution pheophorbide Additional problems are the instability of these salts during storage and their partial solubility after storage in solid form.

Also known is a method of obtaining motorstv the m alkali solution. The extract obtained various chlorin derivative is treated with acid, the product is filtered and washed with hexane, dissolved in ethanol and subjected to alkaline hydrolysis in a vacuum, followed by neutralization. The precipitation containing chlorine treated with a solution of NaOH or KOH [Albizia O. N., The ashmarov centuries, Meshcheryakov, A. L. the Method of obtaining 18-carboxy-20-(carboxymethyl)-8-ethynyl-13-ethyl-2,3-dihydro-3,7,12, 17-tetramethyl-21H, 23H-porphin-2-propionic acid or its salts. MCI C 07 D 487/22, application N 93-036361/13 (036894) from 07/1993].

The described method is tedious, time-consuming, accompanied by low output and low frequency of the target product. The resulting derivative is characterized by low stability when stored as dissolved, and in powder form.

As the closest analogue is the selected method of obtaining trinational salt chlorine by reacting solution trimethylboron ether chlorine in tetrahydrofuran with 1N NaOH solution. The reaction mass is stirred for 2 days at room temperature in a nitrogen atmosphere and add water. The organic solvent is extracted with methylene chloride, removing the last traces by bubbling through a solution of salt chlorine nitrogen is and obtain the target product and its instability during storage in the form of powder, and in the form of a solution are an obstacle to create a dosage form.

The task of the invention is to provide opportunities for sustainable water-soluble chlorin.

The problem is solved by the proposed method, which consists in the interaction of the source of chlorine with a solution of the base. According to the invention as a source of chlorine use compounds of General formula (3) or (4):

< / BR>
where R1= -CH(OAlk)CH3, -CH=CH2, -CHO, -C(O)CH3;

R2=H or lower alkyl;

R3=-CH3, -CHO;

and as the basis of linear or cyclic primary or secondary amine, amino acid or the salt of the amino acids.

The original pre-chlorin periostat by gradually adding water to its acetone solution, and a stoichiometric amount of base is added directly to the wet precipitate of chlorine and subsequent sterowanie 1.5 - 2% aqueous solution of the desired product with a concentrated solution of the appropriate base or concentrated HCl to pH 7.5 - 8.5.

As bases there may be used, for example, 4-aminobutanol, N-methylglucamine, glucosamine, interacting with carboxyglutamic chlorine and mating system of the tetrapyrrole macrocycle.

Used a range of pH values is due to the fact that the bottom boundary pH 7.5 is the lower limit of the solubility of chlorine in aqueous solutions to produce concentrations, natural, for use in the pharmaceutical industry, without adding solubilization. The upper limit of this range pH 8.5 is a biological limit portability concentration of hydroxide ions [OH-].

The range of concentrations of chlorine 1.5 - 2% due to the fact that the use of more dilute solutions is connected with the necessity to introduce into the body a large amount of solution, which is undesirable. Concentration of about 2.5% is the upper limit of solubility.

Free from impurities original chlorines General formula (3) and (4) can be derived from chlorophyll or bacteriochlorophyll quickly and with high yield [Lotjonen , S., P. H. Hynninen, An improved method for the preparation of (10R)- and (10S)-pheophytins and Synthesis. 1983, 705 - 708; Hynninen, P. H. , Lotjonen, S., Preparation of phorbin derivatives from chlorophyll mixture utilizing the principle of selective hydrolysis. Synthesis. 1980, 539 - 541; Lotjonen, S., P. H. Hynninen, A convenient method for the preparation of chlorin and rhodin g7trimethyl esters. Synthesis, 1980, 541 - 543].

P is emer, after drying), but in the form of solutions ready for injection, while the classical K+and Na+salt chlorine are not stable derivatives. The use of stabilization using volumetric ligand for chlorine to date was not known. The invention is illustrated by examples of specific performance.

Example 1. Chlorophyll biomass microalgae Spirulina Platensis is turned into chlorine according to standard methods [Lotjonen, S., P. H. Hynninen, An improved method for the preparation of (10R)- and (10S)-pheophytins and Synthesis. 1983, 705 - 708; Hynninen, P. H., Lotjonen, S., Preparation of phorbin derivatives from chlorophyll mixture utilizing the principle of selective hydrolysis. Synthesis. 1980, 539 - 541; Lotjonen, S., P. H. Hynninen , A convenient method for the preparation of chlorin and rhodin g7trimethyl esters. Synthesis, 1980, 541 - 543] with a total output of more than 50% of the original chlorophyll and then the product periostat by gradually adding water to its acetone solution. The precipitation is centrifuged and washed 3 times with water. To the precipitate chlorine is added 2 g-EQ. N-methylglucamine and water enough to get a 1.7% solution of the chlorine. Dissolve without residue, to which a control for the pH meter lead to the solution of N-methylglucamine to pH 8.5 and filtered through a porous glass filter # 4. The yield of water-soluble complex of chlorine is):

(in CDCL3+ 5% CF3COOD, conc. solution) 9.67 (s, meso, 9.23 (s, meso), 8.22 (s, meso), 7.58, 7.62 (2D, =CH2), 6.25, 6.13 (2D, CH= ), 4.80 (m, protoner. 8), 4.45 (m, 7), 4.04 (m, protoner. 7), 3.96 (m, 8), 3.90 (t, 8), 3.86 (s, -mesozoan), 3.83 (K, 4CH3), 3.26 (s, 5), 3.09 (s, 1), 3.03 (s, 3), 2.43, 2.20 (2m, 7COOH), 1.47 (m, 4-CH2C);

((CD3)2SO, conc. solution) 9.75 (s, meso), 9.67 (s, meso, 9.07 (s, meso), 8.29, 8.24 (2D, =CH2), 6.43, 6.16 (2D, CH=), 5.405, 5.29 (2C, -meso-CCOOH), 4.60 (m, 7), 4.45 (m, 8), 3.79 (K, 4CH3), 3.58 (s, 5), 3.51 (s, 1-

), 3.28 (s, 3), 2.44, 2.28 (2m, 7CH2COOH), 2.60, 2.14 (2m, 7-CH2COOH), 2.08 (V. 8), 1.68 (m, 4-CH2), -1.63, -1.91 (2C ).

Example 2. The amino acid is suspended in a mixture of chloroform/ethanol 1 : 1 (50 ml of a mixture of 1 g amino acids) and add a stoichiometric amount of ateleta sodium (potassium). Thus forming a salt entirely passes into solution. This solution is heated at 50 - 60oC under reflux for 1 hour, the solvents are removed in vacuo and add this amount of distilled water to obtain a solution of known concentration. This solution containing 2 g-EQ. the grounds add to wet collapse of chlorine to dissolve the latter, then bring the aqueous solution of the target product using conc. solution of this base or conc. HCl to pH 8.3 and Rabba is.

Example 3. Prepare concentrated aqueous solution of amino acids or other organic derivatives of known content base. This solution containing 2 g-EQ. the grounds add to wet collapse of chlorine to dissolve the latter, then bring the aqueous solution of the target product using conc. HCl to pH 7.5 and diluted with distilled water to the required concentration for chlorine (1.5%). Product specifications see Table.1.

Example 4. For comparison were obtained potassium and sodium salt of chlorin . A solution containing 2 g-EQ. alkali (112 mg, 2 mmol KOH or 80 mg, 2 mmol NaOH) in water (1 mg of chlorin 0.2 ml of water), add to wet collapse of chlorine (625 mg, 1 mmol) and stirred at 40oC 12 hours, ensuring maximum dissolution of chlorine, after which the mixture was adjusted using conc. HCl to pH 8.3 and filtered. The quantity of dissolved chlorine is determined by the weight of the residue. Product specifications see Table.1.

Example 5. Dissolve 2-devinyl-2-[(1-ethoxy)ethyl] chlorin (R1= CH(OC2H5)CH3, R2= -H and R3= -CH3) in an aqueous solution containing 2 g-EQ. N-methyl-D-glucosamine, as in example 3. Product features see Table. 4.

Example 7. Dissolve Rodin g7(R1= -CH=CH2, R2= -H and R3= -CHO) in an aqueous solution containing 2 g-EQ. N-methyl-D-glucosamine, as in example 3. Product features see Table.4.

Example 8. Dissolve 2-devinyl-2-formyl-3,4-dihydroxylated (R1= -CHO, R2= -H and R3= -CH3) in an aqueous solution containing 2 g-EQ. N-methyl-D-glucosamine, as in example 3. Product features see Table.4.

Example 9. Dissolve bacteriochlorin (R1= -C(O)CH3, R2= -H and R3= -CH3) in an aqueous solution containing 2 g-EQ. N-methyl-D-glucosamine, as in example 3. Product features see Table.4.

The stability of the obtained compounds was studied on the example of N-methylglucamine complex of the chlorin

Solutions of known concentration were kept in the dark in hermetically sealed flasks at three temperature regimes: freezer (-15 - 20oC), refrigerator (0 + 5oC) and room (+20 + 25oC). Every 2 misjah waves 502, 532 and 654 nm. The measurements were carried out on writing spectrophotometer Hitachi 557. The ratio 502/532 for pure N-methylglucamine complex of chlorine in water is 3. With the collapse of the chlorin macrocycle to linear tetrapyrroles decreases the magnitude of the absorption at a wavelength of 654 nm (n, accordingly, decreases the specific extinction) and simultaneously decreasing the ratio 502/532 due to the simultaneous growth of absorption at both wavelengths (grows background absorption in this wavelength range). The change data control units for 2 months. and for the year are presented in Table.2.

It is known that the decrease of extinction during storage by approximately 10% per year for drug substances allowed. In this example, the storage conditions of the freezing chamber leads to a decrease in both measures approximately 5% per year and in the refrigerator for approximately 13% for the year. This saves the filterability connections (not precipitate formed), which is important for the pharmaceutical industry.

Data on the stability of a 2% aqueous solution of chlorine during storage at different temperatures depending on the time shown in Table 3.

Along with the measurement of the optical properties of solutions of N-methylglucamine took aliquots of 0.1 ml, neutralized 1 N hydrochloric acid to pH 5 - 6, fallen chlorine was extracted with diethyl ether, the extract was dried 1 hour anhydrous magnesium sulfate and chlorine was atrificial with diazomethane, getting chromatographic mobile broadcast. TLC was performed on plates Kieselgel 60 F254 Merck in the system chloroform/acetone 20 : 1. According to this analysis, all samples for the first 4 months of storage significantly reduced the number of impurity pheophorbide and slightly increased the number of purpurine, and in the next 6 months decreased the number of purpurine and increased the number of chlorine Appreciable formation of polar products yellow or yellow-green color began with a 4-month storage and to the greatest extent was observed in the samples, which were carried out at room temperature. This process correlates with the fall of extinction.

Not all of the obtained complexes are able to give a 2% solution by chlorine at physiological pH values (table 1). So, only complexes with lysate of sodium and Argentum sodium give such a solution at pH 8.3, and this is not the limit of their solubility. 4-Aminobutanol, glucosamine and N-methylglucamine can form a saturated 2% solution only at pH 8.5.

Glade amounted to 1.25%, and sodium - 0.97% and these values are among the lowest (see Table 1), along with the neutral amino acid salts and free bases miloslavich amino acids.

Therefore, these salts are not suitable for derivation of dosage forms because, first, low concentration limits, and secondly, because of the increased propensity for aggregation and the formation of water-insoluble products.

The advantage of the proposed method is that the target products are water-soluble complexes of chlorine is obtained in a sustainable manner and with high yield by simplifying terms and conditions of allocation. It provides previously unknown chemical compounds. We offer the option of stabilizing chlorine opens the way to obtaining effective and accessible FS. Assumes the use of new compounds as photosensitizers for PDT of cancer and photodegradation desolate vessels of the cornea. The proposed method will be implemented in industrial production of medicines "Chlorine" and "Photoditazine".

1. The method of obtaining water-soluble chlorine by reacting a source of chlorine with a solution of base, different>/BR>where R1= -CH(OAIk)CH3, -CH=CH2, -CHO, -C(O)CH3;

R2= H or lower alkyl;

R3= -CH3, -CHO,

and as the basis of linear or cyclic primary or secondary amine, amino acid or the salt of the amino acids, the original pre-chlorin periostat by gradually adding water to its acetone solution, and a stoichiometric amount of base is added directly to the wet precipitate source of chlorine and subsequent sterowanie 1,5 - 2%-aqueous solution of the desired product with a concentrated solution of the appropriate base or concentrated HCl to pH of 7.5 - 8.5.

2. The method of obtaining water-soluble chlorin under item 1, characterized in that the base use 4-aminobutanol, N-methylglucamine, glucosamine, arginine, lysine, histidine, sodium or potassium salts of arginine, lysine or histidine.

 

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