RU2144538C1 - Method of preparing water-soluble chlorines - Google Patents

Abstract

FIELD: synthesis of biologically-active compounds. SUBSTANCE: new water-soluble chlorines that can find use as photosensitizers in photodynamic tumor therapy are prepared by interaction of initial chlorines with base solution. Initial chlorines are compounds with general formulas 1 or 2:

(1) (2) wherein -CH(OAlk)CH₃, -CH=CH₂, -CHO, -C(O)CH₃ is R₂; R₃ hydrogen or lower alkyl; and -CH₃, methyl or aldehyde group. Bases involved in reaction can be linear or cyclic primary or secondary amines, amino acids, or amino acid salts. Initial chlorine is preliminarily reprecipitated by gradually adding water to its acetone solution. Stoichiometric quantity of base is directly added to wet chlorine precipitate, after which pH value of 1.5-2% aqueous solution of desired product is adjusted to 7.5-8.5 by adding concentrated solution of alkali or hydrochloric acid. Compounds are stable and fit to prepare injection solution. EFFECT: enlarged choice of antitumor agents. 2 cl, 4 tbl, 9 ex

Description

 The invention relates to the chemistry of biologically active compounds, specifically to a method for producing new water-soluble chlorins of the general formula (1) and (2).

где R₁ = -CH(OAlk)CH₃, -CH=CH₂, -CHO, -C(O)CH₃;
R₂ =H или низший алкил;
R₃=-CH₃, -CHO;
L = объемный лиганд (линейный или циклический первичный или вторичный амин, аминокислота или соль аминокислоты), а n ≥ 1.

where R ₁ = —CH (OAlk) CH ₃ , —CH = CH ₂ , —CHO, —C (O) CH ₃ ;
R ₂ = H or lower alkyl;
R ₃ = —CH ₃ , —CHO;
L = bulky ligand (linear or cyclic primary or secondary amine, amino acid or amino acid salt), and n ≥ 1.

 These compounds can be used as photosensitizers (PS) for the photodynamic therapy (PDT) of cancer and fluorescent labels.

в виде натриевой соли [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; Зорина Т.Е. Фотодинамическая активность производных хлорина

молекулярные и клеточные аспекты. Дисс. на соиск. уч.ст. канд. биол. наук. Минск, 1992], тетранатриевая соль моно-L-аспартил хлорина

[Bommer J.C., Burnham B.F.Tetrapyrrol Polyaminomonocarboxylic Acid Therapeutic Agents. U.S. Cl. 514/410. Pat. No. 4,977,177, 11/1990] , тринатриевая соль лизил-хлорина

[Leach M.W., Higgins R.J., Boggan J.E., 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]. Имеются сообщения об использовании в качестве потенциальных фотосенсибилизаторов производных феофорбидов [Nakazato M. Method of Producing Water-soluble Sodium Pheophorbide

U.S. Cl.540/145. Pat. N 5378835, 01/1995] и бактериохлоринов [Dougherty T.J. Bacteriochlorophyll-a derivatives useful in photodynamic therapy. U.S. Cl. 514/410. Pat. N 5173504, 12/1992].Chlorine-type FSs with high tropism for malignant tumors, for example, chlorin, are known.

as sodium salt [Kostenich GA, Zhuravkin IN, Zhavrid EA, 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 T.E. Photodynamic activity of chlorin derivatives

molecular and cellular aspects. Diss. for a job. part Cand. biol. sciences. Minsk, 1992], tetrasodium salt of mono-L-aspartyl chlorin

[Bommer JC, Burnham BFTetrapyrrol Polyaminomonocarboxylic Acid Therapeutic Agents. US Cl. 514/410. Pat. No. 4,977,177, 11/1990], trisodium salt of lysyl chlorine

[Leach MW, Higgins RJ, Boggan JE, Lee S. -J., Autry S., Smith KM Effectiveness of a Lysylchlorin

/ Chlorin

mixture in Photodynamic Therapy of the Subcutaneous 9L Glioma in the Rat. Cancer Research 1992, V.52, 1235-1239]. There are reports of the use of derivatives of pheophorbides as potential photosensitizers [Nakazato M. Method of Producing Water-soluble Sodium Pheophorbide

US Cl. 540/145. Pat. N 5378835, 01/1995] and bacteriochlorins [Dougherty TJ Bacteriochlorophyll-a derivatives useful in photodynamic therapy. US Cl. 514/410. Pat. N 5173504, 12/1992].

заключающийся во взаимодействии метилового эфира пурпурина-18, полученного по реакции алломеризации метилфеофорбида

с водным раствором лизина в хлористом метилене в присутствии пиридина. Смесь перемешивают 12 часов при комнатной температуре и удаляют растворители в высоком вакууме. Полученный сырой продукт очищают с помощью обращенно-фазовой ВЭЖХ и растворители удаляют лиофильной сушкой. С целью получения инъекционного раствора для ФДТ препарат растворяют в фосфатном буфере, прибавляют 0.1N раствор NaOH, доводят раствор до физиологического значения pH 7.35 с помощью 0.1N NCl и фильтруют через микропористый фильтр [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 known method of producing trisodium salt of lysyl chlorine

consisting in the interaction of methyl ester of purpurin-18 obtained by the reaction of the allomerization of methylpheophorbide

with an aqueous solution of lysine in methylene chloride in the presence of pyridine. The mixture was stirred for 12 hours at room temperature and the solvents removed in high vacuum. The resulting crude product was purified by reverse phase HPLC and the solvents were removed by freeze drying. In order to obtain an injection solution for PDT, the drug is dissolved in phosphate buffer, 0.1N NaOH solution is added, the solution is adjusted to physiological pH 7.35 with 0.1N NCl and filtered through a microporous filter [Smith KM, Lee S.-J. Long-wavelength Water Soluble Chlorin Photosensitizers Useful for Photodynamic Therapy and Diagnosis of Tumors. USCl. 424/9. Pat. N 5330741, 07/1994].

/ 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].The disadvantages of this method include poor reproducibility, complexity and the use of highly toxic reagents, which makes it unsuitable for pharmaceutical production. In addition, the resulting water-soluble target product is stable in aqueous solution only 24 hours at 4 ^o C in the dark, and in solid form up to 4 months at 4 ^o C in the dark [Leach MW, Higgins RJ, Boggan JE, Lee S. -J., Autry S., Smith KM 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 KM, Lee S.-J. Long-wavelength Water Soluble Chlorin Photosensitizers Useful for Photodynamic Therapy and Diagnosis of Tumors. USCl. 424/9. Pat. N 5330741, 07/1994].

Согласно этому способу феофорбид

растворяют в диэтиловом эфире и к раствору медленно, по каплям, при перемешивании прибавляют очень разбавленный раствор щелочи в н-пропаноле, изопропаноле или в смеси этих растворителей. Реакцию проводят до полного выпадения соли феофорбида

в осадок, который отделяют центрифугированием и сушат в вакууме. Продукт растворяют в воде, получая раствор с концентрацией 0.5% и pH 9.2 - 9.5, который затем разбавляют фосфатным буфером с pH 7.4 - 7.8 [Nakazato M. Method of Producing Water-soluble Sodium Pheophorbide

U.S. Cl.540/145. Pat. N 5378835,01/1995].A known method of obtaining a water-soluble sodium salt of pheophorbide

According to this method, pheophorbide

dissolved in diethyl ether and a very dilute alkali solution in n-propanol, isopropanol or a mixture of these solvents is slowly added dropwise to the solution with stirring. The reaction is carried out until the pheophorbide salt is completely

to a 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 a pH of 9.2 - 9.5, which is then diluted with phosphate buffer with a pH of 7.4 - 7.8 [Nakazato M. Method of Producing Water-soluble Sodium Pheophorbide

US Cl. 540/145. Pat. N 5378835.01 / 1995].

Дополнительными проблемами являются неустойчивость этих солей при хранении и их неполная растворимость после хранения в твердом виде.The disadvantage of this method is that at physiological pH values it is impossible to obtain a sufficiently concentrated (> 1%) injection solution of pheophorbide salt

Additional problems are the instability of these salts during storage and their incomplete solubility after storage in solid form.

включающий обработку биомассы цианобактерий водно-спиртовым раствором щелочи. Полученный экстракт различных хлориновых производных обрабатывают кислотой, продукт отфильтровывают и промывают гексаном, растворяют в этаноле и подвергают щелочному гидролизу в вакууме с последующей нейтрализацией. Выпавший осадок, содержащий хлорин

подвергают обработке раствором NaOH или KOH [Альбицкая О.Н., Ашмаров В.В., Мещерякова А. Л. Способ получения 18-карбокси-20-(карбоксиметил)-8-этенил-13-этил-2,3-дигидро-3,7,12, 17-тетраметил-21H, 23H-порфин-2-пропионовой кислоты или ее солей. МКИ C 07 D 487/22, заявка N 93-036361/13 (036894) от 07/1993].There is also known a method of obtaining water-soluble trisodium and tripotassium salts of chlorin

including treatment of biomass of cyanobacteria with an aqueous-alcoholic alkali solution. The obtained extract of various chlorin derivatives is treated with acid, the product is filtered and washed with hexane, dissolved in ethanol and subjected to alkaline hydrolysis in vacuo, followed by neutralization. Chlorine precipitated

subjected to treatment with a solution of NaOH or KOH [Albitskaya ON, Ashmarov VV, Meshcheryakova A. L. A method for producing 18-carboxy-20- (carboxymethyl) -8-ethenyl-13-ethyl-2,3-dihydro- 3,7,12,17-tetramethyl-21H, 23H-porphin-2-propionic acid or its salts. MKI C 07 D 487/22, application N 93-036361 / 13 (036894) from 07/1993].

 The described method is time-consuming, lengthy, accompanied by a low yield and low frequency of the target product. The resulting derivative is characterized by low storage stability both in the dissolved state and in powder form.

путем взаимодействия раствора триметилового эфира хлорина

в тетрагидрофуране с 1N раствором NaOH. Реакционную массу перемешивают 2 суток при комнатной температуре в атмосфере азота и добавляют воду. Органический растворитель экстрагируют хлористым метиленом, удаляя следы последнего барботированием через раствор соли хлорина

азота [Pandey R.K., Dougherty T. J. Photosensitizing Agents. U. S. Cl. 514/410. Pat. No. 5, 002, 962, 03/1991(прототип)].As the closest analogue, the method of producing trisodium salt of chlorin was selected

by reacting a solution of chlorine trimethyl ether

in tetrahydrofuran with 1N NaOH. The reaction mass is stirred for 2 days at room temperature in a nitrogen atmosphere and water is added. The organic solvent is extracted with methylene chloride, removing traces of the latter by bubbling through a solution of chlorine salt

nitrogen [Pandey RK, Dougherty TJ Photosensitizing Agents. US Cl. 514/410. Pat. No. 5, 002, 962, 03/1991 (prototype)].

 However, the duration of the process of obtaining the target product and its instability during storage both in powder form and in the form of a solution are an obstacle to creating a dosage form from it.

 The objective of the invention is to provide the possibility of obtaining stable water-soluble chlorins.

где R₁ = -CH(OAlk)CH₃, -CH=CH₂, -CHO, -C(O)CH₃;
R₂=H или низший алкил;
R₃=-CH₃, -CHO;
а в качестве основания - линейный или циклический первичный или вторичный амин, аминокислоту или соль аминокислоты.The problem is solved by the proposed method, which consists in the interaction of the initial chlorin with a solution of the base. According to the invention, compounds of the general formula (3) or (4) are used as the starting chlorin:

where R ₁ = —CH (OAlk) CH ₃ , —CH = CH ₂ , —CHO, —C (O) CH ₃ ;
R ₂ = H or lower alkyl;
R ₃ = —CH ₃ , —CHO;
and as the base, a linear or cyclic primary or secondary amine, amino acid or amino acid salt.

 In this case, the initial chlorine is pre-precipitated by gradually adding water to its acetone solution, and a stoichiometric amount of the base is added directly to the wet chlorine precipitate, followed by adjustment of a 1.5 - 2% aqueous solution of the target product with a concentrated solution of the corresponding base or concentrated HCl to pH 7.5 - 8.5 .

 As bases, for example, 4-aminobutanol, N-methylglucosamine, glucosamine, arginine, lysine, histidine, sodium or potassium salts of arginine, lysine or histidine and other bulk molecules interacting with both chlorine carboxy groups and the conjugated system can be used. tetrapyrrole macrocycle.

The pH range used is due to the fact that its lower pH 7.5 is the lower solubility of chlorins in aqueous solutions to obtain concentrations that are natural for use in pharmaceuticals without the addition of solubilizers. The upper limit of this pH range of 8.5 is the limit of the biological tolerance of the concentration of hydroxide ions, [OH ^- ].

 The range of chlorin concentrations of 1.5 - 2% is due to the fact that the use of more dilute solutions is associated with the need to enter large volumes of the drug solution into the body, which is undesirable. A concentration of about 2.5% is the upper limit of solubility.

или бактериохлорофилла

быстро и с высоким выходом [Lotjonen S. , Hynninen P.H., 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., Hynninen P.H., A convenient method for the preparation of chlorin

and rhodin g₇ trimethyl esters. Synthesis, 1980, 541 - 543].Free from impurities, the initial chlorins of the general formula (3) and (4) can be obtained starting from chlorophyll

or bacteriochlorophyll

fast and high yield [Lotjonen S., Hynninen PH, An improved method for the preparation of (10R) - and (10S) -pheophytins

and

Synthesis 1983, 705–708; Hynninen PH, Lotjonen S., Preparation of phorbin derivatives from chlorophyll mixture utilizing the principle of selective hydrolysis. Synthesis 1980, 539-541; Lotjonen S., Hynninen PH, A convenient method for the preparation of chlorin

and rhodin g ₇ trimethyl esters. Synthesis, 1980, 541-543].

The proposed method allows to obtain stable derivatives of chlorins not only in an anhydrous state (for example, after lyophilization), but also in the form of solutions ready for injection, while the classical K ⁺ and Na ⁺ salts of chlorins are not stable derivatives. The use of stabilization with a volumetric ligand for chlorins has not been known to date. The invention is illustrated by examples of specific performance.

из биомассы микроводоросли Spirulina Platensis превращают в хлорин

согласно стандартным методикам [Lotjonen S., Hynninen P.H., 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., Hynninen P.H. , A convenient method for the preparation of chlorin

and rhodin g₇ trimethyl esters. Synthesis, 1980, 541 - 543] с общим выходом более 50% на исходный хлорофилл

а затем продукт переосаждают путем постепенного прибавления воды к его ацетоновому раствору. Выпавший осадок центрифугируют и 3 раза промывают водой. К осадку хлорина

прибавляют 2 г-экв. N-метилглюкозамина и воды столько, чтобы получить 1.7%-ный раствор по хлорину. Растворяют без остатка, для чего при контроле по pH-метру доводят раствор N-метилглюкозамином до pH 8.5 и фильтруют через пористый стеклянный фильтр N 4. Выход водорастворимого комплекса хлорина

- количественный. Электронный спектр продукта см. в Табл.1.Example 1. Chlorophyll

from the biomass of the microalga Spirulina Platensis turn into chlorine

according to standard procedures [Lotjonen S., Hynninen PH, An improved method for the preparation of (10R) - and (10S) -pheophytins

and

Synthesis 1983, 705–708; Hynninen PH, Lotjonen S., Preparation of phorbin derivatives from chlorophyll mixture utilizing the principle of selective hydrolysis. Synthesis 1980, 539-541; Lotjonen S., Hynninen PH, A convenient method for the preparation of chlorin

and rhodin g ₇ trimethyl esters. Synthesis, 1980, 541-543] with a total yield of more than 50% on the initial chlorophyll

and then the product is reprecipitated by the gradual addition of water to its acetone solution. The precipitate was centrifuged and washed 3 times with water. To the chlorine precipitate

add 2 g-equiv. N-methylglucosamine and water enough to get a 1.7% solution of chlorine. Dissolve without residue, for which, by monitoring according to a pH meter, bring the solution with N-methylglucosamine to pH 8.5 and filter through a porous glass filter N 4. The output of the water-soluble chlorin complex

- quantitative. The electronic spectrum of the product is shown in Table 1.

("Brucker", 360 MHz; δ, м.д.):
(в CDCL₃ + 5% CF₃COOD, конц. раствор) 9.67 (с, β-мезо

, 9.23 (с, α-мезо

), 8.22 (с, δ-мезо

), 7.58, 7.62 (2д,

=CH₂), 6.25, 6.13 (2д, CH=

), 4.80 (м, протонир. 8

), 4.45 (м, 7

), 4.04 (м, протонир. 7

), 3.96 (м, 8

), 3.90 (т, 8

), 3.86 (с, γ-мезо

COOH), 3.83 (к, 4

CH₃), 3.26 (с, 5

), 3.09 (с, 1

), 3.03 (с, 3

), 2.43, 2.20 (2м, 7

COOH), 1.47 (т, 4-CH₂C

);
(в (CD₃)₂SO, конц. раствор) 9.75 (с, β-мезо

), 9.67 (с, α-мезо

, 9.07 (с, δ-мезо

), 8.29, 8.24 (2д,

=CH₂), 6.43, 6.16 (2д, CH=

), 5.405, 5.29 (2с, γ-мезо-C

COOH), 4.60 (м, 7

), 4.45 (м, 8

), 3.79 (к, 4

CH₃ ), 3.58 (с, 5

), 3.51 (с, 1-

), 3.28 (с, 3

), 2.44, 2.28 (2м, 7

CH₂COOH), 2.60, 2.14 (2м, 7-CH₂

COOH), 2.08 (д. 8

), 1.68 (т, 4-CH₂

), -1.63, -1.91 (2с,

).PMR spectra of chlorin

("Brucker", 360 MHz; δ, ppm):
(in CDCL ₃ + 5% CF ₃ COOD, conc. solution) 9.67 (s, β-meso

9.23 (s, α-meso

), 8.22 (s, δ-meso

), 7.58, 7.62 (2d,

= CH ₂ ), 6.25, 6.13 (2d, CH =

), 4.80 (m, protonir. 8

), 4.45 (m, 7

), 4.04 (m, protonir. 7

), 3.96 (m, 8

), 3.90 (t, 8

), 3.86 (s, γ-meso

COOH), 3.83 (q, 4

CH ₃ ), 3.26 (s, 5

), 3.09 (s, 1

), 3.03 (s, 3

), 2.43, 2.20 (2m, 7

COOH), 1.47 (t, 4-CH ₂ C

);
(in (CD ₃ ) ₂ SO, conc. solution) 9.75 (s, β-meso

), 9.67 (s, α-meso

, 9.07 (s, δ-meso

), 8.29, 8.24 (2d,

= CH ₂ ), 6.43, 6.16 (2d, CH =

), 5.405, 5.29 (2s, γ-meso-C

COOH), 4.60 (m, 7

), 4.45 (m, 8

), 3.79 (q, 4

CH ₃ ), 3.58 (s, 5

), 3.51 (s, 1-

), 3.28 (s, 3

), 2.44, 2.28 (2m, 7

CH ₂ COOH), 2.60, 2.14 (2m, 7-CH ₂

COOH), 2.08 (d. 8

), 1.68 (t, 4-CH ₂

), -1.63, -1.91 (2s,

)

до полного растворения последнего, после чего доводят водный раствор целевого продукта при помощи конц. раствора данного основания или конц. HCl до pH 8.3 и разбавляют дистиллированной водой до необходимой концентрации по хлорину (2%). Характеристики продуктов см. в Табл.1.Example 2. The amino acid is suspended in a 1: 1 chloroform / ethanol mixture (50 ml of the mixture per 1 g of amino acid) and a stoichiometric amount of sodium (potassium) ethylate is added. In this case, the salt forming completely passes into the solution. This solution was heated at 50-60 ^° C under reflux for 1 hour, the solvents were removed in vacuo and such an amount of distilled water was added to obtain a solution of known concentration. This solution containing 2 g-eq. bases are added to the wet sediment of chlorin

to complete dissolution of the latter, after which the aqueous solution of the target product is adjusted using conc. a solution of this base or conc. HCl to pH 8.3 and diluted with distilled water to the desired chlorine concentration (2%). Product specifications are shown in Table 1.

до полного растворения последнего, после чего доводят водный раствор целевого продукта при помощи конц. HCl до pH 7.5 и разбавляют дистиллированной водой до необходимой концентрации по хлорину (1.5%). Характеристики продуктов см. в Табл.1.Example 3. A concentrated aqueous solution of an amino acid or other organic amino derivative with a known base content is prepared. This solution containing 2 g-eq. bases are added to the wet sediment of chlorin

to complete dissolution of the latter, after which the aqueous solution of the target product is adjusted using conc. HCl to pH 7.5 and diluted with distilled water to the desired chlorine concentration (1.5%). Product specifications are shown in Table 1.

. Раствор, содержащий 2 г-экв. щелочи (112 мг, 2 ммоль KOH или 80 мг, 2 ммоль NaOH) в воде (на 1 мг хлорина 0.2 мл воды), прибавляют к влажному осадку хлорина

(625 мг, 1 ммоль) и перемешивают при 40^oC 12 часов, добиваясь максимального растворения хлорина, после чего смесь доводят при помощи конц. HCl до pH 8.3 и фильтруют. Количество растворившегося хлорина

определяют по весу остатка. Характеристики продуктов см. в Табл.1.Example 4. For comparison, potassium and sodium salts of chlorin were obtained.

. A solution containing 2 g-eq. alkalis (112 mg, 2 mmol KOH or 80 mg, 2 mmol NaOH) in water (per 1 mg of chlorin 0.2 ml of water) are added to the wet precipitate of chlorin

(625 mg, 1 mmol) and stirred at 40 ^o C for 12 hours, achieving maximum dissolution of chlorin, after which the mixture was brought with conc. HCl to pH 8.3 and filtered. The amount of dissolved chlorin

determined by the weight of the residue. Product specifications are shown in Table 1.

(R₁ = CH(OC₂H₅)CH₃, R₂ = -H и R₃ = -CH₃) в водном растворе, содержащем 2 г-экв. N-метил-D-глюкозамина, как в примере 3. Характеристики продукта см. в Табл. 4.Example 5. Dissolve 2-desvinyl-2 - [(1-ethoxy) ethyl] chlorin

(R ₁ = CH (OC ₂ H ₅ ) CH ₃ , R ₂ = —H and R ₃ = —CH ₃ ) in an aqueous solution containing 2 g-equiv. N-methyl-D-glucosamine, as in example 3. Product characteristics, see table. 4.

(R₁ = -CH=CH₂, R₂ = -C₂H₅ и R₃ = -CH₃) в водном растворе, содержащем 2 г-экв. N-метил-D-глюкозамина, как в примере 3, за исключением того, что разбавляют дистиллированной водой до концентрации 0.5% по хлорину. Характеристики продукта см. в Табл.4.Example 6. Dissolve chloro monoethyl ether

(R ₁ = —CH = CH ₂ , R ₂ = —C ₂ H ₅ and R ₃ = —CH ₃ ) in an aqueous solution containing 2 g-equiv. N-methyl-D-glucosamine, as in example 3, except that it is diluted with distilled water to a concentration of 0.5% in chlorine. Product specifications are shown in Table 4.

Example 7. Dissolve the motherland g ₇ (R ₁ = —CH = CH ₂ , R ₂ = —H and R ₃ = —CHO) in an aqueous solution containing 2 g-equiv. N-methyl-D-glucosamine, as in example 3. Product characteristics, see Table 4.

(R₁ = -CHO, R₂ = -H и R₃ = -CH₃) в водном растворе, содержащем 2 г-экв. N-метил-D-глюкозамина, как в примере 3. Характеристики продукта см. в Табл.4.Example 8. Dissolve 2-desvinyl-2-formyl-3,4-dihydroxychlorin

(R ₁ = —CHO, R ₂ = —H and R ₃ = —CH ₃ ) in an aqueous solution containing 2 g-equiv. N-methyl-D-glucosamine, as in example 3. Product characteristics, see Table 4.

(R₁ = -C(O)CH₃, R₂ = -H и R₃ = -CH₃) в водном растворе, содержащем 2 г-экв. N-метил-D-глюкозамина, как в примере 3. Характеристики продукта см. в Табл.4.Example 9. Dissolve bacteriochlorin

(R ₁ = —C (O) CH ₃ , R ₂ = —H and R ₃ = —CH ₃ ) in an aqueous solution containing 2 g-equiv. N-methyl-D-glucosamine, as in example 3. Product characteristics, see Table 4.

Растворы определенной концентрации хранили в темноте в герметически укупоренных склянках при трех температурных режимах: морозильной камеры (-15 - 20^oC), холодильника (0 + 5^oC) и комнаты (+20 + 25^oC). Каждые 2 месяца отбирали аликвоты 0.1 мл в 50 мл боратного буферного раствора (с pH 9.18) и измеряли оптическую плотность на длинах волн 502, 532 и 654 нм. Измерения производились на пишущем спектрофотометре Hitachi 557. Соотношение 502/532 для чистого N-метилглюкозаминового комплекса хлорина

в воде составляет 3. При распаде хлоринового макроцикла до линейных тетрапирролов уменьшается величина поглощения на длине волны 654 нм (н, соответственно, уменьшается удельная экстинкция) и одновременно уменьшается соотношение 502/532 за счет одновременного роста поглощения на обеих длинах волн (растет фоновое поглощение в этом диапазоне длин волн). Изменение данных контрольных величин за 2 мес. и за год представлено в Табл.2.The stability of the obtained compounds was studied by the example of the N-methylglucosamine complex of chlorin

Solutions of a certain concentration were stored in the dark in hermetically sealed vials at three temperature conditions: freezer (-15 - 20 ^o C), refrigerator (0 + 5 ^o C) and room (+20 + 25 ^o C). Every 2 months, 0.1 ml aliquots of 50 ml of borate buffer solution (pH 9.18) were taken and the absorbance was measured at wavelengths of 502, 532 and 654 nm. The measurements were carried out on a Hitachi 557 spectrophotometer. Ratio 502/532 for pure chlorin N-methylglucosamine complex

in water is 3. When the chlorine macrocycle decays to linear tetrapyrroles, the absorption at a wavelength of 654 nm decreases (n, accordingly, the specific extinction decreases) and at the same time the ratio 502/532 decreases due to a simultaneous increase in absorption at both wavelengths (the background absorption increases this wavelength range). Change in control data over 2 months. and for the year are presented in Table 2.

 It is known that a decrease in extinction during storage by about 10% per year for medicinal substances is permissible. In this example, storage in a freezer leads to a decrease in both indicators by about 5% per year, and in a refrigerator, by about 13% per year. At the same time, the filterability of the compounds is preserved (no precipitate forms), which is important for pharmaceuticals.

при хранении в различных температурных условиях в зависимости от времени приведены в Таблице 3.Data on the stability of a 2% aqueous solution of chlorin

when stored in different temperature conditions, depending on the time are shown in Table 3.

в процессе хранения проводили анализ образцов методом тонкослойной хроматографии. Для этого брали аликвоты 0.1 мл, нейтрализовали 1 N соляной кислотой до pH 5 - 6, выпавший хлорин

экстрагировали диэтиловым эфиром, экстракт сушили 1 час безводным сульфатом магния и хлорин

этерифицировали диазометаном, получая хроматографически подвижный эфир. ТСХ проводили на пластинках Kieselgel 60 F254 "Merck" в системе хлороформ/ацетон 20 : 1. Согласно этому анализу, во всех образцах за первые 4 месяца хранения значительно уменьшилось количество примесного феофорбида

и несколько увеличилось количество пурпурина, а в последующие 6 месяцев уменьшалось количество пурпурина и возрастало количество хлорина

Заметное образование полярных продуктов желтого или желто-зеленого цвета началось с 4-го месяца хранения и в наибольшей степени отмечалось в образцах, хранение которых осуществлялось при комнатной температуре. Этот процесс коррелирует с падением экстинкции.Along with measuring the optical properties of solutions of the chlorin N-methylglucosamine complex

during storage, samples were analyzed by thin layer chromatography. For this, 0.1 ml aliquots were taken, neutralized with 1 N hydrochloric acid to pH 5-6, precipitated chlorin

extracted with diethyl ether, the extract was dried for 1 hour with anhydrous magnesium sulfate and chlorine

esterified with diazomethane to give chromatographically mobile ether. TLC was performed on Kieselgel 60 F254 Merck plates in the chloroform / acetone 20: 1 system. According to this analysis, the amount of impurity pheophorbide significantly decreased in all samples during the first 4 months of storage

and the amount of purpurine increased slightly, and in the next 6 months, the amount of purpurine decreased and the amount of chlorin increased

A noticeable formation of polar products of yellow or yellow-green color began from the 4th month of storage and was most pronounced in samples stored at room temperature. This process correlates with a decrease in extinction.

 Not all of the complexes obtained are capable of producing a 2% chlorin solution at physiological pH values (Table 1). So, only complexes with sodium lysate and sodium arginate give such a solution at pH 8.3, and this is not the limit of their solubility. 4-Aminobutanol, glucosamine and N-methylglucosamine are able to form a saturated 2% solution only at pH 8.5.

При pH 8.3 предельная концентрация калиевой соли в воде составила 1.25%, а натриевой - 0.97% причем эти значения являются одними из наименьших (см. Таблицу 1), наряду с солями нейтральных аминокислот и свободными основаниями малоосновных аминокислот.For comparison, potassium and sodium salts of chlorin were obtained

At pH 8.3, the limiting concentration of potassium salt in water was 1.25%, and sodium - 0.97%, and these values are one of the lowest (see Table 1), along with salts of neutral amino acids and free bases of slightly basic amino acids.

 Therefore, such salts are unsuitable for the preparation of dosage forms due, firstly, to low limit concentrations, and secondly, because of the increased tendency to aggregate and form water-insoluble products.

открывает путь к получению эффективного и доступного ФС. Предполагается использование полученных новых соединений в качестве фотосенсибилизаторов для ФДТ рака и при фотодеструкции запустелых сосудов роговицы глаза. Предложенный способ будет реализован при промышленном производстве лекарственных средств "Хлорин" и "Фотодитазин".The advantage of the proposed method lies in the fact that the target products — water-soluble complexes of chlorins — are obtained in a stable form and with a high yield due to simplification of the selection conditions. It allows you to get previously not described chemical compounds. Our Chlorin Stabilization Option

paves the way for efficient and affordable FS. It is supposed to use the obtained new compounds as photosensitizers for PDT cancer and in the photodestruction of desolate vessels of the cornea of the eye. The proposed method will be implemented in the industrial production of medicines "Chlorin" and "Photoditazine".

Claims (2)

1. The method of obtaining water-soluble chlorins by reacting the source of chlorin with a solution of the base, characterized in that as the source of chlorine use a compound of General formula (1)

or 2)

where R ₁ = —CH (OAIk) CH ₃ , —CH = CH ₂ , —CHO, —C (O) CH ₃ ;
R ₂ = H or lower alkyl;
R ₃ = —CH ₃ , —CHO,
and as the base, a linear or cyclic primary or secondary amine, amino acid or amino acid salt, the initial chlorine is pre-precipitated by the gradual addition of water to its acetone solution, and a stoichiometric amount of the base is added directly to the wet precipitate of the initial chlorine, followed by an adjustment of 1.5 - 2% aqueous solution of the target product with a concentrated solution of the appropriate base or concentrated HCl to a pH of 7.5 to 8.5.  2. The method of producing water-soluble chlorins according to claim 1, characterized in that 4-aminobutanol, N-methylglucosamine, glucosamine, arginine, lysine, histidine, sodium or potassium salts of arginine, lysine or histidine are used as the base.

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