RU2807293C1 - Method of obtaining photosensitizer for photodynamic destruction of tumor cells - Google Patents

Abstract

FIELD: organic and pharmaceutical chemistry.

SUBSTANCE: invention relates to a method of producing an organic compound for the photodynamic destruction of tumor cells. A method of producing a photosensitizer for the photodynamic destruction of tumor cells with the specified structural formula is disclosed, the said method includes the interaction of 1 molar equivalent of (2R,3R,4S,5R,6R)-2-(acetoxymethyl)-6-(2-azidoethoxy)tetrahydro-2H-pyran- 3,4,5-triyl triacetate with 1 molar equivalent of 6-(4-ethynylphenyl)-1-(1-(4-nitrophenyl)ethyl)-2,4-diphenyl-1,4-dihydro-1,2,4,5-tetrazin-3(2H)-one in a deoxygenated mixture of tetrahydrofuran and water, obtained at a ratio of 6:3 ml per 1 mmol (2R,3R,4S,5R,6R)-2-(acetoxymethyl)-6-( 2-azidoethoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate in the presence of 0.8 molar equivalents of sodium ascorbate and 0.4 molar equivalents of copper (II) sulfate at a temperature of 70°C with constant stirring in an argon atmosphere for 2 hours, after which the reaction mass is cooled to room temperature, the solvent is removed in vacuo, the residue is dissolved in water and extracted with ethyl acetate, the organic layer is separated, dried using anhydrous magnesium sulfate and then evaporated, the product reactions are isolated by column chromatography on silica gel using hexane and ethyl acetate, then 1 molar equivalent of the resulting product is dissolved in 100 ml of anhydrous methanol per 1 mmol of tetraacetate 1-(1-(4-nitrophenyl)ethyl)-2,4-diphenyl-6-(4-(1-(2-(((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethyl)-1H-1,2,3-triazol-4-yl)phenyl)-1,4-dihydro-1,2,4,5-tetrazin-3(2H)-one and add 1.5 molar equivalents of sodium methoxide, stir at room temperature for 2 hours, the pH of the solution is adjusted to 7 using an ion exchange resin, then the solution is filtered from the ion exchange resin, the resin on the filter is washed with methanol 3 times and the combined methanol solution is evaporated to dryness in vacuum on a rotary evaporator, the target product is isolated by column chromatography on silica gel using dichloromethane and methanol, resulting in 1-(1-(4-nitrophenyl)ethyl)-2,4-diphenyl-6-(4-(1-(2-(((2R,3R,4S, 5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethyl)-1H-1,2,3-triazol-4-yl)phenyl)- 1,4-dihydro-1,2,4,5-tetrazin-3(2H)-one with the general formula C₄₄H₄₄N₈O₁₂.

EFFECT: invention provides a new photosensitizer that exhibits cytotoxic activity when exposed to light with a wavelength of 395–410 nm for 20 minutes, and its solubility in an aqueous solution is 400 μM.

1 cl, 1 dwg, 1 ex

Description

The invention relates to the field of pharmaceutical chemistry and oncology, namely to methods for producing organic compounds for photodynamic destruction of tumor cells.

Photosensitizers are compounds that, upon absorption of light, undergo a chemical and physical change in state, which leads to the appearance of active molecules or fragments that have a damaging biological effect. Substances from different chemical groups have photosensitizing properties, so there are different ways to obtain them.

A known method for producing a photosensitizer [RU 2416614 C2 IPC (2006.01)C07D 487/22, A61P 35/00, A61K 31/409, publ.: 04/20/2011], which consists of treating a suspension of freeze-dried spirulina in methanol or absolute alcohol to obtain alkyl pheophorbide and reacting the latter, for example, with ethylenediamine, preferably in an organic solvent, isolating the corresponding ester of chlorine monoamide e 6, dissolving it in alcohol and adding adipic acid in molar ratio 2:1.

There is a known method for producing a sensitizer for the photodynamic destruction of tumor cells [RU 2771237 C1, IPC (2006.01) C07B 257/08, A61K 31/395, A61K 41/00, A61P 35/00, publ. 04/28/2022], which is 1-(1-(4-nitrophenyl)ethyl)-2,4,6-triphenyl-1,4-dihydro-1,2,4,5-tetrazine-3(2H)- it has the structural formula:

To obtain such a sensitizer, add 76 mg of deoxygenated suspension of copper powder and 86 mg of copper (I) bromide in 30 ml of benzene into a 100 ml two-neck round-bottom flask, and then add 125 μl of N,N,N′,N′′,N′′ -pentamethyldiethylenetriamine. To the resulting mixture is added a deoxygenated solution obtained by dissolving 327 mg of 1,3,5-triphenylverdazyl radical and 276 mg of 1-(1-bromoethyl)-4-nitrobenzene in 20 ml of benzene. The resulting reaction mass is boiled for 8 hours with a Dimroth reflux condenser in an atmosphere of argon supplied from the Schlenk line. After cooling in air to room temperature, the resulting reaction mass is passed through a 3 cm layer of silica gel, and then the silica gel is additionally washed with 15 ml of benzene to completely transfer the product into the mother liquor. The resulting mother liquor is evaporated in a vacuum in a round-bottomed flask using a rotary evaporator to 3-4 ml of the final solution. After this, 50 ml of hexane is added to the flask with the concentrated solution to precipitate the product. The precipitated product is filtered on a Schott filter attached to a Bunsen flask under an absolute pressure of 800 mbar, washed with 100 ml of hexane and air-dried in the dark.

The result is a pale yellow crystalline substance with the formula C ₂₈ H ₂₃ N ₅ O _3.

The photosensitizing properties of this compound appear when exposed to light with a wavelength of 395-410 nm.

However, this sensitizer has low solubility, which limits its potential biological effectiveness.

The technical result of the claimed invention is to expand the arsenal of means for photodynamic destruction of tumor cells

Method for producing a photosensitizer for photodynamic destruction of tumor cells with the structural formula:

,

involves the interaction of 1 molar equivalent of (2R,3R,4S,5R,6R)-2-(acetoxymethyl)-6-(2-azidoethoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate with 1 molar equivalent of 6- (4-ethynylphenyl)-1-(1-(4-nitrophenyl)ethyl)-2,4-diphenyl-1,4-dihydro-1,2,4,5-tetrazin-3(2H)-one in a deoxygenated mixture tetrahydrofuran and water, obtained at a ratio of 6:3 ml per 1 mmol (2R,3R,4S,5R,6R)-2-(acetoxymethyl)-6-(2-azidoethoxy)tetrahydro-2H-pyran-3,4, 5-triyl triacetate) in the presence of 0.8 molar equivalents of sodium ascorbate and 0.4 molar equivalents of copper (II) sulfate at a temperature of 70°C with constant stirring in an argon atmosphere for 2 hours. After this, the reaction mass is cooled to room temperature, the solvent is removed in vacuo, the residue is dissolved in water and extracted with ethyl acetate. The organic layer is separated, dried using anhydrous magnesium sulfate, and then evaporated. The reaction product is isolated by column chromatography on silica gel using hexane and ethyl acetate. Next, 1 molar equivalent of the resulting product is dissolved in 100 ml of anhydrous methanol per 1 mmol of tetraacetate 1-(1-(4-nitrophenyl)ethyl)-2,4-diphenyl-6-(4-(1-(2-(((2R ,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethyl)-1H-1,2,3-triazol-4- yl)phenyl)-1,4-dihydro-1,2,4,5-tetrazin-3(2H)-one and add 1.5 molar equivalents of sodium methoxide, stir at room temperature for 2 hours, the pH of the solution is adjusted to 7, using ion exchange resin. Then the solution is filtered from the ion exchange resin, the resin on the filter is washed with methanol 3 times and the combined methanol solution is evaporated to dryness in vacuum on a rotary evaporator. The target product is isolated by column chromatography on silica gel using dichloromethane and methanol. The result is 1-(1-(4-nitrophenyl)ethyl)-2,4-diphenyl-6-(4-(1-(2-(((2R,3R,4S,5S,6R)-3,4 ,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethyl)-1H-1,2,3-triazol-4-yl)phenyl)-1,4-dihydro-1, 2,4,5-tetrazin-3(2H)-one with the general formula C ₄₄ H ₄₄ N ₈ O ₁₂ .

The resulting photosensitizer exhibits cytotoxic activity when exposed to light with a wavelength of 395 - 410 nm for 20 minutes.

The solubility of the resulting photosensitizer in an aqueous solution is 400 μM, which significantly exceeds this parameter of the sensitizer obtained by the prototype method, the solubility of which is 25 μM.

In fig. Figure 1 shows the viability of tumor cells after photodynamic exposure in the presence of a photosensitizer.

To obtain the photosensitizer, 501 mg of (2R,3R,4S,5R,6R)-2-(acetoxymethyl)-6-(2-azidoethoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (417 mg) was added to the flask ) and 6-(4-ethynylphenyl)-1-(1-(4-nitrophenyl)ethyl)-2,4-diphenyl-1,4-dihydro-1,2,4,5-tetrazine-3(2H)- it was evacuated for 1 hour at 1 mbar and then filled with argon using a Schlenk line. 6 ml of tetrahydrofuran and 3 ml of water, previously deoxygenated by bubbling argon, were added into the flask through a septum with a syringe, then 159 mg of sodium ascorbate and 100 mg of copper (II) sulfate were added with stirring, the resulting reaction mass was kept at a temperature of 70°C with constant stirring in an argon atmosphere within 2 hours. After this, the reaction mass was cooled to room temperature, the solvent removed in vacuo on a rotary evaporator, the residue was dissolved in water and extracted with ethyl acetate 4 times 100 ml, the organic layer was separated, dried using anhydrous magnesium sulfate and then evaporated on a rotary evaporator in vacuum to a volume of 5 ml. The reaction product was isolated using silica gel column chromatography in a solvent gradient from hexane/ethyl acetate = 2/1 to hexane/ethyl acetate = 1/5. After evaporation and vacuum, a gray powder weighing 573 mg was obtained, which is 62% of the theoretical yield of 1-(1-(4-nitrophenyl)ethyl)-2,4-diphenyl-6-(4-(1-(2- (((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethyl)-1H-1,2,3- triazol-4-yl)phenyl)-1,4-dihydro-1,2,4,5-tetrazin-3(2H)-one.

Next, the resulting powder weighing 460 mg was dissolved in 50 ml of anhydrous methanol and 40 mg of sodium methoxide was added, the resulting solution was stirred at room temperature for 2 hours. After this, the pH of the solution was adjusted to 7 using Amberlist® H15 ion exchange resin, after which the solution was filtered on a Schott filter from the ion exchange resin, the resin on the filter was washed with methanol 3 times 20 ml each, after which the combined methanol solution was evaporated to dryness in vacuum on a rotary evaporator .

Isolation of the target product was carried out using column chromatography on silica gel in a solvent gradient from dichloromethane/methanol 10/1 to dichloromethane/methanol 5/1, which after evaporation and vacuum gave a gray powder weighing 377 mg, which is 86% of the theoretical yield of 1-( 1-(4-nitrophenyl)ethyl)-2,4-diphenyl-6-(4-(1-(2-(((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6 -(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethyl)-1H-1,2,3-triazol-4-yl)phenyl)-1,4-dihydro-1,2,4,5- tetrazin-3(2H)-one with the general formula C ₄₄ H ₄₄ N ₈ O ₁₂ .

The photosensitizing properties of the resulting product were assessed by the level of cytotoxic effects on the culture of human prostate cancer tumor cells (PC-3 line) when exposed to light with a wavelength of 395-410 nm. To do this, cells were seeded into a 96-well plate at a concentration of 5000 cells per well in phenol-free nutrient medium RPMI 1640, supplemented with 10% fetal bovine serum and antibiotics. Next, the cells were incubated for 24 hours at 37°C in an environment of 5% carbon dioxide. Then the resulting product was added. Serial dilutions of the resulting product were preliminarily prepared in the RPMI 1640 nutrient medium for the cultivation of cell cultures in the concentration range of 250-25 μg/ml. Next, the nutrient medium in the plate with cells was replaced with a similar one, but containing the resulting product at concentrations of 250, 150, 100, 50, 25 μg/ml in 6 repetitions. Wells with cells in which the medium was changed to fresh without adding the compound were used as a control group. Cell-free medium was used as a positive control.

Next, the plate with cells and the added product was exposed to light for 20 minutes. Emission source: LED matrix consisting of 5 columns of 7 LEDs per column with interdiode distances of 18 mm in rows and columns, emission range 395-410 nm (Thorlabs LED395L). The distance from the LED to the well with tumor cells was 2 cm. The radiation power at the cell level was 5.5 mW/cm ² , the maximum radiation was 395 nm.

A second similar plate containing cells was not exposed to light, but was subjected to all other culture procedures to assess the intrinsic toxicity of the resulting product.

After light exposure, the cells were again placed in a CO ₂ incubator at 37°C in an environment of 5% carbon dioxide for 24 hours.

After completion of cell incubation, culture viability was assessed using the MTT test. To do this, the nutrient medium was removed from each well and 100 μl of a solution of MTT reagent (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) in RPMI 1640 nutrient medium was added, then the plates were placed in CO ₂ incubator at 37°C for 4 hours. The media was then completely removed from the plates and 100 μL of dimethyl sulfoxide was added to each well to dissolve the formed formazan crystals. After this, the optical density of the samples was measured using a microplate photocolorimeter (MULTISCAN FC) at a wavelength of 570 nm. Cell viability was calculated using the formula:

viability%= (OP _reverse -OP _empty )/(OP _counter -OP _empty )*100,

where, OD _arr is the average optical density of cell samples with added photosensitizer;

OP _{is empty} - the average optical density of the medium without cells;

OD _control - average optical density of control (cells not exposed to the sensitizer).

The results of determining the level of viability of PC-3 cell culture after photodynamic irradiation using the resulting product and a control group without irradiation are presented in Fig. 1. The percentage of viable cells after photodynamic exposure in the presence of the resulting photosensitizer at concentrations of 250-25 μg/ml is statistically lower than the control level without a photosensitizer, and the corresponding control points with the addition of a photosensitizer, but without exposure to light (p<0.001).

The solubility of the photosensitizer obtained by the proposed method and the sensitizer obtained by the prototype method was determined as follows: a 20 mg sample of the compound was placed in a 50 ml volumetric flask, the flask was filled with distilled water to the mark, and then placed in an ultrasonic bath for 20 minutes. After this, the dispersion was poured into a centrifugation tube and the undissolved photosensitizer or the prototype sensitizer was precipitated in a centrifuge at 10,000 rpm. The supernatant was carefully poured into a volumetric flask to remove any undissolved sediment from the flask, after which centrifugation was repeated. After removing the supernatant, the precipitate was dried first in air and then at a pressure of 0.5 mbar, after which the precipitate was weighed and the concentration of the dissolved substance was determined. The solubility of the photosensitizer obtained by the proposed method in an aqueous solution was 400 μM, and the solubility of the sensitizer obtained by the prototype method was 25 μM.

Claims (3)

Method for producing a photosensitizer for photodynamic destruction of tumor cells with a structural formula , involving the interaction of 1 molar equivalent of (2R,3R,4S,5R,6R)-2-(acetoxymethyl)-6-(2-azidoethoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate with 1 molar equivalent of 6- (4-ethynylphenyl)-1-(1-(4-nitrophenyl)ethyl)-2,4-diphenyl-1,4-dihydro-1,2,4,5-tetrazin-3(2H)-one in a deoxygenated mixture tetrahydrofuran and water, obtained at a ratio of 6:3 ml per 1 mmol (2R,3R,4S,5R,6R)-2-(acetoxymethyl)-6-(2-azidoethoxy)tetrahydro-2H-pyran-3,4, 5-triyl triacetate) in the presence of 0.8 molar equivalents of sodium ascorbate and 0.4 molar equivalents of copper (II) sulfate at a temperature of 70°C with constant stirring in an argon atmosphere for 2 hours, after which the reaction mass is cooled to room temperature, the solvent is removed in vacuo, the residue is dissolved in water and extracted with ethyl acetate, the organic layer is separated, dried using anhydrous magnesium sulfate and then evaporated, the reaction product is isolated by column chromatography on silica gel using hexane and ethyl acetate, then 1 molar equivalent of the resulting product is dissolved in 100 ml of anhydrous methanol per 1 mmol of tetraacetate 1-(1-(4-nitrophenyl)ethyl)-2,4-diphenyl-6-(4-(1-(2-(((2R,3R,4S,5S,6R) -3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethyl)-1H-1,2,3-triazol-4-yl)phenyl)-1,4- dihydro-1,2,4,5-tetrazin-3(2H)-one and add 1.5 molar equivalents of sodium methoxide, stir at room temperature for 2 hours, adjust the pH of the solution to 7 using an ion exchange resin, then filter the solution from the ion exchange resin, the resin on the filter is washed with methanol 3 times and the combined methanol solution is evaporated to dryness in vacuum on a rotary evaporator, the target product is isolated by column chromatography on silica gel using dichloromethane and methanol, resulting in 1-(1-(4-nitrophenyl) ethyl)-2,4-diphenyl-6-(4-(1-(2-(((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H -pyran-2-yl)oxy)ethyl)-1H-1,2,3-triazol-4-yl)phenyl)-1,4-dihydro-1,2,4,5-tetrazine-3(2H)- it has the general formula C ₄₄ H ₄₄ N ₈ O ₁₂ .