RU2670087C1 - Photosensitizer for photodynamic therapy of prostate cancer and method fr manufacturing thereof - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: present invention relates to a photosensitizer for photodynamic therapy of prostate cancer. Photosensitizer has the structural formula (1)

,

where as R₁ can be hydrogen (H), sodium (Na), potassium (K), C₁-C₂ – alkyl, as R₂ can be a compound of general formula C_xH_2x, where x=4÷17, as R₃ may be hydrogen (H), sodium (Na) or potassium (K). Method for preparing a photosensitizer and a medicament for treating prostate cancer are also provided.

EFFECT: invention makes it possible to obtain a photosensitizer that is highly effective in carrying out PDT of prostate cancer.

5 cl, 1 tbl, 3 ex

Description

The present invention relates to the pharmaceutical industry, in particular to the development of new anti-cancer drugs.

Prostate cancer is one of the most common malignancies in men. Over 400,000 cases of prostate cancer are diagnosed worldwide every year; in a number of countries, it takes the 2nd or 3rd place in the structure of oncological diseases after lung cancer and stomach cancer (for example, in Europe in 2004 the incidence of prostate cancer was 214 cases per 100,000 men, as a result of which prostate cancer occupied 2- e is among the leading causes of cancer death in men, ahead of lung cancer and colorectal cancer). According to global statistics, in 2012 prostate cancer was reported in 1.1 million men, and at the same time led to 307 thousand deaths. Prostate cancer causes nearly 10% of cancer deaths in men and is one of the main causes of death in older men. In the United States, prostate cancer is the third leading cause of death from malignant tumors [https://ru.wikipedia.org/wiki/Prostate cancer].

Photodynamic therapy (PDT) is a method of treating cancer, some skin diseases or infectious diseases, based on the use of photosensitive substances - photosensitizers (including dyes), and, as a rule, visible light of a certain wavelength [https: //ru.wikipedia .org / wiki / Photodynamic therapy]. The sensitizer is injected into the body most often intravenously, but can be used orally or orally. Substances for PDT have the property of selective accumulation in a tumor or other target tissues (cells). Then, the tissues affected by the pathological process are irradiated with light with a wavelength corresponding to the maximum absorption of the dye. Currently, laser installations are used as a light source, which make it possible to emit light of a certain wavelength and high intensity. The absorption of light quanta by the photosensitizer molecules in the presence of oxygen leads to a photochemical reaction, as a result of which molecular triplet oxygen turns into singlet oxygen, and also a large number of highly active radicals are formed. Singlet oxygen and radicals cause necrosis and apoptosis in the tumor cells (two variants of cell death). PDT also leads to malnutrition and death of the tumor due to damage to its microvessels. Photo dynamic therapy - technology of the XXI century // Physiotherapy, balneology and rehabilitation: journal. - 2013. - No. 1. - S. 36-43.].

At present, photosensitizers (PS) of various classes (porphyrins and their metal complexes, chlorins, benzoporphyrins, phthalocyanines, etc.) are used in the clinic or are at different stages of clinical trials. Of particular interest are natural chlorophylls and their derivatives with intense absorption in the near infrared region of the spectrum. Light with a wavelength of 650-700 nm penetrates into tissues to a depth of 20-25 mm, this significantly expands the possibilities of the currently used methods of treatment of large and deep-lying tumors. The basis of most modern photosensitizers is the structure of chlorin eb, obtained from natural raw materials.

One common photosensitizer is the commercially available Photofrin II® [Dougherty, T.J. Hematoporphyrin as a photosensitizer of tumors / T.J. Dougherty // Photochem. Photobiol. - 1983. - V.38, No. 3 - P. 377-79]. This porphyrin sensitizer actually consists of several porphyrin dimers and chains. The drug is activated at several wavelengths, but 630 nm is clinically optimal for tissue penetration.

The domestic counterpart of Photofrin is Photogem [RF Patent No. 2128993, 1999]. Another clinically successful porphyrin photosensitizer used in gynecology is 5-aminolevulinic acid, which, when administered, changes heme metabolism and leads to the production of protoporphyrin IX [Musiol R, Serda M, Polanski J (2011). "Prodrugs in photodynamic anticancer therapy." Curr. Pharm. Des. 17 (32): 3548-59.]. In Russia it is issued under the name "Alasens". Another domestic drug is "Photoditazine", which is a salt of chlorin e 6 with alkali metals and N-methyl-D-glucamine [RF patent 2276976, publ. 2006].

The synthetic chlorin-based photosensitizer is Foscan (Temoporfin) is also used successfully in clinical and biological research [Bonnett, R. Hydroporphyrins of the mesotetra (hydroxyphenyl) poprhyrin series as tumor photosensitizers / R. Bonnett, R.D. White, U.J. Winfield, C. Berenbaum // Biochem. J. - 1989. - V. 261. - P. 277-80].

Third-generation drugs include Tookad and Tookad Soluable (WST11), which are derivatives of natural bacteriochlorophyll a and can be used for a wide range of deep-seated neoplasms [Taneja, S.S. Final Results of a Phase I / II Multicenter Trial of WST11 Vascular Targeted Photodynamic Therapy for Hemi-Ablation of the Prostate in Men with Unilateral Low Risk Prostate Cancer Performed in the United States / S.S. Taneja, J. Bennett, J. Coleman // J Urol. - 2016. - V. 196, No. 4. - P. 1096-104].

The prior art also knows the use of small vector ligands that provide targeted delivery of FS to tumor tissues. Thus, the PSMA ligand is known, which is a vector peptidomimetic for PSMA receptors overexpressed on the surface of a number of prostate tumors. Based on it, targeted radiopharmaceuticals are known for radiotherapy and diagnostics [Patent WO 2015171792 A1, Metal / radiometal-labeled psma inhibitors for psma-targeted imaging and radiotherapy].

The closest analogue of the claimed invention is the photosensitizer "Radachlorin" (Radafarma LLC) [https://www.rlsnet.ru/tn_index_id_24446.htm], which is an aqueous solution of a mixture of various chlorins of variable composition, in which the content of the main product is trisodium salt of chlorin e ₆ accounts for 80-90% of the total amount of all chlorins [RF patent No. 2183956].

The disadvantages of the drug "Radachlorin" should include low efficiency. This drawback is due to the low selectivity of the accumulation of the drug in a tumor of the prostate gland.

Thus, the objective of the claimed invention is the creation of a photosensitizer that is highly effective in PDT of prostate cancer, as well as expanding the arsenal of this class of drugs.

The solution to this problem provides a photosensitizer for photodynamic therapy with the formula (1):

where R ₁ can be hydrogen (H), sodium (Na), potassium (K), C ₁ -C ₂ alkyl, R ₂ can be a compound of the general formula C _x H _2x , where x = 4 ÷ 17, hydrogen (H), sodium (Na) or potassium (K) may be R ₃ .

The effectiveness of the claimed photosensitizer is due to a synergistic effect due to the presence in the structure of the molecule of a vector fragment that binds to PSMA receptors in prostate cancer cells in the body, as well as a high level of photoinduced activity of the claimed compound.

The PSMA ligand and the chlorin e ₆ molecule are connected to each other via a spacer that separates the photoactive and target components in space. This spacer can be of a different nature (alkyl, peptide, polyethylene glycol, etc.) and does not significantly affect the effectiveness of the inventive photosensitizer.

To a large extent, the solution of the problem provides a method of obtaining the above photosensitizer, including:

- a cyclopentane ring opening reaction of a compound with the structural formula (2)

in an organic solvent by heating to obtain a compound of formula (3);

- adding zinc acetate to the compound with the structural formula (3) to obtain a compound with the structural formula (4);

- adding compounds with structural formula (5)

to the compound (6)

in an organic solvent in the presence of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide to give a compound with structural formula (7);

- exposure to a compound with structural formula (7) trifluoroacetic acid in an organic solvent to obtain a compound with structural formula (S);

- the reaction of a compound with structural formula (4) with a compound with structural formula (S) in the presence of copper iodide to obtain a compound with structural formula (9);

- exposure to a compound with structural formula (9) with trifluoroacetic acid in dimethyl sulfoxide to give a crude compound with structural formula (1);

- washing the crude compound with the structural formula (1) to obtain the claimed photosensitizer.

where R ₁ can be hydrogen (H), sodium (Na), potassium (K), C ₁ -C ₂ alkyl, R ₂ can be a compound of the general formula C _x H _2x , where x = 4 ÷ 17, hydrogen (H), sodium (Na) or potassium (K) may be R ₃ .

Alternatively, the claimed method may further include the step of freeze drying the resulting photosensitizer to obtain its crystalline form.

The synthesis strategy is to realize the reaction of a copper-catalyzed azide-alkyne cycloaddition. This approach allows introducing a spacer group into the composition of the molecule and obtaining the target product with high yields. The zinc complex of dimethyl ether 13 ¹ - (propargylcarbomoyl) isochlorin e ₄ was obtained as an alkyne component. The azide component was obtained by acylating the terminal amino group of the aforementioned PSMA ligand, prepared according to the previously described method, starting from the protected derivatives of lysine and glutamic acid using triphosgene. After removal of the gareg-butyl groups, a click reaction was carried out to give the compound. The main difficulty in the click reaction was the choice of solvent, since the PSMA ligand is poorly soluble in both non-polar and polar solvents, and is easily esterified in the presence of alcohols. The use of highly polar solvents when working with chlorins leads to oxidation and, as a result, to contamination of the product. The reaction was carried out in DMF, which was thoroughly degassed. After the reaction, the target product was crystallized using acetonitrile, followed by washing the crystals with a mixture of acetonitrile, water and hexane.

The choice of natural pigment for the creation of PS is determined by a number of factors, including their prevalence in nature, the intensity of absorption in the long-wavelength region of the spectrum, and structural proximity to endogenous porphyrins. The presence of these properties suggests a relatively low level of toxicity of such compounds and their rapid excretion from the body.

The claimed invention also provides a medicament for the treatment of prostate cancer, including the claimed photosensitizer and pharmaceutically acceptable additives.

Alternatively, as a pharmaceutically acceptable additive, the claimed drug may include an isotonic solution.

The invention is illustrated by the following examples.

Example 1. Obtaining the claimed photosensitizer.

Pheophorbide a methyl ester (40 mg) and propargylamine (200 μl) were dissolved in methylene chloride (2 ml) with the addition of diisopropylethylamine (100 μl) and stirred for 48 hours. Upon completion of the reaction, the mixture was diluted with methylene chloride (50 ml) and washed with 6% hydrochloric acid solution. The organic layer was separated, dried over sodium sulfate and evaporated under reduced pressure. Purification was carried out using silica gel column chromatography (chloroform / methanol, 50/1, v / v) to obtain 43 mg of methyl ester 13 ¹ - (propargylcarbamoyl) isochlorin e ₄ . The resulting product was dissolved in methylene chloride (2 ml) with the addition of a suspension of zinc acetate (50 mg) in methanol (500 μl). The mixture was stirred for 30 minutes. After that, saturated sodium chloride solution was added to the reaction, and the mixture was filtered through a Schott filter. The mixture was diluted with dichloromethane (100 ml) and washed with distilled water. The organic layer was dried over sodium sulfate and evaporated under reduced pressure. As a result, 34 mg of the zinc complex of methyl ester 13 ¹ - (propargylcarbamoyl) isochlorin e ₄ was obtained.

To a solution of 11-azidoundecanoic acid (173 mg) in DMF (4 ml) was added EDC (78.5 mg) and N-hydroxy succinimide (58.3 mg) with stirring. After 60 minutes, (S) -di-tert-butyl-2- (3 - ((S) -6-amino-1- (tert-butoxy) -1-oxohexane) ureido) pentadioate (70 mg) was added to the mixture. . The reaction was carried out for 48 hours, after which the mixture was diluted with methylene chloride (70 ml) and washed with sodium chloride solution. The organic layer was dried over sodium sulfate and evaporated on a rotary evaporator. The product was purified by silica gel column chromatography (hexane / ethyl acetate, 3/2, v / v) to obtain 73 mg of (S) -di-tert-butyl-2- (3 - ((S) -6- (11- azidooundecananamido) -1- (tert-butoxy) -1-oxohexane) ureido) pentadioate, which was subsequently dissolved in 11% solution of trifluoroacetic acid in methylene chloride (5 ml). The mixture was stirred for 16 hours and then evaporated and purified by reverse phase flash chromatography (acetonitrile-water gradient). 48 mg of (S) -2- (3 - ((S) -5- (11-azidoundecananamido) -1-carboxypentyl) ureido) pentanedioic acid were obtained.

The cycloaddition reaction of the zinc complex of methyl ester 13 ¹ - (propargylcarbamyl) isochlorin e ₄ (33.7 mg) and (S) -2- (3 - ((S) -5- (11-azidoundecananamido) -1-carboxypentyl) ureido) pentanedioic acid (26 mg;) was carried out in DMF supplemented with DIPEA. The reaction was catalyzed by copper iodide (0.4 mg) with stirring for 16 hours. The end of the reaction was fixed by analytical TLC. The reaction mixture was evaporated and the dry residue was washed with acetonitrile, a mixture of acetonitrile - water and hexane. The resulting product was redissolved in a 10% solution of trifluoroacetic acid in DMSO and stirred for 30 minutes. Then the mixture was diluted with two volumes of water and the precipitate was separated by centrifugation. The precipitate was washed three times with distilled water, and then dried under low pressure. The yield was 59 mg.

1 H NMR (400 MHz, CDCl 3, δ, ppm): 9.52 (s, 1H), 9.48 (s, 1H), 9.32 (br s, 1H), 8.65 (s, 1H), 8.23 (w, 3H), 7.72 (br s, 1H), 6.25-6.18 (dd, 1H), 6.02-5.98 (dd, 1H), 5.42-5.35 (d, 1H), 5.14-5.07 (d, 1H), 4.45 (m, 4H), 4.30-4.27 (d, 1H), 3.81-3.79 (s, 3H), 3.64 (s, 3H), 3.53 (s, 3H), 2.98 (br s, 2H), 2.73 (s, 1H), 2.38-1.83 (m, 10H), 1.66 (t, 6H), 1.59 (d, 6H), 1.54-1.42 (m, 8H), 1.28 (m, 20H) 1.15 (s, 3H), 0.88-0.76 (m, 2H).

MALDI MS: m / z calculated [M + H] 1190.41; found 1190.52

UV / VIS (H2O), λmax, nm (ε, M-1 cm-1): 390 (132000), 502 (12000), 530 (4800), 607 (4700), 662 (46000)

Thus, the claimed photosensitizer was obtained.

Example 2. Obtaining the claimed photosensitizer.

Pheophorbide a (50 mg) and propargylamine (200 μl) were dissolved in methylene chloride (4 ml) with the addition of diisopropylethylamine (100 μl) and stirred for 72 hours. Upon completion of the reaction, the mixture was diluted with methylene chloride (70 ml) and washed with 6% hydrochloric acid solution. The organic layer was separated, dried over sodium sulfate and evaporated under reduced pressure. Purification was carried out using silica gel column chromatography (chloroform / methanol, 25/1, v / v) to obtain 49 mg of 13 ¹ - (propargylcarbamoyl) isochlorin e ₄ . The resulting product was dissolved in methylene chloride (3 ml) with the addition of a suspension of zinc acetate (50 mg) in methanol (500 μl). The mixture was stirred for 25 minutes, after which saturated NaHCO ₃ solution was added to the reaction, and the mixture was filtered through a Schott filter. The mixture was diluted with dichloromethane (100 ml) and washed with distilled water. The organic layer was dried over sodium sulfate and evaporated on a rotary evaporator. As a result, 44 mg of the zinc complex 13 ¹ - (propargylcarbamoyl) isochlorin e ₄ was obtained.

To a solution of 6-azidohexanoic acid (184 mg) in DMF (4 ml) was added EDC (117.8 mg) and N-hydroxy succinimide (87.5 mg) with stirring. After 60 minutes, (S) -di-tert-butyl-2- (3 - ((S) -6-amino-1- (tert-butoxy) -1 oxohexane) ureido) pentadioate (105 mg) was added to the mixture. The reaction was carried out for 48 hours, after which the mixture was diluted with methylene chloride (100 ml) and washed with sodium chloride solution. The organic layer was dried over sodium sulfate and evaporated on a rotary evaporator. The product was purified by silica gel column chromatography (hexane / ethyl acetate, 3/2, v / v) to obtain 95 mg of (S) -di-tert-butyl-2- (3 - ((S) -6- (6- azidogeksanamido) -1- (tert-butoxy) -1-oxohexane) ureido) pentadioata, which subsequently was dissolved in 12% by _volume solution of trifluoroacetic acid in methylene chloride (6 ml). The mixture was stirred for 16 hours and then evaporated and purified by reverse phase flash chromatography (acetonitrile-water gradient). 48 mg of (S) -2- (3 - ((S) -5- (6-azidohexanamido) -1-carboxypentyl) ureido) pentanedioic acid were obtained. Zinc complex cycloaddition reaction 13 ¹ - (propargylcarbamoyl) isochlorin e ₄ (44 mg) and (S) -2- (3 - ((S) -5- (6-azidohexanamido) -1-carboxypentyl) ureido) pentanedioic acid (21 mg) was carried out in DMF supplemented with DIPEA. The reaction was catalyzed by copper iodide (0.5 mg) with stirring for 16 hours. The end of the reaction was fixed by analytical TLC. The reaction mixture was evaporated, the dry residue was washed with acetonitrile, a mixture of acetonitrile-water and hexane. The resulting product was redissolved in a 10% solution of trifluoroacetic acid in DMSO and stirred for 30 minutes. Then the mixture was diluted with two volumes of water and the precipitate was separated by centrifugation. The precipitate was washed three times with distilled water, and then dried under low pressure. The yield was 59 mg.

¹ H NMR (400 MHz, CDCl 3, δ, ppm): 9.52 (s, 1H), 9.48 (s, 1H), 9.32 (br s, 1H), 8.65 (s, 1H), 8.23 (m, 3H), 7.72 (br s, 1H), 6.25-6.18 (dd, 1H), 6.02-5.98 (dd, 1H), 5.42-5.35 (d, 1H), 5.14-5.07 (d, 1H), 4.45 (m, 4H) 4.30-4.27 (d, 1H), 3.81-3.79 (s, 3H), 3.64 (s, 3H), 3.53 (s, 3H), 2.98 (br s, 2H), 2.73 (s, 1H), 2.38- 1.83 (m, 8H), 1.78 (m, 1H), 1.66 (t, 6H), 1.59 (d, 6H), 1.54-1.42 (m, 6H), 1.28 (m, 10H) 1.15 (s, 3H ), 0.88-0.76 (m, 2H).

MALDI MS: m / z calculated [M + H] 1106.25; found 1106.28

UV / VIS (H ₂ O), λ _max , hm (ε, M ^-1 cm ^-1 ): 390 (134000), 502 (13000), 530 (4700), 607 (4600), 662 (49000)

Thus, the claimed photosensitizer was obtained.

Example 3. The effectiveness of the claimed photosensitizer against tumor cells of the prostate (22Rv1).

Purpose of the study

The aim of the study is to evaluate the effectiveness of the claimed photostabilizer in vitro.

Materials and methods

To assess the effectiveness of the claimed photostabilizer in vitro, a prostate tumor cell culture (22Rv1) was used. This culture exhibits overexpression of PSMA receptors on the surface.

Cells were scattered into the wells of a flat-bottomed 96-well microplate (Costar, USA) in the amount of 15 × 10 (22Rv1) cells per well. Test compounds — the claimed photosensitizer (group 1) and the Radachlorin preparation as a positive control (group 2) were introduced 24 hours after seeding, varying the concentration of the declared photosensitizer from 0.07 to 17 μM, and the Radachlorin preparation from 1.9 up to 30 μM. To assess the effectiveness after 0.5, 1, 2, 4 and 6 hours of incubation with photosensitizers, the cells were irradiated with a halogen lamp through a KS-10 broadband filter (λ≥620 nm). The power density was 16.2 ± 0.7 mW / cm ² , the calculated light dose was 10 J / cm ² . After irradiation, the cells were incubated under standard conditions for a day.

Negative controls were unexposed cells. Cell survival was evaluated using a colorimetric MTT assay [Carmichael J., DeGraff W.G., Gazdar A.F., Minna J.D., Mitchell J. Evaluation of a tetrazolium-based semiautomated colorimetric assay: assessment of chemosensitivity testing. Canser res. 47: 936-942; 1987], according to the results of which the IC50 value was calculated, the PS concentration at which 50% cell death was observed after exposure. Quantitative parameters were calculated according to the results of three independent tests.

For statistical analysis of the obtained results, Student's criterion was used.

results

When studying the effectiveness of the claimed photosensitizer in comparison with the drug "Radachlorin" it was found that both PS are active against prostate tumor cells (Table 1). The maximum effectiveness of the claimed photosensitizer (group 1) was determined after 6 hours of incubation of FS with cells, the drug "Radachlorin" (group 2) after 4-6 hours of incubation. The IC ₅₀ for group 1 for 22Rv1 cells was 1.2 ± 0.1 μM, which is probably due to the high expression of PSMA receptors in this cell culture. It should be noted that the drug "Radachlorin" (group 2) showed lower efficiency on 22Rv1 cells (IR ₅₀ = 3.0 ± 0.2 μM) compared with the claimed photosensitizer.

* - significant difference (p <0.5) from group 2 Conclusions

Based on the data obtained, it can be concluded that the claimed photosensitizer with respect to prostate tumor cells (22Rv1) exhibiting overexpression of PSMA receptors is highly effective (compared with the Radachlorin preparation), which indicates the high efficiency of the claimed photosensitizer during PDT of prostate cancer .

Claims (26)

1. Photosensitizer for photodynamic therapy of prostate cancer with the structural formula (1)

where R ₁ can be hydrogen (H), sodium (Na), potassium (K), C ₁ -C ₂ is alkyl, R ₂ can be a compound of the general formula C _x H _2x , where x = 4 ÷ 17 , as R ₃ can be hydrogen (H), sodium (Na) or potassium (K). 2. A method of producing a photosensitizer according to claim 1, including: - a cyclopentane ring opening reaction of a compound with the structural formula (2)

in an organic solvent by heating to obtain a compound of formula (3);

- adding zinc acetate to the compound with the structural formula (3) to obtain a compound with the structural formula (4);

- adding compounds with structural formula (5)

to the compound (6)

in an organic solvent in the presence of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide to give a compound with structural formula (7);

- exposure to a compound with structural formula (7) trifluoroacetic acid in an organic solvent to obtain a compound with structural formula (8);

- the reaction of a compound with structural formula (4) with a compound with structural formula (8) in the presence of copper iodide to obtain a compound with structural formula (9);

- exposure to a compound with structural formula (9) with trifluoroacetic acid in dimethyl sulfoxide to give a crude compound with structural formula (1); - washing the crude compound with structural formula (1) to obtain a photosensitizer according to claim 1, where R ₁ can be hydrogen (H), sodium (Na), potassium (K), C ₁ -C ₂ is alkyl, R ₂ can be a compound of the general formula C _x H _2x , where x = 4 ÷ 17 , as R ₃ can be hydrogen (H), sodium (Na) or potassium (K). 3. The method according to p. 2, characterized in that it includes the step of freeze drying the resulting photosensitizer according to p. 1 to obtain its crystalline form. 4. A drug for the treatment of prostate cancer, comprising the photosensitizer according to claim 1 and pharmaceutically acceptable additives. 5. The drug according to claim 4, characterized in that an isotonic solution is used as pharmaceutically acceptable additives.