RU2789401C1 - CARBORANE DERIVATIVES OF 4,4-DIFLUORO-4-BORA-3α,4α-DIASE-s-INDACENE SHOWING ANTI-TUMOR ACTIVITY - Google Patents

Abstract

FIELD: carborane derivatives.

SUBSTANCE: invention relates to new carborane derivatives of 4,4-difluoro-4-boron-3α,4α-diaza-s-indacene of general formula I, where

EFFECT: invention consists in obtaining new carborane-containing derivatives of 4,4-difluoro-4-boron-3α,4α-diaza-s-indacene, exhibiting antitumor activity, and expanding the arsenal of agents for PDT and BNCT.

2 cl, 1 tbl, 6 ex

Description

The invention relates to new compounds, namely to 4,4-difluoro-4-boron-3α,4α-diaza-s-indacene derivatives of general formula I, in particular to their carborane derivatives exhibiting antitumor activity:

,

,

Where

These compounds can be most effectively used as photo/radiosensitizers in photodynamic therapy (PDT) and boron neutron capture therapy (BNCT) of oncological diseases.

These compounds of formula I, their properties and method of obtaining in the literature are not described.

PDT is a type of chemotherapy and consists in the local activation of a photosensitizer (PS) introduced into the tumor with visible red light, which, in the presence of tissue oxygen, leads to the development of a photochemical reaction that destroys tumor cells. PDT avoids systemic (general) effects on the body, which are typical for conventional chemotherapy.

Photodynamic therapy has been known since 1975 [Dougherty, T.G.; Grindey, G.B.; Fiel, R. J. Natl. Cancer Inst., 1975, 55, 115-121], and in subsequent years was widely used in the USA, Europe, China and Japan. PDT has been used in the Russian Federation since 1996.

The successful application of the PDT method for the treatment of malignant neoplasms stimulates the search for new photosensitizers with improved properties.

J. Photochem. Photobiol. C: Photochem. Rev., 2019, 40,21-48].It is known that some derivatives of 4,4-difluoro-4-boron-3α,4α-diaza-s-indacene can significantly increase the generation of reactive oxygen species with higher quantum yields and lifetime of active particles, compared with derivatives of porphyrins and chlorins, currently used in the clinic for PDT [A. Kamkaew, S. H. Lim, H. B. Lee, LV Kiew, LY Chung, K. Burgess. Chem. soc. Rev., 2013, 42, 77-88]. It is known that some derivatives of 4,4-difluoro-4-boron-3α,4α-diaza-s-indacene can significantly increase the generation of reactive oxygen species with higher quantum yields and lifetime of active particles, compared with derivatives of porphyrins and chlorins, currently used in the clinic for PDT [A. Kamkaew, S. H. Lim, H. B. Lee, LV Kiew, LY Chung, K. Burgess. Chem. soc. Rev., 2013, 42, 77-88]. Further in this application, the generally accepted technical name of 4,4-difluoro-4-boron-3α,4α-diaza-s-indacene - BODIPY [Tram K.; Yan H.; Jenkins n. A.; Vassiliev S.; Bruce D. Dyes and Pigments, 2009, 82, 392-395]. In vivo studies published to date show that a number of BODIPY derivatives have an increased ability to selectively suppress tumor growth without significant damage to healthy tissues [M. L. Agazzi, M. B. Ballatorea, A. M. Durantini, E. N. Durantini,

J. Photochem. photobiol. C: Photochem. Rev., 2019, 40,21-48].

BNCT is a promising method for the therapeutic treatment of malignant tumors by accumulating a stable isotope of boron-10 in them and subsequent irradiation with thermal neutrons. The high-energy α-particles and ⁷ Li recoil nuclei (200 and 350 keV µm ^-1 , respectively) generated by the ¹⁰ Β(n,α) ⁷ Li reaction with a free path comparable to the cell diameter cause lethal damage to tumor cells that have accumulated a sufficient amount of boron-containing substance. Damage to surrounding non-tumor cells is minimal. In order to successfully implement the unique possibilities of BNCT in clinical practice, it is necessary to solve a complex of complex chemical, biological, medical, and technical problems; first of all, it is necessary to create boron-containing compounds capable of selectively delivering a therapeutic amount of boron to malignant tumors (20 μg/g of a tumor) with a concentration gradient of 3: 1 (tumor to normal tissue) or higher, ensuring its optimal microdistribution and the ability to remain in the tumor for the time required for irradiation [RF Barth, Μ. G. H. Vicente, O. K. Harling, W. S. Kiger III, K. J. Riley, P. J. Binns, F. M. Wagner, M. Suzuki, T. Aihara, I. Kato, S. Kawabata. Radiat. Oncol, 2012, 7, 146].

Currently, the standard therapeutic drug used for BNZT is L-Boronfenilalanine (Do) [R. F. Barth, P. Mi, W. Yang, Cancer Commun., 2018, 38, 35]. A significant disadvantage of L-BPA is the low content of boron, so of particular interest are carboranes, which contain 75% boron and are actively used to modify biomolecules that ensure the delivery of boron to the tumor [J. F. Valliant, K. J. Guenther, A. S. King, P. Morel, P. Schaffer, O. O. Sogbein, K. A. Stephenson, Coord. Chem. Rev., 2002, 232, 173-230].

It is known that the introduction of fluorine into the molecules of substances of drugs in some cases can increase their resistance to metabolism, bioavailability, binding indicators and provide optimal lipophilic characteristics of the final products [S. Purser, P. R. Moore, S. Swallow, V. Gouvemeur. Chem. soc. Rev., 2008, 37, 320-330], and thus, the synthesis of compounds modified by fluorine atoms can lead to the creation of promising drugs for FDT and BNZT.

Formula II is known, the structure of which is close to the structure of the claimed compounds [J. N. GIBBS, η. Wang, ν. V. S. D. K. Bhupathiraju, F. R. Fronczek, K. M. Smith, M. G. H. Vicente. J. Organomet. Chem., 2015, 798, 209-213].

In compound II, ortho-carborane is used as the carborane component, which is linked to the BODIPY fragment via a phenyl spacer at the carbon atom of the carborane polyhedron. This compound has low dark cytotoxicity and low photoinduced cytotoxicity (IC ₅₀ >100 μM) on tumor cells, which significantly limits its use in PDT.

The task of the present invention is the creation of Bodipy derivatives with a high content of boron, applicable in photodynamic therapy (FDT) and borneetron therapy (BNZT) of oncological diseases.

EFFECT: obtaining new carborane-containing derivatives of 4,4-difluoro-4-boron-3α,4α-diaza-s-indacene, exhibiting antitumor activity, expanding the arsenal of agents for PDT and BNCT.

The problem is solved by the claimed compounds Ia-Ie, exhibiting antitumor properties.

,

,

Where

Compounds I contain a unique array of pharmacophore groups such as an indacene moiety, a tetrafluorophenyl group, and a carborane polyhedron, with BODIPY bonded to the carborane polyhedron via a fluorophenyl spacer and a sulfur atom. The indicated set of pharmacophore elements in new compounds can provide a cytotoxic effect on cancer cells.

It turned out that the conjugation of ortho- and meta-mercaptocarboranes with pentafluorophenyl derivatives of 4,4-difluoro-4-boron-3α,4α-diaza-s-indacene through the boron atom of the carborane polyhedron or the introduction of water-soluble 1-mercapto-1-carba-closo- cesium dodecaborate into such a molecule, as well as the presence of a fluorophenyl substituent in similar structures, significantly increases both dark cytotoxicity and photoinduced cytotoxicity of such compounds (IC ₅₀ <50 μM).

Thus, the claimed compounds can be used as chemotherapeutic agents, agents for PDT, and an increased content of boron atoms in the molecule (>20%) can allow the use of such structures as agents for BNCT.

Starting Bodipy (8- (2.3.4.5,6-pentiforophenil) -4.4-diaphor-4-boiled-3α, 4α-Diaza-Sindacen and 8- (2.3.4.5, 6-pentiforophenil) -1,3,5,7-tetrammethyl-4.4-diaphor-4-column-3α, 4α-Diaza-S-induced) are obtained according to well-known methods [N. R. A. Golf, N.-u. Reissig, A. Wiehe. Org. Lett., 2015, 77, 982-985; G. Vives, C. Giansante, R. Bofinger, G. Raffy, A. Del Guerzo, B. Kauffmann, P. Batat, GJonusauskas, N. D. McClenaghan. Chem. Commun., 2011, 47, 10425-10427].

IA-IR compounds are obtained by the interaction of 8- (2.3.4.5,6-pentiforophenil) -4.4-diaphor-4-column-3α, 4α-diaza-S-indacen or 8- (2.3.4 , 5.6-pentiforophenil) -1,3,5,7-tetrammethyl-4.4-diaphor-4-column-3α, 4α-diaza-s-indacen with 9-mercapto-meter-carboat or 9-mercapto- Orto-carboard in dimethyl formamam in the presence of sodium acetate and subsequent standard cleaning.

Compounds Ie and Ie are obtained by the interaction of 8-(2,3,4,5,6-pentafluorophenyl)-4,4-difluoro-4-boron-3α,4α-diaza-s-indacene or 8-(2,3,4 ,5,6-pentafluorophenyl)-1,3,5,7-tetramethyl-4,4-difluoro-4-boron-3α,4α-diaza-s-indacene with cesium 1-mercapto-1-carba-closo-dodecaborate in dimethylformamide in the presence of potassium carbonate and subsequent standard purification (Scheme 1).

Compounds of formula I according to the present invention are available substances: they are obtained in high yields from available reagents using a one-step and technologically advanced synthesis.

All obtained derivatives are dark red microcrystals, readily soluble in chloroform (compounds Ia-Ig), acetone, ethyl acetate, dimethyl sulfoxide, dimethylformamide, water (compounds Id-Ie).

The invention is illustrated by specific examples of its implementation and the results of tests of the cytotoxicity of the claimed compounds Ia-Ie below.

General procedure for the preparation of carborane derivatives BODIPY Ia-Ir

To a solution of 0.2 mmol 8-(2,3,4,5,6-pentafluorophenyl)-4,4-difluoro-4-boron-3α,4α-diaza-s-indacene or 8-(2,3,4,5 ,6-pentafluorophenyl)-1,3,5,7-tetramethyl-4,4-difluoro-4-boron-3α,4α-diaza-s-indacene in dimethylformamide, 1.0 mmol of 9-mercapto-meta-carborane or 9- mercapto-ortho-carborane and 0.2 mmol anhydrous sodium acetate. The reaction mixture is mixed in the atmosphere of the argon for 60 minutes at room temperature in the dark. The reaction mixture is poured into 200 ml of water, 100 ml of ethylacetate is extracted, ethylacetate is washed 3 times 100 ml with water. The solvent is evaporated in a vacuum, the product is placed on a column with silica gel, chromatography on a 40x60 silica gel, an eluent - hexan - ethyl acetate (7: 3).

Example 1

8-[4-(m-carboran-9-ylthio)-2,3,5,6-tetrafluorophenyl]-4,4-difluoro-4-boron-3α,4α-diaza-s-indacene (Ia). Yield 68 mg (66.1%). MS (MALDI): m/z [M ⁺ ] for C ₁₇ H ₁₇ B ₁₁ N ₂ F ₆ S calculated 514.211; found 514.215. IR spectrum (KBr), ν, cm ^-1 : 3069 (CH carborane), 2616 (BH carborane), 1567, 1471 (C=C, C=N), 1190, 1254 (BF ₂ ). Electronic spectrum ((CH ₃ ) ₂ CO), λ _max , nm, (ε 10 ^-3 ): 513 (42.8). ¹ H-NMR spectrum (400 MHz, CDC1 ₃ ) δ, ppm: 7.99 (s, 2Н, Η pyrrole), 6.84 (d, J=3.81 Hz, 2Н, Η pyrrole), 6.59 (d, J= 3.81 Hz, 2Н, Η of pyrrole). 3.05 (br. s, 2H, carborane CH). ¹¹ B-NMR spectrum (128 MHz, CDCl ₃ ) δ, ppm: 0.21 (t, J=28.39 Hz, 1B, BF ₂ ,), -1.19 (br.s, 1IB), -6.25 ( d, J=158.5 Hz, 2B), -9.54 (d, J=156.1 Hz, 1B) -12.34 - -14.28 (m, 5B), -17.36 (d, 1B, J=182.1 Hz ). ¹⁹ F-NMR (376 MHz, CDC1 ₃ ); δ, ppm: -129.71 (dd, J=24.42, 9.16 Hz, 2F, opmo-F), -137.48 (dd, J=21.36, 9.16 Hz, 2F, meta-F), -144.83 (dd, J=57.98, 30.52 Hz, 2F, BF ₂ ).

Example 2

8-[4-(m-carboran-9-ylthio)-2,3,5,6-tetrafluorophenyl]-1,3,5,7-tetramethyl-4,4-difluoro-4-boron-3α,4α- diaza-s-indacene (Ib). Yield 78 mg (68.4%). MS (MALDI): m/z [M ⁺ ] for C ₂₁ H ₂₅ B ₁₁ N ₂ F ₆ S calculated 570.274; found 570.271. Electronic spectrum ((CH ₃ ) ₂ CO) λ _max nm (ε 10 ^-3 ): 513 (46.3). IR spectrum (KBr), ν, cm ^-1 3063 (CH carborane), 2612 (BH carborane), 1551, 1471 (C=C, C=N), 1190 (BF ₂ ). ¹ H-NMR spectrum (400 MHz, CDC1 ₃ ) δ, ppm: 6.06 (s, 2H, Η pyrrole), 3.01 (br. s, 2H, CH carborane), 2.59 (s, 6H, CH ₃ ) , 1.64. (s, 6H, CH ₃ ). ¹¹ B-NMR spectrum (128 MHz, CDCl ₃ ) δ, ppm: 0.268 (t, J=30.75 Hz, 1B, BF ₂ ), -1.23 (br.s, 1B), -6.11 (d , J=170.3 Hz, 2B), -9.71 (d, J- 151.4 Hz, 1B), -12.43 - -14.35 (m, 5V), -17.46 (d, J=184.5 Hz, 1V ). ¹⁹ F-NMR spectrum (376 MHz, CDCl ₃ ); δ, ppm: -129.93 (dd, J=24.09, 11.48 Hz, 2F, opmo-F), -140.07 (dd, J=25.24, 12.63 Hz, 2F, meta-F), -146.09 (dd, J=64.25, 32.13 Hz, 2F, BF ₂ ).

Example 3

8-[4-(o-carboran-9-ylthio)-2,3,5,6-tetrafluorophenyl1-4,4-difluoro-4-boron-3α,4α-diaza-s-indacene (Ic). Yield 62 mg, (60.3%). MS (MALDI): m/z [M ⁺ ] for C ₁₇ H ₁₇ B ₁₁ N ₂ F ₆ S calculated 514.211; found 514.212. IR spectrum (KBr), ν, cm ^-1 : 3090 (CH _carborane ), 2605 (BH _carborane ), 1577, 1473 (C=C, C=N), 1228 (BF ₂ ). Electronic spectrum ((CH ₃ ) ₂ CO) λ _max nm (ε 10 ^-3 ): 513 (41.1). ¹ H-NMR spectrum (400 MHz, CDC1 ₃ ) δ, ppm: 7.99 (s, 2H, Η pyrrole), 6.84 (d, J=3.50 Hz, 2H, Η pyrrole), 6.59 (d, J= 3.81 Hz, 2Н, Η of pyrrole). 3.67 (br.s, 1H, carborane CH), 3.55 (br.s, 1H, carborane CH). ¹¹ B-NMR spectrum (128 MHz, CDC1 ₃ ) δ, ppm: 5.42 (br.s, 1V), 0.21 (t, 1B, J=28.4 Hz, BF ₂ ), -2.10 (d, J=144.3 Hz, 1V), -8.59 (d, J=153.7 Hz, 2V), -13.63 --16.06 (m, 6V). ¹⁹ F-NMR spectrum (376 MHz, CDC1 ₃ ); δ, ppm: -129.83 (dd, J=24.41, 9.15 Hz, 2F, opmo-F), -137.82 (dd, J=24.41, 9.15 Hz, 2F, meta-F), -144.83 (dd, J=57.98, 27.47 Hz, 2F, BF ₂ ).

Example 4

8-[4-(o-carboran-9-ylthio)-2,3,5,6-tetrafluorophenyl]-1,3,5,7-tetramethyl-4,4-difluoro-4-boron-3α,4α- diaza-s-indacene (Ig). Yield 73 mg, (64.0%). MS (MALDI): m/z [M ⁺ ] for C ₂₁ H ₂₅ B ₁₁ N ₂ F ₆ S calculated 570.274; found 570.277. IR spectrum (KBr), ν, cm ^-1 : 3073 (CH carborane), 2617 (BH carborane), 1550, 1513, 1467 (C=C, C=N), 1190 (BF ₂ ). Electronic spectrum ((CH ₃ ) ₂ CO) λ _max nm (ε 10 ^-3 ): 513 (49.9). ¹ H-NMR spectrum (400 MHz, CDC1 ₃ ) δ, ppm: 6.06 (s, 2H, Η of pyrrole), 3.64 (br. s, 1H, CH carborane), 3.53 (br. s, 1H, CH carborane), 2.59 (s, 6H, _CH3 ), 1.64. (s, 6H, CH ₃ ). ¹¹ B-NMR spectrum (128 MHz, CDC1 ₃ ) δ, ppm: 5.35 (br.s, 1V), 0.68 (t, J=30.8 Hz, 1B, BF ₂ ), -2.04 (d, J=151.4 Hz, 1V), -8.68 (d, J=153.7 Hz, 2V), -13.76 - -15.97 (m, 6V). ¹⁹ F-NMR spectrum (376 MHz, CDC1 ₃ ); δ, ppm: -129.95 (dd, J=24.75, 11.00 Hz, 2F, opmo-F), -140.16 (dd, J=24.75, 11.00 Hz, 2F, meta-F), -146.07 (dd, J=63.23, 30.24 Hz, 2F, BF ₂ ).

General procedure for the preparation of carborane derivatives BODIPY Id-Ie

To a solution of 0.2 mmol 8-(2,3,4,5,6-pentafluorophenyl)-4,4-difluoro-4-boron-3α,4α-diaza-s-indacene or 8-(2,3,4,5 ,6-pentafluorophenyl)-1,3,5,7-tetramethyl-4,4-difluoro-4-boron-3α,4α-diaza-s-indacene in dimethylformamide, 1.0 mmol of 1-mercapto-1-carba-closo- cesium dodecaborate and 0.2 mmol anhydrous K ₂ CO ₃ . The reaction mixture was stirred under argon for 8 hours at room temperature in the dark. The reaction mixture is poured into 200 ml of water, extracted with 100 ml of ethyl acetate, the ethyl acetate is washed with water 3 times with 100 ml. The solvent is evaporated in vacuo, the product is placed on a column of silica gel, chromatographed on silica gel 40X60, eluent - chloroform - acetone (2: 1).

Example 5

8-[4-(1-carba-closo-dodecaboran-1-ylthio)-2,3,5,6-tetrafluorophenyl]-4,4-difluoro-4-boron-3α,4α-diaza-s-indacene} cesium (Id). Yield 108 mg, (83.6%). MS (MALDI): m/z [M ⁺ ] for C ₁₆ H ₁₇ B ₁₂ N ₂ F ₆ SCs calculated 646.126; found 646.129. IR spectrum (KBr), ν, cm ^-1 : 2540 (BH carborane), 1576, 1470 (C=C, C=N), 1259 (BF ₂ ). Electronic spectrum ((CH ₃ ) ₂ CO) λ _max nm (ε 10 ^-3 ): 513 (37.2). ¹ Η-NMR spectrum (400 MHz, (CD ₃ ) ₂ CO) δ, ppm: 8.16 (s, 2Н, Η pyrrole), 7.18 (d, J=3.67 Hz, 2Н, Η pyrrole), 6.75 ( e, J=4.11 Hz, 2Н, Η of pyrrole). ¹¹ B-NMR spectrum (128 MHz, (CD ₃ ) ₂ CO) δ, ppm: 0.23 (t, J=28.39 Hz, 1B, BF ₂ ), -8.68 (d, J=135 Hz, 1B), -12.64 (d, J=142Hz, 10V). ¹⁹ F-NMR spectrum (376 MHz, (CD ₃ ) ₂ CO); δ, ppm: -129.14 (dd, J=24.75, 11.00 Hz, 2F, opmo-F), -140.52 (dd, J=24.75, 11.00 Hz, 2F, meta-F), -144.38 (dd, J=57.74, 30.24 Hz, 2F, BF ₂ ).

Example 6

8-[4-(1-carba-closo-dodecaboran-1-ylthio)-2,3,5,6-tetrafluorophenyl]-1,3,5,7-tetramethyl-4,4-difluoro-4-boron- 3α,4α-diaza-s-indacene} cesium (Ie). Yield 102 mg (72.6%). MS (MALDI): m/z [M ⁺ ] for C ₂₀ H ₂₅ B ₁₂ N ₂ F ₆ SCs calculated 702.189; found 702.185. IR spectrum (KBr), ν, cm ^-1 : 2536 (BH carborane), 1553, 1512, 1469 (C=C, C=N), 1190, 1193 (BF ₂ ). Electronic spectrum ((CH ₃ ) ₂ CO) λ _max nm (ε 10 ^-3 ): 513 (51.9). ¹ H-NMR spectrum (400 MHz, (CD ₃ ) ₂ CO) δ, ppm: 6.28 (s, 2H, Η pyrrole), 2.55. (s, 6H, CH ₃ ), 1.72. (s, 6H, CH ₃ ). ¹¹ B-NMR spectrum (128 MHz, (CD ₃ ) ₂ CO) δ, ppm: 0.63 (t, J=33.12 Hz, 1B, BF ₂ ), -8.66 (d, J=134.8 Hz, 1B), -12.73 (d, J=141.9 Hz, 10 V). ¹⁹ F-NMR spectrum (376 MHz, (CD ₃ ) ₂ CO); δ, ppm: -128.32 (dd, J=24.75, 11.00 Hz, 2F, opmo-F), -142.77 (dd, J=24.75, 11.00 Hz, 2F, meta-F), -145.58 (dd, J=63.23, 32.99 Hz, 2F, BF ₂ ).

Below are the results of a series of cytotoxic testing in a dark experiment and an experiment with a photograph of the representatives of the declared compounds IA-E.

The test results showed that compounds Ia-Ie exhibit significant activity on cultured malignant cells (dark and photoinduced at therapeutic concentrations), which allows us to consider them promising "drug candidates" for further research as antitumor agents.

Results of studying the dark and phototoxicity of compounds in tumor cell culture

Research drugs

Compounds Ia-Ie were obtained for research. Compounds were dissolved in DMSO to a stock solution concentration of 10 mM.

Cultured cell line

The HCT116 line (human colon carcinoma) was used for the experiments. HCT116 cells were cultured in DMEM medium with the addition of the following components to final concentrations: 5% fetal calf serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (PanEco, Russia), incubation was carried out at 37°C, 5% CO ₂ in a humidified atmosphere. Cells in the logarithmic growth phase were used in the experiments.

ΜTT cytotoxicity test

The cytotoxic effect of the compounds was studied in the MTT test (by the reduction of the yellow salt of 3-4,5-dimethylthiazol-2-yl-2,5-diphenylterazol into dark blue crystalline formazan by the mitochondria of living cells). Based on the results of the study of cytotoxicity, survival curves were constructed and the concentration values necessary for the death of 50% of cells -IC ₅₀ were determined.

Cells were seeded into wells of a 96-well plate (NUNC, USA) (5000 cells in 190 μl of culture medium), incubated for 24 hours at 37°C, 5% CO ₂ , in a humidified atmosphere. 10 μl of a solution of the test substances in the culture medium, prepared by serial dilutions from the initial solution, was added, up to 10 final concentrations.

Cells without preparation (intact) served as the control in the experiment.

Dark cytotoxicity study

Cells with drugs were incubated for 72 h at 37°C, 5% CO ₂ in a humidified atmosphere. 1 hour before the end of incubation, 20 μl of an aqueous solution of MTT (5 mg/ml, PanEco, Russia) were added to the wells and incubated at 37°C, 5% CO ₂ , in a humidified atmosphere for 1.5 hours. After the end of incubation, the culture medium were collected, the cells were resuspended in 100 µl of DMSO, and the optical density of the solution was measured on a Multiscan FC plate spectrophotometer (Thermo Scientific, USA) at a wavelength of 570 nm. The percentage of cells that survived under the action of each dose of the compound was calculated as the quotient of the average optical density in the wells after incubation with a given dose divided by the average optical density of the control wells (the values of the latter were taken as 100%). Based on the results, survival curves were constructed and IC ₅₀ values were determined.

Study of photoinduced cytotoxicity

Cells were seeded into wells of a 96-well plate (NUNC, USA) (5000 cells in 190 μl of culture medium), incubated for 24 hours at 37°C, 5% CO ₂ , in a humidified atmosphere. 5-10 μl of a solution of the test substances in the culture medium, prepared by serial dilutions from the initial solution, was added, up to 10 final concentrations).

Cells with drugs were incubated at 37°C, 5% CO ₂ for 72 hours. Then it was illuminated with a diode source at 530 nm (AFS, Polironic, Russia) with a dose density of 30 J/ ^cm2 . After the end of illumination, 20 µl of an aqueous solution of MTT (5 mg/ml, PanEco, Russia) were added to the wells and incubated at 37°C, 5% CO ₂ , in a humidified atmosphere for 1.5 h. resuspended in 100 μl of DMSO, and the optical density of the solution was measured on a Multiscan FC plate spectrophotometer (Thermo Scientific, USA) at a wavelength of 570 nm. The percentage of cells that survived under the action of each dose of the compound was calculated as the quotient of the average optical density in the wells after incubation with a given dose divided by the average optical density of the control wells (the values of the latter were taken as 100%). Based on the results, survival curves were constructed and IC ₅₀ values were determined.

Claims (5)

1. Compounds of formula I

, Where

2. Compounds according to p. 1, showing antitumor activity.