Phenothiazine leuco photo-caged compounds, methods of synthesis and uses thereof

Abstract

The application discloses a synthesis method and application of a kind of phenothiazine leuco photo-caged compound, and relates to the application in the field of biological medicine.The compound is formed by using oxygen, sulfur and selenium containing phenothiazine leuco as matrix, connecting different functional groups by amide bond, especially STAT3 inhibitor molecules, forming a photo-controllable release platform that can be activated under near infrared light 650 or 660nm.In addition, the dye has good biocompatibility and low cytotoxicity, and experiments prove that it also has excellent imaging and treatment effect on specific tumor cells.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of fine chemical technology, in particular to a phenothiazine cryptophan photocage compound and a synthesis method and application thereof. BACKGROUND

[0002] Photocage molecules, also known as "photoremovable protecting groups", are a class of molecules containing photosensitive groups, which can release specific compounds at a specific time and location through light stimulation to achieve special functions. Due to its time and space resolution, simple operation, easy control and strong modifiability, photocage plays an increasingly important role in chemical biology research and therapeutic applications. In recent years, boron dipyrromethene (BODIPY), porphyrin, cyanine and other photocages in the therapeutic window (650-900 nm) have been developed. The near-infrared light activated by these photocages depends on the near-infrared absorption of the photocage molecules themselves, and the photolysis mechanism mainly includes photochemical SN1 reaction, photooxidation reaction and [2+2] cycloaddition reaction. However, most of these photocage molecules with near-infrared absorption are difficult to synthesize, and it is difficult to monitor the photolysis reaction process by solution colorimetric visualization and achieve the synergistic treatment of specific tumor diagnosis and treatment integration. SUMMARY

[0003] In view of the problems of the prior art, such as the scarcity of photocages excited by near-infrared light, the difficulty in visualizing the monitoring of the photolysis reaction process by photocage molecules, and the difficulty in achieving the synergistic treatment of specific tumor diagnosis and treatment integration, the present application provides a phenothiazine cryptophan photocage compound and a synthesis method and application thereof. The phenothiazine cryptophan photocage compound is obtained by reacting a phenothiazine cryptophan dye with a functional group, and can controllably release a functional compound under 650 or 660 nm light irradiation. It can not only controllably image tumor cells, but also be used for the treatment of specific tumors.

[0004] In order to achieve the above-mentioned purpose, the technical scheme of the present application is as follows: a phenothiazine cryptophan photocage compound, having the structure of general formula I:

[0005]

[0006] In general formula I, R1 is selected from any one of oxygen, sulfur, selenium and tellurium elements;

[0007] R2 is selected from one of the following structural groups:

[0008]

[0009] Further, the phenothiazine cryptophan photocage compound is selected from one of the following compounds:

[0010]

[0011] Further, the absorption wavelength of the phenothiazine leuco photo-cage compound is 250-350 nm, and the photo-activation wavelength is 650 / 660 nm.

[0012] When R1 is oxygen, and R2 is selected from , the photo-activation wavelength is 650 nm.

[0013] When the compound R1 is sulfur or selenium, and R2 is selected from , the photo-activation wavelength is 660 nm corresponding to the light irradiation.

[0014] The synthesis method of the phenothiazine leuco photo-cage compound comprises the following steps:

[0015] (1) In an inert gas environment, the R1-substituted phenothiazine leuco dye of formula Y-1, sodium pyrosulfate and sodium carbonate are reacted in a first organic solvent and water, the reaction temperature is 25-45℃, the reaction time is 0.5-3h, and the compound of formula Y-2 is prepared. The compound of formula Y-2 is cooled with ice water, and triphosgene and a basic solvent dissolved in a second organic solvent are added and continue to react for 3-6h to prepare the corresponding acyl chloride-substituted phenothiazine leuco dye of formula Y-3.

[0016] (2) The Y-3 compound synthesized in step (1), R2 and triethylamine are uniformly mixed in a third organic solvent, and reacted at 0-25℃ for 6-12h to prepare the corresponding phenothiazine leuco photo-cage compound of formula I.

[0017]

[0018] Further, in step (1), the molar ratio of the R1-substituted phenothiazine leuco dye, sodium pyrosulfate and sodium carbonate is 1:2-4:2-4.

[0019] The molar ratio of the compound of formula Y-2 and triphosgene is 1:0.6-1.

[0020] Further, in step (1), the first organic solvent is selected from any one or a combination of mixed solvents of dichloromethane, toluene, benzene;

[0021] The second organic solvent is selected from any one or a combination of solvents of dichloromethane, chloroform, ethyl acetate;

[0022] The basic solvent is selected from at least one of triethylamine, pyridine, and sodium carbonate.

[0023] The inert gas environment is selected from one of nitrogen and argon.

[0024] Further, in step (2), the molar ratio of the compound represented by Y-3, R2 and triethylamine is 1:1-2:3.

[0025] Further, in step (2), the third organic solvent is selected from any one or a combination of dichloromethane, chloroform, tetrahydrofuran.

[0026] The phenothiazine leuco form photo-cage compound is used for cell / tumor light-controlled imaging and triple-negative breast cancer tumor treatment.

[0027] Further, the phenothiazine leuco form photo-cage compound is used for STAT3 signal pathway mediated tumor imaging and treatment in vivo.

[0028] In summary, the present application has the following beneficial effects:

[0029] First, the phenothiazine leuco form photo-cage compound of the present application is synthesized by connecting the phenothiazine leuco form photo-cage compound with different functional groups. When the functional group is selected from a STAT3 inhibitor, the photosensitizer and STAT3 inhibitor can be cleaved and released under light, producing reactive oxygen and down-regulating STAT3 protein, thereby achieving excellent cell imaging and specific tumor killing effect.

[0030] Second, according to experimental tests, the photo-cage compound of the present application can be activated and cleaved by near-infrared light 650 / 660, and does not depend on the ultraviolet absorption of the compound itself, thereby avoiding the damage of ultraviolet light to biological tissues and the low penetration depth, reducing the background fluorescence when the photo-cage compound is applied to biological imaging, and realizing the visual monitoring of the photolysis reaction process.

[0031] Third, the photo-cage compound of the present application can be released under low-dose red light (15mW / cm 2 ), has good biocompatibility, and the tumor inhibition rate of specific tumors reaches 95.3%, indicating that the photo-cage compound can be well applied to the field of tumor treatment. BRIEF DESCRIPTION OF DRAWINGS

[0032] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description. Obviously, the drawings in the following description are some embodiments of the present application, and those skilled in the art can also obtain other drawings according to these drawings without creative labor.

[0033] Figure 1 is the ultraviolet absorption spectrum of compound 1 disclosed in embodiment 1 of the present application in different solvents;

[0034] Figure 2is the UV absorption spectrum of compound 2 disclosed in embodiment 2 of the present application in different solvents;

[0035] Figure 3 is the UV absorption spectrum of compound 5 disclosed in embodiment 5 of the present application in different solvents;

[0036] Figure 4 is the photolysis UV absorption spectrum of compound 1 disclosed in embodiment 1 of the present application under light irradiation;

[0037] Figure 5 is the photolysis UV absorption spectrum of compound 2 disclosed in embodiment 2 of the present application under light irradiation;

[0038] Figure 6 is the photolysis UV absorption spectrum of compound 5 disclosed in embodiment 5 of the present application under light irradiation;

[0039] Figure 7 is the cell uptake imaging diagram of compound 1 disclosed in embodiment 1 of the present application before and after light irradiation;

[0040] Figure 8 is the MTT experiment diagram of compound 1 disclosed in embodiment 1 of the present application under light irradiation and without light irradiation;

[0041] Figure 9 is the MTT experiment diagram of compound 2 disclosed in embodiment 2 of the present application under light irradiation and without light irradiation;

[0042] Figure 10 is the MTT experiment diagram of compound 5 disclosed in embodiment 5 of the present application under light irradiation and without light irradiation;

[0043] Figure 11 is the protein expression diagram in the western blot experiment of compounds 1, 2 and 5 disclosed in the present application. DETAILED DESCRIPTION

[0044] The present application will be further described in conjunction with the examples below, but it should be understood that the protection scope of the present application is not limited by the examples.

[0045] In the present application, unless otherwise explicitly stated, the percentage and percentage content are by mass. Unless otherwise specified, the experimental methods used are conventional methods, and the materials, reagents, etc. used can be purchased from commercial channels.

[0046] The term "MTT" used in the present application refers to a method for detecting cell survival and growth.

[0047] The term "Western blot" used in the present application refers to a method for detecting a certain protein in a complex sample according to the specific binding of antigen and antibody.

[0048] Instruments and equipment employed in the examples:

[0049] In the column chromatography process of the present application, 200-300 mesh, 100-200 mesh column chromatography silica gel purchased from Qingdao Meigao Group Co., Ltd. and 20-40 mesh analytical pure quartz sand purchased from Tianda Chemical Reagent Factory were used.

[0050] In the detection of compounds, nuclear magnetic resonance hydrogen spectrum was detected by Bruker Avance III 500 of American Bruker Company.

[0051] The absorption, emission spectrum and light stability of the dye were measured by Cary 60 ultraviolet visible spectrophotometer and Cary Eclipse fluorescence spectrophotometer of Agilent Company.

[0052] Cell toxicity test was measured by Varioskan LUX Multimode Microplate Reader instrument of American Thermofisher Company.

[0053] All animal experiments involved in the present work were approved by the Animal Protection and Use Committee of Dalian Medical University.

[0054] The phenazine cryptophore photocaged compound represented by general formula I is described in detail below in combination with examples.

[0055] Example 1

[0056] Synthesis of compound 1 with R1 as oxygen and R2 as The synthesis process is as follows:

[0057]

[0058] The synthesis method comprises the following steps:

[0059] S1: 3-dimethylaminophenol (2 g, 14.58 mmol) was dissolved in 5 mL of cold 6M HCl solution, and NaNO2 (1.06 g, 15.31 mmol) was added in batches within 1 h while keeping the solution temperature below 5°C. The reaction mixture was stirred for another 2 h. After the reaction was completed, the precipitate was filtered with a Buchner funnel and washed with a cold 2M HCl solution. The product was dried to obtain an intermediate, a yellow solid, which was directly used in the next step.

[0060] Intermediate (1.5 g, 9.03 mmol) was dissolved in i-PrOH / H2O (9 / 1, 10 mL) and stirred at 80 °C for 30 min. 3-Diaminophenol (1.49 g, 10.83 mmol) and HCIO4 (70%, 35 μL) were mixed homogeneously in 90% i-PrOH (5 mL) and the mixture was added to the above solution in 4 portions over 1 h. The solution was stirred overnight and the solvent was removed by rotary evaporation under reduced pressure. The dark blue solid intermediate 1-1 (600 mg, yield 25%) was obtained by silica gel column chromatography.

[0061] S2: The intermediate 1-1 (500 mg, 10 mmol) prepared in step S1 was dissolved in 15 mL water under a constant nitrogen atmosphere, then 10 mL dichloromethane was added, followed by the addition of sodium pyrosulfate (1.08 g, 6.20 mmol) at 45 °C, then sodium carbonate (0.66 g, 6.20 mmol) was added. After stirring vigorously for 30 min, the reaction mixture turned yellow and both phases were transparent, indicating that the reduced dye was transferred to the dichloromethane phase. Subsequently, the mixture was cooled with ice water and triphosgene (0.6 eq) dissolved in dichloromethane was added dropwise to it, along with 0.5 mL triethylamine. The mixture was stirred for another 3 h. After the reaction was completed, the solution was poured into 200 mL ice water and the resulting mixture was extracted with 3 x 100 mL dichloromethane. The organic layers were combined and dried, and the crude product was recrystallized from acetonitrile three times to obtain the off-white product 1-2 (100 mg, yield 30%).

[0062] S3: The intermediate 1-2 (50 mg, 0.15 mmol) prepared in step S2, triethylamine (3 eq) were dissolved in 10 mL dichloromethane and the mixture was stirred in an ice water bath. STAT3 inhibitor HJC0152 (61.36 mg, 0.16 mmol) dissolved in 5 mL dichloromethane was added dropwise. The reaction was monitored by TLC and was completed in total 5 h at room temperature. Purification by silica gel column chromatography (petroleum ether / ethyl acetate = 4 / 1) gave the grey-green solid (20 mg, yield 30%).

[0063] The structure was confirmed to be correct: 1H NMR (400 MHz, CDC13) δ 10.44 (s, 1H), 8.86 (d, J = 9.2 Hz, 1H), 8.27 (d, J = 2.6 Hz, 1H), 8.23 (d, J = 2.8 Hz, 1H), 8.20 (dd, J = 9.3, 2.6 Hz, 1H), 7.48 (dd, J = 8.9, 2.8 Hz, 1H), 7.18 (d, J = 8.7 Hz, 2H), 7.08 (d, J = 8.9 Hz, 1H), 6.30 (d, J = 12.8 Hz, 4H), 5.70 (t, J = 5.8 Hz, 1H), 4.45 (t, J = 5.5 Hz, 2H), 3.73 (q, J = 5.7 Hz, 2H), 2.87 (s, 12H).

[0064] 13 C NMR (101 MHz, CDC13) δ 162.48, 156.05, 154.96, 151.92, 149.50, 142.99, 141.03, 134.06, 132.55, 127.50, 124.78, 124.56, 123.43, 122.69, 122.25, 121.25, 118.20, 114.44, 107.25, 100.77, 68.84, 40.55, 39.84, 29.38, 22.71, 21.96.

[0065] Example 2

[0066] Synthesis of compound 2 with R1as sulfur, R2as The synthesis process is shown as follows:

[0067]

[0068] The synthesis method comprises the following steps:

[0069] S1 : Methylene blue (3 g, 9.38 mmol) was dissolved in 15 mL of water under constant nitrogen atmosphere, then 10 mL of dichloromethane and sodium pyrosulfate (6.53 g, 37.52 mmol) were added while stirring at 45 °C, sodium carbonate (3.98 g, 37.52 mmol) was added, after 30 min of vigorous stirring the reaction mixture turned yellow, both phases became transparent indicating that the colorless methylene blue had transferred to the dichloromethane phase. Subsequently, the mixture was cooled with an ice water bath, to which bis(trichloromethyl) carbonate (2.78 g, 9.38 mmol) in 20 mL of dichloromethane was added dropwise, along with 0.5 mL of triethylamine, the mixture was stirred for another 3 h. After the end of the reaction the solution was poured into 200 mL of ice water, and the resulting mixture was extracted with 3x100 mL of dichloromethane. The combined extracts were washed with brine and the solvent was removed under reduced pressure, the crude product was recrystallized three times with acetonitrile to obtain the off-white product 2-1 (0.8 g, yield 45%);

[0070] S2: The intermediate product 2-1 (50 mg, 0.15 mmol) prepared in step S1 above, triethylamine (3 eq) were dissolved in 10 mL of dichloromethane, the resulting mixture was stirred in an ice water bath. A solution of HJC0152 (61.36 mg, 0.16 mmol) in 5 mL of dichloromethane was added dropwise. After the addition, the mixture was stirred at room temperature until the completion of the reaction, TLC analysis every 1 h, the reaction was completed in total 6 h. While stirring the reaction mixture was poured into 200 mL of ice water, and the resulting mixture was extracted with 3x150 mL of ethyl acetate. The combined extracts were washed with brine, dried over anhydrous sodium sulfate, and evaporated on a rotary evaporator to obtain an oily residue, which was purified by column chromatography (ethyl acetate / petroleum ether = 1 / 3) to obtain the gray-green solid 2 (25 mg, yield 35%).

[0071] The structure was confirmed to be correct: 1 H NMR (400 MHz, CDC13) δ 10.41 (s, 1H), 8.88 (d, J = 9.2 Hz, 1H), 8.25 (d, J = 2.6 Hz, 1H), 8.23 (d, J = 2.8 Hz, 1H), 8.22 - 8.18 (m, 1H), 7.47 (dd, J = 8.9, 2.8 Hz, 1H), 7.22 (d, J = 8.8 Hz, 2H), 7.09 (d, J = 8.9 Hz, 1H), 6.58 (s, 2H), 6.51 - 6.45 (m, 2H), 5.38 (t, J = 5.7 Hz, 1H), 4.44 (t, J = 5.6 Hz, 2H), 3.70 (q, J = 5.6 Hz, 2H), 2.86 (s, 12H).

[0072] 13C NMR (101 MHz, CDC13) δ 162.53, 156.19, 155.22, 148.97, 142.97, 141.07, 134.27, 134.11, 132.44, 127.88, 127.44, 126.91, 124.69, 123.43, 122.61, 122.16, 121.26, 114.70, 111.07, 110.92, 77.05, 68.98, 58.44, 40.54, 39.96.

[0073] Example 3

[0074] Synthesis of compound 3 with R1as selenium and R2as The synthesis process is shown as follows:

[0075]

[0076] The synthesis method comprises the following steps:

[0077] S1: To a mixture of 3-iodoaniline (5 g, 22.83 mmol) and K2CO3(6.31 g, 45.66 mmol) in CH3CN (60 mL), iodomethane (9.72 g, 68.49 mmol) was added dropwise. The reaction was stirred at reflux for 16 h. After cooling to room temperature, the mixture was treated with water and extracted with ethyl acetate. The organic solvent was dried with Na2SO4. After removing the solvent under reduced pressure, the residue was purified by eluting petroleum ether / ethyl acetate (20 / 1) in a silica gel column chromatography to obtain 3-1 (1.8 g, 32%) as a light yellow oil.

[0078] S2: Under a nitrogen atmosphere, CuI (277.49 mg, 1.46 mmol), K3PO4(3.87 g, 18.21 mmol), Se (1.44 g, 18.21 mmol), the intermediate 3-1 (1.8 g, 7.29 mmol) obtained in step S1 above and 15 mL of DMSO were mixed, and the mixture was reacted at 100°C for 24 h. After the reaction was completed, it was cooled to room temperature, a saturated aqueous solution of sodium chloride was added, and then extracted with ethyl acetate. The combined organic layers were dried with anhydrous sodium sulfate and then concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to obtain 3-2 (2 g, yield 69%) as a dark yellow oil.

[0079] S3: The intermediate 3-2 (0.91 g, 2.0 mmol) from step S2 above was dissolved in HC1 (30 mL, 1 mM) at 0 °C, and an aqueous solution of NaNCte (0.27 g, 4 mmol) was added. A clear yellow solution was observed to quickly turn to a red precipitate. After 10 min, the mixture was extracted with CH2CI2. The collected organic solution was evaporated, and the resulting brown solid 3-3 was used in the next step without purification.

[0080] S4: The product 3-3 (1.70 g, 3.3 mmol) from the previous step was dissolved in 10 mL of trifluoroethanol along with N,N-dimethylaniline (9.6 mmol). After stirring for 10 min, the mixture was heated to reflux for 1 h, resulting in a blue waxy solid. The blue waxy solid was dissolved in a mixture of 1 N aqueous NaOH and dichloromethane. The solution turned bright magenta, indicating deprotonation of the dye. The organic layer was washed twice with brine. The dye was converted to the deep blue chloride salt by adding 0.5 mL of concentrated HC1 to the magenta dichloromethane solution. After removing the solvent and excess HC1 under reduced pressure, the blue solid 3-4 was purified by column chromatography.

[0081] S5: Same as S1 of Example 2, using the intermediate 3-4 (400 mg, 1.09 mmol) from step S4 above in place of methylene blue in Example 2, resulting in the intermediate 3-5 (120 mg, 43% yield) after recrystallization.

[0082] S6: Same as S2 of Example 2, using the intermediate 3-5 (50 mg, 0.13 mmol) from step S5 above in place of 2-1, resulting in compound 3 (27 mg, 34% yield) after purification by column chromatography.

[0083] The structure was confirmed to be correct: 1 H NMR (400 MHz, CDC13) δ 9.97 (s, 1H), 8.32 - 8.27 (m, 2H), 8.19 (dd, J = 8.5, 1.9 Hz, 1H), 7.85 (d, J = 2.5 Hz, 1H), 7.52 (d, J = 2.0 Hz, 2H), 7.48 (dd, J = 7.5, 1.8 Hz, 2H), 7.43 (dd, J = 9.0, 2.4 Hz, 1H), 7.04 (d, J = 9.1 Hz, 1H), 6.80 (t, J = 5.1 Hz, 1H), 6.64 (dd, J = 7.5, 2.0 Hz, 2H), 4.15 (t, J = 4.7 Hz, 2H), 3.62 (q, J = 4.8 Hz, 2H), 2.96 (s, 10H).

[0084] 13C NMR (101 MHz, CDC13) δ 164.36, 157.37, 155.88, 146.99, 142.42, 139.69, 133.25, 132.73, 131.69, 128.88, 127.60, 127.40, 126.52, 126.04 (d, J = 16.2 Hz), 125.44, 124.65, 121.87, 120.86, 116.25, 114.70 (d, J = 12.9 Hz), 67.33, 40.40, 40.23.

[0085] Example 4

[0086] Synthesis of compound 4 with R1as oxygen, R2as

[0087] The synthesis of compound 4 was the same as compound 1, and the only difference was that in step S3, 2-(2-chlorophenyl)ethan-1-amine (24.90 mg, 0.16 mmol) was used to replace the above STAT3 inhibitor HJC0152, and compound 4 (40 mg, yield 78%) was obtained by column chromatography purification, and its structural formula is as follows:

[0088]

[0089] The structure was confirmed to be correct: 1 H NMR (400 MHz, CDC13) δ 7.33 - 7.29 (m, 3H), 7.25 - 7.20 (m, 3H), 6.91 (dd, J = 8.1, 2.3 Hz, 2H), 6.58 (d, J = 2.0 Hz, 2H), 6.53 (t, J = 4.9 Hz, 1H), 3.42 (td, J = 6.0, 4.9 Hz, 2H), 2.97 (s, 14H).

[0090] 13 C NMR (101 MHz, CDC13) δ 156.62, 146.47, 145.67, 137.80, 134.76, 130.94, 129.24, 128.77, 128.41, 122.04, 121.62, 121.54, 121.43, 110.24, 102.57, 40.60, 40.06, 32.20.

[0091] Example 5

[0092] Synthesis of compound 5 with R1as sulfur, R2as

[0093] ​​The synthesis of compound 5 was the same as compound 2, and the difference between example 2 was only that in step S2, 2-(2-chlorophenyl)ethan-1-amine (24.90 mg, 0.16 mmol) was used to replace STAT3 inhibitor HJC0152, and compound 5 (52 mg, yield 75%) was obtained by column chromatography purification, and its structural formula was as follows:

[0094]

[0095] The structure was confirmed to be correct: 1 H NMR (400 MHz, CDC13) δ 7.39 (dd, J = 7.9, 7.0 Hz, 2H), 7.35 - 7.28 (m, 1H), 7.27 - 7.18 (m, 3H), 6.81 (dd, J = 7.9, 2.2 Hz, 2H), 6.73 (d, J = 2.0 Hz, 2H), 6.48 (t, J = 4.9 Hz, 1H), 3.42 (td, J = 6.0, 4.9 Hz, 2H), 2.98 - 2.92 (m, 14H).

[0096] 13 C NMR (101 MHz, CDC13) δ 156.48, 146.63, 137.80, 134.76, 133.13, 132.10, 130.94, 129.24, 128.77, 128.41, 122.99, 122.52, 113.70, 112.36, 40.60, 40.40, 32.20.

[0097] Example 6

[0098] The synthesis of compound 6 with R1as selenium and R2as was as follows:

[0099] The synthesis of compound 6 was the same as compound 3, and the difference between example 3 was only that in step S6, 2-(2-chlorophenyl)ethan-1-amine (24.90 mg, 0.16 mmol) was used to replace STAT3 inhibitor HJC0152 in the above step S6, and compound 6 (65 mg, yield 66%) was obtained by column chromatography purification, and its structural formula was as follows:

[0100]

[0101] The structure was confirmed to be correct: 1H NMR (400 MHz, CDC13) δ 7.52 (d, J = 2.0 Hz, 2H), 7.48 (dd, J = 7.4, 1.8 Hz, 2H), 7.35 - 7.28 (m, 1H), 7.27 - 7.18 (m, 3H), 6.64 (dd, J = 7.5, 2.0 Hz, 2H), 6.40 (t, J = 4.9 Hz, 1H), 3.42 (td, J = 5.9, 4.9 Hz, 2H), 2.95 (s, 14H).

[0102] 13 C NMR (101 MHz, CDC13) δ 155.88, 146.99, 137.80, 134.76, 132.71, 131.87, 130.94, 129.24, 128.77, 128.41, 127.40, 126.11, 125.98, 116.25, 114.65, 40.61, 40.40, 32.20.

[0103] Example 7

[0104] Synthesis of compound 7 with R1 as oxygen, R2 as

[0105] The synthesis of compound 7 was the same as compound 1, and the only difference between Example 1 was that in step S3, N,N-dimethylaniline (21.79 mg, 0.16 mmol) was used to replace STAT3 inhibitor HJC0152 in the above step S3, and compound 7 (45 mg, yield 62%) was obtained by column chromatography purification, and its structural formula is as follows:

[0106]

[0107] The structure was confirmed to be correct: 1 H NMR (400 MHz, CDC13) δ 8.56 (s, 1H), δ 7.39 - 7.34 (m, 3H), 7.33 (d, J = 8.0 Hz, 1H), 6.91 (dd, J = 8.1, 2.2 Hz, 2H), 6.68 - 6.62 (m, 2H), 6.58 (d, J = 2.0 Hz, 2H), 2.98 (d, J = 13.5 Hz, 18H).

[0108] 13 C NMR (101 MHz, CDC13) δ 154.00, 149.49, 146.47, 145.82, 134.75, 123.41, 123.39, 121.62, 121.49, 121.42, 113.61, 110.26, 102.57, 40.28, 40.06. ​

[0109] Example 8

[0110] Synthesis with R1 as sulfur and R2 as sulfur Compound 8:

[0111] The synthesis method of compound 8 is the same as that of compound 2, except that in step S3, the STAT3 inhibitor HJC0152 in step S3 above was replaced with N,N-dimethylaniline (21.79 mg, 0.16 mmol), and compound 8 (52 mg, yield 60%) was obtained by column chromatography purification. Its structural formula is shown below:

[0112]

[0113] The structure has been confirmed to be correct. 1 H NMR (400MHz, CDCl3) δ8.61 (s, 1H), δ7.39–7.33 (m, 4H), 6.81 (dd, J = 7.9, 2.2Hz, 2H), 6.73 (d, J = 2.0Hz, 2H), 6.68–6.62 (m, 2H), 2.98 (d, J = 10.6Hz, 18H).

[0114] 13 C NMR (101MHz, CDCl3) δ153.86,149.49,146.63,134.19,133.70,132.81,122.71,122.48,122.43,121.42,113.73,113.61,112.35,40.40,40.28.

[0115] Example 9

[0116] Synthesis with R1 as selenium and R2 as... Compound 9:

[0117] The synthesis method of compound 9 is the same as that of compound 3, except that in step S6, the STAT3 inhibitor HJC0152 in step S6 was replaced with N,N-dimethylaniline (21.79 mg, 0.16 mmol). Compound 9 (58 mg, 53% yield) was purified by column chromatography, and its structural formula is shown below:

[0118]

[0119] The structure has been confirmed to be correct. 1H NMR (400 MHz, CDCI3) δ 8.36 (s, 1 H), δ 7.54 - 7.45 (m, 4H), 7.39 - 7.33 (m, 2H), 6.68 - 6.61 (m, 4H), 2.99 (s, 6H), 2.96 (s, 12H).

[0120] 13 C NMR (101 MHz, CDCI3) δ 152.83, 148.78, 147.20, 147.17, 136.35, 135.80, 134.29, 125.66, 125.54, 121.85, 121.81, 121.61, 121.56, 118.88, 118.84, 114.73, 114.72, 113.53, 113.50, 40.42, 40.29.

[0121] Performance test

[0122] The dyes synthesized in the above Examples 1-9 after vacuum drying were accurately weighed with a millionth scale, and a 2 mmol / L DMSO compound mother liquor was prepared in a brown sample bottle and stored in a 4°C refrigerator for standby use. The compounds prepared in Examples 1, 2 and 5 were taken as examples for performance testing as follows:

[0123] Test Example 1 Test of ultraviolet absorption spectrum in different solvents

[0124] Test method: The solvents were selected as dichloromethane, tetrahydrofuran, ethyl acetate, methanol, ethanol, N,N-dimethylformamide and dimethyl sulfoxide. 6 μL of the compound mother liquor was taken with a pipette and dissolved in a quartz cuvette containing 3 mL of organic solvent, mixed uniformly to obtain a solution with a concentration of 4 μM for ultraviolet spectrum determination. All tests were completed at 25°C. The test results are shown in Table 1. Figures 1-3 .

[0125] Figure 1 The ultraviolet absorption spectrum of compound 1 in different solvents is shown in Figure 1. Figure 1 It can be seen that the maximum absorption wavelength of compound 1 in different solvents is about 320 nm, and the absorbance is basically unchanged (0.1).

[0126] Figure 2 The ultraviolet absorption spectrum of compound 2 in different solvents is shown in Figure 2. Figure 2 It can be seen that the maximum absorption wavelength of compound 2 in different solvents is 320-330 nm, and the absorbance is slightly different, with the largest absorbance in tetrahydrofuran (0.1).

[0127] Figure 3 The ultraviolet absorption spectrum of compound 5 in different solvents is shown in Figure 5.Figure 3 It can be seen that the maximum absorption wavelength of compound 5 in different solvents is about 260 nm, and the absorbance is slightly different, with the maximum absorbance in tetrahydrofuran (0.48).

[0128] In combination Figures 1-3 It can be seen that different R1 has little effect on the absorbance of the light cage compound, but different linking groups R2 can affect the maximum absorption wavelength and absorbance of the compound, and the absorbance of compound 5 is greater than that of compounds 1 and 2, which is due to the effect of linking group R2 on the molar extinction coefficient of the whole light cage molecule.

[0129] Test Example 2: UV absorption spectrum determination under light

[0130] Test method: Add 3 mL of THF / H2O mixed solvent in a cuvette, and add 6 μL of compound mother liquor respectively. Place the cuvette under the light of 650 / 660 nm LED lamp, and the light density is 20 mw / cm 2 , test the UV absorption spectrum of the solution every 1 min. The test results of compounds 1, 2 and 5 are shown in Figure 4 , Figure 5 and Figure 6 .

[0131] As can be seen from Figure 4 , under 650 nm light, the absorption of compound 1 at 645 nm gradually increases and is almost completely released within 30 min. Figure 5 It can be seen that compound 2 under 660 nm light, the absorption at 660 nm is continuously enhanced, and the release reaches equilibrium within 10 min. Figure 6 It can be seen that compound 5 also occurs photolysis under 660 nm light, and reaches equilibrium within 30 min. The experimental results show that the photolysis rate of compound 2 is the fastest, which may be due to the release of photosensitizer methylene blue under light, which produces singlet oxygen and promotes the photolysis reaction, and also the result of the synergistic effect of R1 and R2.

[0132] Test Example 3: Cell imaging before and after light

[0133] Test method: MDA-MB-231 cells (triple negative breast cancer) were selected as test cell lines, cultured with DMEM high glucose medium, and incubated in a confocal culture dish for 24 h, then 2 μM of compound was added and incubated for 2 h. After 5, 10, 15 and 20 min of light irradiation with 660 nm LED light, confocal imaging was performed, and the control group was not irradiated. Confocal parameters: 60X oil lens, compound 1 laser channel: excitation 650 nm, emission 660-750 nm, test results are shown in Figure 7 .

[0134] From Figure 7It can be seen that under 650nm light irradiation, the channel of compound 1 shows the enhancement of red fluorescence with the increase of irradiation time, while there is no red fluorescence signal without light irradiation, indicating that compound 1 can perform light-controlled visual imaging of triple-negative breast cancer cells.

[0135] Test Example 4 Cytotoxicity (MTT) test

[0136] The cytotoxicity of the compound to cells was evaluated by MTT assay. The principle is that the succinate dehydrogenase in the mitochondria of living cells can reduce exogenous MTT to water-insoluble blue-violet crystalline formazan and deposit in the cells, while dead cells do not have this function. Dimethyl sulfoxide (DMSO) can dissolve formazan in cells, and its light absorption value is measured at 490nm wavelength by an enzyme marker, which can indirectly reflect the number of living cells.

[0137] MDA-MB-231 cells were inoculated in 6 pieces of 96-well plates, and after a period of culture, a certain concentration of compounds 1, 2 and 5 was added to different wells, so that the compound concentration was 0-5μM. After 2h of incubation in the cell incubator, one of the 96-well plates added with compounds 1, 2 and 5 was placed under 660nm LED light irradiation for 20min, and the light density was 20mW / cm2. Then after 12h of incubation in the cell incubator, the cell activity was detected by MTT experiment, and the test results are shown in Figures 8-10 .

[0138] Combination Figures 8-10 It can be seen that without light irradiation, the cell survival rates of compounds 1, 2 and 5 are all higher than 90%, indicating that within the concentration range of 0-5μM, compounds 1, 2 and 5 all have low cytotoxicity. Under light irradiation, the cell survival rate of compound 1 is 82%, the cell survival rate of compound 2 is as low as 40%, and the cell survival rate of compound 5 is 52%, which indicates that compounds 1, 2 and 5 all undergo photolysis under light irradiation, releasing fluorophore / photosensitizer and functional molecules. The phototoxicity of compound 2 is obviously higher than that of compounds 1 and 5, because compound 2 generates singlet oxygen and STAT3 inhibitor under light irradiation, which has a combined killing effect on MDA-MB-231 cells.

[0139] Test Example 5 Western blot protein experiment

[0140] Test method: Compound 1, 2 and 5 were incubated with MDA-MB-231 cells on 6-well plates for 2h, then treated by different treatments (light and no light). The cells were lysed with RIPA cell lysis buffer on ice, and the supernatant was collected after centrifugation at 12000 rpm for 5 min to obtain total protein. Then the protein was separated by SDS-PAGE electrophoresis and transferred to a PVDF membrane. After blocking with western blocking solution, STAT3 and P-STAT3 were incubated overnight at 4°C, then incubated with horseradish peroxidase (HRP) labeled secondary antibody for 2h. Finally, the expression of STAT3 and P-STAT3 was detected by chemiluminescence, and Actin protein was used as an internal reference. The test results are shown in Figure 11 .

[0141] From Figure 11 it can be seen that the expression of STAT3 protein in cells is reduced under light, and compounds 2 and 5 significantly inhibit the expression of STAT3 and phosphorylated STAT3 (pSTAT3-Tyr705) protein. In particular, compound 2, the expression of STAT3 and pSTAT3 protein is almost completely inhibited, indicating that compound 2 reduces the expression of STAT3 protein after light. 1 O2 also reduces the expression of STAT3 protein.

[0142] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present application, and not to limit it; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacement for part or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A phenothiazine leuco photo-cage compound, characterized in that, The phenthiazine leuco photo-caged compound is selected from one of the following compounds: 。 2. The method of synthesis of a phenothiazine leuco photo-cage compound as claimed in claim 1, wherein, The method comprises the following steps: (1) reacting a phenthiazine leuco dye containing R1 substitution, sodium pyrosulfate and sodium carbonate in a first organic solvent and water under an inert gas environment, a reaction temperature of 25-45 ℃, a reaction time of 0.5-3 h, to prepare a compound shown as formula Y-2; cooling the compound shown as formula Y-2 with ice water, and adding triphosgene and an alkaline solvent dissolved in a second organic solvent to continue the reaction for 3-6 h, to prepare a corresponding acyl chloride-substituted phenthiazine leuco dye shown as formula Y-3; the R1 is selected from any one of oxygen, sulfur and selenium; (2) mixing the compound shown as formula Y-3 synthesized in step (1), R2 and triethylamine uniformly in a third organic solvent, and reacting at 0-25 ℃ for 6-12 h, to prepare the phenthiazine leuco photo-caged compound; R2 is selected from the following structural groups: ； 。 3. The method of synthesis of claim 2, wherein, In step (1), the molar ratio of the phenthiazine leuco dye containing R1 substitution, sodium pyrosulfate and sodium carbonate is 1:2-4:2-4; the molar ratio of the compound shown as formula Y-2 and triphosgene is 1:0.6-1.

4. The method of synthesis of claim 2, wherein, In step (1), the first organic solvent is selected from any one or a combination of mixed solvents of dichloromethane, toluene and benzene; The second organic solvent is selected from any one or a combination of solvents of dichloromethane, chloroform and ethyl acetate; The alkaline solvent is selected from at least one of triethylamine, pyridine and sodium carbonate; The inert gas environment is selected from one of nitrogen and argon.

5. The method of synthesis of claim 2, wherein, In step (2), the molar ratio of the compound shown as formula Y-3, R2 and triethylamine is 1:1-2:

3.

6. The method of synthesis of claim 2, wherein, In step (2), the third organic solvent is selected from any one or a combination of mixed solvents of dichloromethane, chloroform and tetrahydrofuran.

7. Use of a phenothiazine leuco photo-caged compound in the preparation of a drug for triple negative breast cancer, characterized in that, The phenthiazine leuco photo-caged compound is selected from one of the following compounds: 。 8. Use according to claim 7, characterized in that, The method is used for tumor treatment mediated by STAT3 signal pathway in vivo.

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