High-activity BODIPY type photosensitizer targeting double organelles as well as preparation method and application of high-activity BODIPY type photosensitizer

Abstract

The invention discloses a high-activity BODIPY type photosensitizer targeting double organelles as well as a preparation method and application of the high-activity BODIPY type photosensitizer, and relates to the technical field of medical photosensitizers. The high-activity BODIPY type photosensitizer is prepared by the following steps: reacting 2, 4-dimethyl pyrrole with 4-bromobutyryl chloride to obtain a compound Br-BY containing carboxyl, then reacting with morpholine to obtain a morpholine substituted compound Mor-BY, and finally reacting with N-iodosuccinimide (NIS) to obtain a single atom I substituted BODIPY compound Mor-IBY, namely the target photosensitizer. The photosensitizer prepared by the invention can target mitochondria and lysosome in cancer cells at the same time, and can be excited by visible light; the compound has good water solubility, can generate a large amount of reactive oxygen species (ROS) under irradiation of green light (520 nm), shows excellent photodynamic activity to tumor cells, and has low toxicity to normal cells.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medical photosensitizers, in particular to the preparation of a high-activity BODIPY-type photosensitizer targeting double organelles. BACKGROUND

[0002] Traditional cancer or malignant tumor treatment methods (surgery, chemotherapy and radiotherapy, etc.) have different limitations and drawbacks. For example, some cancerous tissues cannot be well resected, and chemotherapy and radiotherapy will cause drug resistance and varying degrees of resistance in the later treatment, resulting in reduced sensitivity. Photodynamic therapy has better spatiotemporal selectivity and generally does not cause drug resistance. Therefore, photodynamic therapy (PDT) as a micro (non) invasive therapy is widely considered as a promising method for treating malignant tumors, and is expected to be widely used in the treatment of various cancers in the future.

[0003] The three elements of photodynamic therapy are photosensitizer, oxygen and light, among which the photosensitizer (also called photodynamic drug) is the core. Therefore, one of the current research focuses is to develop new photosensitizers or photosensitizer prodrugs with high efficiency, low toxicity and good biocompatibility.

[0004] The fluoroboron dipyrrin (BODIPY) structural unit has the advantages of large molar absorption coefficient, good light stability and easy structural modification, and is widely used in the design and preparation of new fluorescent probes and the construction of photosensitizers. In order to improve the yield of reactive oxygen species (ROS), i.e. to convert more oxygen ( 3 O2) into singlet oxygen ( 1 O2), it is generally believed that one of the effective ways is to introduce heavy atoms (such as Br, I, etc. halogen atoms) symmetrically on one side or both sides of 3, 5 of BODIPY, and to use the enhanced intersystem crossing (ISC) time to achieve it. The generated ROS will attack nearby DNA, RNA or protein, further causing cancer cell apoptosis, necrosis, autophagy or ferroptosis, etc.

[0005] At present, there are many BODIPY-type photosensitizers containing heavy atoms developed based on the above principle, but many of them do not have organelle targeting ability, causing toxic side effects to normal cells while killing cancer cells. In order to attenuate the photosensitizer and improve its efficiency, the targeting ability of the photosensitizer to organelles (such as mitochondria, lysosomes, Golgi apparatus, endoplasmic reticulum, etc.) in cancer cells can directly improve the damage ability to biological macromolecules such as DNA, RNA and protein in the organelles, and thus greatly promote the killing of cancer cells. At the same time, in normal cells, the photosensitizer only exists locally and is not activated by light, thus weakening its toxic side effects.

[0006] Therefore, designing and preparing photosensitizers with organelle-targeting capabilities is of practical significance for enhancing anticancer activity and reducing toxic side effects, and provides guidance and reference for further developing novel photodynamic anticancer drugs that meet clinical needs. Summary of the Invention

[0007] This invention provides a highly active BODIPY-type photosensitizer targeting two organelles, its preparation method, and its applications. The highly active BODIPY-type photosensitizer provided by this invention exhibits good photostability and water solubility, can selectively accumulate in the mitochondria and lysosomes of cancer cells, and generates singlet oxygen (SO4) under green light (520 nm) irradiation. 1 O2 exhibits excellent photodynamic activity against tumor cells and low dark toxicity to normal cells, and possesses a high photodynamic activity index (PI value). Based on these effects, this photosensitizer can be used for photodynamic inhibition and elimination of tumors, achieving improved tumor treatment efficacy while maintaining low toxicity. This is specifically achieved through the following technology.

[0008] This invention provides a highly active BODIPY-type photosensitizer targeting two organelles, the structural formula of which is shown in Formula I below:

[0009] ; Formula I.

[0010] This invention also provides a method for preparing the above-mentioned highly active BODIPY-type photosensitizer targeting two organelles, wherein the preparation method is carried out in a protective gas atmosphere and includes the following steps:

[0011] 2,4-Dimethylpyrrole and 4-bromobutyryl chloride were added to a first organic solvent to carry out a first reaction until the reaction was complete; the mixture was concentrated, dissolved in toluene and the first organic solvent, and then triethylamine and / or ammonia were added and stirred. Boron trifluoride·diethyl ether was added dropwise to carry out a second reaction until the reaction was complete; the mixture was concentrated and purified to obtain compound Br-BY.

[0012] The compound Br-BY was dissolved in a second organic solvent, morpholine and a carbonate solution were added, and the reaction was carried out for a third time until the reaction was complete; after purification, the compound Mor-BY was obtained; the carbonate was at least one of potassium carbonate, sodium carbonate and cesium carbonate.

[0013] The compound Mor-BY was dissolved in the first organic solvent, and N-iodosuccinimide was added for a fourth reaction until the reaction was complete. After purification, the highly active BODIPY-type photosensitizer was obtained.

[0014] Optionally, in the above preparation method, the protective gas atmosphere can be a nitrogen (N2) atmosphere or an inert gas (helium, argon, etc.) atmosphere.

[0015] Further, the molar ratio of 2,4-dimethylpyrrole, 4-bromobutyryl chloride and boron trifluoride etherate is 2: (1-1.2): (4.5-5.0).

[0016] Further, the molar ratio of 2,4-dimethylpyrrole, 4-bromobutyryl chloride and boron trifluoride etherate is 2: (1-1.2): (4.5-5.0).

[0017] Further, the molar ratio of the compound Br-BY, morpholine and carbonate is 1: (3-4): (6-10).

[0018] Further, the molar ratio of the compound Br-BY, morpholine and carbonate is 1: (3-4): (6-10).

[0019] Further, the molar ratio of the compound Mor-BY and N-iodosuccinimide is 1: (2-6).

[0020] Further, the molar ratio of the compound Mor-BY and N-iodosuccinimide is 1: (2-6).

[0021] Further, the condition of the first reaction is 40-50℃ for 2-4 h.

[0022] Further, the condition of the first reaction is 40-50℃ for 2-4 h.

[0023] Further, the condition of the second reaction is 50-90℃ for 2-6 h.

[0024] Further, the condition of the second reaction is 50-90℃ for 2-6 h.

[0025] Further, the condition of the third reaction is 20-40℃ for 1-8 h.

[0026] Further, the condition of the third reaction is 20-40℃ for 1-8 h.

[0027] Further, the fourth reaction is a room temperature reaction, and the reaction of the compound Mor-BY is detected.

[0028] Further, the first organic solvent is dichloromethane and / or trichloromethane.

[0029] Further, the second organic solvent is any one of acetonitrile, dichloromethane, trichloromethane, acetone, dimethyl sulfoxide and N, N-dimethylformamide.

[0030] The application also provides an application of the high-activity BODIPY-type photosensitizer, which is used for preparing an anticancer / antitumor drug.

[0031] The high-activity BODIPY photosensitizer Mor-IBY provided by the application can target mitochondria and lysosomes of cancer cells, generate singlet oxygen (O2) under green light irradiation, thereby having excellent photodynamic activity on tumor cells and low dark toxicity on normal cells. 1 O2), thereby having excellent photodynamic activity on tumor cells and low dark toxicity on normal cells.

[0032] Compared with the prior art, the application has the advantages that:

[0033] 1. The high-activity BODIPY photosensitizer provided by the application has good stability and water solubility, can selectively gather in mitochondria and lysosomes of cancer cells, has strong singlet oxygen release capacity under 520 nm green light irradiation, and can cause reduction of mitochondrial membrane potential and induction of lysosome rupture.

[0034] 2. In photodynamic toxicity activity experiments (MTT) on colorectal cancer cells HCT116, human bladder transitional cells T24, human laryngeal cancer cells TU686 and human normal intestinal epithelial cells NCM460, the IC 50 of the high-activity BODIPY photosensitizer Mor-IBY provided by the application under light irradiation on the three kinds of cancer cells is 0.06, 2.43 and 3.16 μM respectively; and the IC 50 without light irradiation is 5.29, 15.17 and 16.32 μM respectively. The IC 50 of the normal cells NCM460 under light irradiation and without light irradiation is 7.08 and 54.11 μM respectively. In summary, the photosensitizer Mor-IBY has the best activity on colorectal cancer cells HCT116, and the photodynamic index (PI) is 88. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 The flowchart is for preparation of the photosensitizer Mor-IBY of the examples.

[0036] Figure 2 The nuclear magnetic resonance hydrogen spectrum (CDCl3, 400 MHz) of the intermediate compound Br-BY.

[0037] Figure 3 The nuclear magnetic resonance carbon spectrum (CDCl3, 101 MHz) of the intermediate compound Br-BY.

[0038] Figure 4 The high-resolution mass spectrum of the intermediate compound Br-BY.

[0039] Figure 5 The nuclear magnetic resonance hydrogen spectrum (CDCl3, 400 MHz) of the intermediate compound Mor-BY.

[0040] Figure 6 NMR spectrum of intermediate compound Mor-BY (CDCl3, 101 MHz).

[0041] Figure 7 High resolution mass spectrum of intermediate compound Mor-BY.

[0042] Figure 8 NMR spectrum of intermediate compound Mor-IBY (CDCl3, 400 MHz).

[0043] Figure 9 High resolution mass spectrum of intermediate compound Mor-IBY.

[0044] Figure 10 UV-Vis absorption spectrum (A), fluorescence excitation and emission spectrum (B), and decay plot of DPBF absorbance at 417 nm over time under light (C) of three compounds Br-BY, Mor-BY and Mor-IBY.

[0045] Figure 11 Survival rate plot of Mor-IBY on HCT116 and NCM460 cells under light and dark conditions, respectively.

[0046] Figure 12 Results plot of co-localization experiment of Mor-IBY on mitochondria and lysosome in HCT116 cells.

[0047] Figure 13 Results plot of the effect of Mor-IBY on mitochondrial membrane potential of HCT116 cells (JC-1 staining).

[0048] Figure 14 Effect of Mor-IBY on lysosome of HCT116 cells (OA staining). DETAILED DESCRIPTION

[0049] The technical solutions of the present application will be described clearly and completely below. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0050] The present application provides a high-activity BODIPY-type photosensitizer targeting double organelles, which has the following structural formula I:

[0051] Formula I.

[0052] The application further provides a preparation method of the above-mentioned high-activity BODIPY-type photosensitizer targeting double organelles, which is carried out in a protective gas atmosphere, and comprises the following steps:

[0053] 2,4-dimethylpyrrole and 4-bromobutyryl chloride are added to a first organic solvent to carry out a first reaction until the reaction is complete; toluene and the first organic solvent are added for dissolution, triethylamine and / or ammonia water are added for stirring, and boron trifluoride etherate is added dropwise to carry out a second reaction until the reaction is complete; after concentration, the compound Br-BY is obtained after purification;

[0054] The compound Br-BY is dissolved in a second organic solvent, morpholine and a carbonate solution are added, and a third reaction is carried out until the reaction is complete; after purification, the compound Mor-BY is obtained; the carbonate is at least one of potassium carbonate, sodium carbonate and cesium carbonate;

[0055] The compound Mor-BY is dissolved in the first organic solvent, N-iodosuccinimide is added to carry out a fourth reaction until the reaction is complete, and after purification, the high-activity BODIPY-type photosensitizer is obtained.

[0056] Optionally, in the above preparation method, the protective gas atmosphere can be a nitrogen (N2) or inert gas (helium, argon, etc.) atmosphere.

[0057] Optionally, in the above preparation method, the purification method includes but is not limited to dissolution, washing, filtration, etc. Specifically, the purification process of each step can be adjusted according to the physicochemical properties of the corresponding product and impurities.

[0058] Further, the molar ratio of 2,4-dimethylpyrrole, 4-bromobutyryl chloride and boron trifluoride etherate is 2:(1-1.2):(4.5-5.0).

[0059] Further, the molar ratio of 2,4-dimethylpyrrole, 4-bromobutyryl chloride and boron trifluoride etherate is 2:1:4.76.

[0060] Further, the molar ratio of the compound Br-BY, morpholine and carbonate is 1:(3-4):(6-10).

[0061] Further, the molar ratio of the compound Br-BY, morpholine and carbonate is 1:3.46:8.03.

[0062] Further, the molar ratio of the compound Mor-BY and N-iodosuccinimide is 1:(2-6).

[0063] Further, the molar ratio of the compound Mor-BY and N-iodosuccinimide is 1:6.

[0064] Further, the first reaction condition is 40-50 °C for 2-4 h.

[0065] Further, the first reaction condition is 40-50 °C for 2-4 h.

[0066] Further, the second reaction condition is 50-90 °C for 2-6 h.

[0067] Further, the second reaction condition is 50-90 °C for 2-6 h.

[0068] Further, the third reaction condition is 20-40 °C for 1-8 h.

[0069] Further, the third reaction condition is 20-40 °C for 1-8 h.

[0070] Further, the fourth reaction is at room temperature, and the reaction of the compound Mor-BY is detected to be complete.

[0071] Further, the first organic solvent is dichloromethane and / or chloroform.

[0072] Further, the second organic solvent is any one of acetonitrile, dichloromethane, chloroform, acetone, dimethyl sulfoxide, and N, N-dimethylformamide.

[0073] Example 1

[0074] The preparation method of the high-activity BODIPY-type photosensitizer provided in the embodiment is as follows:

[0075] 1. Synthesis of compound Br-BY

[0076] (1) 2,4-dimethylpyrrole (2.00 g, 21 mmol) was dissolved in 150 mL of dichloromethane (first organic solvent) and poured into a 250 mL three-necked flask; under nitrogen protection, 4-bromobutyryl chloride (1.95 g, 10.5 mmol) was added, and heating and stirring were performed at 50 °C for 2.5 h;

[0077] The reaction was stopped, cooled to room temperature, and rotary evaporated under reduced pressure to obtain dark red oily material.

[0078] (2) 150 mL of toluene and 25 mL of dichloromethane (first organic solvent) were added to re-dissolve, and triethylamine (2.53 g, 25 mmol) was added, and after stirring for 15 min, a clear orange yellow solution was obtained.

[0079] (3) Under nitrogen protection, slowly drop boron trifluoride etherate (7.60 g, 50 mmol) and heat the reaction in a 50°C oil bath for 4 h until the solution becomes opaque green;

[0080] (4) Cool to room temperature, rotary evaporate under reduced pressure, dissolve the residue with 150 mL ethyl acetate and wash with distilled water (3x25 mL); collect the organic layer and dry overnight with anhydrous sodium sulfate;

[0081] Perform suction filtration, evaporate the solvent under reduced pressure to obtain Br-BY red viscous crude product;

[0082] Purify with silica gel column chromatography (petroleum ether / ethyl acetate = 3 / 1) to obtain orange red powder compound Br-BY (774 mg, yield: 20.02%). The nuclear magnetic resonance hydrogen spectrum (CDCl3, 400 MHz) of compound Br-BY is shown in Figure 2 , the nuclear magnetic resonance carbon spectrum (CDCl3, 101 MHz) is shown in Figure 3 , and the high resolution mass spectrum is shown in Figure 4 .

[0083] The nuclear magnetic resonance information of compound Br-BY is as follows:

[0084] 1 H NMR (400 MHz, CDCl3) δ: 6.07 (s, 1H), 3.48 (t, J = 6.4 Hz, 1H), 2.52(s, 1H), 2.44 (s, 1H). 13 C NMR (101 MHz, CDCl3) δ: 172.44, 154.32, 144.73, 140.22, 131.36, 121.86, 77.36, 77.04, 76.72, 64.08, 32.62, 32.26, 30.67, 27.63, 25.09 16.39, 14.47.

[0085] ESI-HRMS (CH3OH): C 20 H 27 O2N2BF2Br + , 455.1319 [Br-BY+H] + (Found: 455.1312).

[0086] 2. Synthesis of skeleton intermediate compound Mor-BY

[0087] (1) The above compound Br-BY (0.58 g, 1.57 mmol) was dissolved in 30 mL of acetonitrile (second organic solvent), poured into a 100 mL three-necked flask, and morpholine (0.55 g, 5.43 mmol) and potassium carbonate solution were added; under nitrogen protection, the reaction was stirred at 30°C for 3 h;

[0088] The potassium carbonate solution was prepared by dissolving 1.74 g (12.61 mmol) of potassium carbonate in 10 mL of distilled water.

[0089] (2) After the reaction system was cooled to room temperature, it was suction filtered, and the solution was rotary evaporated under reduced pressure. The residue was dissolved in 50 mL of dichloromethane (first organic solvent) and washed with distilled water (3 x 10 mL). The organic layer was collected and dried over anhydrous sodium sulfate overnight.

[0090] Suction filtration was performed, and the solvent was rotary evaporated under reduced pressure to obtain a dark red viscous crude product. Silica gel column chromatography (pure dichloromethane) was used to purify it to obtain Mor-BY (0.44 g, yield: 45.36%) in reddish brown. The nuclear magnetic resonance hydrogen spectrum (CDCl3, 400 MHz) of compound Mor-BY is shown in Figure 5 , the nuclear magnetic resonance carbon spectrum (CDCl3, 101 MHz) is shown in Figure 6 , and the mass spectrum is shown in Figure 7 .

[0091] The nuclear magnetic resonance information of compound Mor-BY is as follows:

[0092] 1 H NMR (400 MHz, CDCl3) δ: 6.06 (s, 2H), 4.24~4.21(t, J = 6.2 Hz, 2H),3.71(s, 4H), 3.06~3.02(m, 2H), 2.52(s 6H), 2.46(s, 2H), 2.40(s, 6H), 2.38~2.36(t, J = 7.3 Hz, 2H). 13 C NMR (101 MHz, CDCl3) δ :173.53, 154.45, 144.93,140.31, 131.49, 121.97, 77.48, 77.16, 76.84, 67.07, 63.95, 58.09, 53.73,32.09, 30.89, 25.24, 21.82, 16.49, 14.59.

[0093] ESI-MS (CH3OH): C 24 H 35 O3N3BF2+ 462.272 [Mor-BY+H] + (Found: 462.273).

[0094] 3. Synthesis of photosensitizer Mor-IBY

[0095] The above compound Mor-BY (0.33 g, 0.88 mmol) was added to a 100 mL three-necked flask and dissolved in 30 mL of dichloromethane.

[0096] N-iodosuccinimide (NIS) (1.19 g, 5.28 mmol) was weighed into the three-necked flask and stirred at room temperature under N2protection.

[0097] The reaction progress was monitored by TCL until Mor-BY was completely reacted.

[0098] The mixed solution was washed with distilled water (3 x 10 mL) to remove excess NIS.

[0099] The organic phase was collected, dried with anhydrous sodium sulfate and left overnight.

[0100] It was suction filtered and the solvent was evaporated under reduced pressure to obtain a red viscous crude product. Purification was performed on a silica gel column (petroleum ether / dichloromethane = 1 / 1) to obtain red powder Mor-IBY product (300 mg, yield: 77.1%). The nuclear magnetic resonance hydrogen spectrum of compound Mor-IBY (CDCl3, 400 MHz) is shown in Figure 8 , and the high-resolution mass spectrum is shown in Figure 9 .

[0101] The nuclear magnetic resonance information of compound Mor-IBY is as follows:

[0102] 1 H NMR (400 MHz, CDCl3) δ: 3.74 (s, 1H), 3.10 (dt, J = 16.7, 8.4 Hz, 1H), 2.77 (s, 1H), 2.60 (s, 1H), 2.48 (s, 1H), 2.45 (s, 1H), 2.41 ~ 2.36 (m, 1H), 1.87 (s, 1H).

[0103] ESI-HRMS (CH3OH): C 24 H 34 O3N3BF2I + : 588.1708 [Mor-IBY+H] + , (Found 588.1700).

[0104] 4. UV-Vis absorption spectra and fluorescence spectra

[0105] Both UV-Vis absorption spectra and fluorescence spectra were measured in HEPES buffer (2% DMSO, 10 mM, pH 7.4). The concentrations of Br-BY, Mor-BY and Mor-IBY were all 20 μM.

[0106] As shown in Figure 10 , Figure 10 A represents the UV-Vis absorption spectra of Br-BY, Mor-BY and Mor-IBY, respectively; Figure 10 B represents the fluorescence emission and excitation spectra of Br-BY, Mor-BY and Mor-IBY, respectively.

[0107] From Figure 10 A, it can be seen that the maximum absorption peaks of compound I2BA-Cyclen and I2BA-Cyclen-Cu(II) are located at 501 nm, 495 nm and 511 nm, respectively, which are in the green light absorption region. From Figure 10 B, it can be seen that under the excitation wavelength of 480 nm, the maximum emission peaks of Br-BY, Mor-BY and Mor-IBY are located at 515 nm, 527 nm and 531 nm, respectively. The fluorescence emission wavelength of Mor-IBY containing heavy atom I is obviously red-shifted compared with Br-BY. At the same time, due to the heavy atom effect, the fluorescence is almost completely quenched.

[0108] Example 2

[0109] The preparation method of the high-activity BODIPY type photosensitizer provided in the embodiment is basically the same as the preparation method of Example 1, and the difference lies in that:

[0110] (1) When synthesizing compound Br-BY, the amount of 2,4-dimethylpyrrole used is 21 mmol, and the amount of 4-bromobutyryl chloride used is 12.6 mmol; the reaction condition is heating and stirring at 40℃ for 4 h;

[0111] (2) When synthesizing compound Br-BY, the amount of boron trifluoride etherate added is 47.25 mmol, and the reaction condition after adding boron trifluoride etherate dropwise is heating in a 90℃ oil bath for 2 h;

[0112] (3) When preparing Mor-BY, the amount of compound Br-BY added is 1.57 mmol, the amount of morpholine added is 6.28 mmol, and the amount of potassium carbonate added is 9.42 mmol; the reaction condition under nitrogen protection is stirring at 20℃ for 8 h.

[0113] Example 3

[0114] The preparation method of the high-activity BODIPY-type photosensitizer provided in the embodiment is basically the same as the preparation method of the embodiment 1, and the difference lies in that:

[0115] (1) When the compound Br-BY is synthesized, the reaction conditions after 2,4-dimethylpyrrole and 4-bromobutyryl chloride are added are 50 °C heating and stirring reaction for 6 h;

[0116] (2) When the compound Br-BY is synthesized, the amount of boron trifluoride etherate added is 52.5 mmol, and the reaction conditions after the boron trifluoride etherate is added dropwise are 50 °C heating in an oil bath for 6 h;

[0117] (3) When the Mor-BY is prepared, the added amount of the compound Br-BY is 1.57 mmol, the added amount of morpholine is 4.71 mmol, and the added amount of potassium carbonate is 15.7 mmol; the reaction conditions under nitrogen protection are 40 °C stirring for 1 h.

[0118] Test example 1: ROS release in solution (DPBF experiment)

[0119] DPBF is a commonly used probe for detecting active oxygen (mainly singlet oxygen), and has extremely strong sensitivity. The characteristic absorption peak of DPBF is at 417 nm. When DPBF reacts with 1 O2, the light absorption intensity will decrease significantly.

[0120] In the test example, (1) first, the DPBF stock solution (10 mM) is diluted 200 times by DMSO to form a 50 μM solution, and 3 mL is taken and added to a cuvette; (2) then 6 μL of compound (Br-BY, Mor-BY and Mor-IBY prepared in the embodiment 1) HEPES stock solution (2% DMSO, 10 mM, pH 7.4) with a concentration of 1 mM is added to the cuvette, and mixed uniformly, to prepare a mixed test solution with a concentration ratio of DPBF and compound of 25:1. (3) The above three test solutions are scanned by ultraviolet light every 5 s, and the change of absorbance at 417 nm with time is recorded, and Figure 10 C.

[0121] Therefore, it can be known that Br-BY and Mor-BY almost do not produce 1 O2. With the change of light irradiation time, the compound Mor-IBY induces more and more 1 O2 in the solution. Therefore, it can be inferred that Mor-IBY has the potential to be used as an excellent photosensitizer for anti-cancer.

[0122] Test Example 2: Toxicity and activity of Br-BY, Mor-BY, Mor-IBY and cisplatin on HCT116, T24, TU686 and NCM460 (MTT experiment).

[0123] 1. HCT116 (human colon cancer cell line), T24 (human bladder transitional cell carcinoma cell line), TU686 (human laryngeal carcinoma cell line) and NCM460 (human normal intestinal epithelial cell line) were purchased from Wuhan Pnuo Sai Company. All cell lines were cultured in RPMI-1640 / DMEM culture medium containing 10% calf serum and 100 U / mL streptomycin, and incubated in a 37°C incubator containing 5% CO2 by volume.

[0124] 2. The concentrations of T24, TU686, HCT16 and NCM460 cells were adjusted to 8 x 10 3 cells / well, and 6 replicate wells were established for each concentration. 0, 12.5, 25, 50, 100 and 200 nM drug-containing medium was added at a dose of 100 μL per well, and incubated in a 37°C, 5% CO2 incubator in the dark for 24 h. The drug-containing medium was prepared by purchasing from Gibco Company (USA).

[0125] The light condition was as follows: the same drug concentration was first incubated in a 37°C, 5% CO2 incubator in the dark for 24 h, and then fixed light source light for 20 min (520 nm, 20 mW / cm 2 ).

[0126] 3. Then 10 μL (5 g / L) thiazole blue (MTT) solution was added to each well, and incubated in the dark for 4 h. The old liquid was discarded, 150 μL DMSO was added to each well, mixed well on a shaking bed at room temperature, and the absorbance value was detected at 490 nm wavelength by an enzyme marker instrument. The cell growth curve was drawn by GraphPad Prism 9.0, and the IC 50 value was calculated.

[0127] 4. For cisplatin, physiological saline was used as the solvent, and after dissolution, the drug was administered, and incubated in the dark for 24 h. The concentration gradient was still 0, 12.5, 25, 50, 100 and 200 nM.

[0128] Table 1 is the summary data of the toxicity and activity of Br-BY, Mor-BY, Mor-IBY and cisplatin (a first-line clinical chemotherapy drug) prepared in Example 1 on HCT116, T24, TU686 and NCM460 (IC 50 , unit: μM).

[0129] Table 1

[0130] From Table 1 and Figure 11It can be seen that Mor-BY has only weak photodynamic activity due to the absence of iodine atom, and the IC50 values of Mor-BY against three cancer cells are between 43.68-48.61 μM. Br-I2BY and Mor-IBY have good photodynamic activity due to the presence of iodine atom. Especially for HCT116 cells, Mor-IBY has a low IC50 value of 60 nM under light, indicating its potential as a photodynamic anti-colorectal cancer drug. 50 It can be seen that Mor-BY has only weak photodynamic activity due to the absence of iodine atom, and the IC50 values of Mor-BY against three cancer cells are between 43.68-48.61 μM. Br-I2BY and Mor-IBY have good photodynamic activity due to the presence of iodine atom. Especially for HCT116 cells, Mor-IBY has a low IC50 value of 60 nM under light, indicating its potential as a photodynamic anti-colorectal cancer drug.

[0131] Test Example 3: Distribution of Mor-IBY in HCT116 cells (co-localization test)

[0132] The nuclear probe DAPI, the mitochondrial probe MitoTracker Red and the lysosome probe LysoTracker Red were all purchased from Shanghai Biyuntian Biotechnology Co., Ltd.

[0133] The density of HCT116 cells was adjusted to 3 x 10 4 cells / well, and the compound Mor-IBY prepared in Example 1 was added to a final concentration of 10 μM under dark conditions for 3 h. Then MitoTracker Red or LysoTracker Red was added for incubation for 15 min, and DAPI was added for incubation for 30 min. Finally, three-channel imaging was performed on a laser confocal fluorescence microscope.

[0134] Blue channel: E x =405 nm; E m =430-470 nm. Green channel: E x =488 nm, E m =510-550 nm. Red channel: E x =561 nm; E m =590-630 nm.

[0135] The test results are shown in Figure 12 It can be seen that the green fluorescence and the blue fluorescence in the cells do not overlap, indicating that the photosensitizer Mor-IBY does not enter the nucleus and is only distributed in the cytoplasm. The green fluorescence of Mor-IBY has a good overlap rate with the red fluorescence of MitoTracker Red, indicating that Mor-IBY can selectively distribute in mitochondria. At the same time, the green fluorescence of Mor-IBY also has a good overlap rate with the red fluorescence of LysoTracker Red, indicating that Mor-IBY also selectively accumulates in lysosomes. In summary, the photosensitizer Mor-IBY has both mitochondrial and lysosomal targeting types.

[0136] Test Example 4: Effect of Mor-IBY on mitochondrial membrane potential in HCT116 cells (JC-1 staining)

[0137] JC-1 was purchased from Shanghai Biyun Tian Biotechnology Co., Ltd., and CCCP was a positive control drug attached when purchased.

[0138] Adjust the density of HCT116 cells to 3 x 10 4 / well, and add Mor-IBY prepared in Example 1 to a final concentration of 0, 25, 50, 100 nM under dark and light (520 nm, 20 mW / cm 2 , 20 min) conditions for 24 h; after treatment, add 0.5 mL of positive control and incubate in the incubator for 20 min; discard the old liquid, add 200 μL of cell culture solution and JC-1 working solution per well and incubate for 20 min; discard the old liquid, wash twice with JC-1 staining buffer, and finally add cell culture solution for photography. The incubation conditions of CCCP, such as concentration and time, are set according to the instructions attached when purchased.

[0139] The results of JC-1 staining are shown in Figure 13 , it can be seen that the blank group only has red fluorescence, indicating that JC-1 mainly accumulates in the matrix of mitochondria, forming a polymer, indicating that the mitochondrial membrane potential is high at this state; and under the action of the positive control drug CCCP, only clear green fluorescence can be seen, indicating that JC-1 does not accumulate in the matrix of mitochondria, at this time JC-1 is a monomer, indicating that the cell has a low mitochondrial membrane potential at this time.

[0140] After administration of Mor-IBY, without light, whether it is a low concentration of 25 nM or a high concentration of 100 nM, there is only red fluorescence, indicating that without light activation, ROS cannot be effectively produced, and it does not have the function of reducing mitochondrial membrane potential. Under light conditions, with the increase of the administration concentration from 25 nM to 100 nM, the red fluorescence gradually weakens, proving that the photosensitizer Mor-IBY can efficiently produce ROS under light activation, and there is a positive dependence on the concentration, ROS destroys the mitochondrial membrane, leading to the loss of mitochondrial membrane potential. This is one of the internal mechanisms of Mor-IBY to inhibit proliferation.

[0141] Test Example 5: Effect of Mor-IBY on lysosome integrity in HCT116 cells (AO staining)

[0142] Acridine orange (AO) was purchased from Shanghai Beyotime Biotechnology Co., Ltd. Before use, it was diluted 1000-fold with PBS. The culture medium in the HCT116 cell plates was gently aspirated, and the cells were washed twice with PBS. Acridine orange staining solution was added to each well (1 mL), and staining was performed at 37°C for 10 min. The staining solution was then aspirated, and the cells were washed twice with PBS. Appropriate amounts of cell culture medium and staining buffer were added, and the cells were observed under a microscope.

[0143] Observe green fluorescence: E x =488 nm; E m =510-550 nm. Observe the red fluorescence: E x =561 nm; E m =610-650nm.

[0144] AO staining results as follows Figure 14 As shown, AO emits bright red fluorescence in the acidic microenvironment of lysosomes, but the red fluorescence weakens in damaged lysosomes due to reduced integrity and acidity. The figure shows that, regardless of whether it is in darkness or light, the cells in the control group (without drug administration) exhibit significant red fluorescence, indicating better lysosomal integrity. When Mor-IBY 0.25 μM prepared in Example 1 is administered, the red fluorescence weakens compared to the control group. Furthermore, the red fluorescence is weaker under light conditions compared to darkness, indicating that light promotes the activation of the photosensitizer Mor-IBY, leading to ROS production and subsequent damage to lysosomal integrity. Therefore, lysosomal damage is one of the intrinsic reasons for the photodynamic anticancer activity of Mor-IBY.

[0145] The above detailed embodiments describe the implementation of the present invention; however, the present invention is not limited to the specific details described in the above embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. A high activity BODIPY-type photosensitizer targeting double organelles, characterized in that, The structural formula is shown as formula I below. ； Formula I.

2. A process for the preparation of the high activity BODIPY-type photosensitizer targeted to two organelles of claim 1, characterized by, The preparation method is carried out in a protective gas atmosphere, comprising the following steps: 2,4-dimethylpyrrole and 4-bromobutyryl chloride are taken into a first organic solvent to carry out a first reaction until the reaction is complete; concentrated, toluene and the first organic solvent are dissolved, triethylamine and / or ammonia water are stirred, and boron trifluoride ether is added dropwise to carry out a second reaction until the reaction is complete; concentrated, and after purification, compound Br-BY is obtained; The compound Br-BY is dissolved in a second organic solvent, morpholine and a carbonate solution are added, and a third reaction is carried out until the reaction is complete; after purification, compound Mor-BY is obtained; the carbonate is at least one of potassium carbonate, sodium carbonate and cesium carbonate; The compound Mor-BY is dissolved in the first organic solvent, N-iodosuccinimide is added, a fourth reaction is carried out until the reaction is complete, and after purification, the high-activity BODIPY type photosensitizer is obtained.

3. The method of claim 2, wherein the method is characterized by, The molar ratio of 2,4-dimethylpyrrole, 4-bromobutyryl chloride and boron trifluoride ether is 2:(1-1.2):(4.5-5.0); Further, the molar ratio of 2,4-dimethylpyrrole, 4-bromobutyryl chloride and boron trifluoride ether is 2:1:4.

76.

4. The method of claim 2, wherein the method is characterized by, The molar ratio of the compound Br-BY, morpholine and carbonate is 1:(3-4):(6-10); Further, the molar ratio of the compound Br-BY, morpholine and carbonate is 1:3.46:8.

03.

5. The method of claim 2, wherein the method is characterized by, The molar ratio of the compound Mor-BY and N-iodosuccinimide is 1:(2-6); Further, the molar ratio of the compound Mor-BY and N-iodosuccinimide is 1:

6.

6. The method of claim 2, wherein the method is characterized by, The condition of the first reaction is 40-50 DEG C for 2-4 h; The condition of the second reaction is 50-90 DEG C for 2-6 h; The condition of the third reaction is 20-40 DEG C for 1-8 h; The fourth reaction is a room temperature reaction, and the reaction of the compound Mor-BY is detected.

7. The method of preparation of a high activity BODIPY-type photosensitizer targeted to two organelles according to claim 6, characterized in that, The condition of the first reaction is 50 DEG C for 2.5 h; The condition of the second reaction is 50 DEG C for 4 h; The condition of the third reaction is 30 DEG C for 3 h.

8. The method of claim 2, wherein the method is characterized by, The first organic solvent is dichloromethane and / or trichloromethane; The second organic solvent is any one of acetonitrile, dichloromethane, trichloromethane, acetone, dimethyl sulfoxide and N,N-dimethylformamide.

9. Use of the high activity BODIPY photosensitizer according to claim 1 or of the high activity BODIPY photosensitizer prepared according to the preparation method of any one of claims 2 to 8, characterized in that, The high-activity BODIPY type photosensitizer is used for preparing an anticancer / antitumor drug.

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