Photosensitizer, hypoxia response activation type photosensitive compound and application of photosensitizer and hypoxia response activation type photosensitive compound

Abstract

The invention provides a photosensitizer, a hypoxia response activation type photosensitive compound and application thereof, and relates to the technical field of medicinal chemistry. The photosensitizer disclosed by the invention is a photosensitizer based on a BODIPY compound; the hypoxia response activated photosensitive compound or the pharmaceutically acceptable salt thereof is formed by connecting a photosensitizer or the pharmaceutically acceptable salt thereof with a chemotherapeutic drug through a covalent bond capable of generating response fracture in a tumor tissue microenvironment, combines photodynamic therapy and chemical drug therapy, and can be used for tumor treatment.

Description

[0001] This application claims the entire priority of a Chinese patent application with the filing date of April 28, 2024, application number 202410523341.0, and invention title "A hypoxia-responsive activated photosensitizer and its chemotherapeutic drug prodrug", the relevant content of which is incorporated herein by reference. Technical Field

[0002] The present invention belongs to the technical field of medicinal chemistry and relates to a photosensitizer, a hypoxia-responsive activated photosensitive compound and their applications. Background Art

[0003] For tumors, traditional chemotherapeutic drugs have significant efficacy, but have large off-target toxic and side effects, so their role in the field of tumor treatment is restricted. Photodynamic Therapy (PDT) has the characteristics of low toxic and side effects and no drug resistance, making it a potential new method for cancer treatment and widely used in the treatment of various malignant tumors. However, the low oxygen content in deep tumor tissues is often one of the important problems restricting the efficacy of PDT. When tumors grow rapidly, the demand for oxygen by tumor cells will increase, while the internal blood supply of tumors is insufficient, making it difficult to meet the oxygen demand for tumor cell proliferation and metabolism, and ultimately resulting in a significantly hypoxic state in part of the intratumoral microenvironment. Therefore, hypoxia is a common feature of all solid tumors, and most tumors have a certain internal hypoxic microenvironment during growth.

[0004] Photosensitizers, excitation light of a specific wavelength, and molecular oxygen (ROS) are the three elements of photodynamic therapy. If the tissue oxygen content is too low, the converted ROS will decrease. At the same time, the converted ROS will consume tissue oxygen, further exacerbating hypoxia, and ultimately affecting the photodynamic therapy effect.

[0005] To improve the therapeutic efficacy of tumors, combined methods based on multiple therapies, namely therapy combinations, have been established. Among many therapy combinations, the photodynamic therapy-chemotherapy combined therapy is a method with positive efficacy and is one of the current research hotspots in tumor treatment. The photodynamic therapy-chemotherapy combined therapy combines the advantages of photodynamic therapy and chemotherapy, has low toxic and side effects and no drug resistance, and can reduce or eliminate the influence of the hypoxic state of the tumor tissue microenvironment on the efficacy. Summary of the Invention

[0006] Boron dipyrromethene compounds are potential photosensitizers for photodynamic therapy. Boron dipyrromethene compounds, abbreviated as BODIPY (4,4-Difluoro-4-borata-3a-azonia-4a-aza-s-indacene), have the following structural formula (IV):

[0007]

[0008] The applicant combines BODIPY with chemotherapeutic drugs and hydrophilically modifies BODIPY, and discovers a hypoxia-responsive activated photosensitive compound with good curative effect on tumors and good biocompatibility. Based on this, the present invention provides a photosensitizer, a hypoxia-responsive activated photosensitive compound and their applications.

[0009] The technical solution of the present invention is as follows:

[0010] A photosensitizer, the structure of the photosensitizer or its pharmaceutically acceptable salt is shown in formula (I):

[0011]

[0012] A hypoxia-responsive activated photosensitive compound, the hypoxia-responsive activated photosensitive compound or its pharmaceutically acceptable salt is connected by a covalent bond that can undergo responsive cleavage in the tumor tissue microenvironment from the photosensitizer or its pharmaceutically acceptable salt described in the above embodiments and a chemotherapeutic drug.

[0013] Preferably, the chemotherapeutic drug is paclitaxel.

[0014] Preferably, the covalent bond specifically responds to azoreductase and undergoes responsive cleavage.

[0015] More preferably, the covalent bond contains a nitrogen-nitrogen double bond.

[0016] More preferably, the covalent bond contains an azobenzene structure.

[0017] Preferably, the structure of the photosensitive compound is shown in the following formula (Ⅱ):

[0018]

[0019] Preferably, the method of connecting the covalent bond is the reaction of the azide group on the photosensitizer or its pharmaceutically acceptable salt with an alkynyl group.

[0020] An application of the photosensitizer described in the above embodiments or the hypoxia-responsive activated photosensitive compound described in any one of the above embodiments, as a therapeutic drug for tumors, as one of the components of a tumor therapeutic drug composition, or for preparing a drug for treating tumors.

[0021] Preferably, the tumor therapeutic drug composition further comprises a pharmaceutically acceptable carrier.

[0022] The beneficial effects of the present invention are:

[0023] (1) In the molecular structure of BODIPY of the present invention, multiple hydrophilic polyoxyethylene chain segments are introduced, which can greatly improve the hydrophilicity and biocompatibility of the photosensitizer. Moreover, an azide group is introduced into the molecular structure of BODIPY, and the azide group has high reactivity with alkynyl groups, which is beneficial to the subsequent application of the photosensitizer (I).

[0024] (2) The hypoxia-responsive activated photosensitive compound (II) obtained in the present invention has the following characteristics: 1) Good photophysical and chemical properties, with the efficacy of photodynamic therapy; 2) It can release chemotherapeutic drugs in a hypoxic environment. The lower the oxygen concentration in the tissue or microenvironment, the higher the release efficiency of the chemotherapeutic drug, which can make up for the decline in the photodynamic therapy effect caused by the decrease in oxygen concentration and maintain the overall drug efficacy; 3) By introducing multiple hydrophilic polyoxyethylene chain segments on the BODIPY molecule, the water solubility, bioavailability and targeted delivery ability of the photosensitive compound are improved. The photosensitive compound can be enriched in tumor tissues and increase the local drug concentration, while reducing the systemic off-target toxicity of chemotherapeutic drugs (such as paclitaxel), playing a synergistic role of enhancing efficacy and reducing toxicity.

[0025] (3) The compound of the present invention has the characteristics of targeted enrichment in tumor tissues, easy metabolism and difficult accumulation, and no damage to animal organs, and has good safety. Description of the Drawings

[0026] Figure 1 It is the ultraviolet-visible absorption spectrum of the photosensitizer (I) in Test 1.

[0027] Figure 2 It is the ultraviolet-visible absorption spectrum of the photosensitive compound (II) in Test 1.

[0028] Figure 3 It is the ultraviolet-visible absorption spectrum of the photosensitive compound (III) in Test 1.

[0029] Figure 4 It is the comparison of the comprehensive electronic absorption spectra of the photosensitizer (I), the photosensitive compound (II), the photosensitive compound (III), Compound 5b and PTX.

[0030] Figure 5 It is the fluorescence emission spectra of the photosensitizer (I), the photosensitive compound (II) and the photosensitive compound (III) in DMSO in Test 2.

[0031] Figure 6 It is the change diagram of the electronic absorption spectrum of DPBF in DMSO with the illumination time in the photosensitizer (I) in Test 3.

[0032] Figure 7 It is the change diagram of the electronic absorption spectrum of DPBF in DMSO with the illumination time in the photosensitive compound (II) in Test 3.

[0033] Figure 8 It is a graph showing the change of the electronic absorption spectrum of DPBF in DMSO with the illumination time for the photosensitive compound (III) in Test 3.

[0034] Figure 9 It is a graph showing the degradation rate of DPBF by each compound under illumination conditions in Test 3.

[0035] Figure 10 It is for testing the drug release performance of the photosensitive compound (II) in Test 4.

[0036] Figure 11 It is for comparing the dark toxicity of the compound under different oxygen concentrations in Test 5-1.

[0037] Figure 12 It is for testing the PTX release behavior of the compound under different oxygen concentrations in Test 5-2.

[0038] Figure 13 It is for testing the detection results of intracellular ROS (reactive oxygen species) in Test 5-3.

[0039] Figure 14 It is for studying the PTX release induced by photodynamic action of the compound in Test 5-5.

[0040] Figure 15 It is for testing the drug tissue distribution map of the photosensitive compound (II) in Test 6-1.

[0041] Figure 16 It is for testing the drug tissue distribution map of the photosensitive compound (III) in Test 6-1.

[0042] Figure 17 It is for testing the change trend of tumor volume in Test 6-2.

[0043] Figure 18 It is for testing the change trend of tumor weight in Test 6-2.

[0044] Figure 19 It is for testing the change trend of tumor size in Test 6-2.

[0045] Figure 20 It is for testing the change trend of the body weight of mice in Test 6-2. Detailed implementation manners

[0046] Definitions and Explanations:

[0047] Unless otherwise indicated, the following terms and phrases used herein are intended to have the following meanings. A particular term or phrase should not be considered indefinite or unclear if it is not specifically defined, but should be understood in its ordinary sense. When a trade name appears in this text, it is intended to refer to the corresponding product or its active ingredient.

[0048] As used herein, the term "pharmaceutically acceptable" pertains to those compounds, materials, compositions, and / or dosage forms that, within the scope of sound medical judgment, are suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0049] The term "pharmaceutically acceptable salt" refers to salts of the compounds of the present invention, prepared from compounds having specific substituents found in the present invention with relatively non-toxic acids or bases. When a compound of the present invention contains a relatively acidic functional group, the base addition salt can be obtained by contacting the neutral form of such a compound with a sufficient amount of base in a pure solution or a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amine, or magnesium salts or similar salts. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts. Certain specific compounds of the present invention contain both basic and acidic functional groups and can thus be converted into either base or acid addition salts.

[0050] The salt is contacted with a base or acid in a conventional manner, and the parent compound is then separated to regenerate the neutral form of the compound. The parent form of the compound differs from its various salt forms in certain physical properties, such as solubility in polar solvents.

[0051] In addition to the salt form, prodrug forms of the compounds provided by the present invention also exist. The prodrugs of the compounds described herein readily undergo chemical changes under physiological conditions to be converted into the compounds of the present invention. Furthermore, prodrugs can be converted into the compounds of the present invention by chemical or biochemical methods in the in vivo environment.

[0052] Certain compounds of the present invention can exist in non-solvated or solvated forms, including hydrate forms. Generally, the solvated forms are equivalent to the non-solvated forms and are all included within the scope of the present invention.

[0053] Certain compounds of the present invention can have asymmetric carbon atoms (optical centers) or double bonds. Racemates, diastereoisomers, geometric isomers, and individual isomers are all included within the scope of the present invention.

[0054] The compounds of the present invention may exist in specific geometric or stereoisomeric forms. The present invention contemplates all such compounds, including cis- and trans-isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereoisomers, (D)-isomers, (L)-isomers, and their racemic mixtures and other mixtures, such as enantiomer- or diastereomer-enriched mixtures, all of which mixtures are within the scope of the present invention. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers and their mixtures are included within the scope of the present invention.

[0055] The term "pharmaceutically acceptable carrier" refers to any formulation or carrier medium that can deliver an effective amount of the active substance of the present invention, does not interfere with the biological activity of the active substance, and is non-toxic and side-effect-free to the host or patient. Representative carriers include water, oils, vegetables and minerals, paste bases, lotion bases, ointment bases, microspheres, capsules, liposomes, etc. These bases include suspending agents, thickening agents, transdermal promoters, etc. Their formulations are well known to those skilled in the art of the cosmetic or topical drug fields.

[0056] For a drug or a pharmacological active agent, the term "effective amount" or "therapeutically effective amount" refers to a sufficient amount of the drug or agent that is non-toxic but can achieve the desired effect. For the oral dosage forms in the present invention, the "effective amount" of an active substance in the composition refers to the amount required to achieve the desired effect when used in combination with another active substance in the composition. The determination of the effective amount varies from person to person, depending on the age and general condition of the recipient, and also depends on the specific active substance. The appropriate effective amount in a particular case can be determined by those skilled in the art through routine tests.

[0057] The terms "active ingredient", "therapeutic agent", "active substance" or "active agent" refer to a chemical entity that can effectively treat a target disorder, disease or condition.

[0058] "Optionally", "preferred" or "optionally" means that the subsequently described event or condition may but does not necessarily occur, and this description includes the cases where the described event or condition occurs and the cases where the described event or condition does not occur.

[0059] The solvents used in the present invention are commercially available. The following abbreviations are used in the present invention: DCM represents dichloromethane; DMF represents N,N-dimethylformamide; DMSO represents dimethyl sulfoxide; EtOH represents ethanol; MeOH represents methanol; HOAc represents acetic acid; THF represents tetrahydrofuran; DDQ represents 2,3-dichloro-5,6-dicyano-p-benzoquinone; DMAP represents 4-dimethylaminopyridine; PTX represents paclitaxel; PBS represents phosphate buffer solution; DPBF represents 1,3-diphenylisobenzofuran; CuSO4·5H2O represents copper(II) sulfate pentahydrate; NaVC represents sodium ascorbate; BDP-COOH (B-COOH) represents carboxyl-substituted BODIPY.

[0060] Compounds are named by the conventional naming methods in the art, and commercially available compounds use the supplier catalog names.

[0061] The technical solutions of the present invention are further described and illustrated below by specific embodiments. The compounds of the present invention can be prepared by a variety of synthesis methods well-known to those skilled in the art, including the specific embodiments listed below, the embodiments formed by their combination with other chemical synthesis methods, and equivalent substitution methods well-known to those skilled in the art. Preferred embodiments include, but are not limited to, the examples of the present invention.

[0062] On the one hand, the present invention provides a photosensitizer, and the structure of the photosensitizer or its pharmaceutically acceptable salt is shown in formula (I):

[0063]

[0064] For the above photosensitizer (I) of the present invention, multiple polyoxyethylene ether segments are introduced into the structure of BODIPY, which improves the hydrophilicity, biocompatibility and transportability in vivo of BODIPY; and an azide (-N3) group is introduced, which can further undergo chemical reactions.

[0065] For the above photosensitizer (I), there is no particular limitation on the synthesis method, and one synthesis method can be carried out according to the following steps (I-1)-(I-4):

[0066] (I-1):

[0067]

[0068] (I-2):

[0069]

[0070] (I-3):

[0071]

[0072] (I-4):

[0073]

[0074] On the other hand, the present invention provides a hypoxia-responsive activated photosensitive compound or a pharmaceutically acceptable salt thereof. The hypoxia-responsive activated photosensitive compound or a pharmaceutically acceptable salt thereof is formed by connecting a photosensitizer or a pharmaceutically acceptable salt thereof described in the above embodiments with a chemotherapeutic drug through a covalent bond that can undergo responsive cleavage in the tumor tissue microenvironment.

[0075] In some embodiments, the chemotherapeutic drug is paclitaxel. Paclitaxel (PTX) has good anti-tumor effects, especially effective against ovarian cancer, uterine cancer, breast cancer, etc. with a relatively high incidence of cancer. Although paclitaxel shows good effects on a variety of solid tumor cells, its off-target side effects are also very significant. The most common side effects of paclitaxel are myelosuppression, such as neutropenia and leukopenia, and it can also cause gastrointestinal adverse reactions, such as nausea and vomiting. Some people will have allergic reactions, cardiovascular system reactions, and relatively severe neurotoxicity, leading to peripheral neuropathy after using paclitaxel. In the present invention, paclitaxel is connected with the photosensitizer (I) through a covalent bond, which can significantly reduce the off-target toxicity of paclitaxel, facilitate improving the therapeutic efficacy against tumors and avoiding side effects.

[0076] In some embodiments, the covalent bond specifically responds to azoreductase and undergoes responsive cleavage. Azoreductase is a reducing enzyme widely distributed in hypoxic tumor tissues, and the concentration of azoreductase in hypoxic tumor tissues is much higher than that in normal tissues. Thus, the covalent bond only undergoes responsive cleavage and releases the chemotherapeutic drug in hypoxic tumor tissues, simultaneously achieving the effect of combined photodynamic therapy and chemotherapy, and having good safety.

[0077] In some embodiments, the covalent bond contains a nitrogen-nitrogen double bond. The nitrogen-nitrogen double bond (-N=N-) can specifically respond to azoreductase and break.

[0078] In some embodiments, the covalent bond contains an azobenzene structure. The azobenzene structure (-Ph-N=N-Ph-, Ph represents a benzene ring) can specifically respond to azoreductase and break.

[0079] In some embodiments, the structure of the photosensitive compound is shown in the following formula (Ⅱ):

[0080]

[0081] In some embodiments, the method of covalent bond connection is the reaction of an azide group on the photosensitizer or a pharmaceutically acceptable salt thereof with an alkyne group. Specifically, a synthesis method of the photosensitive compound (Ⅱ) of the present invention can be carried out according to the following steps (Ⅱ-1)-(Ⅱ-2):

[0082] (Ⅱ-1):

[0083]

[0084] (Ⅱ-2):

[0085]

[0086] On the other hand, the present invention also provides an application of the photosensitizer described in the above embodiments or the hypoxia-responsive activation photosensitive compound described in any one of the above embodiments, as a therapeutic drug for tumors, as one of the components of a tumor therapeutic drug composition, or for preparing a drug for treating tumors, which combines photodynamic therapy and chemotherapy drug therapy, having both good curative effects and avoiding or reducing the toxic and side effects brought by chemotherapy drugs.

[0087] In some embodiments, the tumor therapeutic drug composition further comprises a pharmaceutically acceptable carrier.

[0088] The technical solutions of the present invention will be further described and illustrated according to the following examples.

[0089] Example 1

[0090] Synthesize photosensitizer (Ⅰ)

[0091] Step 1-1: Synthesis of compound 1a

[0092]

[0093] Accurately weigh triethylene glycol monomethyl ether (15.0 g, 91.35 mmol) and dissolve it in a certain amount of dichloromethane solution. Add triethylamine (11.09 g, 109.62 mmol) and stir in an ice bath for 15 min. Weigh p-toluenesulfonyl chloride (20.90 g, 109.62 mmol), dissolve it in dichloromethane, transfer it to a constant pressure dropping funnel, and slowly add it dropwise to the reaction flask under ice bath conditions. React overnight under nitrogen protection. After the reaction is completed, directly extract with ammonium chloride aqueous solution and dichloromethane for 3 times, combine the organic phases, dry over anhydrous sodium sulfate, filter under reduced pressure, and rotary evaporate to obtain the crude product. Then purify it by silica gel column chromatography and elute with dichloromethane:ethyl acetate (1:1, v / v) to obtain a colorless oily liquid, which is compound 1a (20.94 g, yield 72%).

[0094] Compound 1a: 11H NMR (400 MHz, CDCl3): δ = 7.80 (d, J = 7.2 Hz, 2H, ArH), 7.34 (d, J = 7.6 Hz, 2H, ArH), 4.16 (t, J = 4.8 Hz, 2H, OCH2), 3.69 (t, J = 4.8 Hz, 2H, OCH2), 3.62 - 3.54 (m, 6H, OCH2), 3.52 (t, J = 2.4 Hz, 2H, OCH2), 3.37 (s, 3H, OCH3), 2.45 (s, 3H, CH3).

[0095] Step 1-2: Synthesis of Compound 2a

[0096]

[0097] Accurately weigh o-phenylenedihydroxybenzaldehyde (1.04 g, 8.56 mmol) and dissolve it in a certain amount of N,N-dimethylformamide (DMF). Weigh Compound 1a (3 g, 9.42 mmol) from the above Step 1-1 and add it to the reaction flask and stir to dissolve. Then add anhydrous potassium carbonate (5.93 g, 0.04 mol) to the reaction flask and react overnight under nitrogen protection at 80 °C. After the reaction is completed, turn off the heating and cool to room temperature. Collect the filtrate by suction filtration with a Buchner funnel under reduced pressure. Rotate to dry the filtrate to remove DMF, and then extract with water and dichloromethane three times. Combine the organic phases, dry over anhydrous sodium sulfate, filter under reduced pressure, and rotate to dry to obtain the crude product. The crude product is further purified by silica gel column chromatography and eluted with petroleum ether:ethyl acetate (1:1, v / v) to obtain a pale yellow oily liquid, which is Compound 2a (1.89 g, yield 82%).

[0098] Step 1-3: Synthesis of Compound 1b

[0099]

[0100] Using p-hydroxybenzaldehyde and dibromomethane as raw materials, using absolute ethanol as the solvent, adding potassium carbonate, and reacting under argon protection at 70 °C. The product reacts with sodium azide in DMF at 100 °C for 10 h to obtain Compound 1b, with a yield of 40%.

[0101] Step 1-4: Synthesis of Compound 2b

[0102]

[0103] Accurately weigh Compound 1b (0.85 g, 4.45 mmol) in the above Steps 1 - 3 and dissolve it in a certain amount of redistilled dichloromethane solution. After stirring until fully dissolved, add 2,4 - dimethylpyrrole (1.15 g, 12.22 mmol) to the reaction solution, add a drop of trifluoroacetic acid, and stir at room temperature overnight under nitrogen protection. Measure 2,3 - dichloro - 5,6 - dicyano - p - benzoquinone (DDQ) (1.21 g, 5.33 mmol) and dissolve it in redistilled dichloromethane. Ultrasonicate to dissolve it, and adopt the strategy of adding it in small portions. Add DDQ to the reaction flask and stir at room temperature for 4 h. Under ice - bath conditions, add triethylamine (12 mL) dropwise and stir for 30 min. Then measure boron trifluoride - diethyl ether complex (12 mL) and slowly add it dropwise to the reaction round - bottom flask, and stir overnight under nitrogen protection. Treatment of the reaction: Filter the reaction solution through silica gel, then extract it 3 times with dichloromethane (60 mL × 3) and an appropriate amount of saturated sodium bicarbonate aqueous solution. Combine the organic phases, dry over anhydrous sodium sulfate, filter under reduced pressure, and rotary - evaporate to obtain the crude product. The crude product is purified by silica gel column chromatography, and eluted with petroleum ether:dichloromethane (1:1, v / v) to obtain a red solid, which is Compound 2b (1.22 g, yield 32%).

[0104] Compound 2b: 1 H NMR (500 MHz, CDCl3) δ = 7.20 (d, J = 8.5 Hz, 2H), 7.04 (d, J = 8.5 Hz, 2H), 5.98 (s, 2H), 4.21 (t, J = 5.0 Hz, 2H), 3.66 (t, J = 5.0 Hz, 2H), 2.55 (s, 6H), 1.43 (s, 6H).

[0105] Steps 1 - 5: Synthesis of Compound 3b

[0106]

[0107] Accurately weigh Compound 2b (0.21 g, 0.51 mmol) in the above Steps 1 - 4 and dissolve it in 150 mL of dichloromethane. After ultrasonication, stirring, and heating until the solid is completely dissolved, dissolve N - iodosuccinimide (NIS) (0.14 g, 0.62 mmol) in methanol and then add it to the round - bottom flask. React at 60 °C under nitrogen protection for 2 h. Rotary - evaporate the reaction solvent, then extract it 3 times with an appropriate amount of sodium chloride aqueous solution and dichloromethane (50 mL × 3). Combine the organic phases, dry over anhydrous sodium sulfate, filter under reduced pressure, and rotary - evaporate to obtain the crude product. The crude product is purified by silica gel column chromatography, and eluted with petroleum ether:dichloromethane (1:1, v / v) to obtain a red solid, which is Compound 3b (0.29 g, yield 81%).

[0108] Compound 3b: 11H NMR (500 MHz, CHLOROFORM-D) δ (ppm) 7.16 (d, J = 8.5 Hz, 2H), 7.06 (d, J = 8.5 Hz, 2H), 4.23 (t, J = 5.0 Hz, 2H), 3.68 (t, J = 5.0 Hz, 2H), 2.64 (s, 6H), 1.44 (s, 6H).

[0109] Steps 1-6: Synthesis of photosensitizer (Ⅰ)

[0110]

[0111] Measure the compound 3b (0.15 g, 0.24 mmol) from the above Steps 1-5 and the compound 2a (1.01 g, 2.60 mmol) from Steps 1-2 and place them in a 100 mL two-necked round-bottom flask. Then add 50 mL of redistilled toluene and stir until the solid is completely dissolved. Then add piperidine (1.2 mL), glacial acetic acid (1.0 mL), and a small spoonful of anhydrous magnesium perchlorate in sequence. Use a water separator and a reflux device to remove the water generated in the reaction to improve the yield. Wrap the reaction flask with tin foil and reflux at 130 °C under dark conditions until the color in the reaction flask changes from red to green. Monitor the reaction by TLC until the reaction is complete, and then stop heating. Cool to room temperature, rotary evaporate the toluene solvent, extract with an appropriate amount of aqueous sodium chloride solution and dichloromethane (50 mL × 3) three times, combine the organic phases, dry over anhydrous sodium sulfate, filter under reduced pressure, and rotary evaporate to obtain the crude product. The crude product is purified by silica gel column chromatography, eluted with dichloromethane:methanol (30:1, v / v) to obtain a green solid, and then purified by GPC to obtain a green solid, which is photosensitizer (Ⅰ) (0.11 g, yield 40%).

[0112] Photosensitizer (Ⅰ): 1 1H NMR (500 MHz, CDCl3) δ = 8.04 (d, J = 16.5 Hz, 2H), 7.52 (d, J = 16.5 Hz, 2H), 7.27 (dd, J = 9.0, 2.0 Hz, 2H), 7.19 (d, J = 8.5 Hz, 2H), 7.14 (d, J = 2.0 Hz, 2H), 7.06 (d, J = 8.5 Hz, 2H), 6.94 (d, J = 8.5 Hz, 2H), 4.28 - 4.15 (m, 10H), 3.88 (m, 8H), 3.79 - 3.70 (m, 8H), 3.71 - 3.60 (m, 18H), 3.58 - 3.49 (m, 8H), 3.37 (s, 6H), 3.34 (s, 6H), 1.49 (s, 6H).

[0113] 1313C NMR (151 MHz, CDCl3) δ = 159.19, 150.47, 150.36, 148.93, 148.90, 145.70, 139.31, 138.41, 133.33, 130.41, 129.79, 128.01, 121.64, 117.16, 115.43, 114.27, 114.19, 83.19, 83.17, 77.27, 77.06, 76.85, 71.94, 71.92, 70.87, 70.85, 70.68, 70.56, 70.52, 69.77, 69.66, 69.07, 68.73, 67.05, 59.05, 59.02, 50.22, 17.78, 14.06。

[0114] Example 2

[0115] The photosensitive compound (II) was synthesized and its structural formula is as shown below (II):

[0116]

[0117] Step 2-1: Synthesis of compound 4b

[0118]

[0119] Dissolve p-aminobenzyl alcohol (1.00 g, 7.99 mmol) in 30 mL of a mixed solvent (THF:H2O = 1:1). Add 3 mL of a 3 M HCl solution to the above solution, and stir the reaction solution in an ice-water bath for 30 min. Then add NaNO2 (0.66 g, 9.59 mmol), 1 mL of a THF solution of 3-hydroxy-N,N-dimethylaniline (1.10 g, 7.99 mmol), and sodium acetate (1.31 g, 15.98 mmol) to the reaction flask. The resulting reaction solution was continuously stirred at room temperature for 18 h. After the reaction was completed, the solvent was removed by rotary evaporation under reduced pressure. The residue was purified by silica gel column chromatography using dichloromethane / methanol (50:1, v / v) as the eluent to obtain a yellow solid, which is compound 4b (0.29 g, yield 31%).

[0120] Compound 4b: 1 1H NMR (400 MHz, CDCl3): δ = 14.63 (s, 1H, Phenol-OH), 7.69 (d, J = 8.0 Hz, 2H), 7.55 (d, J = 8.8 Hz, 1H), 7.44 (d, J = 8.0 Hz, 2H), 6.42 (d, J = 8.8 Hz, 1H), 6.12 (s, 1H), 4.73 (s, 2H), 3.11 (s, 6H), 1.89 (s, 1H).

[0121] 13 13C NMR (150.9 MHz, CDCl3): δ = 159.78, 154.65, 148.48, 147.26, 140.94, 134.95, 127.85, 120.41, 106.42, 98.60, 65.00, 40.32.

[0122] Step 2-2: Synthesis of Compound 5b

[0123]

[0124] Dissolve the compound 4b (0.20 g, 0.74 mmol) from the above Step 2-1, 3-bromopropyne (0.13 g, 1.11 mmol), and anhydrous potassium carbonate (0.51 g, 3.69 mmol) in 50 mL of acetone in a round-bottom flask, and heat under reflux for 12 h. After the reaction is completed, filter off the anhydrous potassium carbonate, and rotary evaporate the solvent in the filtrate. The crude product is purified by silica gel column chromatography using dichloromethane / ethyl acetate (30:1, v / v) as the eluent to obtain a yellow solid, which is compound 5b (0.16 g, yield 96%).

[0125] Compound 5b: 1 1H NMR (400 MHz, CDCl3): δ = 7.83 (d, J = 8.0 Hz, 2H), 7.80 (d, J = 18.8 Hz, 1H), 7.44 (d, J = 8.0 Hz, 2H), 6.45 (s, 1H), 6.41 (d, J = 8.8 Hz, 1H), 4.98 (s, 2H, CH2), 4.74 (s, 2H, CH2), 3.09 (s, 6H), 2.56 (s, 1H), 1.78 (br s, 1H, OH).

[0126] 13 13C NMR (150.9 MHz, CDCl3): δ = 157.45, 153.75, 153.08, 141.87, 134.17, 127.45, 122.57, 118.14, 106.35, 98.95, 79.00, 75.91, 65.10, 58.52, 40.30.

[0127] Step 2-3: Synthesis of Compound 6b

[0128]

[0129] Accurately weigh the compound 5b (0.10 g, 0.32 mmol) and p-nitrophenyl chloroformate (0.08 g, 0.4 mmol) from the above step 2-2, dissolve them in 25 mL of redistilled dichloromethane solution, add DMAP (50 mg, 0.4 mmol) and 0.5 mL of triethylamine. After the reaction solution reacts at room temperature for 24 h under nitrogen protection, add PTX (0.3 g, 0.35 mmol) to the reaction solution, and react overnight at room temperature under nitrogen protection. Rotate to dry the solvent, extract with dichloromethane and water three times, combine the organic phases, dry with anhydrous sodium sulfate, filter, and purify by silica gel column chromatography with dichloromethane: petroleum ether (10:1, v / v) as the eluent to obtain a pale yellow solid, which is compound 6b (0.21 g, yield 53.2%).

[0130] Compound 6b: 1 H NMR (500 MHz, CDCl3) δ = 8.14 (dd, J = 9.5, 6.0 Hz, 2H), 7.82 (d, J = 8.0 Hz, 1H), 7.73 (dd, J = 8.5, 1.5 Hz, 2H), 7.61 (d, J = 7.5 Hz, 1H), 7.53 - 7.47 (m, 2H), 7.44 - 7.33 (m, 5H), 6.95 (d, J = 9.0 Hz, 1H), 6.47 - 6.40 (m, 1H), 6.32 - 6.27 (m, 1H), 5.99 (dd, J = 9.5, 2.5 Hz, 1H), 5.69 (d, J = 7.0 Hz, 1H), 5.46 (d, J = 3.0 Hz, 1H), 5.21 (dd, J = 12.5, 6.5 Hz, 2H), 5.00 - 4.95 (m, 3H), 4.48 - 4.42 (m, 1H), 4.33 (d, J = 8.0 Hz, 1H), 4.21 (d, J = 8.0 Hz, 1H), 3.82 (d, J = 7.0 Hz, 1H), 3.11 (s, 4H), 2.60 - 2.51 (m, 2H), 2.46 (s, 3H), 2.41 (dd, J = 15.3, 9.5 Hz, 1H), 2.23 (s, 3H), 2.22 - 2.17 (m, 1H), 1.93 (d, J = 1.5 Hz, 3H), 1.92 - 1.86 (m, 1H), 1.69 (s, 3H), 1.64 (s, 3H), 1.25 (s, 3H), 1.14 (s, 2H).

[0131] 1313C NMR (101 MHz, CDCl3) δ = 203.88, 171.29, 169.91, 167.90, 167.30, 167.00, 157.67, 154.15, 154.02, 153.83, 142.63, 136.69, 134.91, 134.12, 133.69, 133.53, 132.88, 132.07, 130.25, 129.27, 129.17, 129.02, 128.77, 128.74, 128.59, 127.19, 126.63, 122.62, 118.21, 106.31, 98.63, 84.49, 81.09, 79.08, 78.93, 77.42, 77.10, 76.91, 76.78, 76.47, 76.06, 75.63, 75.16, 72.15, 70.47, 58.51, 58.34, 52.83, 45.60, 43.23, 40.30, 35.66, 35.58, 26.85, 22.71, 22.20, 20.85, 14.83, 9.64。

[0132] Step 2-4: Synthesis of the photosensitive compound (II)

[0133] Accurately weigh copper sulfate pentahydrate (10 mg, 0.04 mmol) and NaVC (20 mg, 0.10 mmol) and place them in a 10 mL round-bottom flask. Add 0.5 mL of deionized water and stir until the solution turns light yellow; dissolve the photosensitizer (I) from Example 1 (60 mg, 0.041 mmol) and the compound 6b from the above Step 2-3 (40 mg, 0.033 mmol) in 2 mL of redistilled dichloromethane and add it to the reaction flask, then add 2 mL of tert-butanol to make the solvents miscible. React under nitrogen protection in the dark at room temperature for 24 h. Extract directly with water and dichloromethane (25 mL × 3) three times, collect the organic phase, dry it over anhydrous sodium sulfate, filter it under reduced pressure, and rotary evaporate to obtain the crude product; the crude product is purified by silica gel column chromatography and GPC using dichloromethane:methanol (30:1, v / v) as the eluent to obtain a dark green solid, which is the photosensitive compound (II) (58 mg, yield 64.5%).

[0134] Photosensitive compound (II): 11H NMR (400 MHz, CDCl3) δ = 8.14 (d, J = 7.2 Hz, 2H), 8.05 (d, J = 16.8 Hz, 2H), 7.91 (s, 1H), 7.81 (d, J = 8.0 Hz, 2H), 7.79 (d, J = 8.0 Hz, 1H), 7.75 - 7.71 (m, 2H), 7.64 - 7.57 (m, 1H), 7.54 (s, 1H), 7.53 - 7.49 (m, 3H), 7.48 - 7.46 (m, 1H), 7.45 - 7.41 (m, 2H), 7.41 - 7.37 (m, 6H), 7.37 - 7.32 (m, 1H), 7.28 (d, J = 3.0 Hz, 1H), 7.16 - 7.12 (m, 4H), 6.97 - 6.92 (m, 5H), 6.49 (s, 1H), 6.37 (d, J = 8.0 Hz, 1H), 6.29 (m, 2H), 5.99 (dd, J = 9.2, 2.8 Hz, 1H), 5.69 (d, J = 6.8 Hz, 1H), 5.55 (s, 2H), 5.47 (d, J = 2.8 Hz, 1H), 5.26 - 5.16 (m, 2H), 5.05 - 4.94 (d, J = 8.0 Hz, 1H), 4.80 (t, J = 4.0 Hz, 2H), 4.44 (t, J = 4.0 Hz, 3H), 4.32 (d, J = 8.4 Hz, 1H), 4.22 (m, 8H), 3.89 (m, 8H), 3.81 (d, J = 7.2 Hz, 1H), 3.76 (m, 8H), 3.70 - 3.61 (m, 18H), 3.58 - 3.51 (m, 8H), 3.38 (s, 6H), 3.35 (s, 6H), 3.08 (s, 6H), 2.66 - 2.51 (m, 2H), 2.45 (s, 3H), 2.40 (m, 1H), 2.22 (s, 3H), 1.94 - 1.89 (m, 5H), 1.68 (s, 3H), 1.43 (s, 6H), 1.24 (s, 3H), 1.14 (s, 2H).

[0135] 1313C NMR (600 MHz, CDCl3) δ = 203.85, 171.26, 169.88, 167.90, 167.14, 167.03, 158.74, 154.12, 150.49, 150.38, 148.89, 145.58, 144.81, 142.62, 139.36, 138.08, 136.68, 134.82, 133.71, 133.48, 133.25, 132.84, 132.06, 130.38, 130.23, 129.87, 129.20, 129.16, 129.07, 128.76, 128.72, 128.59, 128.38, 127.17, 126.64, 124.39, 122.53, 121.67, 118.38, 117.13, 115.41, 114.26, 114.20, 105.88, 98.06, 84.46, 83.20, 81.08, 79.13, 77.26, 77.04, 76.83, 76.46, 75.61, 75.09, 72.13, 71.94, 71.92, 70.85, 70.83, 70.67, 70.55, 70.51, 69.75, 69.64, 69.08, 68.72, 66.39, 64.21, 59.06, 59.02, 58.52, 52.85, 49.79, 45.60, 43.22, 40.39, 35.60, 29.71, 26.85, 22.73, 22.16, 20.86, 17.80, 14.86, 11.22, 9.64。

[0136] Comparative Example 1

[0137] Synthesis of photosensitive compound (III)

[0138] The structure of the synthesized photosensitive compound (III) is shown in the following formula (III).

[0139]

[0140] Accurately weigh copper sulfate pentahydrate (10 mg, 0.04 mmol) and NaVC (20 mg, 0.10 mmol) and place them in a 10 mL round-bottom flask. Add 0.5 mL of deionized water and stir until the solution turns light yellow; dissolve the photosensitizer (Ⅰ) (30 mg, 0.02 mmol) from Example 1 and compound 5b (10 mg, 0.032 mmol) from Example 2 in 2 mL of redistilled dichloromethane and add it to the reaction flask, then add 2 mL of tert-butanol to mix the solvents. React under nitrogen protection at room temperature in the dark for 24 h. Extract directly with water and dichloromethane (25 mL × 3) three times, collect the organic phase, dry it over anhydrous sodium sulfate, filter under reduced pressure, and rotary evaporate to obtain the crude product; the crude product is purified by silica gel column chromatography and GPC using dichloromethane:methanol (30:1, v / v) as the eluent to obtain a dark green solid, which is the photosensitive compound (Ⅲ) (32 mg, yield 89.2%).

[0141] Photosensitive compound (Ⅲ): 1 H NMR (400 MHz, CDCl3) δ = 8.05 (d, J = 16.8 Hz, 2H), 7.91 (s, 1H), 7.82 (d, J = 8.4 Hz, 2H), 7.78 (d, J = 9.4 Hz, 1H), 7.52 (d, J = 16.4 Hz, 2H), 7.44 (d, J = 8.4 Hz, 2H), 7.30 - 7.26 (d, J = 2.0 Hz, 1H), 7.15 (d, J = 2.4 Hz, 2H), 7.13 (d, J = 8.8 Hz, 2H), 6.95 (d, J = 8.4 Hz, 2H), 6.93 (t, J = 8.0 Hz, 1H), 6.47 (d, J = 3.2 Hz, 1H), 6.37 (dd, J = 9.2, 2.6 Hz, 1H), 4.80 (t, J = 5.2 Hz, 2H), 4.72 (s, 2H), 4.43 (t, J = 5.2 Hz, 2H), 4.23 (p, J = 4.8 Hz, 8H), 3.91 - 3.87 (m, 8H), 3.77 - 3.74 (m, 8H), 3.66 (m, 18H), 3.56 - 3.54 (m, 4H), 3.53 - 3.51 (m, 4H), 3.38 (s, 6H), 3.35 (s, 6H), 3.07 (s, 6H), 1.42 (s, 6H).

[0142] 1313C NMR (600 MHz, CDCl3) δ = 158.68, 158.00, 153.93, 153.09, 150.47, 150.42, 148.93, 145.62, 144.97, 141.90, 139.36, 138.12, 134.13, 133.24, 130.39, 129.88, 128.36, 127.45, 124.26, 122.47, 121.65, 118.34, 117.12, 115.41, 114.32, 114.30, 105.89, 98.16, 83.19, 77.25, 77.04, 76.83, 71.95, 71.93, 70.88, 70.87, 70.70, 70.57, 70.54, 69.79, 69.67, 69.12, 68.76, 66.38, 64.98, 64.28, 59.06, 59.03, 49.74, 40.38, 31.60, 22.67, 17.80, 14.14。

[0143] Performance test

[0144] Test 1: Measure the UV-Vis absorption spectra of photosensitizer (I), photosensitive compound (II) and photosensitive compound (III) in DMSO, and preliminarily evaluate the ability of the photosensitizer for photodynamic therapy.

[0145] 1.1 Experimental instruments:

[0146] UV-Vis spectrophotometer (instrument model Lambda 365).

[0147] 1.2 Experimental methods and procedures:

[0148] Accurately weigh 1 mg of the compound to be tested, add an appropriate volume of DMSO, and ultrasonically dissolve it to obtain a stock solution with a concentration of 1 mM. Use a pipette to separately pipette 3 μL, 6 μL, 9 μL, 12 μL, and 15 μL of the stock solution into a quartz cuvette containing 3 mL of DMSO solution, and pipette to mix the solution evenly. The concentrations of the compound solutions to be tested are 1 μM, 2 μM, 3 μM, 4 μM, and 5 μM in sequence. Using DMSO solvent as a reference, scan the electronic absorption spectrum of the compound to be tested in the wavelength range of 300 - 800 nm. The experimental data is processed using Origin 8.0 software. Plot the absorbance on the vertical axis and the wavelength on the horizontal axis to obtain the electronic absorption spectra of the compound to be tested at different concentrations. Take the absorbance value of the Q band and plot it against its concentration c to obtain the standard curve of the compound. According to the Lambert-Beer law A = εbc, the slope of the standard curve is the molar extinction coefficient of the compound to be tested in DMSO. Finally, measure the electronic absorption spectra of photosensitizer (Ⅰ), photosensitive compound (Ⅱ), photosensitive compound (Ⅲ), compound 5b, and PTX at wavelengths of 190 - 800 nm to determine whether the introduction of the Linker and chemotherapeutic drugs affects the spectral properties of the BODIPY prodrug.

[0149] 1.3 Experimental Results

[0150] An ideal photosensitizer should have a strong molar absorptivity within the photodynamic therapy window (Q band, 650 - 800 nm) and weak absorption in the "Soret" band (B band, 400 - 600 nm). The test results of this experiment are shown in Table 1 below:

[0151] Table 1 Photophysical and Photochemical Parameters

[0152]

[0153] Remarks: [a]: Excited at 610 nm

[0154] The relationships between the ultraviolet-visible absorption spectra of photosensitizer (Ⅰ), photosensitive compound (Ⅱ), and photosensitive compound (Ⅲ) in DMSO and concentration are shown in Appendices Figure 1 、 2 、3 respectively. Photosensitizer (Ⅰ), photosensitive compound (Ⅱ), and photosensitive compound (Ⅲ) all have sharp Q band absorption peaks at a wavelength of 671 nm, and there is a good linear relationship between the absorbance value of the compound and the concentration of the compound. According to the Lambert-Beer law, it shows that the above three compounds exist in the form of monomers in DMSO. Calculate the molar extinction coefficients (ε) of photosensitizer (Ⅰ), photosensitive compound (Ⅱ), and photosensitive compound (Ⅲ) to be 105000, 98300, and 94100 L·mol -1 ·cm -1, indicating that the maximum absorption wavelength of the compound is located in the ideal photodynamic window and the molar extinction coefficient is very high, meeting the characteristics of an ideal photosensitizer.

[0155] Attached Figure 4 For the comparative comprehensive electronic absorption spectra of photosensitizer (Ⅰ), photosensitive compound (Ⅱ) and photosensitive compound (Ⅲ), compound 5b and PTX, it can be seen that the spectra of photosensitive compound (Ⅱ) and photosensitive compound (Ⅲ) are the adducts of photosensitizer (Ⅰ), compound 5b (as the linker structure linker) and chemotherapeutic drug PTX and their respective spectral properties do not change, indicating that the introduction of the linker between photosensitizer (Ⅰ) and PTX in photosensitive compound (Ⅱ) has no effect on the absorption spectral properties of the photosensitizer and chemotherapeutic drug.

[0156] Test 2: Test the fluorescence emission of photosensitizer (Ⅰ), photosensitive compound (Ⅱ) and photosensitive compound (Ⅲ) in DMSO to obtain fluorescence properties, including the maximum emission wavelength and fluorescence quantum yield (Φ F ).

[0157] 2.1 Experimental instruments:

[0158] Fluorescence spectrophotometer (Model: F-4600).

[0159] 2.2 Experimental methods and procedures:

[0160] Fluorescence emission spectrum: Add 30 μL of the mother liquor of the compound to be tested (1 mM) to a quartz cuvette containing 3 mL of DMSO respectively. After mixing evenly, a 10 μM solution of the compound to be tested is obtained. The excitation wavelength is the maximum absorption wavelength determined by the electronic absorption spectrum, and the fluorescence emission spectrum of the compound in the range of 615 - 900 nm is measured.

[0161] Fluorescence quantum yield: In this experiment, carboxyl-substituted BODIPY (BDP-COOH) is used as the standard sample, and the relative fluorescence quantum yield of the compound to be tested is measured by the indirect comparison method. To reduce errors, first adjust the concentrations of BDP-COOH and the sample to be tested so that their electronic absorption at 610 nm is between 0.04 - 0.05. Uniformly use 610 nm to excite the samples to obtain the fluorescence emission spectra of each compound, and then calculate the relative fluorescence quantum yield of the sample to be tested according to the following formula (2-1):

[0162]

[0163] In the above formula (2-1): Φ F(x) and are the fluorescence quantum yields of the compound to be tested and the standard sample BDP-COOH respectively; A x and A stdThey are the absorbance values of the compound to be measured and the standard BDP-COOH at 610 nm; F x and F std They are the integrated areas at the maximum emission wavelengths of the compound to be measured and the standard BDP-COOH; η x and η std They are the refractive indices of the compound to be measured and the standard sample solvent, respectively.

[0164] 2.3 Experimental Results

[0165] Table 2 Photophysical and Photochemical Parameters

[0166]

[0167] Note: [a]: Excitation at 610 nm; [b] Using unsubstituted B-COOOH (Φ F = 0.130) as a reference

[0168] The fluorescence emission spectra of photosensitizer (I), photosensitive compound (II) and photosensitive compound (III) in DMSO are as shown in the appendix Figure 5 as shown. The fluorescence quantum yields are as shown in Table 2 above. The results show that: under the same conditions (the compound concentrations are all 10 μM and the excitation wavelength is 610 nm), the maximum absorption wavelengths of photosensitizer (I), photosensitive compound (II) and photosensitive compound (III) are all 671 nm, the maximum emission wavelengths are all 710 nm, and the fluorescence quantum yields Φ F are 0.063, 0.056 and 0.057, respectively.

[0169] Test 3: Singlet Oxygen Yield Study

[0170] Experimental Purpose: Using 1,3-diphenylisobenzofuran (DPBF) as a singlet oxygen scavenger, the ability of photosensitizer (I), photosensitive compound (II) and photosensitive compound (III) to photosensitize the generation of singlet oxygen in DMSO was detected by an indirect measurement method.

[0171] 3.1 Experimental Instruments and Reagents

[0172] 1,3-diphenylisobenzofuran (DPBF), analytical pure, manufacturer: Sigma-Aldrich Chemical Technology (Shanghai) Co., Ltd.;

[0173] 660 nm laser (model: DPSSL DRIVER II).

[0174] 3.2 Experimental Procedures:

[0175] Weigh 1 mg of the compound to be tested and 1 mg of DPBF separately, add a certain volume of DMSO, and sonicate to completely dissolve the samples. Finally, prepare a stock solution with a concentration of 1 mM for the compound to be tested and 10 mM for DPBF. Use a pipette to aspirate 3 mL of DMSO into a quartz cuvette, and then measure 30 μL of the stock solution of the compound to be tested and 30 μL of the stock solution of DPBF and add them to the quartz cuvette and mix well. Use a UV-visible spectrophotometer to scan the electronic absorption spectrum of the mixed solution in the range of 300 - 800 nm at 0 min. Place a 660 nm laser 2 cm away from the quartz cuvette and irradiate the mixed solution with a power of 1 mW. Every 30 s of irradiation, scan the electronic absorption spectrum of the mixed solution in the range of 300 - 800 nm. This process lasts for 6 min, and the entire test is carried out in the dark to avoid the influence of light on the degradation of DPBF. The obtained data is processed using Origin 8.0 software, and finally, a degradation change graph of the mixed solution at 415 nm with respect to the irradiation time is obtained.

[0176] Singlet oxygen yield: The determination of the singlet oxygen yield of the compound also uses the indirect comparison method. Using BDP-COOH as the standard, the compound to be tested and BDP-COOH are subjected to the same light irradiation treatment. Under the same conditions, the degradation rates of the compound and BDP-COOH on DPBF are obtained, and the singlet oxygen yield of the compound to be tested is calculated according to the following formula (2-2).

[0177]

[0178] In the above formula (2-2): Φ Δ(x) and are the singlet oxygen yields of the sample to be tested and the standard BDP-COOH respectively; K x and K std are the DPBF degradation rate constants in the sample to be tested and the standard BDP-COOH respectively; and are the light absorption rates of the sample to be tested and the standard BDP-COOH respectively.

[0179] Among them, for the light absorption rates I of the sample to be tested and BDP-COOH, I = 1 - 10 -OD , the OD value is the absorbance value corresponding to each compound at the maximum absorption; the singlet oxygen yield of BDP-COOH in DMSO is 0.83.

[0180] 3.3 Experimental results

[0181] Singlet oxygen ( 1O2) is a type of reactive oxygen species with high reactivity and high oxidizing property. It is the most important active substance for photosensitizers to achieve PDT treatment. The higher the singlet oxygen quantum yield, the stronger the killing ability of the photosensitizer to diseased tissue cells, and the more significant the treatment effect.

[0182] The graphs of the change in the electronic absorption spectrum of DPBF in DMSO with the irradiation time for photosensitizer (Ⅰ), photosensitive compound (Ⅱ), and photosensitive compound (Ⅲ) are shown in Appendix Figure 6 , Appendix Figure 7 , and Appendix Figure 8 respectively. The graphs of the degradation rate of DPBF by each compound under irradiation conditions (λ ex = 660 nm, power is 1 mW / cm 2 , and the concentration of the photosensitizer is 10 μM) are shown in Appendix Figure 9 .

[0183] The results show that in DMSO, photosensitizer (Ⅰ), photosensitive compound (Ⅱ), and photosensitive compound (Ⅲ) all have a high degradation rate for DPBF under irradiation conditions, indicating that they have a strong ability to produce singlet oxygen through photosensitization by themselves, and there is no obvious difference among the three, indicating that the introduction of the linker and the chemotherapeutic drug does not affect the singlet oxygen quantum yield of the photosensitizer. Moreover, the absorbance values of photosensitizer (Ⅰ), photosensitive compound (Ⅱ), and photosensitive compound (Ⅲ) themselves remain stable all the time, indicating that the three have good photostability. Through formula calculation, the singlet oxygen quantum yields Φ Δ of photosensitizer (Ⅰ), photosensitive compound (Ⅱ), and photosensitive compound (Ⅲ) are 0.37, 0.40, and 0.39 respectively, as shown in Table 3 below.

[0184] Table 3 Photophysical and photochemical parameters

[0185]

[0186] Note: [a]: Excited at 610 nm; [b]: Using unsubstituted B-COOOH (Φ Δ = 0.83) as a reference

[0187] Test 4: Drug release study

[0188] [[ID=__38]]Experimental purpose: Whether the prodrug formed by connecting the BODIPY photosensitizer and PTX through a hypoxia-responsive azobenzene linker can break and release PTX under the stimulation of hypoxia signals is the key to realizing photodynamic-chemotherapy combined treatment. In this experiment, Na2S2O4 was used as a chemical mimic of azoreductase to test the release of PTX in photosensitive compound (Ⅱ) under simulated hypoxia conditions.

[0189] Test principle: Azobenzene is a chemical group sensitive to sodium dithionite (Na2S2O4) and anaerobic azoreductase. In the presence of the above stimulants, the azo structure in the prodrug molecule is reductively cleaved, thereby releasing paclitaxel (PTX). The release of PTX is analyzed by HPLC after the reaction of sodium dithionite with the prodrug molecule.

[0190] 4.1 Experimental procedure:

[0191] Accurately weigh 1 mg of the compound to be tested, add an appropriate volume of DMSO, and sonicate to completely dissolve it to obtain a stock solution with a concentration of 1 mM. Weigh 10 mg of solid Na2S2O4 and dissolve it in an appropriate volume of ultrapure water to prepare a 50 mM Na2S2O4 stock solution. Take 10 μL of the 50 mM Na2S2O4 stock solution and dilute it to 1 mL to prepare a 500 μM Na2S2O4 stock solution. Take 100 μL of the stock solution of photosensitive compound (II), add it to 300 μL of a mixed solution of acetonitrile:PBS = 1:1, and then add 100 μL of the 50 mM Na2S2O4 stock solution to make the concentration of Na2S2O4 in the mixed solution reach 5 mM. Prepare a mixed solution with concentrations of photosensitive compound (II) and Na2S2O4 of 10 μM and 50 μM respectively according to the same operation. React the mixed solution at 37 °C for 12 h and then lyophilize it. Dissolve the lyophilized powder in 0.5 mL of acetonitrile, centrifuge, take the supernatant, and sample 5 μL for HPLC detection.

[0192] 4.2 HPLC chromatographic conditions:

[0193] Chromatographic column: SinoChrom ODS-BP C8 column (5 μm, 4.6×150 mm); Mobile phase: pure water (phase A, containing 0.05% trifluoroacetic acid), acetonitrile (phase B, containing 0.05% trifluoroacetic acid); Gradient elution method: 0 - 15 min, 30% B; Column temperature: 25 °C; Injection volume: 5 μL; Detection wavelength: 230 nm.

[0194] 4.3 Experimental results

[0195] The experimental results are as attached Figure 10As shown, where (a) is the PTX standard sample; (b) is the photosensitive compound (II) standard sample; (c) is the spectrum at a Na2S2O4 concentration of 50 μM; (d) is the spectrum at a Na2S2O4 concentration of 5 mM; the results show that: the characteristic peak of PTX is at 9.55 min, and the characteristic peak of the photosensitive compound (II) is at 2.95 min. In the experimental group without Na2S2O4, the characteristic peak of PTX did not appear. In the experimental group with 50 μM Na2S2O4, the peak area of the characteristic peak of the photosensitive compound (II) at 2.95 min decreased, and the characteristic peak corresponding to PTX appeared at 9.55 min, indicating that the azo structure in the photosensitive compound (II) was destroyed and PTX was released. In the experimental group with 5 mM Na2S2O4, the characteristic peak area of PTX increased compared with the experimental group with 50 μM Na2S2O4, and at the same time, the characteristic peak of the photosensitive compound (II) basically disappeared, indicating that the photosensitive compound (II) can be basically completely activated under 5 mM Na2S2O4 to release PTX.

[0196] Test 5-1: Cell experiment, study on the dark toxicity of compounds under different oxygen concentrations

[0197] Experimental purpose: To evaluate the survival / killing of cells after administration of photosensitive compound (II), photosensitive compound (III) and PTX by double staining with Calcein-AM and PI, and to confirm the response of the compounds to the tumor hypoxic microenvironment.

[0198] (1) Experimental reagents and instruments:

[0199] Dimethyl sulfoxide (DMSO): Shanghai Chemical Reagent Company, China National Pharmaceutical Corporation.

[0200] Mouse breast cancer cells (4T1): Cell Bank of Shanghai Institute of Life Sciences, Chinese Academy of Sciences.

[0201] Phosphate buffer solution (PBS): Jiangsu KeyGen Biotech Co., Ltd.

[0202] DMEM cell culture medium: Thermo Fisher Scientific Biochemicals (Beijing) Co., Ltd.

[0203] DMEM cell culture medium (without phenol red): solarbio.

[0204] 1640 culture medium: Beijing Dingguo Changsheng Biotechnology Co., Ltd.

[0205] Trypsin (+EDTA): Beijing Dingguo Changsheng Biotechnology Co., Ltd.

[0206] Penicillin and streptomycin solution: Beijing Dingguo Changsheng Biotechnology Co., Ltd.

[0207] Fetal bovine serum (FBS): Thermo Fisher Scientific Biochemicals (Beijing) Co., Ltd.

[0208] 96-well sterile cell culture plates: Thermo Fisher Scientific Biochemicals (Beijing) Co., Ltd.

[0209] Cell counter: Shanghai Ruiyu Biotechnology Co., Ltd.

[0210] Calcein-AM: Beyotime Biological Research Institute.

[0211] Propidium iodide (PI): Beyotime Institute of Biology.

[0212] (2) Experimental steps:

[0213] a. Cell Culture: Select healthy mouse breast cancer 4T1 cells and incubate them in culture flasks using DMEM or 1640 medium supplemented with 1% penicillin and streptomycin and 10% fetal bovine serum. Incubate the cells in a 5% CO2 incubator until they completely fill the flask.

[0214] b. Plating confocal microplate: Wash 4T1 cells in culture flask twice with PBS, digest with trypsin, centrifuge, resuspend and count. Finally, the concentration was 1.5×10 5 cell / mL were plated in a laser confocal dish and placed in a cell culture incubator for 12 h to allow the cells to adhere;

[0215] c. Drug Addition: Prepare 5 μM cell culture medium containing photosensitizing compound (II), photosensitizing compound (III), and PTX (containing 0.05% Tween 80). Remove the old culture medium from the confocal dish and wash twice with PBS. Gently add the prepared drug-containing culture medium to the confocal dish. Then, incubate the confocal dish in each group under normoxic (21% O2), microoxic (8% O2), and hypoxic (0.1% O2) conditions in the dark for 24 hours.

[0216] d. Add live and dead cell probes: Pipette 1 μL of calcein-AM solution and 1 μL of propidium iodide (PI) into 1 mL of DMEM medium and mix to obtain live and dead cell staining solutions. Prepare live and dead cell staining solutions containing 1640 medium in the same manner. Wash the confocal laser scanning confocal dish twice with PBS after drug incubation. Finally, add the live and dead cell staining solutions to the confocal laser scanning confocal dish and continue incubating in the cell culture incubator for 1 hour.

[0217] e. Photographing: Use a confocal laser scanning microscope for scanning and photographing. (Calcein-AM: excitation wavelength is 488 nm, detection wavelength is 510 nm - 570 nm; PI: excitation wavelength is 543 nm, detection wavelength is 550 nm - 620 nm).

[0218] (3) Experimental results

[0219] As shown in the Figure 11 appendix, under normal oxygen (21% O2) conditions, there was no cell death in the photosensitive compound (II) group, indicating that the expression of azoreductase was low under normal oxygen environment, which was not sufficient to break the azo structure of the Linker, and there was no release of PTX, so no chemotoxicity was exhibited. There was no cell death in each photosensitive compound (III) control group, indicating that its dark toxicity was very low and was independent of oxygen concentration. The PTX group exhibited normal chemotoxicity, which was independent of oxygen concentration; under hypoxic (8% O2) conditions, due to the start of azoreductase expression, cell death occurred in the photosensitive compound (II) group, indicating that azoreductase could break the azo structure of the Linker, thereby releasing PTX and exhibiting certain chemotoxicity; as the oxygen concentration decreased to the microoxic (0.1% O2) range, more cell death occurred in the photosensitive compound (II) group, showing more significant chemotoxicity, indicating that with the increase in azoreductase expression, more PTX was released. The above experimental results proved that the photosensitive compound (II) produced good chemotherapeutic effects under hypoxic (low / microoxic) conditions, which could well make up for the deficiencies of photodynamic therapy. In addition, under hypoxic conditions, the chemotherapeutic effect produced by the photosensitive compound (II) was better than that of direct chemotherapy with PTX, indicating that the photosensitive compound (II) had higher bioavailability.

[0220] Test 5-2: Cell experiment, PTX release of the compound at different oxygen concentrations

[0221] Experimental purpose: In this experiment, the microtubules were stained with Tubulin-Tracker Green fluorescent probe to detect the changes in microtubule morphology after drug administration, so as to judge whether the azo bond was broken and whether PTX was released.

[0222] (1) Experimental instruments and reagents:

[0223] Tubulin-Tracker: Biotium.

[0224] DAPI nuclear probe: Biotium.

[0225] (2) Experimental steps:

[0226] a. Cell Culture: Select healthy mouse breast cancer 4T1 cells and incubate them in culture flasks using DMEM or 1640 medium supplemented with 1% penicillin and streptomycin and 10% fetal bovine serum. Incubate the cells in a 5% CO2 incubator until they completely fill the flask.

[0227] b. Spread on a 96-well plate: Mouse breast cancer 4T1 cells were cultured at a cell density of 1×10 5 cells / mL were seeded into 96-well plates and allowed to adhere;

[0228] c. Drug Addition: Prepare 2 μM cell culture medium containing photosensitizing compound (II), photosensitizing compound (III), and PTX (containing 0.05% Tween 80). Remove the old culture medium from the 96-well plate and wash twice with PBS. Add the prepared drug solution to the 96-well plate. Then, incubate the 96-well plate in the dark for 24 hours under normoxic (21% O2), microoxic (8% O2), and hypoxic (0.1% O2) conditions.

[0229] d. Probe loading: Prepare 2 mL of Tubulin-Tracker Green staining solution and 2 mL of DAPI nuclear staining solution. Remove the drug from the 96-well plate and wash twice with PBS. Add the prepared Tubulin-Tracker Green staining solution to the 96-well plate and incubate in a cell culture incubator for 20 minutes. Then, wash twice with staining enhancement solution and add DAPI nuclear staining solution and incubate for 15 minutes.

[0230] e. Photography: High-content scanning was used to observe changes in microtubule morphology in cells (Tubulin-Tracker Green microtubule green fluorescent probe: excitation wavelength 488 nm, detection wavelength 500-600 nm; DAPI cell nuclear probe: excitation wavelength 405 nm, detection wavelength 425-475 nm).

[0231] (3) Experimental results:

[0232] See attached for the results Figure 12, in 4T1 cells, the cells in the photosensitive compound (II) group maintained a normal filamentous microtubule network structure under normal oxygen concentration (21% O2). As the O2 concentration decreased, the microtubule structure in the photosensitive compound (II) group changed significantly compared with that in the photosensitive compound (III) group, changing from a filamentous structure to an elliptical structure, which was the same as the result of the PTX group, indicating that the photosensitive compound (II) could release PTX in 4T1 cells depending on the decrease in O2 concentration. In the control group of photosensitive compound (III), since it did not contain PTX, it maintained a normal microtubule structure regardless of the oxygen concentration. In addition, through DAPI dye staining, it was also observed that the cell nucleus was slightly fragmented with the release of PTX, further proving that the released chemotherapeutic drug could cause damage to tumor cells. This result also corroborated the results of the study on the dark toxicity of cells under different oxygen concentrations above, indicating that the dark toxicity at this time was the chemotoxic effect of PTX.

[0233] Test 5-3: Cell experiment, detection of intracellular ROS (reactive oxygen species)

[0234] Experimental purpose: In this experiment, the production of reactive oxygen species by photosensitive compound (II) and photosensitive compound (III) in cells was observed by quantitatively detecting DCF fluorescence with a flow cytometer.

[0235] (1) Experimental instruments and reagents

[0236] Polyoxyethylene castor oil (CEL): Beijing Dingguo Changsheng Biotechnology Co., Ltd.

[0237] Reactive oxygen species detection kit (DCFH-DA, 10 mM): Beijing Dingguo Changsheng Biotechnology Co., Ltd.

[0238] BD flow cytometer: BD Company, USA.

[0239] (2) Experimental method: Flow cytometer quantitative method.

[0240] (3) Experimental steps:

[0241] a. Seed 12-well plates: After 4T1 cells were digested, centrifuged, resuspended, and counted in sequence, they were diluted with culture medium to a density of 1×10 5 cell / mL. After being evenly pipetted, 1 mL / well of the cell culture medium mixture was accurately aspirated and inoculated into 12-well plates, and cultured in a cell incubator for 12 h to allow the cells to adhere to the wall;

[0242] b. Add drugs: Prepare cell culture media containing 1 μM of photosensitive compound (II) and photosensitive compound (III) respectively (containing 0.05% Tween 80) for drug addition. Add 1 mL of the drug culture medium solution to each well and continue to incubate in a cell incubator for 24 h;

[0243] c. Preparation of DCFH-DA working solution: At room temperature, take 1 μL of the DCFH-DA kit solution and dilute it into 1 mL of the culture medium, and mix well.

[0244] d. Loading the probe: Wash away the old culture medium with PBS, add 1 mL of the DCFH-DA working solution to each well, and continue to culture in the cell incubator for 1 h.

[0245] e. Irradiation: After aspirating the culture medium in the wells and washing twice with PBS, replace it with 1 mL of the culture medium, place the well plate under a 660 nm LED light panel (20 mW / cm 2 ) and irradiate for 2 min, then place it in the incubator and continue to incubate for 15 min. The non-irradiated group is also incubated for 15 min.

[0246] f. Fluorescence detection: After irradiation, wash three times with PBS to remove the unabsorbed drug. Add 200 μL of 0.25% trypsin to each well for digestion, add 1 mL of the culture medium to each well, pipette the cells evenly, and transfer them to a 1.5 mL centrifuge tube. Centrifuge at 1000 r / min for 3 min, discard the supernatant, and pipette the cells evenly with 1 mL of the culture medium. Detect the intracellular drug fluorescence intensity with a BD flow cytometer: Select the first channel (excitation wavelength 488 nm, emission wavelength 525 nm), the injection volume each time is 50 μL, inject slowly, and perform 10000 events for each sample. The detection results are presented as a histogram, where the abscissa is the fluorescence intensity of DCF in the cells and the ordinate is the number of cells.

[0247] g. Use Graphpad Prism 6 software to plot the DCF fluorescence intensity values corresponding to each compound for the measured data, compare the abilities of each photosensitizer to generate reactive oxygen species, perform parallel experiments three times, and express the results as Mean±SD. P<0.05 is considered significantly different, and P<0.01 is considered extremely significantly different.

[0248] (4) Experimental results:

[0249] As shown in the appendix Figure 13 (where the L group represents the irradiated group), regardless of whether there is irradiation, almost no DCF fluorescence is generated in the 4T1 cells in the blank group, indicating that the intracellular ROS is very low, and single irradiation will not increase the ROS production; under dark conditions, after the cells are treated with photosensitive compound (Ⅱ) and photosensitive compound (Ⅲ), no obvious fluorescence is detected either, indicating that the two compounds do not generate ROS in the dark; under light conditions, after the cells are treated with photosensitive compound (Ⅱ) and photosensitive compound (Ⅲ), the intracellular DCF fluorescence intensity is significantly enhanced, indicating that under laser irradiation, both photosensitive compound (Ⅱ) and photosensitive compound (Ⅲ) can generate ROS and have significant phototoxic effects on tumor cells.

[0250] Test 5-4: Cell experiment, study on the toxic effect of compounds on tumor cells

[0251] Experimental purpose: To test the cytotoxic effects of photosensitive compound (II) and photosensitive compound (III) on tumor cells by the MTT method and to reflect the strength of the cytotoxic effects on tumor cells by calculating the cell survival rate (I%). The "toxicity" to tumor cells described in this experiment refers to having corresponding effects or curative effects.

[0252] (1) Experimental instruments and reagents:

[0253] MTT dry powder: Sigma

[0254] Dimethyl sulfoxide (DMSO): Shanghai Chemical Reagent Company, Sinopharm Chemical Reagent Co., Ltd.

[0255] Microplate reader: Molecular Devices (Shanghai) Co., Ltd.

[0256] HERA carbon dioxide incubator: Thermo Scientific

[0257] (2) Experimental procedures:

[0258] a. Cell seeding: Select 4T1 cells in good growth state, pour out the old culture medium in the culture flask, wash it twice with PBS, add 1 mL of trypsin, put it in the incubator for digestion for 2 min, then take it out, continue to add 2 mL of cell culture medium to terminate the digestion, and carefully pipette the cells on the flask wall until the flask wall becomes transparent. Continue to pipette the cell suspension to make it evenly mixed, and then transfer it equally to 3 1.5 mL centrifuge tubes for centrifugation. Pour out the old culture medium and add 1 mL of culture medium to resuspend the cells and count. Dilute the cells to a density of 8×10 5 cells / mL, and evenly add the cells to the 96-well plate with a multi-channel pipette. Set 6 replicates for each concentration data, and add 100 μL of cell suspension to each well;

[0259] b. Drug addition: Prepare DMSO stock solutions (containing 10% CEL) with drug concentrations of 1 mM, 0.316 mM, 0.1 mM, 0.0316 mM, 0.01 mM, 0.00316 mM, and 0.001 mM respectively. Pipette 10 μL of the stock solution and dilute it to 1 mL of culture medium. The diluted concentrations are 10 μM, 3.16 μM, 1 μM, 0.316 μM, 0.1 μM, 0.0316 μM, and 0.01 μM respectively, and finally obtain drug solutions with logarithmic concentrations {lg[conc.(M)]} of -5.0, -5.5, -6.0, -6.5, -7.0, -7.5, and -8.0 in sequence. Use a multi-channel pipette to aspirate the old culture medium in the 96-well plate, wash it twice with PBS, and then add 100 μL of the corresponding concentration drug to each well. Place the 96-well plate under normal oxygen (21% O2), micro-oxygen (8% O2), and hypoxic (0.1% O2) conditions and continue to culture for 24 h.

[0260] c. Phototoxicity and dark toxicity experiments: Aspirate the old culture medium and wash it twice with PBS, and then add 100 μL of fresh culture medium to each well. In the phototoxicity experiment, irradiate the cells with a 660 nm LED lamp (20 mW / cm 2 ) for 2 min and then place them in a 37 °C constant temperature incubator to cultivate overnight. In the control group for dark toxicity experiment, after washing with PBS, replace the old culture medium with fresh one and continue to cultivate overnight in the incubator;

[0261] d. OD value detection: After the cultivation is completed, add 10 μL of MTT solution (5 mg / mL) to each well with a pipette, and continue to place the 96-well plate in the incubator to cultivate for 4 h. Aspirate the culture medium, add 100 μL of DMSO to each well to lyse the cells and dissolve the formazan inside the cells. Place the 96-well plate on a shaker and shake for 15 min to fully dissolve the formazan, and then measure the OD value of the solution at 570 nm with an enzyme-linked immunosorbent assay (ELISA) reader;

[0262] e. Data processing: Process the obtained data with the software Graphpad Prism 6, plot the cell survival rate against the logarithmic concentration of the photosensitizer drug, and calculate the half-maximal inhibitory concentration IC 50 value. The formula for calculating the cell survival rate I is shown in Equation (2-3):

[0263] I% = (A - A0) / (A1 - A0) × 100%

[0264] (2-3)

[0265] where A is the OD value of the experimental group; A0 is the OD value of the blank control group; A1 is the OD value of the cell control group.

[0266] (3) Experimental results:

[0267] Under different O2 concentrations, the IC of 4T1 cells for different treatment groups under light (±L) conditions is as follows 50 shown in Table 4 below.

[0268] Table 4 IC of 4T1 cells for different treatment groups under light (±L) conditions at different O2 concentrations 50 value

[0269]

[0270] Under dark conditions, the cell survival rates of the photosensitive compound (Ⅱ) group and the photosensitive compound (Ⅲ) group are both around 90% at 21% O2 concentration, showing neither phototoxicity nor chemotoxicity. Under light conditions, the photosensitive compound (Ⅱ)+L group and the photosensitive compound (Ⅲ)+L group exhibit phototoxicity. As the concentration increases, the cell survival rates of both groups show a similar decline, and the IC 50 values are 0.254 μM and 0.369 μM respectively.

[0271] Under dark conditions, as the oxygen concentration decreases to 8% O2, the photosensitive compound (Ⅱ) group begins to show certain cytotoxicity to 4T1 cells, and the IC 50 value is 1.748 μM. When the oxygen concentration decreases to 0.1% O2, the cytotoxicity of the photosensitive compound (Ⅱ) group is stronger, and the IC 50 value becomes 1.361 μM. However, the cell survival rate of the photosensitive compound (Ⅲ) group remains around 90% all the time, indicating that the photosensitive compound (Ⅱ) can release the chemotherapeutic drug PTX under hypoxic or microoxic conditions to induce cell death and produce chemotoxic effects.

[0272] Under light and 8% O2 conditions, both the photosensitive compound (Ⅱ)+L group and the photosensitive compound (Ⅲ)+L group have high cytotoxicity, and the IC 50 values are 0.283 μM and 1.252 μM respectively. Since the photosensitive compound (Ⅱ) can release PTX, and with the superposition of phototoxicity and chemotoxicity, its toxic effect is higher than that of the photosensitive compound (Ⅲ). This value is also very close to the IC 50 value of 0.221 μM for the photosensitive compound (Ⅲ):PTX (1:1)+L mixed group under light conditions, indicating that both its phototoxicity and chemotoxicity can be fully released.

[0273] Under light conditions, as the oxygen concentration changes from normoxia (21% O2) to hypoxia (8% O2), the IC of the photosensitive compound (Ⅱ)+L group 50The value changes from 0.254 μM to 0.283 μM, and the cytotoxic effect remains basically unchanged, indicating that under hypoxic conditions, the phototoxic effect of photosensitive compound (III) decreases; while photosensitive compound (II) is also limited by the decrease in oxygen concentration, resulting in a weakened phototoxic effect. However, the hypoxic environment promotes the release of PTX, generating chemotoxic effects and making up for the deficiencies of photodynamic therapy, thus maintaining the cytotoxic effect.

[0274] Under micro-oxygen (0.1% O2) conditions, for the photosensitive compound (II) + L group with light irradiation and the photosensitive compound (II) group without light irradiation, the IC 50 values are 1.292 μM and 1.361 μM respectively. It can be seen that photodynamic therapy almost fails under insufficient O2 conditions. At this time, the cytotoxic effect of photosensitive compound (II) completely comes from the chemotoxicity of PTX, which is very close to the IC 50 value of PTX, which is 1.453 μM.

[0275] Test 5-5: Cell experiment: Study on photodynamic induction of PTX release

[0276] Experimental purpose: To prove that with the decrease in oxygen concentration, photosensitive compound (II) can induce hypoxia through the photodynamic process and release the chemotherapeutic drug PTX.

[0277] (1) Experimental instruments and reagents:

[0278] Tubulin-Tracker: Beyotime Institute of Biotechnology.

[0279] (2) Experimental methods and procedures:

[0280] a. 4T1 cell culture: The specific operation details are as described in the above tests.

[0281] b. Laying the confocal dish: Wash the 4T1 cells in the culture flask twice with PBS, then add trypsin for digestion, centrifuge, resuspend and count. Finally, seed the cells at a concentration of 1.5×10 5 cells / mL in a laser confocal dish and place it in a cell culture incubator for 12 h to allow the cells to adhere;

[0282] c. Adding drugs: To avoid the killing of cancer cells by photodynamic therapy, the cells in the dish are first treated with the reactive oxygen species scavenger vitamin C (VC, 100 μM) for 1 h. Prepare cell culture media containing 2 μM of photosensitive compound (II), photosensitive compound (III) and PTX respectively (containing 0.05% Tween 80). Then wash the VC-treated cells twice with PBS, add the drug-containing medium, and incubate in a 37 °C constant temperature incubator for 24 h;

[0283] d. Cover the glass slide: Wash the incubated confocal dish twice with PBS and add fresh culture medium. Then cover the culture medium with a sterile glass slide to block the entry of oxygen in the air. Subsequently, irradiate the confocal dish with a laser (660 nm, 20 mW / cm 2 ) for the corresponding time (0 min, 2 min, 5 min, 10 min) respectively. After the light irradiation, place the confocal dish in a 37°C incubator and continue to culture for 6 h;

[0284] e. Load the probe: Wash the confocal dish twice with PBS. Then add 1 mL of Tubulin-TrackerGreen staining working solution and incubate it in a cell culture incubator for 20 min. Wash the incubated confocal dish twice with PBS, and then add 1 mL of Hypoxia red probe staining working solution and incubate it in a cell culture incubator for 10 min;

[0285] f. Take pictures: Use a confocal microscope to scan and take pictures. (Tubulin-Tracker Green: excitation wavelength is 488 nm, detection wavelength is 510 nm - 570 nm; Hypoxia probe: excitation wavelength 543 nm, detection wavelength 550 nm - 620 nm).

[0286] (3) Experimental results:

[0287] As shown in the appendix Figure 14 , in both the photosensitive compound (II) group and the photosensitive compound (III) group, a certain degree of hypoxia occurred after 2 min of laser irradiation, and with the increase of the light irradiation time, the hypoxia effect was enhanced, manifested as gradually enhanced Hypoxia red fluorescence. While the PTX group did not produce a hypoxia effect under light irradiation conditions. This indicates that the hypoxia phenomenon is caused by the photosensitive compounds (II) and (III) consuming oxygen through photodynamic action. In addition, with the increase of the light irradiation time, the microtubule structure of the cells in the photosensitive compound (II) group changed from filamentous to elliptical and approached that of the PTX group. This shows that the photodynamic process can lead to hypoxia, induce the release of the chemotherapeutic drug PTX by the photosensitive compound (II), and cause the aggregation and death of cancer cell microtubules.

[0288] Test 6-1: Mouse experiment, study on the tissue distribution of the compound

[0289] Experimental purpose: In this experiment, the distribution of the drug in tumors and various organs was preliminarily investigated by means of organ fluorescence imaging.

[0290] (1) Experimental instruments and reagents:

[0291] Small animal imager: Perkin-Elmer.

[0292] (2) Experimental methods and procedures:

[0293] a. Establishment of tumor-bearing mouse model: Take 4T1 cells in good growth state. After digestion, centrifugation and washing twice with PBS, resuspend the cells and adjust the cell density to 6×10 7 cells / mL. Select 9 female balb / c mice at 4 weeks old with a body weight of about 20 g. After disinfecting the back of each mouse with 75% alcohol, subcutaneously inject 100 μL of cell suspension. Wait until the average size of the mouse tumor grows to 80 - 100 mm 3 , and randomly divide the mice into three groups, with 3 mice in parallel in each group.

[0294] b. Administration: Prepare 100 μM saline solutions of photosensitive compound (Ⅱ) and photosensitive compound (Ⅲ) (containing 1% DMSO and 0.1% CEL) for drug addition. The saline group is not treated. The three groups of mice are respectively injected with 100 μL of saline solution of photosensitive compound (Ⅱ), saline solution of photosensitive compound (Ⅲ) and saline via the tail vein.

[0295] c. Fluorescence imaging of mouse organs: 12 h after the mice are injected with the drug, decapitate and sacrifice each group of mice by cervical dislocation. Dissect the mice and take out the tumors, heart, liver, spleen, lungs and kidneys. Take pictures of the removed tumors and organs with a small animal imager to observe the fluorescence intensity of the drug in the three-dimensional organs and tumors.

[0296] (3) Experimental results:

[0297] The tissue distributions of photosensitive compound (Ⅱ) and photosensitive compound (Ⅲ) are shown in Appendix Figure 15 and 16 respectively. There are significant differences in the fluorescence signals between the photosensitive compound (Ⅱ) group, the photosensitive compound (Ⅲ) group and the Saline group. It can be found that the fluorescence signals are mainly concentrated in the tumor location, as well as the liver and kidneys. Among them, the fluorescence intensity of the tumor tissue is the highest, indicating that photosensitive compound (Ⅱ) and photosensitive compound (Ⅲ) have obvious tumor enrichment tendency; followed by the kidneys, and then the liver, suggesting that photosensitive compound (Ⅱ) and photosensitive compound (Ⅲ) may be metabolized through the kidneys and liver at 12 h; the drug fluorescence signals of photosensitive compound (Ⅱ) and photosensitive compound (Ⅲ) in the main organs are all lower than those of the tumor tissue, and there is no abnormal accumulation. Therefore, during photodynamic therapy, the damage to normal tissues can be minimized.

[0298] Test 6-2: Mouse experiment, research on the anti-tumor activity of the compound

[0299] (1) Experimental reagents and instruments

[0300] Dimethyl sulfoxide (DMSO): Shanghai Chemical Reagent Company, China National Pharmaceutical Corporation.

[0301] Polyoxyethylene castor oil (CEL): Beijing Dingguo Changsheng Biotechnology Co., Ltd.

[0302] Paraformaldehyde: Beijing Dingguo Changsheng Biotechnology Co., Ltd.

[0303] Paraffin: Shanghai Huayong Paraffin Co., Ltd.

[0304] Hematoxylin: Beijing Solarbio Science & Technology Co., Ltd.

[0305] Eosin: Beijing Solarbio Science & Technology Co., Ltd.

[0306] LED photodynamic beauty instrument: MBG Technologies Inc.

[0307] Laminar flow bench: ESCO Corporation, Singapore.

[0308] (2) Experimental methods and procedures:

[0309] a. Establishment of 4T1 tumor-bearing mouse model: Take 4T1 cells in good growth state. After digestion, centrifugation and washing twice with PBS, resuspend the cells and adjust the cell density to 6×10 7 cells / mL. Select 35 female balb / c mice at 4 weeks old with a body weight of about 20 g. After disinfecting the back of each mouse with 75% alcohol, subcutaneously inoculate 100 μL of cell suspension;

[0310] b. Preparation of drug stock solutions: Prepare DMSO stock solutions (10 mM, containing 10% CEL) of photosensitive compound (II), photosensitive compound (III) and PTX respectively. Take 10 μL and dilute it to 1 mL of physiological saline to obtain physiological saline solutions of photosensitive compound (II), photosensitive compound (III) and PTX with a final concentration of 100 μM each (containing 1% DMSO, 0.1% CEL). The physiological saline group is not treated. Among them, the preparation of the photosensitive compound (III):PTX = 1:1 group is as follows: Prepare DMSO stock solutions (20 mM, containing 10% CEL) of photosensitive compound (III) and PTX respectively. Take 10 μL and dilute it to 1 mL of physiological saline to obtain physiological saline solutions of photosensitive compound (III) and PTX with a final concentration of 200 μM each (containing 1% DMSO, 0.1% CEL), and then mix the two according to a volume ratio of 1:1 to obtain a drug solution of photosensitive compound (III):PTX = 1:1 (where the final concentrations of photosensitive compound (III) and PTX are both 100 μM);

[0311] c. Grouping and drug administration: When the tumor volume grows to 60 - 80 mm 3, the mice were randomly divided into 8 groups (photosensitive compound (Ⅱ), photosensitive compound (Ⅲ) group, PTX group, photosensitive compound (Ⅲ):PTX = 1:1 group, photosensitive compound (Ⅱ)+L group, photosensitive compound (Ⅲ)+L group, photosensitive compound (Ⅲ):PTX = 1:1+L group, PBS group), with five mice in each group in parallel. Each group was given the corresponding drug (100 μL) by tail vein injection;

[0312] d. Light treatment: The first administration was on day 0, and the administration frequency was once every 3 days. The light treatment group was irradiated with a 660 nm laser on the tumor site of the mice for 10 min (100 mW / cm 2 ) 12 h after administration;

[0313] e. Data measurement and recording: After light treatment, the body weight of the mice was weighed regularly every day and the tumor volume was measured, which was repeated for 15 days.

[0314] (3) Experimental results:

[0315] The anti-tumor activity results of photosensitive compound (Ⅱ) and photosensitive compound (Ⅲ) are as shown in the appendix Figures 17 - 19 . Whether irradiated or not, the tumors of the mice in the PBS blank control group grew rapidly and uncontrollably, and the final tumor volume increased by about 12 times compared with the initial volume, indicating that light had no inhibitory effect on tumor growth.

[0316] Under the condition of avoiding light, photosensitive compound (Ⅲ) did not show phototoxicity or chemotoxicity; the PTX group was similar to the photosensitive compound (Ⅲ):PTX = 1:1 group, only playing the role of a chemotherapeutic drug, and the tumor volume and weight decreased compared with the blank control group, and the final tumor volume increased by about 9 times compared with the initial volume. The photosensitive compound (Ⅱ) group also only played the role of chemotoxicity, but the drug effect was significantly improved, indicating that the bioavailability of photosensitive compound (Ⅱ) was higher than that of the PTX parent drug.

[0317] Under the condition of light irradiation, the photosensitive compound (Ⅲ) group showed single phototoxicity; the photosensitive compound (Ⅲ):PTX = 1:1+L group had both phototoxicity and chemotoxicity at the same time, and the drug effect of this group was slightly better than that of the photosensitive compound (Ⅲ) group; the photosensitive compound (Ⅱ)+L group had the best therapeutic effect, and was significantly better than the photosensitive compound (Ⅲ):PTX = 1:1+L group, indicating that the effect of photosensitive compound (Ⅱ) was not equivalent to the simple addition of photosensitizer (Ⅰ) and PTX, but played a synergistic effect of "1+1>2" and had the best effect, and obvious ablation of the tumor occurred. It was proved that the introduction of photosensitive compound (Ⅲ) played the role of "a prodrug carrier with photodynamic drug effect", enhanced the targeted enrichment of the drug in tumor tissues, and improved the bioavailability.

[0318] From the appendixFigure 20 It can be seen that there were no significant changes in the body weights of the mice in each group such as the photosensitive compound (II) + L group, etc., indicating the high biosafety of the photosensitive compound (II), the photosensitive compound (III) and the photosensitizer (I), and no adverse effects on the normal growth of the mice.

[0319] Appendix Figures 17 - 20 In it, Compound 1 represents the photosensitive compound (II), Compound 1-2 represents the photosensitive compound (III), and Compound 1-1 represents the photosensitizer (I).

[0320] As described above, the basic principles, main features and advantages of the present invention have been shown and described. Those skilled in the art should understand that the present invention is not limited by the above embodiments. The above embodiments are only preferred embodiments of the present invention, and the scope of implementation of the present invention cannot be limited accordingly. That is, equivalent changes and modifications made according to the scope of the present invention patent and the content of the specification should still fall within the scope covered by the present invention. The scope of protection required by the present invention is defined by the appended claims and their equivalents.

Claims

1. A photosensitizer, characterized in that, The structure of the photosensitizer or its pharmaceutically acceptable salt is shown in formula (I):

2. A hypoxia-responsive activation type photosensitive compound, characterized in that, The hypoxia-responsive activation type photosensitive compound or its pharmaceutically acceptable salt is formed by connecting the photosensitizer or its pharmaceutically acceptable salt described in claim 1 with a chemotherapeutic drug through a covalent bond that can undergo responsive cleavage in the tumor tissue microenvironment.

3. The hypoxia-responsive activation type photosensitive compound according to claim 2, wherein The chemotherapeutic drug is paclitaxel.

4. The hypoxia-responsive activation type photosensitive compound according to claim 2, characterized in that, The covalent bond specifically responds to azoreductase and undergoes responsive cleavage.

5. The hypoxia-responsive activated photosensitive compound according to claim 4, wherein The covalent bond contains a nitrogen-nitrogen double bond.

6. The hypoxia-responsive activation type photosensitive compound according to claim 4, wherein The covalent bond contains an azobenzene structure.

7. The hypoxia-responsive activation type photosensitive compound according to any one of claims 2-6, characterized in that, The structure of the photosensitive compound is shown in the following formula (Ⅱ):

8. The hypoxia-responsive activated photosensitive compound according to claim 2, wherein The method of covalent bond connection is the reaction of the azide group on the photosensitizer or its pharmaceutically acceptable salt with an alkyne group.

9. Use of the photosensitizer according to claim 1 or the hypoxia-responsive activation type photosensitive compound according to any one of claims 2-8, characterized in that, As a therapeutic drug for tumors, as one of the components of a tumor therapeutic drug composition, or for preparing a drug for treating tumors.

10. The application according to claim 9, wherein The tumor therapeutic drug composition further comprises a pharmaceutically acceptable carrier.

Images