Preparation and application of aggregation-induced emission type photosensitizer with dihydrodibenzophenazine structure

Abstract

The invention provides an aggregation-induced emission type photosensitizer with a dihydrodibenzophenazine structure as well as a preparation method and application of the aggregation-induced emission type photosensitizer with the dihydrodibenzophenazine structure, and the aggregation-induced emission type photosensitizer with the dihydrodibenzophenazine structure is obtained by connecting an aggregation-induced emission element with multiple rotors to a framework of a vibration-induced emission dihydrodibenzophenazine derivative. The photosensitizer with the dihydrodibenzophenazine structure integrates the advantages of aggregation-induced emission and vibration-induced emission, and can be applied to fluorescence imaging of biological tissues; the photosensitizer with the dihydrodibenzophenazine structure has viscosity responsiveness and photosensitivity, so that the photosensitizer with the dihydrodibenzophenazine structure can be applied to blood viscosity detection, thrombus imaging and tumor light diagnosis and treatment, and particularly, the photosensitizer with the dihydrodibenzophenazine structure has good performance in blood viscosity detection, thrombus, particularly in-vivo imaging of cerebral thrombosis and tumor light diagnosis and treatment.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of fluorescent probes, in particular to a dihydrodibenzophenazine structure aggregation-induced emission luminophore and a preparation method and application thereof, in particular application in real-time monitoring of thrombosis and tumor diagnosis and treatment scenarios. BACKGROUND

[0002] Thrombosis is a blood clot formed by abnormal hemostasis of blood vessels, and is closely related to various diseases such as pulmonary embolism, myocardial infarction and stroke. Early diagnosis and treatment of thrombosis is the key to reducing the high risk of thrombotic diseases. Due to the characteristics of small volume, instability, wide regional distribution and rapid formation of early thrombosis, it puts forward very high requirements for detection technology, and an method capable of simultaneously meeting high signal-to-noise ratio, early dynamic monitoring and rapid deep tissue imaging is urgently needed.

[0003] In the field of biological imaging, sensing, diagnosis and treatment, fluorescent probes exhibit irreplaceable application value due to their high sensitivity and specificity. Aggregation-induced emission luminophores have obvious advantages, such as strong light stability and good biocompatibility. More importantly, the twisted three-dimensional structure in the aggregated state can effectively block the non-radiative relaxation channel, enhancing the fluorescence imaging effect while improving the triplet quantum yield and stability, and thus significantly improving the generation efficiency of reactive oxygen species (ROS), providing a good basis for thrombus detection.

[0004] Although aggregation-induced emission luminophores have many advantages in thrombus detection, they still have some defects, which are difficult to fully meet the high requirements of early thrombus diagnosis and treatment: In the aspect of thrombus imaging, aggregation-induced emission luminophores are difficult to break through the technical bottleneck of brain thrombus in vivo imaging. Aggregation-induced emission luminophores generally lack viscosity responsiveness, while the local microenvironment viscosity in the brain thrombus region is significantly increased due to blood flow stagnation, and such probes cannot specifically enhance the signal through viscosity changes, resulting in low signal-to-noise ratio of imaging and difficulty in distinguishing thrombus from normal brain tissue. At the same time, most probes have no clear targeting, and are easily diffused non-specifically in the complex network of brain blood vessels, further reducing the imaging accuracy; and the single fluorescence emission mode is seriously interfered by the autofluorescence of brain tissue, which cannot achieve clear delineation of the thrombus boundary, especially difficult to meet the in vivo monitoring requirements of brain thrombus with small volume and deep location.

[0005] In the field of tumor phototherapy, existing AIE photosensitizers suffer from the dual limitations of poor functional synergy and insufficient targeting. Although traditional AIE photosensitizers can generate reactive oxygen species (ROS), they lack responsiveness to the tumor microenvironment. The efficiency of ROS generation fluctuates greatly due to environmental influences, making it difficult to achieve precise photodynamic therapy. Furthermore, most molecules lack mitochondrial targeting capabilities, and since mitochondria are the core of tumor cell energy metabolism, the inability to target and accumulate them significantly reduces the therapeutic effect.

[0006] Therefore, there is an urgent need for optimized aggregation-induced emission photosensitizers that can be used for thrombosis imaging and phototherapy of tumors. Summary of the Invention

[0007] The purpose of this invention is to provide a dihydrodibenzophenazine-structured aggregation-induced emission photosensitizer, its preparation method, and its application. Compared with traditional aggregation-induced emission photosensitizers, it has higher viscosity responsiveness, photosensitivity, and mitochondrial targeting, enabling the application of aggregation-induced emission photosensitizers in thrombosis imaging and phototherapy of tumors.

[0008] To achieve the above objectives, firstly, this technical solution provides a dihydrodibenzophenazine-structured aggregation-induced emission photosensitizer having the following general structural formula:

[0009] Where X and Y are any one of C, N, O, and S. m , n , k The numbers are natural numbers between 0 and 3, and not all of them are 0. x R1 and R2 are natural numbers from 1 to 6, and are selected from hydrogen atoms, alkyl groups, or alkoxy groups.

[0010] It should be noted that dihydrodibenzophenazine derivatives possess a unique vibrational induced emission (VIE) mechanism. These molecules can exhibit red and blue fluorescence in different ratios under single-wavelength excitation. Traditionally, dihydrodibenzophenazine compounds have been used as hole transport materials. However, our team discovered that these compounds also possess unique photophysical properties, namely dual fluorescence emission, large Stokes shift, and environmental sensitivity. Therefore, based on the advantages of dihydrodibenzophenazine derivatives and aggregation-induced emission (AIE) molecules, our team has proposed a further research on vibrational induced emission... By attaching aggregation-induced emission units with multiple rotors to the skeleton of a dihydrodibenzophenazine derivative, a dihydrodibenzophenazine-structured aggregation-induced emission photosensitizer is obtained. This dihydrodibenzophenazine-structured aggregation-induced emission photosensitizer integrates the advantages of aggregation-induced emission and vibration-induced emission, and can be applied to fluorescence imaging of biological tissues. Furthermore, this dihydrodibenzophenazine-structured aggregation-induced emission photosensitizer has viscosity responsiveness and photosensitivity, so it can be applied to blood viscosity detection, thrombosis imaging, and phototherapy of tumors. In particular, it performs well in in vivo imaging of thrombosis, especially cerebral thrombosis, and in phototherapy of tumors.

[0011] In some embodiments, the structure of the dihydrodibenzophenazine-structured aggregation-induced luminescence photosensitizer is any one of the following formulas I-1 to I-36: ; .

[0012] Secondly, this solution provides a method for preparing a dihydrodibenzophenazine-structured aggregation-induced emission photosensitizer, including the following steps: S1: Under inert gas protection, bromoarylacetonitrile and aryl formaldehyde are added to an organic solvent and an alkaline substance is added as a catalyst. The mixture is refluxed at 80-120 °C to obtain intermediate product IV, wherein the molar ratio of bromoarylacetonitrile to aryl formaldehyde is 1:(1-4). S2: Under inert gas protection, intermediate IV and pyridineboronic acid are dissolved in an alkaline solution of palladium catalyst and refluxed at 80-120 °C to obtain intermediate V, wherein the molar ratio of intermediate IV to pyridineboronic acid is 1:(1.1-1.5). S3: Under inert gas protection, intermediate V is reacted with 9-(4-(4-bromoalkoxy)phenyl)-14-phenyl-9,14-dihydrodibenzo[ a , c The phenazine salt formation reaction yielded the target product I.

[0013] The reaction equation for the preparation method of this dihydrodibenzophenazine aggregation-induced emission photosensitizer is shown below: .

[0014] In S1 of this scheme, bromoarylacetonitrile undergoes a Kronowenger reaction with aryl formaldehyde. The basic substance abstracts the active hydrogen from the methylene group of the bromoarylacetonitrile to form a carbanion, which then nucleophilically attacks the carbonyl carbon of the aryl formaldehyde. After addition and dehydration, an α,β-unsaturated nitrile compound containing a carbon-carbon double bond is generated. In some embodiments, the molar amount of the alkaline substance is 0.1 to 0.5 equivalents, and the alkaline substance is selected from any one or a mixture of several of pyridine, piperidine, potassium carbonate, sodium carbonate, potassium hydroxide, sodium hydroxide, sodium alkoxide, and potassium alkoxide.

[0015] In some embodiments, bromoarylacetonitrile and aryl formaldehyde are dissolved in an organic solvent, wherein the organic solvent is selected from any one or a mixture of several of ethanol, isopropanol, tetrahydrofuran, and acetonitrile.

[0016] In some embodiments, the inert gas is selected from either nitrogen or argon.

[0017] In S2 of this scheme, intermediate product IV undergoes a Suzuki-Miyaura cross-coupling reaction with pyridineboronic acid in an alkaline solution with a palladium catalyst. Under the conditions of palladium catalyst and alkaline conditions, intermediate product IV cross-couples with pyridineboronic acid to form a new carbon-carbon bond, thereby introducing the pyridine group into the molecular structure to obtain intermediate product V.

[0018] In some embodiments, the palladium catalyst is selected from tetra(triphenylphosphine)palladium, palladium acetate, tris(dibenzylideneacetone)palladium, and 1,1′-bis(diphenylphosphine)ferrocene palladium dichloride.

[0019] In some embodiments, the alkaline substance in the alkaline solution of the palladium catalyst is one of potassium carbonate, sodium carbonate, cesium carbonate, and tetrabutylammonium fluoride.

[0020] In S3 of this scheme, intermediate V reacts with 9-(4-(4-bromoalkoxy)phenyl)-14-phenyl-9,14-dihydrodibenzo[ a , c ]Phenazine (IX) undergoes a salt-forming reaction, 9-(4-(4-bromoalkoxy)phenyl)-14-phenyl-9,14-dihydrodibenzo[ a , c The bromoalkoxy chain in the phenazine molecule contains an active bromine atom (leaving group), and if the intermediate product V contains a nitrogen-containing heterocyclic structure such as a pyridine ring (with a nucleophilic nitrogen atom), then the nitrogen atom can act as a nucleophile to attack the electrophilic carbon atom of the bromoalkane, thereby replacing the bromide ion to form a stable quaternary ammonium salt structure, i.e., the target product I.

[0021] In some embodiments, intermediate V is reacted with 9-(4-(4-bromoalkoxy)phenyl)-14-phenyl-9,14-dihydrodibenzo[a , c The molar ratio of phenazine is 1:(1-5).

[0022] In some embodiments, intermediate V is reacted with 9-(4-(4-bromoalkoxy)phenyl)-14-phenyl-9,14-dihydrodibenzo[ a , c The inert gas used in the phenazine reaction is nitrogen.

[0023] In some embodiments, intermediate V is reacted with 9-(4-(4-bromoalkoxy)phenyl)-14-phenyl-9,14-dihydrodibenzo[ a , c Phenidine was dissolved in an organic solvent and refluxed at 90–150 °C for 12–72 hours to give target product I. The organic solvent was selected from 1,4-dioxane, acetonitrile, etc. N , N - One or a mixture of dimethylformamide, dimethyl sulfoxide, acetone or toluene.

[0024] In some embodiments, target product I is cooled to room temperature and then concentrated under reduced pressure to obtain a crude product. After adding a low-carbon alcohol or a low-carbon ester to the crude product and washing it multiple times, a dihydrodibenzophenazine-structured aggregation-induced emission photosensitizer is obtained.

[0025] Regarding the 9-(4-(4-bromoalkoxy)phenyl)-14-phenyl-9,14-dihydrodibenzo[ a , c The preparation method of phenazine is as follows: A: 9,10-Phenanthroquinone and aniline were dissolved in a solvent, and titanium tetrachloride was added under ice bath conditions. After the reaction was completed at room temperature, the solvent was removed, and a mixed solvent was added to dissolve the mixture. Subsequently, sodium borohydride was added in batches and refluxed. After post-treatment, the desired product was obtained. N , N -Diphenylphenanthrene-9,10-diamine; B: Will N , N 9,10-Diphenylphenanthrene-9,10-diamine, 1-iodo-4-methoxybenzene, potassium carbonate, and copper trifluoromethanesulfonate were dissolved in a solvent, refluxed until complete, and then post-treated to give 9-(4-methoxyphenyl)-14-phenyl-9,14-dihydrodibenzo[ a , c Phenidine; C: 9-(4-methoxyphenyl)-14-phenyl-9,14-dihydrodibenzo[ a , c ]Phenidine was dissolved in a solvent and boron tribromide was added under ice bath conditions. After the reaction was completed at room temperature, the solvent was removed and post-treatment was performed to obtain 4-(1,4-phenyldibenzo[ a ,c ]Phenomazine-9 (14 H )-yl)phenol; D: 4-(14-phenyldibenzo[ a , c ]Phenomazine-9 (14 H 9-(4-(4-bromoalkoxy)phenyl)-14-phenyl-9,14-dihydrodibenzo[] a , c Phenyrazine.

[0026] In some embodiments, the molar ratio of 9,10-phenanthrenequinone to aniline is 1:(2-4), and 4 equivalents of titanium tetrachloride are added under ice bath conditions, and the molar ratio of sodium borohydride to 9,10-phenanthrenequinone is 1:(1.01-2).

[0027] In step A, the solvent is toluene, and the mixed solvent is a mixture of tetrahydrofuran and ethanol in a volume ratio of (1~2):(2~1).

[0028] In some embodiments, N , N The molar ratio of 1-diphenylphenanthrene-9,10-diamine, 1-iodo-4-methoxybenzene, potassium carbonate, and copper trifluoromethanesulfonate is 1:(1.1–2):(1.5–2.5):(1.5–2.5).

[0029] In step B, the solvent is 1,3,5-trichlorobenzene.

[0030] In some embodiments, 9-(4-methoxyphenyl)-14-phenyl-9,14-dihydrodibenzo[ a , c The molar ratio of phenazine to boron tribromide is 1:(1.5 to 2.5).

[0031] In step C, the solvent is dichloromethane.

[0032] In some embodiments, 4-(14-phenyldibenzo[] a , c ]Phenomazine-9 (14 H The molar ratio of 4-(1,4-phenyldibenzo[]phenol to 1,4-dibromoalkane is 1:(1.01-2), 4-(1,4-phenyldibenzo[]phenol to 1,4-dibromoalkane. a , c ]Phenomazine-9 (14 H The molar ratio of phenol to potassium carbonate is 1:(1.5-2.5).

[0033] In step D, the solvent is acetonitrile or acetone or N , NAny of the following: -dimethylformamide.

[0034] Thirdly, this solution provides an application of a dihydrodibenzophenazine-structured aggregation-induced luminescence photosensitizer as a fluorescent probe for blood viscosity detection, a thrombosis imaging contrast agent, a photodynamic therapy reagent, a fluorescent contrast agent, or an imaging-guided photodynamic therapy reagent.

[0035] In other words, this solution provides a fluorescent imaging developer, which is a dihydrodibenzophenazine structure aggregation-induced emission photosensitizer as described above, wherein the dihydrodibenzophenazine structure photosensitizer has both aggregation-induced emission and vibration-induced emission mechanisms.

[0036] This solution provides a blood viscosity detection probe or thrombosis imaging contrast agent, which is a dihydrodibenzophenazine aggregate-induced emission photosensitizer, wherein the dihydrodibenzophenazine aggregate-induced emission photosensitizer has viscosity responsiveness.

[0037] This solution provides a photodynamic therapy reagent, which is a dihydrodibenzophenazine aggregation-induced emission photosensitizer. The dihydrodibenzophenazine aggregation-induced emission photosensitizer is photosensitized. This photodynamic therapy reagent can be used as a live tumor photodynamic therapy reagent, a fluorescence imaging contrast agent, or an imaging-guided photodynamic therapy reagent, and can be used for fluorescence diagnosis and phototherapy of tumors. Compared with existing technologies, this solution has the following characteristics and beneficial effects: The dihydrodibenzophenonezine-based aggregation-induced emission (AIE) photosensitizer provided in this invention combines the advantages of both AIE and VIE mechanisms. This photosensitizer exhibits AIE properties with a maximum emission wavelength in the near-infrared region (>650 nm), making it suitable for near-infrared fluorescence imaging. Furthermore, this type of photosensitizer specifically targets mitochondria within cells, efficiently generating reactive oxygen species (ROS) under illumination, exceeding the efficiency of the classic photosensitizer dihydroporphyrin E6 (Ce6), thus demonstrating excellent photodynamic therapy efficacy. Simultaneously, this photosensitizer exhibits viscosity sensitivity and low cytotoxicity, enabling real-time monitoring of thrombus formation. It holds potential clinical application value in near-infrared fluorescence imaging-guided photodynamic therapy and early thrombus diagnosis. Verification has shown that the photosensitizer in this invention can achieve photodynamic therapy in live mice, ablating tumor tissue. Moreover, this photosensitizer can effectively penetrate the blood-brain barrier, enabling real-time monitoring of cerebral thrombosis in rats, possessing advantages such as excellent tissue penetration and a high signal-to-noise ratio. Furthermore, the photosensitizer is simple to synthesize and the raw materials are readily available. Attached Figure Description

[0038] Figure 1 The UV-Vis absorption spectrum and fluorescence emission spectrum of I-1 prepared in Example 1 in dimethyl sulfoxide (10 μM); Figure 2The fluorescence emission spectrum and relative fluorescence intensity diagram of I-1 prepared in Example 1 in a mixed system of dimethyl sulfoxide and glycerol are shown. Figure 3 The fluorescence enhancement curves of DCFH-DA are shown in the presence of different photosensitizers. Figure 4 This is a mitochondrial colocalization test diagram of I-1 in HeLa cells in Example 5 of the present invention; wherein, (a) is the fluorescence signal of I-1; (b) is the fluorescence signal of a commercial mitochondrial dye; (c) is the mixed field of (a) and (b); (d) is the overlap coefficient of (a) and (b); the scale bar is 10 micrometers; Figure 5 In Example 6 of this invention, the survival rate of 4T1 cells was measured when they coexisted with different concentrations (0, 1, 2, 5, 10, 20, 50, 100 μmol / L) of I-1 under white light or dark conditions. Figure 6 This is a graph showing the fluorescence signal changes of I-1 in HeLa cells when I-1 was co-incubated with different concentrations (0, 1, 5, 10, 20, 30 μmol / L) of nystatin in Example 7 of the present invention. In the graph, (a) is the fluorescence signal of I-1; (b) is the bright field fluorescence signal; and (c) is the mixed field of (a) and (b). The scale bar is 10 micrometers. Figure 7 This is a curve comparing the effects of different treatment groups on photodynamic tumor treatment in 4T1 tumor-bearing mice in Example 8 of the present invention. Figure 8 This is a graph showing the changes in fluorescence signals over time in the brain regions of SD rats with cerebral artery embolism and normal SD rats in Example 9 of the present invention. (a) is the fluorescence signal in the brain of SD rats with cerebral artery embolism without probes; (b) is the fluorescence signal in the brain of normal SD rats; and (c) is the fluorescence signal in the brain of SD rats with cerebral artery embolism with probes. Detailed Implementation

[0039] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention are within the scope of protection of the present invention.

[0040] For those skilled in chemistry, biochemistry, or related fields, many modifications and variations can be made without departing from the spirit and scope of the claims of this invention, and all of these fall within the scope of protection of this invention.

[0041] Statement: All raw materials used in this invention are commercially available.

[0042] This invention employs the following synthetic route to prepare a class of aggregation-induced emission photosensitizers with a dihydrodibenzophenazine structure:

[0043] Since the selection of alkyl or alkoxy groups on the dihydrodibenzophenazine backbone from R1 and R2 does not affect the properties of the resulting aggregation-induced emission photosensitizer, similarly, the alkoxy group connecting the dihydrodibenzophenazine unit and the AIE unit being methoxy, ethoxy, propoxy, butoxy, pentoxy, or hexoxy also does not affect the properties of the resulting aggregation-induced emission photosensitizer. Therefore, with both R1 and R2 being hydrogen atoms, x The preparation of an aggregation-induced emission photosensitizer with a 4-dihydrodibenzophenazine structure is taken as an example.

[0044] Regarding 9-(4-(4-bromobutoxy)phenyl)-14-phenyl-9,14-dihydrodibenzo[ a , c Preparation of phenazine: (1) N , N Synthesis of -diphenylphenanthrene-9,10-diamine (compound shown in formula VI):

[0045] VI 9,10-Phenanthroquinone (5.0 g, 24.0 mmol) and aniline (8.0 mL, 84.3 mmol) were placed in a reaction flask, and 150 mL of toluene was added and stirred until homogeneous. Titanium tetrachloride (4.0 mL) was added dropwise under ice bath conditions, and the reaction was carried out at room temperature for 12 hours. Thin-layer chromatography (TLC) was used to monitor the reaction until the starting material was exhausted. After removing the toluene by rotary evaporation, 100 mL of a mixed solvent of tetrahydrofuran and ethanol was added. v / v = 1 / 1), then sodium borohydride (1.0 g, 26.4 mmol) was added in portions at room temperature and the mixture was refluxed for two hours. After the reaction was complete, water was added, and the mixture was extracted three times with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, evaporated to dryness, and separated by column chromatography to give 4.1 g of pale yellow solid (the compound shown in formula VI), with a yield of 47%.

[0046] (2) 9-(4-methoxyphenyl)-14-phenyl-9,14-dihydrodibenzo[ a , c Synthesis of phenazine (the compound shown in formula VII):

[0047] VII The compound shown in Formula VI (500 mg, 1.4 mmol) was dissolved in 10 mL of 1,3,5-trichlorobenzene, and 1-iodo-4-methoxybenzene (491 mg, 2.1 mmol), potassium carbonate (387 mg, 2.8 mmol), and copper trifluoromethanesulfonate (127 mg, 2.8 mmol) were added. The mixture was refluxed at 80 °C for 6 hours. The reaction was monitored by TLC until the starting material was exhausted. The solvent was evaporated under reduced pressure, cooled to room temperature, and then extracted three times with water and dichloromethane. The combined organic layers were dried over anhydrous sodium sulfate, evaporated to dryness, and separated by column chromatography to give 288 mg of solid (the compound shown in Formula VII), with a yield of 62%.

[0048] (3) 4-(14-phenyldibenzo[ a , c ]Phenomazine-9 (14 H Synthesis of α-hydroxyphenol (the compound shown in formula VIII):

[0049] VIII The compound shown in Formula VII (500 mg, 1.1 mmol) was dissolved in 10 mL of dichloromethane, and boron tribromide (500 mg, 2.2 mmol) was added under ice bath conditions. The reaction was carried out at room temperature for 12 hours. The reaction was monitored by TLC until the starting material was exhausted. The solvent was evaporated under reduced pressure, water was added, and the mixture was extracted three times with dichloromethane. The combined organic layers were dried over anhydrous sodium sulfate, evaporated to dryness, and separated by column chromatography to give 194 mg of solid (the compound shown in Formula VIII), with a yield of 43%.

[0050] (4) 9-(4-(4-bromobutoxy)phenyl)-14-phenyl-9,14-dihydrodibenzo[ a , c Synthesis of phenazine (the compound shown in formula IX):

[0051] IX The compound shown in Formula VIII (500 mg, 1.1 mmol) and 1,4-dibromobutane (473 mg, 2.2 mmol) were dissolved in acetonitrile (30 mL), and potassium carbonate (304 mg, 2.2 mmol) was slowly added. The mixture was refluxed at 90 °C for 48 hours. The reaction was monitored by TLC until the starting material was exhausted. The solvent was evaporated under reduced pressure, water was added, and the mixture was extracted three times with dichloromethane. The combined organic layers were dried over anhydrous sodium sulfate, evaporated to dryness, and separated by column chromatography to give 140 mg of solid (the compound shown in Formula IX), with a yield of 24%.

[0052] Example 1 ( Z)-4-(4-(2-cyano-2-(4-(dimethylamino)phenyl)vinyl)phenyl)-1-(4-(4-(14-phenyldibenzo[ a , c ]Phenomazine-9 (14 H )yl)phenoxy)butyl)pyridine-1-onium (I-1): (1) 588 mg of p-bromophenylacetonitrile (II-1, 3.0 mmol) and 149 mg of 4-(dimethylamino)benzaldehyde (III-1, 1.0 mmol) were added to a three-necked flask, followed by 20 mL of anhydrous ethanol. 20 mg of sodium hydroxide was dissolved in 5 mL of anhydrous ethanol, and the mixture was added to a constant-pressure dropping funnel. After evacuating and purging with nitrogen three times, the constant-pressure dropping funnel was slowly opened, and the mixture was stirred at room temperature for 12 h. The reaction solution was extracted three times with 50 mL of dichloromethane. The combined organic phases were washed three times with saturated brine and dried over anhydrous sodium sulfate. The organic solvent was removed by vacuum distillation, and the crude product was purified by silica gel column chromatography with a petroleum ether:ethyl acetate ratio of 50:1 (v / v) to obtain 183 mg of solid ( Z 3-(4-bromophenyl)-2-(4-(dimethylamino)phenyl)acrylonitrile (Ⅳ-1), yield: 56%.

[0053] The reaction formula for this step is as follows: ; (2) Under nitrogen protection, 148 mg of 4-pyridineboronic acid (1.2 mmol), 327 mg of intermediate (Ⅳ-1, 1.0 mmol), and tetraphenylphosphine palladium (0) were added to a mixture of potassium carbonate, tetrahydrofuran, and water. The mixture was stirred and refluxed at 85–100 °C for 10–12 hours. After the reaction was completed, the reaction solution was extracted three times with 50 mL of dichloromethane. The combined organic phases were washed three times with saturated brine and dried over anhydrous sodium sulfate. The organic solvent was removed by vacuum distillation. The crude product was purified by silica gel column chromatography with petroleum ether:ethyl acetate = 2:1 (volume ratio) as the eluent, yielding 195 mg of solid ( Z 3-(4-bromophenyl)-2-(4-(dimethylamino)phenyl)acrylonitrile (Ⅳ-1), yield: 60%.

[0054] The reaction formula for this step is as follows: ; (3) Under nitrogen protection, 325 mg of the first-step product (Ⅳ-1, 1.0 mmol) and 700 mg of the intermediate product (Ⅸ, 1.2 mmol) were refluxed in acetonitrile for 48–96 hours. After the reaction was completed, the reaction solution was extracted three times with 50 mL of dichloromethane. The combined organic phases were washed three times with saturated brine and dried over anhydrous sodium sulfate. The organic solvent was removed by vacuum distillation. The crude product was purified by silica gel column chromatography with dichloromethane:methanol = 50:1 (v / v) as the eluent, yielding 325 mg of solid as the final product, with a yield of 36%.

[0055] The proton NMR spectral characterization data of compound I-1 are as follows: 1 H NMR (400 MHz, DMSO) delta 9.11 (dd, J = 13.5, 6.9 Hz, 2H), 9.11 (dd, J = 13.5, 6.9 Hz, 2H), 8.91 (d, J = 8.2 Hz, 2H), 8.91 (d, J = 8.2 Hz, 2H), 8.56 (t, J = 8.8 Hz, 2H), 8.23 ​​– 8.14 (m, 2H), 8.07 (s,1H), 7.97 – 7.92 (m, 3H), 7.86 – 7.82 (m, 1H), 7.66 (ddd, J = 18.6, 15.2, 7.5Hz, 3H), 7.55 (t, J = 7.6 Hz, 1H), 7.42 – 7.33 (m, 2H), 7.11 (t, J = 8.4 Hz, 3H), 6.96 (d, J = 8.0 Hz, 1H), 6.84 (t, J = 7.5 Hz, 2H), 6.70 (d, J = 9.1 Hz, 1H), 4.61(t, J = 7.2 Hz, 2H), 4.11 (q, J = 5.3 Hz, 4H), 3.88 (t, J = 6.0 Hz, 2H), 3.06 (s,4H), 2.05 (dd, J= 14.2, 7.1 Hz, 2H), 1.74 – 1.62 (m, 2H), 1.50 – 0.98 (m, 4H). The reaction formula for this step is as follows: .

[0056] Example 2 ( Z )-4-(4-(2-cyano-2-(4'-(dimethylamino)-[1,1'-biphenyl]-4-yl)vinyl)phenyl)-1-(4-(4-(14-phenyldibenzo[ a , c ]Phenomazine-9 (14 H )-yl)phenoxy)butyl)pyridine-1-onium(I-2): Repeat Example 1, except that 4-(dimethylamino)benzaldehyde (Ⅲ-1) in step (1) above is replaced with 4'-(dimethylamino)-[1,1'-biphenyl]-4-carboxaldehyde (Ⅲ-2), while other conditions remain unchanged.

[0057] The structure is as follows: .

[0058] Example 3 ( E )-4-(4-(2-cyano-2-(5-(4-(dimethylamino)phenyl)thiophene-2-yl)vinyl)phenyl)-1-(4-(4-(14-phenyldibenzo[ a , c ]Phenomazine-9 (14 H )yl)phenoxy)butyl)pyridine-1-onium (I-3): The difference in repeating Example 1 is that 4-(dimethylamino)benzaldehyde (Ⅲ-1) in step (1) above is replaced with 5-(4-(dimethylamino)phenyl)thiophene-2-carboxaldehyde (Ⅲ-3), while other conditions remain unchanged.

[0059] The structure is as follows: .

[0060] Example 4: Evaluation of photophysical properties and the capacity of photoactive oxygen species (1) Photophysical properties study The photosensitizer I-1 generated in Example 1 will be used as an example for illustration. The research on other products is the same as in this example and will not be repeated.

[0061] The synthesized photosensitizers were dissolved in DMSO solution to prepare solutions with a concentration of 10. –3 The mother liquor of M is then diluted to 10.–5 The solution of M was used to test the changes in ultraviolet absorption spectra and fluorescence emission spectra in DMSO / glycerol mixed solvents with different solvent volume ratios. For example... Figure 1 As shown in the UV-Vis absorption spectrum, I-1 has a broad absorption band, with the maximum absorption peak located at 450 nm. Figure 1 As shown, the emission spectrum of I-1 is distributed between 550 and 800 nm, with a maximum emission wavelength around 680 nm, falling within the near-infrared luminescence range. The fluorescence emission peak is weak in DMSO. However, with the gradual addition of glycerol, as the glycerol proportion increases, the probe gradually forms aggregates, restricting intramolecular motion, effectively blocking non-radiative channels and activating their radiative transitions, resulting in a significant increase in the fluorescence intensity of the photosensitizer. Figure 2 As shown, when the glycerol content reaches 99%, the maximum emission wavelength of the photosensitizer is located at 680 nm. The results indicate that the fluorescent molecules all exhibit typical AIE characteristics. Furthermore, the generated fluorescent molecules have a Stokes shift exceeding 200 nm, avoiding interference from excitation light and self-absorption of emitted light during biomedical imaging.

[0062] (2) The ability of photoactive oxygen species The photosensitizer I-1 generated in Example 1 will be used as an example for illustration. The research on other products is the same as in this example and will not be repeated.

[0063] To evaluate the ability of the generated photosensitizer to generate ROS under light irradiation, the commercially available 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) was used as an indicator, and the widely used photosensitizer dihydroporphyrin E6 (Ce6) was selected as a standard reference to detect the ROS generation efficiency. When the photosensitizer was present, a concentration of 25 mW / cm² was used. 2 After 10 minutes of white light irradiation, the fluorescence signal of DCFH-DA at 525 nm showed a rapid upward trend. The faster the fluorescence signal of DCFH-DA decreased, the faster the photosensitizer generated ROS. Figure 3 As shown in the DCFH-DA up-ratio curve, it is evident that the ROS generation rate of the I-1 photosensitizer is much higher than that of the commercially available photosensitizer dihydroporphyrin E6. This implies that this type of photosensitizer has good therapeutic potential in photodynamic therapy.

[0064] Example 5: Assessment of mitochondrial colocalization ability The photosensitizer I-1 generated in Example 1 will be used as an example for illustration. The research on other products is the same as in this example and will not be repeated.

[0065] Cervical cancer cells (HeLa) were seeded in confocal culture dishes and incubated overnight in a 37 °C, 5% CO2 incubator. Subsequently, two groups of cells treated with I-1 were co-stained using a commercial mitochondrial probe (Mito-Tracker Red) to determine the enrichment sites of the photosensitizer in the cancer cells. The degree of overlap between the two fluorescence signals was observed using laser confocal microscopy. Figure 4 The results showed that the red fluorescent signal from the photosensitizer fused well with the green signal (false color) from the mitochondrial dye. This indicates their ability to target and image cancer cell mitochondria, which is beneficial for improving PDT efficiency.

[0066] Example 6: Evaluation of the killing effect of photosensitizer on mouse breast cancer cells (4T1) The photosensitizer I-1 generated in Example 1 will be used as an example for illustration. The research on other products is the same as in this example and will not be repeated.

[0067] Cells in the logarithmic growth phase were digested with trypsin, diluted into a cell suspension with complete culture medium, and then subjected to a 1×10⁻⁶ incubator. 4 The samples were seeded at a density of 1 sample per well in 96-well plates and incubated at 37 °C in a 5% CO2 incubator. After 12 h, different concentrations of I-1 were added to achieve final sample concentrations of 0, 1, 5, 10, 20, 50, and 100 μmol / L. The plates were then incubated for 8 h, followed by white light irradiation (100 mW / cm²). 2 For 30 minutes, a dark toxicity study was conducted simultaneously in the experimental group under the same conditions but without light exposure. After culturing for another 12 hours, the medium containing the photosensitizer was removed, and the cells were then cultured in fresh MTT medium in the dark for 3 hours. The absorbance (OD) at 450 nm was then measured using a microplate reader. The corresponding cell viability was calculated using the following formula: Cell viability (%) = (OD) / (OD) 样品 –OD 背景 ) / (OD 对照 –OD 背景 ) × 100%.

[0068] The cytotoxic effects of different concentrations of photosensitizers on 4TI cells, such as Figure 5 As shown, under light conditions, the cytotoxicity of I-1 against 4TI cells is concentration-dependent. When the concentration of the photosensitizer is as low as 50 μmol / L, the cell viability reaches below 20%, indicating that the prepared photosensitizer exhibits highly efficient photodynamic therapy on 4TI cells.

[0069] Example 7: Evaluation of the viscosity sensitivity of photosensitizers in cervical cancer cells The photosensitizer I-1 generated in Example 1 is used as an example for illustration. The research on other products is the same as this example and will not be repeated.

[0070] (1) Real-time monitoring of mitochondrial viscosity during photosensitizer metabolism Statins (such as nystatin) can be used to disrupt mitochondrial ion homeostasis and alter mitochondrial viscosity. By interfering with mitochondrial ion homeostasis, nystatin (an ion carrier) can induce mitochondrial dysfunction and viscosity changes. Cells were incubated with I-1 (10 μmol / L) at 37 °C for 8 hours, followed by co-incubation with 100 μL of different concentrations (0, 1, 5, 10, 20, 30 μmol / L) of nystatin for 60 minutes. Cells were washed twice with PBS, and fluorescence imaging was recorded using laser confocal microscopy. Figure 6 As shown, the fluorescence signal of I-1 gradually increases with increasing nystatin concentration. This photosensitizer enables real-time monitoring of mitochondrial viscosity during cellular metabolism.

[0071] (2) Real-time monitoring of mitochondrial viscosity by photosensitizers during inflammation Inflammation is a multi-cascade process that leads to increased viscosity. Lipopolysaccharide (LPS), a cell wall component of Gram-negative bacteria, has been shown to trigger cellular inflammation. HeLa cells were incubated with a photosensitizer (10 μmol / L) at 37 °C for 8 hours, followed by co-incubation with 100 μL of different concentrations (0, 1, 50, 100, 200, 300 μmol / L) of astrococcal spores for 60 minutes. Cells were washed twice with PBS, and fluorescence imaging was recorded using laser confocal microscopy. As shown in the figure, the fluorescence signal of I-1 gradually increased with increasing nystatin concentration. This photosensitizer enables real-time monitoring of mitochondrial viscosity during inflammation.

[0072] (3) Real-time monitoring of mitochondrial viscosity by photosensitizers during apoptosis Structural alterations or swelling of mitochondria can lead to inactivation, thereby increasing mitochondrial viscosity. HeLa cells were incubated with a photosensitizer (10 μmol / L) at 37 °C for 8 hours, followed by co-incubation with 100 μL of different concentrations (0, 0.5, 1, 20, 50 μmol / L) of astrococcal spores for 60 minutes. Cells were washed twice with PBS, and fluorescence imaging was recorded using laser confocal microscopy. As shown in the figure, the fluorescence signal of I-1 gradually increased with increasing nystatin concentration. This photosensitizer enables real-time monitoring of mitochondrial viscosity during apoptosis.

[0073] (4) Real-time monitoring of mitochondrial viscosity by photosensitizers during autophagy HeLa cells, a photosensitizer (10 μmol / L), commercial mitochondria, and a commercial lysosomal probe were co-incubated in a 37 ℃, 5% CO2 incubator. Then, rapamycin (10 μmol / L) was added to the cells to induce mitochondrial autophagy. During autophagy, different time points were set: 0, 10, 20, 30, 40, 50, 60, and 90 min. Fluorescence imaging was recorded using a laser confocal microscope at the corresponding time points. As shown in the figure, the fluorescence signal of I-1 gradually increased with time, the fluorescence signal of mitochondria gradually decreased with time, and the fluorescence signal of lysosomes gradually increased with time, indicating that the cell viscosity gradually increased during mitochondrial autophagy.

[0074] Nutritional deficiency may impair mitochondria due to metabolic inhibition, leading to mitophagy. HeLa cells, a photosensitizer (10 μmol / L), commercial mitochondria, and a commercial lysosomal probe were continuously cultured in nutrient-rich normal medium, starvation medium (HBSS), and autophagy-inhibiting medium (HBSS plus 3-methyladenine), respectively. Fluorescence changes were observed under a laser confocal microscope at 0, 30, 60, and 90 minutes. The fluorescence intensity of HeLa cells remained essentially unchanged under normal and autophagy-inhibiting medium conditions; only under starvation medium conditions did the fluorescence gradually increase over time, indicating a gradual increase in viscosity during mitophagy.

[0075] Example 8: Photodynamic therapy of photosensitizers in mice The photosensitizer I-1 generated in Example 1 will be used as an example for illustration. The research on other products is the same as in this example and will not be repeated.

[0076] 4T1 tumor-bearing mice were randomly divided into four groups of eight mice each: a PBS group, a PBS + light therapy group, a probe group, and a probe + light therapy group. Mice were injected with 100 μL of I-1 (5 mg / kg) or PBS solution via tail vein injection. The light therapy group was then treated with white light for 20 minutes. Tumor size and body weight were recorded daily after each treatment. Tumor volume was measured using calipers and calculated according to the formula V = (tumor length × tumor width). 2 ) / 2. To further evaluate the effect of phototherapy, mice were sacrificed 13 days after treatment, and tumor tissues from different treatment groups were removed, and the tumor volume was measured and weighed using calipers.

[0077] The results of photodynamic therapy on tumors in 4 groups of 4T1 tumor-bearing mice are as follows: Figure 7As shown, only the treatment group (probe + light irradiation group) exhibited the effect of inhibiting tumor growth and even tumor ablation. The body weight of mice in different treatment groups did not change significantly, indicating that I-1 has low dark toxicity. The tumor volume in the treatment group was significantly reduced, with some cases showing complete cure. This demonstrates that I-1 has the ability to effectively perform photodynamic therapy (PDT) in complex biological systems.

[0078] Example 9: Real-time monitoring of photosensitizers in in vivo arterial thrombosis SD rats were used to model carotid artery embolism (MCAO) in rats and divided into three groups: one normal rat (injected with the probe), one modeling rat (not injected with the probe), and three modeling rats (injected with the probe). Immediately after modeling, the probe was injected via the tail vein, and behavioral observation was conducted. In vivo imaging was performed at 0, 0.5, 1, 1.5, 2, 3, 4, 5, 6, 9, 12, and 24 hours after probe injection. Figure 8 As shown, no fluorescence signal was observed in the modeling rats (without probe injection) from 0 to 24 hours, while weak non-specific fluorescence appeared in the normal rats (with probe injection) at 12 hours. In the modeling rats (with probe injection), a weak fluorescence signal was clearly observed in the brain region starting at 0.5 hours, gradually increasing in intensity over time, reaching a peak at 12 hours, and decreasing at 24 hours. Non-specific fluorescence appeared at 12 hours. This demonstrates good drug retention and accumulation capabilities in the brain region. These results indicate that I-1 can achieve real-time near-infrared fluorescence imaging of rat cerebral arterial thrombosis.

[0079] Those skilled in the art should understand that the technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments have been described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0080] The above embodiments are merely illustrative of several implementation methods of this application, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of this application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this application should be determined by the appended claims.

Claims

1. A dihydrodibenzophenazine-structured aggregation-induced emission photosensitizer, characterized in that, It has the following general structural formula: ； Where X and Y are any one of C, N, O, and S. m , n , k The numbers are natural numbers between 0 and 3, and not all of them are 0. x R1 and R2 are natural numbers from 1 to 6, and are selected from hydrogen atoms, alkyl groups, or alkoxy groups.

2. The dihydrodibenzophenazine-structure aggregation-induced emission photosensitizer according to claim 1, characterized in that, The structure is any one of the following formulas I-1 to I-36: ； 。 3. A fluorescent imaging developer, characterized in that, This is a dihydrodibenzophenazine-structured aggregation-induced emission photosensitizer, wherein the dihydrodibenzophenazine-structured aggregation-induced emission photosensitizer possesses both aggregation-induced emission and vibration-induced emission mechanisms, and the dihydrodibenzophenazine-structured aggregation-induced emission photosensitizer has the following general structural formula: ； Where X and Y are any one of C, N, O, and S. m , n , k The numbers are natural numbers between 0 and 3, and not all of them are 0. x R1 and R2 are natural numbers from 1 to 6, and are selected from hydrogen atoms, alkyl groups, or alkoxy groups.

4. A blood viscosity detection probe or thrombosis imaging contrast agent, characterized in that, The photosensitizer is a dihydrodibenzophenazine-structured aggregation-induced emission type, wherein the dihydrodibenzophenazine-structured aggregation-induced emission type photosensitizer has viscosity responsiveness, and wherein the dihydrodibenzophenazine-structured aggregation-induced emission type photosensitizer has the following general structural formula: ； Where X and Y are any one of C, N, O, and S. m , n , k The numbers are natural numbers between 0 and 3, and not all of them are 0. x R1 and R2 are natural numbers from 1 to 6, and are selected from hydrogen atoms, alkyl groups, or alkoxy groups.

5. A photodynamic therapy reagent, characterized in that, The photosensitizer is a dihydrodibenzophenazine-structured aggregation-induced emission type, wherein the dihydrodibenzophenazine-structured aggregation-induced emission type photosensitizer is photosensitizing, and wherein the dihydrodibenzophenazine-structured aggregation-induced emission type photosensitizer has the following general structural formula: ； Where X and Y are any one of C, N, O, and S. m , n , k The numbers are natural numbers between 0 and 3, and not all of them are 0. x R1 and R2 are natural numbers from 1 to 6, and are selected from hydrogen atoms, alkyl groups, or alkoxy groups.

6. The photodynamic therapy reagent according to claim 5, characterized in that, Photodynamic therapy reagents are in vivo tumor photodynamic therapy reagents, fluorescence imaging contrast agents, or imaging-guided photodynamic therapy reagents, which can be used for fluorescence diagnosis and phototherapy of tumors.

7. A method for preparing a dihydrodibenzophenazine-structured aggregation-induced emission photosensitizer, characterized in that, Includes the following steps: S1: Under inert gas protection, bromoarylacetonitrile and aryl formaldehyde are added to an organic solvent and an alkaline substance is added as a catalyst. The mixture is refluxed at 80-120 °C to obtain intermediate product IV, wherein the molar ratio of bromoarylacetonitrile to aryl formaldehyde is 1:(1-4). S2: Under inert gas protection, intermediate IV and pyridineboronic acid are dissolved in an alkaline solution of palladium catalyst and refluxed at 80-120 °C to obtain intermediate V, wherein the molar ratio of intermediate IV to pyridineboronic acid is 1:(1.1-1.5). S2: Under inert gas protection, intermediate V is reacted with 9-(4-(4-bromoalkoxy)phenyl)-14-phenyl-9,14-dihydrodibenzo[ a , c The phenazine salt formation reaction yielded the target product I.

8. The method for preparing the dihydrodibenzophenazine aggregate-induced emission photosensitizer according to claim 7, characterized in that, After cooling the target product I to room temperature, it was concentrated under reduced pressure to obtain a crude product. After adding a low-carbon alcohol or a low-carbon ester to the crude product and washing it multiple times, a dihydrodibenzophenazine-structured aggregation-induced emission photosensitizer was obtained.

9. The method for preparing the dihydrodibenzophenazine aggregate-induced emission photosensitizer according to claim 7, characterized in that, Intermediate product V in step S3 reacts with 9-(4-(4-bromoalkoxy)phenyl)-14-phenyl-9,14-dihydrodibenzo[ a , c The molar ratio of phenazine is 1:(1-5), and the organic solvent is selected from 1,4-dioxane, acetonitrile, etc. N , N - One or more of dimethylformamide, dimethyl sulfoxide, acetone or toluene; intermediate V in step S3 is reacted with 9-(4-(4-bromoalkoxy)phenyl)-14-phenyl-9,14-dihydrodibenzo[ a , c The target product I was obtained by dissolving phenazine in an organic solvent and refluxing at 90–150 °C for 12–72 hours.

10. The method for preparing the dihydrodibenzophenazine aggregate-induced emission photosensitizer according to claim 7, characterized in that, In step S3, 9-(4-(4-bromoalkoxy)phenyl)-14-phenyl-9,14-dihydrodibenzo[ a , c The preparation method of phenazine is as follows: A: 9,10-Phenanthroquinone and aniline were dissolved in a solvent, and titanium tetrachloride was added under ice bath conditions. After the reaction was completed at room temperature, the solvent was removed, and a mixed solvent was added to dissolve the mixture. Subsequently, sodium borohydride was added in batches and refluxed. After post-treatment, the desired product was obtained. N , N -Diphenylphenanthrene-9,10-diamine; B: Will N , N 9,10-Diphenylphenanthrene-9,10-diamine, 1-iodo-4-methoxybenzene, potassium carbonate, and copper trifluoromethanesulfonate were dissolved in a solvent, refluxed until complete, and then post-treated to give 9-(4-methoxyphenyl)-14-phenyl-9,14-dihydrodibenzo[ a , c Phenidine; C: 9-(4-methoxyphenyl)-14-phenyl-9,14-dihydrodibenzo[ a , c ]Phenidine was dissolved in a solvent and boron tribromide was added under ice bath conditions. After the reaction was completed at room temperature, the solvent was removed and post-treatment was performed to obtain 4-(1,4-phenyldibenzo[ a , c ]Phenomazine-9 (14 H )-yl)phenol; D: 4-(14-phenyldibenzo[ a , c ]Phenomazine-9 (14 H 9-(4-(4-bromoalkoxy)phenyl)-14-phenyl-9,14-dihydrodibenzo[] a , c Phenyrazine.

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