Aza-bodipy-based molecules with h-aggregate inducing properties, methods of making and using the same

Abstract

The application provides an Aza-BODIPY-based molecule with H aggregation-induced and a preparation method thereof, the introduction of two planar donors N,N-dimethylaminophenyl on an Aza-BODIPY mother nucleus not only red-shifts the emission wavelength of NBDPH to the near-infrared two region, but also can regulate the aggregate state form of NBDPH in the aggregate state, and NBDPH NPs exhibit H aggregation, the H aggregate can effectively induce the generation of type I ROS, breaks through the limitation of the ability of the molecule itself, and solves the problems of the scarcity of type I photosensitizer molecules with NIR-II emission and the insufficient research on the internal law between the molecular structure / aggregate state and the photo-physical properties from the aggregate state level.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological materials, in particular to an Aza-BODIPY-based molecule with H-aggregation-induced and a preparation method and application thereof. BACKGROUND

[0002] In the diagnosis of cancer, traditional clinical imaging modalities include radionuclide imaging (positron emission tomography and single photon emission computed tomography), computed tomography, thermal imaging, ultrasound and magnetic resonance imaging, the problems of these methods are insufficient specificity and imaging sensitivity, low resolution, which cannot effectively detect early tumors, especially micro-tumors, and are expensive. In addition, surgery and radiotherapy and chemotherapy are the traditional and conventional treatment methods for malignant tumors in current clinical practice, but there are limitations such as high side effects, large trauma, low selectivity, etc., and it is difficult to achieve both tumor eradication and postoperative quality of life, so it is particularly important to develop new tumor diagnosis and treatment methods with clinical application prospects.

[0003] Optical diagnosis and treatment is a new non-invasive diagnosis and treatment mode, which is a new therapy in rapid development, including optical imaging, photodynamic therapy (PDT) and photothermal therapy (PTT). Compared with traditional therapy, light therapy has unique advantages such as low toxicity, high selectivity, minimal invasiveness, and no drug resistance.

[0004] Photodynamic therapy is a method of exposing non-toxic photosensitizers to light of corresponding wavelength (visible light or near-infrared), generating reactive oxygen species (ROS) with cytotoxicity, causing organelle oxidative damage or affecting cell function, leading to tumor cell apoptosis and necrosis. In addition, PDT can cause vascular damage and thrombosis in tumors, block the uptake of nutrients by tumor cells, and then remove the lesion. According to the difference in the generation of ROS, PDT therapy can be divided into type I and type II therapy. In the process of type I PDT, photosensitive molecules in the triplet state react with water molecules or O2 and other substrates through electron transfer reaction and hydrogen abstraction reaction to generate ROS including ·OH, O2· - , H2O2, and then effectively kill tumor cells. Type II PDT transfers the energy of triplet photosensitive molecules to oxygen to generate singlet oxygen ( 1 O2). However, more and more studies in recent years have shown that type I PDT has lower O2 dependence, can recycle O2 through Haber-Weiss, Fenton and other reactions, and is more suitable for photodynamic therapy of solid tumors with hypoxic characteristics.

[0005] Photothermal therapy (PTT) is considered as a promising alternative to traditional cancer treatment due to its non-invasive, low trauma, low toxicity and low drug resistance. The essence of PTT is a photophysical process, in which the photothermal agent is passively targeted to the tumor site by the enhanced permeability and retention (EPR) effect, and generates high heat in situ under light excitation to directly ablate tumor cells. Photoacoustic imaging (PAI) is a new biomedical imaging method that is non-invasive and non-ionizing. When pulsed laser irradiates into biological tissue, it induces thermal expansion of the tumor tissue to generate an ultrasonic signal, which carries the light absorption characteristic information of the tissue and can reconstruct the light absorption distribution image of the tissue. PAI combines the advantages of high-contrast optical imaging and high-resolution ultrasonic imaging, and even at a larger penetration depth, it can achieve superior tomographic imaging quality compared to traditional pure optical and / or pure ultrasonic imaging methods. Among various therapeutic and diagnostic techniques reported so far, PAI combined with PTT has been found to be a powerful light therapy and diagnostic tool for cancer diagnosis and treatment applications. Since both PAI and PTT are based on the principle of photo-thermal activation, contrast agents with high light absorption and high photo-thermal conversion efficiency have been proven to be an ideal choice for PAI / PTT therapeutic and diagnostic applications.

[0006] As a new optical imaging technology, fluorescence imaging (FLI) has the advantages of high sensitivity, high contrast and simple operation, and is one of the indispensable modern biological imaging methods, which has a wide range of applications in biomedical basic research and disease diagnosis and treatment. The fluorescence imaging window is divided into visible light region (400-700 nm), near-infrared region I (NIR-I, 700-1000 nm) and near-infrared region II (NIR-II, 1000-1700 nm). Due to the lower tissue absorption, scattering and autofluorescence effect in the NIR-II spectral region, NIR-II fluorescence imaging has higher signal-to-noise ratio and deeper tissue imaging capability, and has great application prospect in tumor intraoperative navigation.

[0007] Currently, compared with inorganic materials and organic semiconductor conjugated polymers, organic small molecules have the characteristics of fast metabolism, clear structure and other characteristics, which are more suitable for clinical conversion requirements. However, due to the difficulty in accurately regulating the energy dissipation path, it is still difficult to construct an organic small molecule with NIR-II emission and type I photodynamic behavior, and there is a lack of reasonable design strategy, especially in the exploration of the aggregation state. On the one hand, compared with type II photosensitizers, the number of type I photosensitizer molecules reported so far (Small 2021, 2006742; VIEW 2022, 3, 20200121; Acc. Chem. Res. 2022, 55, 3253.) is relatively small, mainly because the design rules of type I photosensitizer are not clear, and the internal relationship between molecular structure and photophysical properties still needs to be further explored. On the other hand, hydrophobic organic photosensitizers need to be further modified for biological diagnosis and treatment, and the properties of the obtained nanoparticles (aggregation state) are often subject to change, and the photophysical properties under the aggregation state are often difficult to regulate; in addition, most of the organic small molecules with NIR-II fluorescence imaging capability are constructed by constructing donor-acceptor-donor (D-A-D) structure to extend their absorption / emission wavelength to NIR-II band. Due to the relatively low photon energy of NIR-II band, the excited state energy of the molecule after absorbing these photons is often insufficient to sensitize oxygen to produce singlet oxygen (the required energy is not less than 0.98 eV), which limits its photodynamic therapy performance.

[0008] Chinese Patent Publication No. CN118745797A discloses a near-infrared two-region type I photosensitizer based on aza-BODIPY and its preparation method and application. The photosensitizer is a triphenylamine derivative group with alkoxy substitution as a donor connected to the aza-BODIPY parent, and the fluorescence wavelength successfully reaches the near-infrared two-region, which can be used for visual imaging of tumor sites. Although the photosensitizer can form nanoparticles by nanoprecipitation method, and under the irradiation of a single 808 nm laser, it can perform near-infrared two-region imaging and guide type I photodynamic / photothermal combined therapy, but its ability to generate type I ROS still needs the corresponding organic small molecule itself to have such ability, which cannot break through the limitation of the molecule itself, resulting in the need to spend a lot of manpower and material resources to obtain a superior molecule. SUMMARY

[0009] The present application aims to overcome the shortcomings of the prior art and provides a novel organic small molecule with NIR-II emission and type I photodynamic behavior. Through molecular structure design and aggregation state regulation, an Aza-BODIPY-based organic small molecule with H aggregation characteristics is synthesized.

[0010] According to the first aspect of the present application, there is provided a H-aggregation induced Aza-BODIPY based molecule, denoted as NBDPH, having a chemical structure as shown in Formula I:

[0011]

[0012] As an optional embodiment, the method comprises the following steps:

[0013] S1. In a nitrogen atmosphere, 4-cyanobenzaldehyde and 4-octyloxyacetophenone are dissolved in a mixed medium of methanol and water, potassium hydroxide is used as an inorganic base, and an aldol condensation reaction is carried out under the required reaction conditions to obtain a chalcone compound;

[0014] S2. In a nitrogen atmosphere, the chalcone compound and nitromethane are placed in a methanol solvent, triethylamine is used as an organic base, and a Michael addition reaction occurs under the required reaction conditions to obtain a first intermediate;

[0015] S3. In a nitrogen atmosphere, the first intermediate and ammonium acetate are dissolved in a n-butanol medium, and a reaction is carried out under the required reaction conditions to obtain a second intermediate;

[0016] S4. In a nitrogen atmosphere, the second intermediate and boron trifluoride etherate are placed in a 1,2-dichloroethane solvent, N,N-diisopropylethylamine is used as an organic base, and a reaction is carried out under the required reaction conditions to obtain a fluoroboration product;

[0017] S5. In a nitrogen atmosphere, the fluoroboration product and N-bromosuccinimide (NBS) are dissolved in a mixed medium of chloroform and acetic acid, and a reaction is carried out under the required reaction conditions to obtain a bromination product;

[0018] S6. In a nitrogen atmosphere, the bromination product and 4-(N,N-dimethylamino)phenylboronic acid pinacol ester are dissolved in a mixed medium of toluene and ethanol, tetrakis(triphenylphosphine)palladium is used as a catalyst, and potassium carbonate is used as an inorganic base, and a reaction is carried out under the required reaction conditions to obtain an Aza-BODIPY based molecule, denoted as NBDPH.

[0019] As an optional embodiment, in step S1, the molar ratio of 4-cyanobenzaldehyde, 4-octyloxyacetophenone, and potassium carbonate is 1:1:(1-2), and the required reaction conditions are to react at a temperature of 30-35°C for 18-24h.

[0020] As an optional embodiment, in step S2, the molar ratio of the chalcone compound, nitromethane, and triethylamine is 1:(2-3):(2-3), and the required reaction conditions are to react at a temperature of 70-75°C for 24-30h.

[0021] As an optional embodiment, in the step S3, the molar ratio of the first intermediate, ammonium acetate is 1:(50-60), and the required reaction condition is to react at a temperature of 120-130℃ for 24-30h.

[0022] As an optional embodiment, in the step S4, the molar ratio of the second intermediate, boron trifluoride ether, N, N-diisopropyl ethylamine is 1:(12-16):(10-12), and the required reaction condition is to react at a temperature of 50-60℃ for 12-16h.

[0023] As an optional embodiment, in the step S5, the molar ratio of the fluoroboration product, NBS is 1:(2-3), and the required reaction condition is to react at a temperature of 30-40℃ for 4-6h.

[0024] As an optional embodiment, in the step S6, the molar ratio of the bromination product, 4-(N, N-dimethylamino) phenylboronic acid pinacol ester, tetrakis triphenyl phosphine palladium, potassium carbonate is 1:(2-4):(0.15-0.45):(6-8), and the required reaction condition is to react at a temperature of 90-100℃ for 16-20h.

[0025] As the second aspect of the object of the present application, the application of the aforementioned Aza-BODIPY-based molecule with H aggregation induction in the preparation of a photodiagnosis and treatment reagent for NIR-I photoacoustic and NIR-II fluorescence imaging mediated tumor photodynamic and photothermal therapy is provided.

[0026] As the third aspect of the object of the present application, a photodiagnosis and treatment reagent with NIR-II emission and type I photodynamic force is also provided, which is prepared by using the aforementioned Aza-BODIPY-based molecule with H aggregation induction.

[0027] The Aza-BODIPY-based molecule NBDPH with H aggregation induction provided by the present application has a large conjugated system, exhibits good NIR-I absorption and NIR-II emission, and the nanomaterial prepared by using the molecule NBDPH and the amphiphilic polymer F127 self-assembly exhibits H aggregation. The aggregate can effectively induce the generation of type I ROS, has excellent O2· - generation ability under 730nm laser excitation and a high photothermal conversion efficiency of 55.4%, can be used for precise and efficient NIR-I photoacoustic / NIR-II fluorescence imaging mediated photodynamic / photothermal combined therapy of tumor tissue, reduces the damage and side effects of tumor treatment, improves the treatment efficiency and accuracy, and has good application prospect in precise diagnosis and treatment of cancer.

[0028] The application proposes a new strategy for preparing type I photosensitizer with NIR-II emission by regulating the aggregation form of the molecule, the introduction of two planar donors N,N-dimethylaminophenyl on the Aza-BODIPY mother nucleus not only red shifts the emission wavelength of NBDPH to the near-infrared two region, but also regulates the aggregate state form of NBDPH under the aggregation state, NBDPH NPs exhibit H aggregation, and the H aggregate can effectively induce type I ROS generation, breaking through the limitation of the ability of the molecule itself, and solving the problems of the scarcity of type I photosensitizer molecules with NIR-II emission and the insufficient research on the internal regularity between molecular structure / aggregate state-photophysical properties from the aggregate state level. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 is a synthetic reaction line diagram of NBDPH of the application.

[0030] Figure 2 is an H-NMR spectrum of NBDPH of the application. 1

[0031] Figure 3 is a C-NMR spectrum of NBDPH of the application. 13

[0032] Figure 4 is a mass spectrum of NBDPH of the application.

[0033] Figure 5 is a dynamic light scattering particle size distribution test diagram of NBDPH nanoparticles of the application.

[0034] Figure 6 is an ultraviolet absorption / fluorescence emission spectrum of NBDPH nanoparticles of the application in water.

[0035] Figure 7 is an H aggregation test diagram of NBDPH nanoparticles of the application.

[0036] Figure 8 is a total ROS production condition diagram of NBDPH nanoparticles of the application.

[0037] Figure 9 is a type I ROS production condition diagram of NBDPH nanoparticles of the application.

[0038] Figure 10 is a photothermal conversion efficiency fitting curve of NBDPH nanoparticles of the application.

[0039] Figure 11 is the relative survival rate of different concentrations of NBDPH nanoparticles incubated with mouse breast cancer cells (4T1) for 24 hours under light or dark conditions.​​

[0040] Figure 12 This is an NIR-I photoacoustic imaging image of NBDPH nanoparticles as an example of the present invention in a mouse model of breast cancer.

[0041] Figure 13 This is an NIR-II fluorescence imaging image of the NBDPH nanoparticles of this invention in a mouse model of breast cancer.

[0042] Figure 14 This is a photothermal imaging image of NBDPH nanoparticles in a breast cancer model mouse tumor site, as an example of this invention.

[0043] Figure 15 This is a graph showing the change in tumor volume in a breast cancer model mouse during 14 days of treatment with NBDPH nanoparticles as described in this invention. Detailed Implementation

[0044] To better understand the technical content of the present invention, specific embodiments are described below in conjunction with the accompanying drawings.

[0045] Various aspects of the invention are described in this disclosure with reference to the accompanying drawings, in which numerous illustrative embodiments are shown. The embodiments of this disclosure are not necessarily intended to encompass all aspects of the invention. It should be understood that the various concepts and embodiments described above, as well as those described below in more detail, can be implemented in any of a number of ways.

[0046] Aza-BODIPY-based molecules with H-aggregation induced

[0047] In an exemplary embodiment of the present invention, an Aza-BODIPY-based molecule with H aggregation induction is provided, denoted as NBDPH, and its chemical structure is shown in Formula I:

[0048]

[0049] The Aza-BODIPY-based molecules in the aforementioned examples have a large conjugated system and exhibit good NIR-I absorption and NIR-II emission.

[0050] Preparation

[0051] Combination Figure 1 As shown, the aforementioned method for preparing Aza-BODIPY-based molecules with H-aggregation induction uses 4-cyanobenzaldehyde and 4-octyloxyacetophenone as starting materials, according to... Figure 1 The reaction is prepared using the reaction route described above.

[0052] In one exemplary embodiment, the method for preparing the aforementioned Aza-BODIPY-based molecule with H aggregation induction includes the following steps:

[0053] 1) Reaction 1: Under a nitrogen atmosphere, 4-cyanobenzaldehyde (compound 1) and 4-octoxyacetophenone (compound 2) are dissolved in methanol / water medium, and potassium hydroxide is used as an inorganic base. Under the required reaction conditions, an aldol condensation reaction is carried out to obtain chalcone compounds (compound 3).

[0054] 2) Reaction 2: Under a nitrogen atmosphere, chalcone compounds and nitromethane are placed in methanol solvent, with triethylamine as the organic base, and Michael addition reaction occurs under the required reaction conditions to give the first intermediate (compound 4).

[0055] 3) Reaction 3: Under a nitrogen atmosphere, compound 4 and ammonium acetate were dissolved in n-butanol medium and reacted under the required reaction conditions to obtain the second intermediate (compound 5).

[0056] 4) Reaction 4: Under a nitrogen atmosphere, compound 5 and boron trifluoride diethyl ether were placed in 1,2-dichloroethane solvent, with N,N-diisopropylethylamine as the organic base, and the reaction was carried out under the required reaction conditions to obtain the fluorinated boron product (compound 6).

[0057] 5) Reaction 5: Under a nitrogen atmosphere, the fluorinated borate product and N-bromosuccinimide (NBS) are dissolved in a mixed medium of chloroform and acetic acid, and the reaction is carried out under the required reaction conditions to obtain the brominated product (compound 7).

[0058] 6) Reaction 6: Under a nitrogen atmosphere, the brominated product and 4-(N,N-dimethylamino)phenylboronic acid pinacol ester were dissolved in a mixed medium of toluene and ethanol, with tetrakis(triphenylphosphine)palladium as the catalyst and potassium carbonate as the inorganic base. The reaction was carried out under the required reaction conditions to obtain Aza-BODIPY-based organic small molecule NBDPH.

[0059] As an optional example, the molar ratio of 4-cyanobenzaldehyde, 4-octyloxyacetophenone, and potassium carbonate is 1:1:(1-2), and the required reaction conditions are to react at a temperature of 30℃-35℃ for 18h-24h.

[0060] As an optional example, the molar ratio of chalcone compounds, nitromethane, and triethylamine is 1:(2-3):(2-3), and the required reaction conditions are a reaction at 70℃-75℃ for 24-30 hours.

[0061] As an optional example, the molar ratio of the first intermediate to ammonium acetate is 1:(50-60), particularly preferably 1:60, and the required reaction conditions are to react at a temperature of 120℃-130℃ for 24h-30h.

[0062] As an optional example, the molar ratio of the second intermediate, boron trifluoride ether, and N,N-diisopropylethylamine is 1:(12-16):(10-12), particularly preferably 1:16:11, and the required reaction conditions are a reaction at 50°C-60°C for 12-16 hours.

[0063] As an optional example, the molar ratio of fluorinated borate product to NBS is 1:(2-3), and the required reaction conditions are to react at a temperature of 30℃-40℃ for 4h-6h.

[0064] As an optional example, the molar ratio of the brominated product, 4-(N,N-dimethylamino)phenylboronic acid pinacol ester, tetrakis(triphenylphosphine)palladium, and potassium carbonate is 1:(2-4):(0.15-0.45):(6-8), and the required reaction conditions are to react at a temperature of 90℃-100℃ for 16-20h.

[0065] In other alternative examples, the aforementioned preparation method further includes:

[0066] After the first reaction was completed, the crude product was filtered and washed with methanol to obtain a white solid chalcone compound.

[0067] After reaction 2 was completed, the solvent in the reaction mixture was removed, and the crude product was purified by column chromatography to obtain a white solid compound 4.

[0068] After reaction 3 was completed, the crude product was washed with ethanol, filtered, and purified by column chromatography to obtain black solid compound 5.

[0069] After reaction four was completed, the solvent in the reaction mixture was removed, and the crude product was purified by column chromatography to obtain a black solid fluorinated borate product 6.

[0070] After reaction 5 is completed, the solvent in the reaction mixture is removed, and the crude product is purified by column chromatography to obtain brown solid brominated product 7.

[0071] After reaction 6 was completed, the solvent in the reaction mixture was removed, and the crude product was purified by column chromatography to obtain black solid NBDPH.

[0072] Application

[0073] In another exemplary embodiment of the present invention, the application of the aforementioned H-aggregation-induced Aza-BODIPY-based organic small molecule is provided in the preparation of phototherapeutic reagents for NIR-I photoacoustic and NIR-II fluorescence imaging-mediated tumor photodynamic and photothermal therapy.

[0074] In another exemplary embodiment of the present invention, a phototherapy reagent with both NIR-II emission and type I photodynamics is also provided, which is prepared using the aforementioned Aza-BODIPY-based molecule with H aggregation induction. The phototherapy reagent is a nanoparticle prepared by self-assembly of NBDPH and the amphiphilic polymer F127, which can effectively generate superoxide anion free radicals under 730nm laser excitation, and its photothermal conversion efficiency is more than 50%.

[0075] The Aza-BODIPY-based molecular structure of this invention is well-defined, the synthesis process is simple, and the cost of the raw materials is low. The nanoparticles prepared from this molecule have uniform particle size, good stability, and excellent NIR-I absorption and NIR-II emission. They can induce the generation of type I ROS through H aggregation, and their photothermal conversion efficiency is higher than that of most organic small molecules reported to date.

[0076] To facilitate better understanding, the present invention will be further illustrated below with several specific examples, but the preparation process is not limited to these examples, and the content of the present invention is not limited to these examples.

[0077] Unless otherwise specified, the materials in the examples are prepared according to existing methods or purchased directly from the market.

[0078] Example 1

[0079] Synthesis of Chalcone Compounds

[0080] In a dry 100 mL double-necked flask, 4-cyanobenzaldehyde (2.484 g, 10 mmol), 4-octyloxyacetophenone (2.482 g, 10 mmol), potassium hydroxide (0.112 g, 2 mmol), and ethanol / water (4:1 v / v, 50 mL) were added. The mixture was stirred at room temperature for 24 h. The crude product was filtered and washed with methanol to obtain a white solid chalcone compound (3.07 g, 85%).

[0081] 1 H NMR (400MHz, CDCl3): δppm 8.02(d,J=8.8Hz,2H,Ar-H),7.76(d,J=15.6Hz,1H,CH-C=O),7.71(d,J=2Hz,4H,Ar-H),7.62(d,J=15.6Hz,1H,Ar-CH=),6.98(d,J=8.8Hz, 2H,Ar-H),4.04(t,J=6.4Hz,2H,OCH2),1.88-1.75(m,2H,CH2),1.51-1.43(m,2H,CH2),1.33-1.22(m,8H,CH2),0.89(t,J=6.4Hz,3H,CH3).

[0082] 13 C NMR (100MHz, CDCl3): δppm 187.84,163.46,141.14,139.44,132.63,130.92,130.22,128.58,124.96,118 .44,114.43,113.19,68.35,31.77,29.29,29.19,29.05,25.95,22.62,14.08.

[0083] The molecular structure of chalcone compounds can be determined to be Formula II.

[0084]

[0085] [Synthesis of Compound 4]

[0086] In a dry 100 mL double-necked flask, chalcone compounds (3.615 g, 10 mmol), nitromethane (3.052 g, 50 mmol), triethylamine (2.530 g, 25 mmol), and methanol (40 mL) were added. The mixture was heated under reflux for 24 h under a nitrogen atmosphere. After the reaction cooled, the reaction mixture was acidified with dilute hydrochloric acid (2 M), extracted with dichloromethane (CH2Cl2), and the solvent was removed by vacuum distillation. The crude product was purified by silica gel elution chromatography with CH2Cl2 / petroleum ether (1:2) to give the white product compound 4 (3.000 g, 71%).

[0087] 1 H NMR (400MHz, CDCl3): δppm 7.85(d,J=8.8Hz,2H,Ar-H),7.58(d,J=8.4Hz,2H,Ar-H),7.41(d,J=8.4Hz,2H,Ar-H),6.89( d,J=8.8Hz,2H,Ar-H),4.85(dd,J=6.0,12.8Hz,1H,CH2-NO2),4.69(dd,J=8.8,13.2Hz,1H,C H2-NO2),4.28-4.24(m,1H,CH),3.99(t,J=6.4Hz,2H,OCH2),3.44-3.33(m,2H,CH2),1.82-1 .74(m,2H,CH2),1.49-1.41(m,2H,CH2),1.35-1.21(m,8H,CH2),0.88(t,J=6.8Hz,3H,CH3).

[0088] 13C NMR (100MHz, CDCl3): δppm 194.31,163.54,144.77,132.56,130.16,128.65,128.38,118.29,114.23,111.5 0,78.72,68.22,40.44,39.15,31.62,29.13,29.03,28.87,25.78,22.48,13.94.

[0089] The molecular structure of compound 4 can be determined to be formula III.

[0090]

[0091] [Synthesis of Compound 5]

[0092] Compound 4 (0.844 g, 2 mmol), ammonium acetate (9.250 g, 120 mmol), and n-butanol (15 mL) were added to a dry 100 mL double-necked flask. The mixture was heated under reflux for 24 h in a nitrogen atmosphere. After the reaction cooled to room temperature, the crude product was filtered and washed with cold ethanol to obtain the purple product compound 5 (0.226 g, 30%).

[0093] 1 H NMR (400MHz, CDCl3): δppm 7.89(d,J=8.4Hz,4H,Ar-H),7.60(d,J=8.8Hz,4H,Ar-H),7.48(d,J=8.4Hz,4H,Ar-H),6.94(s,2H,pyrrole-H),6.92(s,4H,Ar-H), 4.05(t,J=6.8Hz,4H,OCH2),1.89-1.82(m,4H,OCH2),1.55-1.48(m,4H,CH2),1.41-1.31(m,16H,CH2),0.91(t,J=6.4Hz,6H,CH3).

[0094] 13 C NMR (100MHz, CDCl3): δppm 161.38,154.32,149.32,139.02,137.85,131.67,128.84,128.05,123.64,118.96 ,115.28,115.10,110.66,68.39,31.83,29.40,29.26,29.22,26.04,22.67,14.12.

[0095] The molecular structure of compound 5 can be determined to be formula IV.

[0096]

[0097] Synthesis of fluorinated borate products

[0098] Compound 5 (0.378 g, 0.5 mmol), dried 1,2-dichloroethane (20 mL), N,N-diisopropylethylamine (0.711 g, 0.55 mmol), and boron trifluoride ether (1.136 g, 8 mmol) were added to a dry 100 mL double-necked flask. The mixture was stirred at 50 °C for 16 h under a nitrogen atmosphere. After the reaction was cooled to room temperature, the organic layer was collected by dichloromethane extraction. The solvent was removed by vacuum distillation. The crude product was further purified by CH2Cl2 / petroleum ether (1:1) silica gel elution chromatography to obtain the purple product, fluorinated borate 6 (0.201 g, 50%).

[0099] 1 H NMR (400MHz, CDCl3): δppm 8.08(d,J=6.8Hz,4H,Ar-H),8.06(d,J=6.4Hz,4H,Ar-H),7.71(d,J=8.4Hz,4H,Ar-H),7.12(s,2H,pyrrole-H),7.01(d,J=8.8Hz,4H,A r-H),4.05(t,J=6.8Hz,4H,OCH2),1.88-1.76(m,4H,CH2),1.51-1.41(m,4H,CH2),1.38-1.22(m,16H,CH2),0.90(t,J=6.8Hz,6H,CH3).

[0100] 13 C NMR (100MHz, CDCl3): δppm 162.24,158.49,145.23,140.18,136.43,132.15,131.90,129.32,123.02,120.00 ,118.61,114.95,112.32,68.31,31.80,29.31,29.22,29.11,25.99,22.65,14.10.

[0101] To determine the molecular structure of the fluorinated borate product as formula V,

[0102]

[0103] Synthesis of bromide products

[0104] In a dry 100 mL double-necked flask, the fluorinated borate product (160 mg, 0.2 mmol), N-bromosuccinimide (43 mg, 0.24 mmol), and chloroform / acetic acid (3:1 v / v, 24 mL) were added. The mixture was stirred at 30 °C for 5 h under a nitrogen atmosphere. After the reaction was completed, the reaction mixture was extracted with dichloromethane, washed with sodium thiosulfate and sodium bicarbonate solution, and the solvent was removed by vacuum distillation. The crude product was purified by CH2Cl2 / petroleum ether (1:2) silica gel elution chromatography to give brown solid brominated product 7 (120 mg, 63%).

[0105] 1 H NMR (400MHz, CDCl3): δppm 7.89(d,J=8.4Hz,4H,Ar-H),7.76(d,J=8.8Hz,4H,Ar-H),7.73(d,J=8.4Hz,4H,Ar-H),6.98(d,J=9.2Hz,4H,Ar-H),4.01(t ,J=6.8Hz,4H,CH2),1.85-1.75(m,4H,OCH2),1.51-1.41(m,4H,CH2),1.38-1.22(m,16H,CH2),0.89(t,J=6.4Hz,6H,CH3).

[0106] 13 C NMR (100MHz, CDCl3): δppm 161.94,158.19,144.10,140.12,134.98,132.61,131.67,131.03,120.85,118.48 ,114.17,99.89,112.89,68.16,31.79,29.29,29.21,29.12,26.01,22.64,14.10.

[0107] The molecular structure of the bromination product can be determined to be formula VI.

[0108]

[0109] Synthesis of NBDPH

[0110] In a dry 100 mL double-necked flask, the brominated product (96 mg, 0.1 mmol) and 4-(N,N-dimethylamino)phenylboronic acid pinacol ester (100 mg, 0.4 mmol), potassium carbonate (112 mg, 0.8 mmol), tetrakis(triphenylphosphine)palladium (23 mg, 0.02 mmol), and anhydrous toluene / ethanol (3:1, v / v, 20 mL) were added. The mixture was heated to 90 °C under a nitrogen atmosphere and reacted for 20 hours. After cooling to room temperature, the reaction mixture was extracted with dichloromethane, and the solvent was removed by vacuum distillation. The crude product was eluted with CH2Cl2 / petroleum ether (2:1) silica gel to give NBDPH (30 mg, 29%).

[0111] like Figure 2 As shown, 1 H NMR (400MHz, CDCl3): δppm 7.52(s,8H,Ar-H),7.44(d,J=8.8Hz,4H,Ar-H),6.79(d,J=8.8Hz,2H,Ar-H),6.74(d,J=8.8Hz,4H,Ar-H),6.53(d,J=7.6Hz,4H,Ar-H),3.94(t ,J=6.4Hz,4H,OCH2),2.95(s,12H,NCH3),1.81-1.74(m,4H,CH2),1.46-1.41(m,4H,CH2),1.36-1.22(m,16H,CH2),0.89(t,J=6.4Hz,6H,CH3).

[0112] like Figure 3 As shown, 13 C NMR (100MHz, CDCl3): δppm 160.77,158.83,149.51,145.08,137.05,136.16,135.03,132.65,131.31,131.24,122.49,118.96,11 3.80,112.08,111.12,103.14,98.69,67.93,40.27,31.78,29.32,29.20,26.02,22.63,19.00,14.09.

[0113] like Figure 4 As shown, MALDI-TOF mass spectrometry (m / z): [M+H] + Calculated for C 66 H 70 BF2N7O2:1042.5652,found:1042.6089.

[0114] The molecular structure of NBDPH can be determined as Formula I.

[0115]

[0116] Example 2

[0117] [Preparation of NBDPH Nanoparticles]

[0118] NBDPH nanoparticles were prepared via a nanoprecipitation method, as detailed below:

[0119] NBDPH (1 mg) (prepared according to the method of Example 1) and Pluronic F-127 (10 mg) were dissolved in tetrahydrofuran (1 mL) and sonicated for 5 min.

[0120] Then, the solution was quickly injected into deionized water (10 mL) and sonicated for 1 h. After stirring and evaporating to completely remove tetrahydrofuran, it was filtered with a 0.22 μm filter to obtain a clear and transparent dark blue NBDPH nanoparticle solution, which was stored at 4 °C for later use.

[0121] like Figure 5 As shown, the dynamic light scattering particle size distribution test results of NBDPH nanoparticles show that their hydrated particle size is 124.9 nm, which meets the requirements of enhanced permeability and retention (EPR) effect.

[0122] like Figure 6 As shown, NBDPH nanoparticles have a maximum absorption peak of 583 nm in water, with the tail of the absorption peak extending to 1000 nm; they also have a maximum emission value of 1045 nm, with the tail of the emission peak extending to 1350 nm.

[0123] like Figure 7 As shown, NBDPH nanoparticles exhibit different absorption curves in water / tetrahydrofuran systems with different volume ratios. With the increase of water volume fraction, the maximum absorption gradually blue-shifts, which is a typical characteristic of H aggregation. After the surface NBDPH is prepared into nanoparticles, H aggregates are formed.

[0124] Example 3

[0125] [Photodynamic Performance Test of NBDPH Nanoparticles]

[0126] Dichlorodihydrofluorescein (DCFH) and dihydrorhodamine 123 (DHR123) were used as the total ROS and O2· - The probe was used with a 730nm laser (100mW cm⁻¹). -2The NBDPH nanoparticle aqueous solution (20 μg / mL), NBDPH, deionized water and the above indicator probe were irradiated respectively. The fluorescence change of the solution at 525 nm was recorded using a fluorescence spectrometer. The photodynamic properties of the NBDPH nanoparticles were evaluated based on the fluorescence change.

[0127] like Figure 8-9 As shown, under irradiation by a 730 nm laser, NBDPH molecules generate almost no ROS in organic solvents, while after being prepared as nanoparticle aggregates NBDPH NPs, they exhibit significant type I ROS (O2). ·- The formation performance indicates that the formation of H aggregates can promote the formation of ROS, demonstrating that the NBDPH nanoparticle aqueous solution of the present invention exhibits excellent O2... ·- Generation capability.

[0128] Reactive oxygen species (ROS) performance tests showed that NBDPH exists as a single molecule in organic solvents with almost no ROS generation. However, after being prepared into NBDPH nanoparticles (NPs) with aggregated behavior, it exhibited significant type I ROS generation performance. Furthermore, theoretical calculations showed that the excited state energy levels in the aggregated state were effectively regulated, satisfying the ROS generation conditions. Theoretically, it was determined that type I ROS is induced by H aggregation.

[0129] As can be seen from the above, in this invention, H aggregation can effectively regulate the excited state energy levels of molecules, reduce the singlet-triplet excited state gap, and thus improve the intersystem crossing rate. Compared with single molecules, it exhibits the ability to generate ROS from nothing.

[0130] Example 4

[0131] [Photothermal Performance Test of NBDPH Nanoparticles]

[0132] Using a 730nm laser (1W cm) -2 Irradiate NBDPH nanoparticle aqueous solution (1 mL, 50 μg / mL) and deionized water (1 mL), heat for 10 minutes and then cool naturally for 12 minutes to return to room temperature. Calculate the photothermal conversion efficiency of NBDPH nanoparticle aqueous solution using the cooling period.

[0133] like Figure 10 As shown, the photothermal conversion efficiency of the NBDPH nanoparticle aqueous solution of the present invention is 55.4%.

[0134] As can be seen from Examples 2-4, the Aza-BODIPY-based molecules of the present invention possess both NIR-II emission and type I photodynamic behavior.

[0135] Example 5

[0136] [Cytotoxicity assay of NBDPH nanoparticles]

[0137] 4T1 cells were loaded at 5.0 × 10⁶ cells per well. 3 NBDPH nanoparticles were seeded at different densities in two 96-well plates and incubated at 37°C in a dark environment with 5% CO2 for 24 hours. Different concentrations of NBDPH nanoparticles (0, 2.5, 5, 10, 15, 20, 25 μg / mL) were then added to the plates. -1 Add to the well plate and incubate for 12 hours.

[0138] After incubation for 12 hours, the sample was subjected to a 730nm laser (0.8W cm⁻¹) at room temperature. -2 One well of the plate was irradiated for 5 minutes per well, while the other well was incubated in the dark to test the cytotoxicity of NBDPH nanoparticles.

[0139] After illumination, the wells were incubated in darkness for 12 hours. Then, 200 μL of MTT solution was added to each well, and the cells were incubated in darkness for 4 hours. The culture medium was removed, and 200 μL of dimethyl sulfoxide was added to dissolve the blue-purple formazan crystals. The absorbance at 492 nm was read using a microplate reader, and the viability of 4T1 cells under different concentrations of NBDPH nanoparticles was calculated. The cell viability was calculated as: (Average absorbance of the treatment group / Average absorbance of the control group) × 100%.

[0140] like Figure 11 As shown, even under low light conditions, the concentration of NBDPH nanoparticles is as high as 25 μg / mL. -1 The survival rate of 4T1 cells remained above 95%; however, under light conditions, 20 μg / mL -1 The NBDPH nanoparticles can kill nearly 50% of 4T1 cells, indicating that the NBDPH nanoparticles of the present invention have excellent biocompatibility and high photocytotoxicity, and can significantly kill cancer cells under light conditions.

[0141] Example 6

[0142] [NIR-I photoacoustic imaging of NBDPH nanoparticles in mice]

[0143] NBDPH nanoparticles (200 μL, 500 μg mL) were added. -1 The tumor was injected into mice via the tail vein, and photoacoustic images of the tumor site were recorded at different time points (pre, 3, 9, 12, 16, 20, and 24 hours before injection).

[0144] like Figure 12As shown, after intravenous injection of NBDPH nanoparticles into mice, the photoacoustic signal at the tumor site gradually increased over time, reaching a maximum value at 20 hours, and then gradually weakened.

[0145] Example 7

[0146] [NIR-II fluorescence imaging of NBDPH nanoparticles in mice]

[0147] NBDPH nanoparticles (200 μL, 500 μg mL) were added. -1 The tumor was injected into mice via the tail vein, and NIR-II fluorescence images of the tumor site were recorded at different time points (10 min, 2, 5, 8, 15, 20, 24 hours).

[0148] like Figure 13 As shown, after intravenous injection of NBDPH nanoparticles into mice, the fluorescence signal at the tumor site gradually increased over time, reaching a maximum at 20 hours, and then gradually weakened.

[0149] As can be seen from Examples 6 and 7, the NBDPH nanoparticles of the present invention can be effectively enriched at the tumor site, and the optimal treatment window is 20 hours after drug injection.

[0150] Example 8

[0151] [In vivo photothermal imaging of NBDPH nanoparticles in mice]

[0152] NBDPH nanoparticles (100 μL, 500 μg mL) -1 ) or PBS buffer (100 μL) was injected into mice via the tail vein, and photothermal images of the tumor site were recorded at different time points (0, 1, 2, 3, 4, 5 minutes). Figure 14 As shown, under irradiation by a 730nm laser, the tumor temperature of mice in the PBS group only increased to 39.0℃ within five minutes, while the tumor temperature of mice in the NBDPH nanoparticle group rapidly increased to 52℃ within five minutes, indicating that the NBDPH nanoparticles of the present invention can be effectively used for photothermal therapy of mouse tumors.

[0153] Example 9

[0154] [Tumor Therapy Experiment with NBDPH Nanoparticles]

[0155] 4T1 tumor-bearing mouse models were established by subcutaneous injection of 4T1 cancer cells until the tumor volume reached 100 mm. 3 After determining the left and right sides, subsequent experiments can be conducted.

[0156] 4T1 tumor-bearing mice were randomly divided into three groups (PBS+L, NPs, and NPs+L, n=5 per group, where L represents laser irradiation). Mice in the PBS+L group were intravenously injected with PBS (100 μL), while mice in the NPs and NPs+L groups were intravenously injected with NBDPH nanoparticles (100 μL, 500 μg / mL). -1 Twenty hours after injection, the tumor sites of mice in the PBS+L group and NPs+L group were treated with a 730nm laser (0.8W cm⁻¹). -2 Irradiate for 5 minutes. After one treatment, the tumor size is measured every 2 days until day 14.

[0157] like Figure 15 As shown, after one treatment, the tumors in the NPs+L group were completely eliminated on the 4th day after treatment and there was no recurrence within 14 days. In contrast, the tumor volume in the PBS+L group and the NPs group increased by 4 to 5 times on the 14th day after treatment. This indicates that the NBDPH nanoparticles of the present invention have significant anti-tumor effects under laser irradiation, demonstrating their potential for combined type I photodynamic and photothermal therapy.

[0158] The above tests demonstrate that the NBDPH of this invention not only exhibits NIR-II emission but also allows for the regulation of the aggregation state of NBDPH nanoparticles. The NBDPH nanoparticles exhibit H aggregation, which can effectively induce the generation of type I ROS. The phototherapy reagents prepared using NBDPH can be used for precise and efficient NIR-II photoacoustic / photothermal imaging-mediated photothermal therapy of deep tumor tissues, reducing tumor treatment damage and side effects, and improving treatment efficiency and accuracy.

[0159] While the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the invention. Those skilled in the art can make various modifications and refinements without departing from the spirit and scope of the invention. Therefore, the scope of protection of the present invention shall be determined by the claims.

Claims

1. A molecule with H aggregation-induced Aza-BODIPY group, characterized in that, The Aza-BODIPY-based molecule is designated NBDPH, and its chemical structure is shown in Formula I: Formula I.

2. A method for preparing an Aza-BODIPY-based molecule with H aggregation induction as described in claim 1, characterized in that, The preparation method follows the reaction route below: ； Includes the following steps: S1. Under a nitrogen atmosphere, 4-cyanobenzaldehyde and 4-octoxyacetophenone are dissolved in a mixed medium of methanol and water, with potassium hydroxide as an inorganic base. Under the required reaction conditions, an aldol condensation reaction is carried out to obtain chalcone compounds. S2. Under a nitrogen atmosphere, chalcone compounds and nitromethane are placed in methanol solvent, with triethylamine as the organic base, and Michael addition reaction is carried out under the required reaction conditions to give the first intermediate; S3. Under a nitrogen atmosphere, the first intermediate and ammonium acetate are dissolved in n-butanol medium and reacted under the required reaction conditions to obtain the second intermediate. S4. Under a nitrogen atmosphere, the second intermediate and boron trifluoride diethyl ether are placed in 1,2-dichloroethane solvent, with N,N-diisopropylethylamine as the organic base, and reacted under the required reaction conditions to obtain the fluorinated boron product. S5. Under a nitrogen atmosphere, the fluorinated borate product and N 1-Brominated succinimide (NBS) is dissolved in a mixture of chloroform and acetic acid and reacted under the desired reaction conditions to give the brominated product; S6. Under a nitrogen atmosphere, the brominated product and 4-(N,N-dimethylamino)phenylboronic acid pinacol ester were dissolved in a mixed medium of toluene and ethanol, with tetrakis(triphenylphosphine)palladium as the catalyst and potassium carbonate as the inorganic base. The reaction was carried out under the required reaction conditions to obtain the Aza-BODIPY-based molecule, denoted as NBDPH.

3. The preparation method according to claim 2, characterized in that, In step S1, the molar ratio of 4-cyanobenzaldehyde, 4-octoxyacetophenone, and potassium carbonate is 1:1:(1-2), and the required reaction conditions are to react at a temperature of 30 ℃-35 ℃ for 18 h-24 h.

4. The preparation method according to claim 2, characterized in that, In step S2, the molar ratio of chalcone compounds, nitromethane, and triethylamine is 1:(2-3):(2-3), and the required reaction conditions are to react at a temperature of 70 ℃-75 ℃ for 24 h-30 h.

5. The preparation method according to claim 2, characterized in that, In step S3, the molar ratio of the first intermediate to ammonium acetate is 1:(50-60), and the required reaction conditions are to react at a temperature of 120 ℃-130 ℃ for 24 h-30 h.

6. The preparation method according to claim 2, characterized in that, In step S4, the molar ratio of the second intermediate, boron trifluoride ethyl ether, and N,N-diisopropylethylamine is 1:(12-16):(10-12), and the required reaction conditions are to react at a temperature of 50 ℃-60 ℃ for 12 h-16 h.

7. The preparation method according to claim 2, characterized in that, In step S5, the molar ratio of the fluorinated borate product to NBS is 1:(2-3), and the required reaction conditions are to react at a temperature of 30 ℃-40 ℃ for 4 h-6 h.

8. The preparation method according to claim 2, characterized in that, In step S6, the molar ratio of the brominated product, 4-(N,N-dimethylamino)phenylboronic acid pinacol ester, tetrakis(triphenylphosphine)palladium, and potassium carbonate is 1:(2-4):(0.15-0.45):(6-8), and the required reaction conditions are to react at a temperature of 90 ℃-100 ℃ for 16 h-20 h.

9. The use of the H-aggregation-induced Aza-BODIPY-based molecule as described in claim 1 in the preparation of phototherapeutic reagents for NIR-I photoacoustic and NIR-II fluorescence imaging-mediated tumor photodynamic and photothermal therapy.

10. A phototherapeutic agent with both NIR-II emission and type I photodynamics, prepared using the H-aggregation-induced Aza-BODIPY-based molecule as described in claim 1.

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