Aza-BODIPY-based molecule with H aggregation induction as well as preparation method and application of Aza-BODIPY-based molecule

Abstract

The invention provides an Aza-BODIPY-based molecule with H aggregation induction and a preparation method of the Aza-BODIPY-based molecule, through introduction of two planar donors N, N-dimethylaminophenyl on an Aza-BODIPY parent nucleus, the emission wavelength of NBDPH is subjected to red shift to a near-infrared second region, the aggregation state form of NBDPH in an aggregation state can be regulated and controlled, NBDPH NPs shows H aggregation, the H aggregate can effectively induce generation of I-type ROS, and the Aza-BODIPY-based molecule has a good application prospect. The limitation of the capability of molecules is broken through, and the problems that the current I-type photosensitizer with NIR-II emission is scarce in molecules and the internal law research between the molecular structure / aggregation state-photophysical properties is insufficient are solved from the aggregation state level.

Description

Technical Field

[0001] The present invention relates to the technical field of biomaterials, and in particular to an Aza-BODIPY-based molecule with H aggregation induction, and a preparation method and application thereof. Background Art

[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 with these methods are that they lack specificity and imaging sensitivity, have low resolution, and cannot effectively detect early tumors, especially microtumors. They are also expensive. In addition, surgery and radiotherapy and chemotherapy are the traditional conventional treatments for clinical malignant tumors, but they have widespread limitations such as high side effects, large trauma, and low selectivity. It is difficult to both cure the tumor and take into account the patient's postoperative quality of life. Therefore, it is particularly important to develop new methods for tumor diagnosis and treatment with clinical application prospects.

[0003] Phototherapy is an emerging non-invasive treatment mode and a rapidly developing new therapy, including optical imaging, photodynamic therapy (PDT) and photothermal therapy (PTT). Compared with traditional therapies, phototherapy 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 wavelengths (visible light or near-infrared) to produce cytotoxic reactive oxygen species (ROS), which can lead to oxidative damage to organelles or affect cell function, causing apoptosis and necrosis of tumor cells. In addition, PDT can cause vascular damage and thrombosis in tumors, blocking the uptake of nutrients by tumor cells and thus clearing lesions. Based on the differences in the types of ROS generated, 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 will directly react with water molecules or substrates such as O2 through electron transfer reactions and hydrogen extraction reactions to generate ·OH, O2· - , H2O2, and other ROS, which effectively kill tumor cells. Type II PDT transfers the energy of triplet photosensitive molecules to oxygen through energy transfer, generating singlet oxygen ( 1 However, in recent years, more and more studies have shown that type I PDT is less dependent on O2 and can recycle O2 with the help of Haber-Weiss and Fenton reactions, making it more suitable for photodynamic therapy of solid tumors with hypoxic characteristics.

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

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

[0007] At present, compared with inorganic materials and organic semiconductor conjugated polymers, organic small molecules have the characteristics of fast metabolism and clear structure, which are more in line with the requirements of clinical transformation. However, since the energy dissipation pathway is difficult to precisely control, it is still difficult to construct organic small molecules with both NIR-II emission and type I photodynamic behavior. There is a lack of reasonable design strategies, especially in the exploration of the aggregation state level. On the one hand, compared with type II photosensitizers, there are fewer types of type I photosensitizer molecules reported so far (Small2021,2006742; VIEW 2022,3,20200121;Acc.Chem.Res.2022,55,3253.), mainly because the design rules of type I photosensitizers are unclear, and the intrinsic rules between molecular structure and photophysical properties still need 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 resulting nanoparticles (aggregate state) often change, and the photophysical properties in the aggregate state are usually difficult to control; in addition, most organic small molecules with NIR-II fluorescence imaging capabilities extend their absorption / emission wavelengths to the NIR-II band by constructing donor-acceptor-donor (DAD) structures. Since the photon energy in the NIR-II band is relatively low, after absorbing these photons, the excited state energy of the molecules is often insufficient to sensitize oxygen to produce singlet oxygen (the required energy is not less than 0.98 eV), which limits their photodynamic therapy performance.

[0008] The Chinese patent with publication number CN118745797A discloses a near-infrared zone II type I photosensitizer based on aza-BODIPY, and its preparation method and application. The photosensitizer is a triphenylamine derivative group substituted by an alkoxy group as a donor connected to the aza-BODIPY matrix, and the fluorescence wavelength successfully reaches the near-infrared zone II, which can be used for visualization imaging of the tumor site. Although the photosensitizer can be used for near-infrared zone II imaging and guided type I photodynamic / photothermal combined therapy under the irradiation of a single beam of 808nm laser after forming nanoparticles by nanoprecipitation, its ability to generate type I ROS still requires the corresponding organic small molecule itself to have such ability, and cannot break through the limitation of the molecule's own ability, resulting in the need to spend a lot of manpower and material resources to obtain molecules with superior performance. Summary of the invention

[0009] The purpose of the present invention is to address the deficiencies of the prior art and propose a new type of organic small molecule with both 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 invention, there is provided an Aza-BODIPY-based molecule having H aggregation induction, the Aza-BODIPY-based molecule is denoted as NBDPH, and the chemical structure is shown in Formula I:

[0011]

[0012] As an optional implementation, the following steps are included:

[0013] S1. Under 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 desired reaction conditions to obtain a chalcone compound;

[0014] S2. Under a nitrogen atmosphere, placing a chalcone compound and nitromethane in a methanol solvent, using triethylamine as an organic base, and subjecting the chalcone compound to a Michael addition reaction under the desired reaction conditions to obtain a first intermediate;

[0015] S3, under a nitrogen atmosphere, dissolving the first intermediate and ammonium acetate in a n-butanol medium, and reacting under desired reaction conditions to obtain a second intermediate;

[0016] S4, under a nitrogen atmosphere, placing the second intermediate and boron trifluoride ether in 1,2-dichloroethane solvent, using N,N-diisopropylethylamine as an organic base, and reacting under the desired reaction conditions to obtain a fluoroboration product;

[0017] S5. Under a nitrogen atmosphere, dissolving the fluoroboration product and N-bromosuccinimide (NBS) in a mixed medium of chloroform and acetic acid, and reacting under desired reaction conditions to obtain a brominated product;

[0018] S6. Under a nitrogen atmosphere, the bromide product and 4-(N,N-dimethylamino)phenylboronic acid pinacol ester are dissolved in a mixed medium of toluene and ethanol, tetrakistriphenylphosphine palladium is used as a catalyst, potassium carbonate is used as an inorganic base, and the reaction is carried out under the desired reaction conditions to obtain an Aza-BODIPY-based molecule, which is recorded 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°C-35°C for 18h-24h.

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

[0021] As an optional embodiment, 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° C.-130° C. for 24 h-30 h.

[0022] As an optional embodiment, in step S4, the molar ratio of the second intermediate, boron trifluoride etherate, and N,N-diisopropylethylamine is 1:(12-16):(10-12), and the required reaction conditions are to react at a temperature of 50°C-60°C for 12h-16h.

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

[0024] As an optional embodiment, in step S6, the molar ratio of the bromide product, 4-(N,N-dimethylamino)phenylboronic acid pinacol ester, tetrakistriphenylphosphine 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°C-100°C for 16h-20h.

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

[0026] As a third aspect of the present invention, there is also provided a photodiagnostic agent having both NIR-II emission and type I photodynamics, which is prepared using the aforementioned Aza-BODIPY-based molecule with H aggregation induction.

[0027] The Aza-BODIPY-based molecule NBDPH with H aggregation induction proposed by the present invention has a large conjugated system and exhibits good NIR-I absorption and NIR-II emission. The nanomaterial prepared by self-assembly of the molecule NBDPH and the amphiphilic polymer F127 exhibits H aggregation, which can effectively induce the generation of type I ROS and has excellent O2· - With its high photothermal conversion efficiency of 55.4%, it can be used for precise and efficient NIR-I photoacoustic / NIR-II fluorescence imaging-mediated photodynamic / photothermal combined therapy of tumor tissues, reducing tumor treatment damage and side effects, improving treatment efficiency and accuracy, and has good application prospects in precision cancer diagnosis and treatment.

[0028] The present invention proposes a new strategy for preparing a type I photosensitizer with NIR-II emission by regulating the aggregation form of molecules. By introducing two planar donors N,N-dimethylaminophenyl groups into the Aza-BODIPY core, not only the emission wavelength of NBDPH is red-shifted to the near-infrared region II, but also the aggregation form of NBDPH in the aggregation state can be regulated. NBDPH NPs show H aggregation, which can effectively induce the generation of type I ROS, breaking through the limitation of the molecular capacity itself, and solving the problems of the scarcity of type I photosensitizer molecules with NIR-II emission and the insufficient research on the intrinsic laws between molecular structure / aggregation state-photophysical properties from the aggregation state level. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 It is the synthetic reaction scheme diagram of NBDPH of the present invention.

[0030] Figure 2 is the NBDPH of the present invention. 1 H-NMR spectrum.

[0031] Figure 3 is the NBDPH of the present invention. 13 C-NMR spectrum.

[0032] Figure 4 is a mass spectrum of NBDPH exemplified in the present invention.

[0033] Figure 5 It is a dynamic light scattering particle size distribution test chart of the NBDPH nanoparticles exemplified in the present invention.

[0034] Figure 6 is the UV absorption / fluorescence emission spectrum of the NBDPH nanoparticles exemplified in the present invention in water.

[0035] Figure 7 It is the H aggregation test diagram of the NBDPH nanoparticles of the present invention.

[0036] Figure 8 Graph showing total ROS production of NBDPH nanoparticles exemplified in the present invention.

[0037] Fig. 9 Graph showing the generation of type I ROS by the NBDPH nanoparticles exemplified in the present invention.

[0038] Fig.10 It is the light-to-heat conversion efficiency fitting curve of the NBDPH nanoparticles exemplified in the present invention.

[0039] Fig.11 Relative survival rate of mouse breast cancer cells (4T1) incubated with different concentrations of NBDPH nanoparticles for 24 h under light or dark conditions.

[0040] Fig.12 This is the NIR-I photoacoustic imaging image of the NBDPH nanoparticles in the breast cancer model mouse.

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

[0042] Fig.14 This is a photothermal imaging image of the NBDPH nanoparticles exemplified in the present invention at the tumor site of a breast cancer model mouse.

[0043] Fig.15 This is a graph showing the changes in tumor volume during the 14-day treatment of breast cancer model mice using the NBDPH nanoparticles exemplified in the present invention. DETAILED DESCRIPTION

[0044] In order to better understand the technical content of the present invention, specific embodiments are given and described as follows in conjunction with the accompanying drawings.

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

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

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

[0048]

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

[0050] preparation

[0051] Combination Figure 1 As shown, the preparation method of the aforementioned Aza-BODIPY-based molecule with H aggregation induction is to use 4-cyanobenzaldehyde and 4-octyloxyacetophenone as starting materials, according to Figure 1 The reaction route is used for reaction preparation.

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

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

[0054] 2) Reaction 2: Under a nitrogen atmosphere, a 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 desired reaction conditions to obtain a first intermediate (compound 4).

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

[0056] 4) Reaction 4: Under a nitrogen atmosphere, compound 5 and boron trifluoride etherate are placed in 1,2-dichloroethane solvent, N,N-diisopropylethylamine is used as an organic base, and the reaction is carried out under the desired reaction conditions to obtain a fluoroboration product (compound 6).

[0057] 5) Reaction 5: Under a nitrogen atmosphere, the fluoroboration product and N-bromosuccinimide (NBS) are dissolved in a mixed medium of chloroform and acetic acid, and reacted under the desired reaction conditions to obtain a brominated product (Compound 7).

[0058] 6) Reaction six: Under a nitrogen atmosphere, the bromide product and 4-(N,N-dimethylamino)phenylboronic acid pinacol ester are dissolved in a mixed medium of toluene and ethanol, tetrakistriphenylphosphine palladium is used as a catalyst, potassium carbonate is used as an inorganic base, and the reaction is carried out under the desired reaction conditions to obtain an 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° C.-35° C. for 18 h-24 h.

[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 to react at a temperature of 70° C.-75° C. for 24 h-30 h.

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

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

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

[0064] As an optional example, the molar ratio of the bromide product, 4-(N,N-dimethylamino)phenylboronic acid pinacol ester, tetrakistriphenylphosphine 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°C-100°C for 16h-20h.

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

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

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

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

[0069] After reaction 4 is completed, the solvent in the reaction mixture is removed, and the obtained crude product is purified by column chromatography to obtain a black solid fluoroboration product 6.

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

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

[0072] application

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

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

[0075] The Aza-BODIPY-based molecule of the present invention has a clear structure, a simple synthesis process, and a low cost of synthetic raw materials. The nanoparticles prepared from the molecule have uniform particle size, good stability, and excellent NIR-I absorption and NIR-II emission. It can induce the generation of type I ROS in the form of H aggregation, and its photothermal conversion efficiency is higher than that of most organic small molecules currently reported.

[0076] For better understanding, the present invention is further described below in conjunction with several specific examples, but the preparation process is not limited thereto, and the content of the present invention is not limited thereto.

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

[0078] Example 1

[0079] [Synthesis of chalcone compounds]

[0080] In a dry 100mL double-necked flask, add 4-cyanobenzaldehyde (2.484g, 10mmol), 4-octyloxyacetophenone (2.482g, 10mmol), potassium hydroxide (0.112g, 2mmol), ethanol / water (4:1v / v, 50mL), stir at room temperature for 24h, and filter the crude product and wash with methanol to obtain a white solid chalcone compound (3.07g, 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] It can be determined that the molecular structure of chalcone compounds is Formula II,

[0084]

[0085] [Synthesis of Compound 4]

[0086] Into a dry 100mL double-necked flask, chalcone compounds (3.615g, 10mmol), nitromethane (3.052g, 50mmol), triethylamine (2.530g, 25mmol), and methanol (40mL) were added, and the mixture was heated to reflux for 24h under a nitrogen atmosphere. After the reaction was cooled, the reaction mixture was acidified with dilute hydrochloric acid (2M), extracted with dichloromethane (CH2Cl2), and the solvent was removed by distillation under reduced pressure. The crude product was purified by silica gel elution chromatography with CH2Cl2 / petroleum ether (1:2) to obtain a white product, compound 4 (3.000g, 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] It can be determined that the molecular structure of compound 4 is 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 bottle and heated to reflux for 24 h under a nitrogen atmosphere. After the reaction was cooled to room temperature, the crude product was filtered and washed with cold ethanol to obtain a 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] It can be determined that the molecular structure of compound 5 is Formula IV,

[0096]

[0097] [Synthesis of fluoroboration products]

[0098] Compound 5 (0.378 g, 0.5 mmol), dry 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 bottle and 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, and the solvent was removed by distillation under reduced pressure. The crude product was further purified by silica gel elution chromatography with CH2Cl2 / petroleum ether (1:1) to obtain a purple product fluoroboration product 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] The molecular structure of the fluoroboration product is determined to be Formula V,

[0102]

[0103] [Synthesis of brominated products]

[0104] In a dry 100 mL double-necked flask, the fluoroboration 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 and 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 solutions, and the solvent was removed by distillation under reduced pressure. The crude product was purified by silica gel elution chromatography with CH2Cl2 / petroleum ether (1:2) to obtain a brown solid bromide 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 brominated product can be determined to be Formula VI,

[0108]

[0109] [Synthesis of NBDPH]

[0110] In a dry 100mL double-necked bottle, add the brominated product (96mg, 0.1mmol) and 4-(N,N-dimethylamino)phenylboronic acid pinacol ester (100mg, 0.4mmol), potassium carbonate (112mg, 0.8mmol), tetrakistriphenylphosphine palladium (23mg, 0.02mmol), anhydrous toluene / ethanol (3:1, v / v, 20mL), heat to 90°C under a nitrogen atmosphere, react for 20 hours and then cool to room temperature. The reaction mixture is extracted with dichloromethane, and the solvent is removed by distillation under reduced pressure. The crude product is eluted by silica gel chromatography with CH2Cl2 / petroleum ether (2:1) to obtain NBDPH (30mg, 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 spectrum (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 to be Formula I.

[0115]

[0116] Example 2

[0117] [Preparation of NBDPH nanoparticles]

[0118] NBDPH nanoparticles were prepared by nanoprecipitation method as follows:

[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 ultrasonicated for 1 h. After stirring and volatilizing 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 in the dynamic light scattering particle size distribution test results of NBDPH nanoparticles, the hydrated particle size is 124.9nm, 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, and the absorption peak tail extends to 1000 nm; have a maximum emission value of 1045 nm, and the emission peak tail extends to 1350 nm.

[0123] like Figure 7 As shown, NBDPH nanoparticles show 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 feature of H aggregation. The surface NBDPH forms H aggregates after being prepared as nanoparticles.

[0124] Example 3

[0125] [Photodynamic performance test of NBDPH nanoparticles]

[0126] Dichlorodihydrofluorescein (DCFH) and dihydrorhodamine 123 (DHR123) were used as the total ROS, O2 - The probe was illuminated by a 730 nm laser (100 mW cm -2) were irradiated with an aqueous solution of NBDPH nanoparticles (20 μg / mL), a mixed solution of NBDPH, deionized water and the above-mentioned indicator probe, and a fluorescence spectrometer was used to record the fluorescence changes of the solution at 525 nm. The photodynamic properties of the NBDPH nanoparticles were evaluated based on the fluorescence changes.

[0127] like Figure 8-9 As shown in the figure, under the irradiation of 730nm laser, NBDPH molecules in organic solvents generate almost no ROS, but after being prepared into nanoparticle aggregated NBDPH NPs, they show significant type I ROS (O2 ·- ) generation performance, indicating that the formation of H aggregates can promote the generation of ROS, indicating that the NBDPH nanoparticle aqueous solution of the present invention exhibits excellent O2 ·- Generative capacity.

[0128] The reactive oxygen performance test shows that in organic solvents, NBDPH exists in the form of a single molecule, and almost no ROS is generated in the test. However, after being prepared into nanoparticles NBDPH NPs with aggregated behavior, it exhibits significant type I ROS generation performance. At the same time, theoretical calculations show that the excited state energy level in the aggregated state has been effectively regulated to meet the ROS generation conditions, which theoretically determines that type I ROS is induced by H aggregation.

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

[0130] Example 4

[0131] [Photothermal performance test of NBDPH nanoparticles]

[0132] The 730nm laser (1W cm -2 ) were irradiated with an aqueous solution of NBDPH nanoparticles (1 mL, 50 μg / mL) and deionized water (1 mL). The temperature was raised for 10 minutes and then naturally cooled for 12 minutes to return to room temperature. The photothermal conversion efficiency of the aqueous solution of NBDPH nanoparticles was calculated using the cooling section.

[0133] like Fig.10 As shown, the light-to-heat 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 have both NIR-II emission and type I photodynamic behavior.

[0135] Example 5

[0136] [Cytotoxicity experiment of NBDPH nanoparticles]

[0137] 4T1 cells were plated at 5.0 × 10 3 The density of each cell was inoculated in two 96-well cell culture plates and incubated at 37°C in a dark environment with 5% CO2 for 24 hours. -1 ) were added to the well plate and incubated for 12 h.

[0138] After 12 h of incubation, the cells were illuminated with a 730 nm laser (0.8 W cm -2 ) One of the wells was irradiated for 5 min per well, and the other well was incubated in the dark to test the dark cytotoxicity of NBDPH nanoparticles.

[0139] After the illumination is completed, the plate is incubated in the dark for 12 hours, and then 200 μL of MTT solution is added to each well and incubated in the dark for 4 hours. The plate culture medium is removed, 200 μL of dimethyl sulfoxide is added to dissolve the blue-purple formazan crystals, and the absorbance value at 492 nm is read with an ELISA reader to calculate the survival rate of 4T1 cells under different concentrations of NBDPH nanoparticles. The cell survival rate is calculated as follows: average absorbance of the treatment group / average absorbance of the control group × 100%.

[0140] like Fig.11 As shown in Figure 2, under dim light, even at a concentration of 25 μg mL -1 , the survival rate of 4T1 cells can still be maintained above 95%; however, under light conditions, 20 μg mL -1 The NBDPH nanoparticles of the present invention 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 -1 ) was injected into mice through the tail vein, and the 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 Fig.12As shown, after mice were intravenously injected with NBDPH nanoparticles, the photoacoustic signal at the tumor site gradually increased with 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 -1 ) was injected into mice through the tail vein, and the NIR-II fluorescence images of the tumor site were recorded at different time points (10 min, 2, 5, 8, 15, 20, and 24 hours).

[0148] like Fig.13 As shown, after mice were intravenously injected with NBDPH nanoparticles, the fluorescence signal at the tumor site gradually increased with time, reached a maximum at 20 hours, and then gradually weakened.

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

[0150] Example 8

[0151] [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 through the tail vein, and the photothermal images of the tumor site at different time points (0, 1, 2, 3, 4, and 5 minutes) were recorded. Fig.14 As shown, under the irradiation of 730nm laser, the tumor temperature of mice in the PBS group only increased to 39.0°C within five minutes, while the tumor temperature of mice in the NBDPH nanoparticle group quickly increased to 52°C 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 treatment experiment of NBDPH nanoparticles]

[0155] 4T1 cancer cells were injected subcutaneously to establish a 4T1 tumor-bearing mouse model. When the tumor volume reached 100 mm 3 After that, you can carry out subsequent experiments.

[0156] The 4T1 tumor-bearing mice were randomly divided into three groups (PBS+L, NPs, and NPs+L, 5 mice in each group, L represents laser irradiation). The mice in the PBS+L group were intravenously injected with PBS (100 μL), and the mice in the NPs group and NPs+L group were intravenously injected with NBDPH nanoparticles (100 μL, 500 μg mL -1 20 h after injection, the tumor sites of mice in the PBS+L group and NPs+L group were illuminated by 730 nm laser (0.8 W cm -2 ) for 5 minutes. After one treatment, the tumor size was measured every 2 days until the 14th day.

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

[0158] Combined with the above tests, it can be proved that the NBDPH of the present invention not only has NIR-II emission, but also can regulate the aggregation form of NBDPH in the aggregation state. NBDPH nanoparticles show H aggregation, and the H aggregates can effectively induce the generation of type I ROS. The photodiagnostic and therapeutic reagents prepared with NBDPH can be used for accurate 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] Although the present invention has been disclosed as above with preferred embodiments, it is not intended to limit the present invention. A person with ordinary knowledge in the technical field to which the present invention belongs may make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention shall be determined by the definition of the claims.

Claims

1. An Aza-BODIPY-based molecule with H aggregation induction, characterized in that The Aza-BODIPY-based molecule is denoted as NBDPH, and its chemical structure is shown in Formula I:

2. A method for preparing an Aza-BODIPY-based molecule with H aggregation induction as claimed in claim 1, characterized in that: The following steps are involved: S1. Under 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 desired reaction conditions to obtain a chalcone compound; S2. Under a nitrogen atmosphere, placing a chalcone compound and nitromethane in a methanol solvent, using triethylamine as an organic base, and subjecting the chalcone compound to a Michael addition reaction under the desired reaction conditions to obtain a first intermediate; S3, under a nitrogen atmosphere, dissolving the first intermediate and ammonium acetate in a n-butanol medium, and reacting under desired reaction conditions to obtain a second intermediate; S4, under a nitrogen atmosphere, placing the second intermediate and boron trifluoride ether in 1,2-dichloroethane solvent, using N,N-diisopropylethylamine as an organic base, and reacting under the desired reaction conditions to obtain a fluoroboration product; S5. Under a nitrogen atmosphere, dissolving the fluoroboration product and N-bromosuccinimide (NBS) in a mixed medium of chloroform and acetic acid, and reacting under desired reaction conditions to obtain a brominated product; S6. Under a nitrogen atmosphere, the bromide product and 4-(N,N-dimethylamino)phenylboronic acid pinacol ester are dissolved in a mixed medium of toluene and ethanol, tetrakistriphenylphosphine palladium is used as a catalyst, potassium carbonate is used as an inorganic base, and the reaction is carried out under the desired reaction conditions to obtain an Aza-BODIPY-based molecule, which is recorded as NBDPH.

3. The preparation method according to claim 2, characterized in that: In the 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° C.-35° C. for 18 h-24 h.

4. The preparation method according to claim 2, characterized in that: 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° C.-75° C. for 24 h-30 h.

5. The preparation method according to claim 2, characterized in that: In the 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° C.-130° C. 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 etherate and N,N-diisopropylethylamine is 1:(12-16):(10-12), and the required reaction conditions are to react at a temperature of 50° C.-60° C. for 12 h-16 h.

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

8. The preparation method according to claim 2, characterized in that: In step S6, the molar ratio of the bromide product, 4-(N,N-dimethylamino)phenylboronic acid pinacol ester, tetrakistriphenylphosphine 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°C-100°C for 16h-20h.

9. Use of the Aza-BODIPY-based molecule with H aggregation induction according to claim 1 in the preparation of photodiagnostic agents for tumor photodynamic and photothermal therapy mediated by NIR-I photoacoustic and NIR-II fluorescence imaging.

10. A photodiagnostic agent having both NIR-II emission and type I photodynamics, prepared using the Aza-BODIPY-based molecule with H aggregation induction according to claim 1.

Images