A cross-shaped near-infrared fluorescent compound, its preparation method and applications

Abstract

The present invention belongs to the field of pharmaceutical technologies, and discloses a cross-shaped near-infrared fluorescent compound, a preparation method thereof and an application thereof. The cross-shaped near-infrared fluorescent compound has a structural general formula as shown in formula (I): wherein, R is selected from hydrogen or an alkoxy group. The emission wavelength of the cross-shaped near-infrared fluorescent compound is located in the second near-infrared region, has a large Stokes shift, excellent performance in generating type I reactive oxygen species and photothermal conversion, and significant effects in photoacoustic / photothermal imaging and photodynamic / photothermal combined therapy.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to a cross-shaped near-infrared fluorescent compound, a preparation method thereof, and an application thereof. Background Art

[0002] Traditional cancer treatments mainly include surgical operation, chemotherapy, and radiotherapy, but they have disadvantages such as large trauma, strong side effects, and easy generation of drug resistance. In recent years, emerging photodynamic therapy (PDT) and photothermal therapy (PTT) have received extensive attention and research in the field of cancer treatment due to their advantages such as small trauma, low side effects, no drug resistance, and prominent treatment effects (Nat Rev Clin Oncol, 2020, 17: 657-674).

[0003] For PDT, due to the hypoxic characteristics of the tumor microenvironment, the generation of reactive oxygen species (ROS) is inhibited, and ROS has a short lifespan and a small diffusion radius. Precise localization of photosensitizers is required to ensure the treatment effect (J Am Chem Soc, 2014, 136: 11707-11715; ACS Nano, 2020, 14: 854-866). In PTT, the heat effect is often enhanced due to the accumulation of photothermal agents in non-tumor tissues, and heat shock is likely to occur. If the two are used in combination, the heat effect of PTT can accelerate the blood circulation speed, increase the oxygen supply of tumor tissues, and thus improve the PDT treatment effect; conversely, PDT can eliminate heat-resistant tumor cells and improve the PTT treatment effect (Adv Mater, 2020, 32: 2003210). Therefore, the combined use of PDT and PTT is regarded as a pioneering strategy in the field of optical diagnosis and treatment (Biomaterials, 2021, 274: 120892; Adv Funct Mater, 2019, 29: 1901480).

[0004] The design, synthesis, and selection of dual-phototherapy functional materials are the key factors determining the efficacy of photodynamic / photothermal combined therapy. Single-molecule optical diagnosis and treatment systems have great research and application values due to their simple composition, easy preparation, precise structure, good repeatability, and complete functions. However, at present, the development of single-molecule photosensitizers and photothermal agents with definite structures has just started, and the design of their structures, the construction of systems, and clinical application research still face great challenges. Summary of the Invention

[0005] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. For this purpose, the present invention provides a cross-shaped near-infrared fluorescent compound, a preparation method and an application thereof. The emission wavelength of the cross-shaped near-infrared fluorescent compound is in the second near-infrared region, with a large Stokes shift, excellent performance in generating type I reactive oxygen species and photothermal conversion, and remarkable effects in photoacoustic / photothermal imaging and photodynamic / photothermal combined therapy.

[0006] In the first aspect of the present invention, there is provided a cross-shaped near-infrared fluorescent compound with the general structural formula shown in formula (I):

[0007]

[0008] Wherein, R is selected from hydrogen or an alkoxy group.

[0009] Preferably, the Ar is selected from a phenyl group or a thiophene group; the R is selected from one of hydrogen, a methoxy group, an ethoxy group or a propoxy group.

[0010] Preferably, the structural formula of the cross-shaped near-infrared fluorescent compound is as follows:

[0011]

[0012]

[0013] Preferably, the cross-shaped near-infrared fluorescent compound has an A-π-D-π-A structure, with triphenylamine or 4',4'-dimethoxytriphenylamine as the electron donor, 1,3-bis(dicyanomethylene)indan as the electron acceptor, and a benzene ring or thiophene as the π-bridge.

[0014] In the second aspect of the present invention, there is provided a preparation method of the above-mentioned cross-shaped near-infrared fluorescent compound, including the following steps:

[0015] Using triphenylamine or 4',4'-dimethoxytriphenylamine as the parent nucleus, reacting with pinacol 4-formylphenylboronate or pinacol 5-formyl-2-thiopheneboronate through Suzuki coupling reaction, and then reacting with 1,3-bis(dicyanomethylene)indan through Knoevenagel condensation reaction to obtain the cross-shaped near-infrared fluorescent compound.

[0016] Preferably, in the preparation method of the cross-shaped near-infrared fluorescent compound, using triphenylamine or 4',4'-dimethoxytriphenylamine as the parent nucleus, reacting with pinacol 4-formylphenylboronate or pinacol 5-formyl-2-thiopheneboronate through Suzuki coupling reaction to obtain compound A2, A3 or B2; the structural formulas of the compound A2, A3 or B2 are as follows:

[0017]

[0018]

[0019] Preferably, the conditions for the Suzuki coupling reaction include: being carried out in the presence of a catalyst, an inorganic base, an organic solvent, and under an inert condition; the reaction temperature is 80 - 100 °C, and the reaction time is 16 - 72 h.

[0020] Preferably, the catalyst includes at least one of tetrakis(triphenylphosphine)palladium, dichlorobis(1,1'-bis(diphenylphosphino)ferrocene)palladium, and cuprous chloride; the inorganic base includes potassium carbonate and / or cesium carbonate; the organic solvent includes at least one of ethanol, toluene, 1,4-dioxane, and N,N-dimethylformamide.

[0021] Preferably, the Knoevenagel condensation reaction is carried out in a mixed solution of 1,3-bis(dicyanomethylene)indanone and acetic anhydride, the reaction temperature is 60 - 80 °C, and the reaction time is 10 - 15 h.

[0022] The preparation process of the above-mentioned cross-shaped near-infrared fluorescent compound is represented by the following chemical reaction equation:

[0023]

[0024] The third aspect of the present invention provides the application of the above-mentioned cross-shaped near-infrared fluorescent compound in the preparation of photoacoustic imaging agents and photothermal agents.

[0025] The fourth aspect of the present invention provides the application of the above-mentioned cross-shaped near-infrared fluorescent compound in the preparation of drugs for photodynamic therapy, photothermal therapy, and photodynamic / photothermal combined therapy of tumors.

[0026] The fifth aspect of the present invention provides a drug, which includes the cross-shaped near-infrared fluorescent compound described in the present invention.

[0027] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0028] The ultraviolet absorption peaks of the cross-shaped near-infrared fluorescent compound of the present invention in solution are located at 580, 626 (PMY); 581, 629 (MePMY); and 585, 631 nm (TPMY) respectively, and the emission peaks are located at 723, 707, and 839 nm respectively. The emission wavelengths of the solid powder of the cross-shaped near-infrared fluorescent compound of the present invention are in the NIR-II region, and the emission peaks are 968, 1090 (PMY); 981, 1107 (MePMY); and 1036, 1161 nm (TPMY) respectively. In vitro detection confirms that the cross-shaped near-infrared fluorescent compound has the performance of generating type I reactive oxygen species, excellent photothermal conversion, photothermal and photoacoustic imaging effects, good biocompatibility and photostability. In vivo experiments show that the cross-shaped near-infrared fluorescent compound has excellent photodynamic / photothermal combined treatment effects, and a single administration and light irradiation can efficiently inhibit tumor growth and even promote complete ablation of tumors. The cross-shaped near-infrared fluorescent compound has great application prospects in the fields of photoacoustic / photothermal imaging-guided photodynamic / photothermal synergistic treatment of tumors and other diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The present invention will be further described below in conjunction with the drawings and embodiments.

[0030] Figure 1 It is the ultraviolet absorption, fluorescence spectra of the cross-shaped near-infrared fluorescent compound (PMY, MePMY, and TPMY) of the present invention in solution, and the fluorescence spectra of the solid powder.

[0031] Figure 2 It is the DLS and TEM test diagrams of the particle sizes of the nanoparticles of the cross-shaped near-infrared fluorescent compound (PMY, MePMY, and TPMY) of the present invention.

[0032] Figure 3 It is the photoacoustic imaging, photothermal conversion diagrams of the nanoparticles of the cross-shaped near-infrared fluorescent compound (PMY, MePMY, and TPMY) of the present invention, and the fluorescence spectra of the TPMY nanoparticles after white light irradiation tested with DCFH, ABDA, SOSG, and HPF as reactive oxygen indicators respectively.

[0033] Figure 4 It is the schematic diagram of the results of the dark toxicity and phototoxicity tests of the nanoparticles of the cross-shaped near-infrared fluorescent compound (TPMY) of the present invention incubated with MCF-7 cells by the CCK-8 method.

[0034] Figure 5 It is the fluorescence image of the intracellular reactive oxygen generation detected with DCFH-DA as the indicator after the nanoparticles of the cross-shaped near-infrared fluorescent compound (TPMY) of the present invention are incubated with MCF-7 cells.

[0035] Figure 6Schematic diagram of the in vitro tumor volume and weight of each group after treating nude mice with the cross-shaped near-infrared fluorescent compound (TPMY) nanoparticles of the present invention. Detailed implementation mode

[0036] In order to make those skilled in the art more clearly understand the technical solutions described in the present invention, the following examples are listed for illustration. It should be noted that the following examples do not limit the scope of protection required by the present invention.

[0037] The experimental methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents, etc. used are all reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0038] Example 1 Preparation of cross-shaped near-infrared fluorescent compound

[0039] The structural formula of the cross-shaped near-infrared fluorescent compound prepared in this example is:

[0040]

[0041] The specific preparation process of the above cross-shaped near-infrared fluorescent compound PMY includes the following steps:

[0042] 1. Synthesis of 2',5'-dibromo-[1,1',4',1”-triphenyl]-4,4”-dicarbaldehyde (A1)

[0043] In a 250 mL flask, add 4-formylphenylboronic acid pinacol ester (2.39 g, 10.3 mmol), 1,4-dibromo-2,5-diiodobenzene (2.0 g, 4.1 mmol), tetrakis(triphenylphosphine)palladium(0) (0.12 g, 0.104 mmol), K2CO3 (2.0 g, 14.49 mmol), 20 mL of ethanol, 60 mL of toluene, and 0.5 mL of deionized water. After deoxygenating by passing N2 for 30 min, react at 95 °C for 72 h under N2 protection. After the reaction is completed, remove the solvent, add water for washing, extract with dichloromethane, and dry with anhydrous magnesium sulfate. Remove the solvent by vacuum distillation, and purify the residue through dichloromethane / petroleum ether (v / v = 1:1) to finally obtain product A1 as a white solid (0.57 g, yield 31.3%). The characterization results of the 1H NMR spectrum of product A1 are as follows: 1 H NMR(400MHz,CDCl3):δ=10.11(s,2H),7.99(d,J=8.1Hz,4H),7.68-7.62(m,6H). 13 C NMR(100MHz,CDCl3):δ=191.61,144.98,142.37,135.88,135.07,129.99,129.54,121.10.

[0044] Synthesis of 2,2',5'-bis(4-(diphenylamino)phenyl)-[1,1':4',1”-terphenyl]-4,4”-dicarbaldehyde (A2)

[0045] In a 50 mL flask, A1 (0.2 g, 0.45 mmol), 4-borotriphenylamine (0.39 g, 1.35 mmol), tetrakis(triphenylphosphine)palladium (0.02 g, 0.017 mmol), K2CO3 (0.276 g, 2.0 mmol), 15 mL of 1,4-dioxane and 1 mL of deionized water were added. After deoxygenation by purging with N2 for 30 min, the reaction was carried out at 95 °C for 24 h under N2 protection. After the reaction was completed, the solvent was removed, washed with water, extracted with dichloromethane, and dried over anhydrous magnesium sulfate. The solvent was removed by distillation under reduced pressure, and the residue was purified by dichloromethane / petroleum ether (v / v = 1:2) to obtain the product A2 as a yellowish-green powder (0.23 g, yield 66.1%). The 1H NMR characterization results of product A2 were as follows: 1 H NMR(400MHz,CDCl3):δ=10.02(s,2H),7.81(d,J=8.2Hz,4H),7.55(s,2H),7.45(d,J=8.1Hz,4H),7.24(d,J=7.5Hz,6H),7.11-7.00(m,18H),6.94(d,J=8.6Hz,4H). 13 C NMR(100MHz,CDCl3):δ=191.96,147.51,147.43,146.95,139.42,139.05,134.74,133.64,132.54,130.57,130.52,129.42,129.27,124.53,124.37,123.10,122.85.

[0046] 3. Synthesis of compound PMY

[0047] 0.1 mmol of A2, 1,3-bis(dicyanomethylene)indane (0.073 g, 0.3 mmol) and 5 mL of acetic anhydride were added to a Schlenk tube, and the mixture was stirred at 70 °C for 12 h. After the reaction was completed, the reaction solution was poured into saturated NaHCO3 solution for washing, extracted with ethyl acetate, washed with deionized water, dried over anhydrous magnesium sulfate, and the residue after removing the solvent by distillation under reduced pressure was recrystallized with chloroform / n-hexane to obtain the final product PMY.

[0048] The compound PMY prepared from A2 was a blackish-brown powder (0.099 g, yield 81.1%). The 1H NMR characterization results of PMY were as follows: 11H NMR (400 MHz, CDCl3): δ = 8.47 (t, J = 7.0 Hz, 2H), 7.80 (d, J = 8.3 Hz, 2H), 7.64 (d, J = 7.6 Hz, 4H), 7.59 - 7.52 (m, 5H), 7.48 (d, J = 7.9 Hz, 3H), 7.36 (s, 4H), 7.23 (t, J = 7.8 Hz, 8H), 7.18 - 7.03 (m, 12H), 6.99 (m, 8H). 13 13C NMR (100 MHz, CDCl3): δ = 147.60, 139.21, 138.83, 137.47, 137.23, 136.13, 133.41, 131.91, 130.92, 130.87, 130.84, 129.20, 129.17, 129.11, 128.55, 125.76, 124.49, 124.22, 123.16, 122.74.

[0049] Example 2 Preparation of Cross - shaped Near - Infrared Fluorescent Compound

[0050] The structural formula of the cross - shaped near - infrared fluorescent compound obtained in this example is:

[0051]

[0052] The specific preparation process of the above - mentioned cross - shaped near - infrared fluorescent compound MePMY includes the following steps:

[0053] 1. Synthesis of 2',5'-dibromo - [1,1',4',1”-triphenyl]-4,4”-dicarbaldehyde (A1)

[0054] The preparation method of A1 is the same as that in Example 1.

[0055] 2. Synthesis of 2',5'-bis(4-(bis(4 - methoxyphenyl)amino)phenyl)-[1,1':4',1”-triphenyl]-4,4”-dicarbaldehyde (A3)

[0056] In a 50 mL flask, A1 (0.2 g, 0.45 mmol), 4-boronic acid-4',4'-dimethoxytriphenylamine (0.47 g, 1.35 mmol), tetrakis(triphenylphosphine)palladium (0.02 g, 0.017 mmol), K2CO3 (0.276 g, 2.0 mmol), 15 mL of 1,4-dioxane and 1 mL of deionized water were added. After deoxygenation by passing N2 for 30 min, the reaction was carried out at 95 °C for 24 h under N2 protection. After the reaction was completed, the solvent was removed, washed with water, extracted with dichloromethane, and dried over anhydrous magnesium sulfate. The solvent was removed by distillation under reduced pressure, and the residue was purified by dichloromethane / petroleum ether (v / v = 1:2) to obtain product A3 as a yellow powder (0.25 g, yield 62.2%). The 1H NMR characterization results of product A3 were as follows: 1 1H NMR (400 MHz, CDCl3): δ = 10.02 (s, 2H), 7.80 (d, J = 8.3 Hz, 2H), 7.51 (s, 1H), 7.45 - 7.43 (m, 3H), 7.06 - 7.02 (m, 12H), 6.95 (d, J = 8.6 Hz, 2H), 6.83 - 6.76 (dd, J = 18.3, 8.8 Hz, 14H), 3.79 (s, 12H). 13 13C NMR (100 MHz, CDCl3): δ = 192.05, 155.96, 155.64, 147.79, 140.57, 130.49, 130.33, 129.45, 126.75, 126.38, 119.58, 114.68, 114.60, 55.47.

[0057] 3. Synthesis of compound MePMY

[0058] 0.1 mmol of A3, 1,3-bis(dicyanomethylene)indane (0.073 g, 0.3 mmol) and 5 mL of acetic anhydride were added to a Schlenk tube, and the mixture was stirred at 70 °C for 12 h. After the reaction was completed, the reaction solution was poured into saturated NaHCO3 solution for washing, extracted with ethyl acetate, washed with deionized water, dried over anhydrous magnesium sulfate, and the residue was recrystallized with chloroform / n-hexane after removing the solvent under reduced pressure to obtain the final product MePMY.

[0059] MePMY prepared from A3 was a black powder (0.110 g, yield 82.0%). The 1H NMR characterization results of MePMY were as follows: 11H NMR (400 MHz, CDCl3): δ = 7.79 (d, J = 7.8 Hz, 1H), 7.70 (s, 2H), 7.48–7.40 (m, 6H), 7.30 (d, J = 7.9 Hz, 3H), 7.04 (d, J = 8.5 Hz, 10H), 6.95 (t, J = 8.1 Hz, 5H), 6.83 - 6.77 (m, 15H), 3.79 (s, 12H). 13 13C NMR (100 MHz, CDCl3): δ = 156.06, 148.46, 141.98, 140.78, 140.46, 135.14, 130.73, 129.86, 127.00, 126.41, 121.30, 118.80, 114.70, 55.44.

[0060] Example 3 Preparation of Cross - shaped Near - infrared Fluorescent Compound

[0061] The structural formula of the cross - shaped near - infrared fluorescent compound obtained in this example is as follows:

[0062]

[0063] The specific preparation process of the above - mentioned cross - shaped near - infrared fluorescent compound TPMY includes the following steps:

[0064] 1. Synthesis of 2',5'-Dibromo - N4,N4,N4”,N4”-tetrakis(4 - methoxyphenyl)-[1,1':4',1”-terphenyl]-4,4”-diamine (B1)

[0065] In a 100 mL flask, add 4 - boronate - 4',4'-dimethoxytriphenylamine (2.15 g, 5.0 mmol), 1,4 - dibromo - 2,5 - diiodobenzene (0.98 g, 2.0 mmol), tetrakis(triphenylphosphine)palladium (0.12 g, 0.104 mmol), K2CO3 (1.0 g, 7.25 mmol), 5 mL of ethanol, 20 mL of toluene and 0.5 mL of deionized water. After deoxygenating with N2 for 30 min, react at 80 °C for 36 h under N2 protection. After the reaction is completed, remove the solvent, add water for washing, extract with dichloromethane, and dry with anhydrous magnesium sulfate. Remove the solvent by vacuum distillation, and purify the residue by dichloromethane / petroleum ether (v / v = 2:3) to obtain the product B1 as a white solid (0.37 g, yield 22.0%). The 1H NMR characterization results of the product B1 are as follows: 11H NMR (400 MHz, CDCl3): δ = 7.61 (s, 2H), 7.24 (d, J = 8.6 Hz, 4H), 7.13 (d, J = 8.8 Hz, 8H), 6.95 (d, J = 8.6 Hz, 4H), 6.86 (d, J = 8.8 Hz, 8H), 3.81 (s, 12H). 13 13C NMR (100 MHz, CDCl3): δ = 156.07, 148.47, 141.99, 140.47, 135.14, 130.75, 129.86, 127.01, 121.31, 118.82, 114.71, 55.44.

[0066] Synthesis of 2.5,5'-(4,4”-bis(bis(4-methoxyphenyl)amino)-[1,1':4',1”-terphenyl]-2',5'-yl)-bis(thiophene-dicarboxaldehyde) (B2)

[0067] In a 25 mL flask, B1 (0.20 g, 0.24 mmol), 5-formyl-2-thiopheneboronic acid pinacol ester (0.17 g, 0.72 mmol), Cs2CO3 (0.31 g, 0.96 mmol), CuCl (0.024 g, 0.24 mmol), [1,1'-bis(diphenylphosphino)ferrocene] palladium dichloride (0.018 g, 0.024 mmol), and 5 mL of DMF were added. After deoxygenation by purging with N2 for 30 min, the reaction was carried out at 100 °C for 16 h. After the reaction solution was cooled to room temperature, it was extracted with ethyl acetate (25 mL × 4), washed twice with 50 mL of water, and then the organic phase was concentrated and purified by petroleum ether / dichloromethane (v / v = 3:2). The product B2 was obtained as an orange powder (0.10 g, yield 46.0%). The 1H NMR characterization results of the product B2 are as follows: 1 1H NMR (400 MHz, CDCl3): δ = 9.85 (s, 2H), 7.59 (s, 4H), 7.10 - 7.04 (dd, J = 17.0, 8.8 Hz, 12H), 6.99 (d, J = 3.9 Hz, 2H), 6.89 - 6.84 (m, 12H), 3.80 (s, 12H). 13 13C NMR (100 MHz, CDCl3): δ = 182.82, 156.03, 148.61, 143.50, 140.55, 139.97, 136.21, 132.70, 130.75, 130.17, 128.22, 126.86, 119.82, 114.74, 55.48.

[0068] 3. Synthesis of compound TPMY

[0069] Add 0.1 mmol of B2, 1,3-bis(dicyanomethylene)indan (0.073 g, 0.3 mmol), and 5 mL of acetic anhydride into a Schlenk tube. Stir the mixture at 70 °C for 12 h. After the reaction is completed, pour the reaction solution into saturated NaHCO3 solution for washing, extract with ethyl acetate, wash with deionized water, dry over anhydrous magnesium sulfate, remove the solvent by distillation under reduced pressure, and recrystallize the residue with chloroform / n-hexane to obtain the final product TPMY.

[0070] The TPMY prepared from B2 is a black powder (0.116 g, yield 85.7%). The characterization results of the 1H NMR spectrum of TPMY are as follows: 1 1H NMR (400 MHz, CDCl3): δ = 8.65 - 8.63 (m, 2H), 8.56 (s, 2H), 8.52 - 8.50 (m, 2H), 7.81 - 7.79 (m, 4H), 7.59 (s, 2H), 7.48 (d, J = 4.1 Hz, 2H), 7.09 (d, J = 8.7 Hz, 12H), 6.99 (d, J = 4.1 Hz, 2H), 6.90 (d, J = 8.5 Hz, 4H), 6.84 (d, J = 8.9 Hz, 8H), 3.80 (s, 12H). 13 13C NMR (100 MHz, CDCl3): δ = 161.27, 160.30, 156.21, 154.98, 140.25, 137.15, 135.58, 134.97, 130.15, 127.12, 119.32, 114.78, 55.49.

[0071] The above detection results confirm that the prepared compound is the cross-shaped near-infrared fluorescent compound shown in formula (I).

[0072] Example 4 UV absorption, fluorescence spectra in solution, and solid fluorescence spectra of the cross-shaped near-infrared fluorescent compound

[0073] Take an appropriate amount of the cross-shaped near-infrared fluorescent compound and prepare a DMSO solution with a concentration of 10 μM. Use a Metash UV-6000 ultraviolet absorption spectrometer to measure the ultraviolet absorption spectrum of the solution, and an Edinburgh FLS 980 steady-state / transient fluorescence spectrometer to measure the fluorescence spectrum of the solution. The results are as shown in Figure 1 (a). The positions of the maximum ultraviolet absorption peaks of PMY, MePMY, and TPMY in solution are located at 580, 581, and 585 nm respectively, and the emission peak positions are at 723, 707, and 839 nm respectively.

[0074] An appropriate amount of the cross-shaped near-infrared fluorescent compound solid powder was placed in a quartz chip groove, and the fluorescence spectrum of the compound solid powder was measured using an Edinburgh FLS 980 steady-state / transient fluorescence spectrometer. The results are as Figure 1 shown in (b). The maximum emission peaks of PMY, MePMY, and TPMY are located at 968, 981, and 1036 nm respectively, belonging to the second near-infrared region.

[0075] Example 5 Preparation of Cross-Shaped Near-Infrared Fluorescent Compound Nanoparticles

[0076] Accurately weigh 10 mg each of PMY, MePMY, and TPMY, and dissolve 150 mg of Pluronic F-127 in 3 mL of THF to obtain a homogeneous mixture. Then, the mixture was dropped into 20 mL of Milli-Q water and sonicated for 15 min to obtain an aqueous solution of nanoparticles. The aqueous solution of nanoparticles was stirred overnight at room temperature to remove THF, and then unbound F-127 was removed by dialysis. Finally, the solution was filtered through a 0.22 μm filter membrane, freeze-dried, and stored in a sealed container at 4 °C for subsequent experiments. The particle size distributions of the prepared PMY, MePMY, and TPMY nanoparticles (NPs) are as Figure 2 shown.

[0077] Example 6 UV Absorption Spectrum, Fluorescence Spectrum, and Photostability Study of TPMY Nanoparticles

[0078] Prepare an aqueous solution of TPMY nanoparticles with a concentration of 100 μg / mL. Take 2 mL of the aqueous solution of nanoparticles and add it to a cuvette. Irradiate it with a white light xenon lamp (400 - 700 nm, 200 mW / cm 2 ) for 10 min, and use a Metash UV-6000 spectrometer to measure the UV absorption spectrum of the solution before and after irradiation. The results show that after strong light irradiation, the compound hardly degrades and has good photostability.

[0079] Example 7 Evaluation of the Photoacoustic Imaging Performance of Cross-Shaped Near-Infrared Fluorescent Compounds

[0080] Prepare aqueous solutions of PMY, MePMY, and TPMY nanoparticles with different concentrations (25, 50, 100 μg / mL), and use an iTheraMedical MSOT inVision 128 photoacoustic tomography system to detect photoacoustic signals. The results are as Figure 3 shown in (a). When the concentration is 100 μg / mL, all three types of nanoparticles have photoacoustic signals, and the signal of TPMY nanoparticles is the strongest; as the concentration decreases, the photoacoustic signals of the nanoparticles gradually weaken. When the concentration is low (25 μg / mL), TPMY nanoparticles can still detect high-intensity photoacoustic signals and have good photoacoustic imaging ability.

[0081] Example 8 Evaluation of Photothermal Conversion Performance of Cross-Shaped Near-Infrared Fluorescent Compounds

[0082] Prepare aqueous solutions of PMY, MePMY, and TPMY nanoparticles with a concentration of 100 μg / mL, and irradiate them with a 660 nm laser (0.8 W / cm 2 ) for 10 min, and record the temperature change every 30 s, as shown in Figure 3 (b). After 6 min of light irradiation, the temperatures of the PMY, MePMY, and TPMY nanoparticle solutions increased to 45.5, 61.9, and 67.5 °C, respectively. It can be seen from the experimental results that MePMY and TPMY nanoparticles have great potential as photothermal agents.

[0083] Example 9 Test of Reactive Oxygen Species Generation Performance of PMY, MePMY, and TPMY Nanoparticles

[0084] Using dihydrofluorescein (DCFH), 9,10-anthracene-bis(methylene)dimalonic acid (ABDA), singlet oxygen green fluorescent probe (SOSG), and hydroxyphenyl fluorescein (HPF) as ROS indicators respectively, prepare DMSO solutions with a concentration of 1 mM. At the same time, prepare PMY (or MePMY, TPMY, RB) nanoparticle solutions with a concentration of 1 mM; among them, commercial rose bengal (RB) is used as a control. Pipette 10 μL of the indicator DMSO solution and 2 μL of the PMY (or MePMY, TPMY, RB) nanoparticle solution and dilute them to 2 mL of ultrapure water to obtain a mixed solution with an indicator concentration of 5 μM and a nanoparticle concentration of 1 μM. Use a white light xenon lamp (400 - 700 nm, 50 mW / cm 2 ) to irradiate the mixed solution, and measure its fluorescence intensity every 30 s. The results are shown in Figure 3 (c - f). It can be seen from the figure that PMY, MePMY, and TPMY nanoparticles have excellent type I reactive oxygen species generation performance.

[0085] Example 10 Cytotoxicity Evaluation of TPMY Nanoparticles

[0086] Take MCF-7 cells in the logarithmic growth phase, digest and count the MCF-7 cells, and add 100 μL to each well in a 96-well plate, with a total of 3×10 4A cell suspension of MCF-7 cells was placed in an incubator at 37 °C and 5% CO2 for 24 h. The medium was aspirated, and a medium containing TPMY nanoparticles at different concentrations (0, 1.56, 3.13, 6.25, 12.5, 25, 50, 100, 200 μg / mL) was added and cultured for 24 h. After washing with PBS, a medium containing 10% CCK-8 reagent was added to each well. After incubation for 2 h, the absorbance of each well at 450 nm was measured using a microplate reader (Biotek ELX80), and the cell viability was calculated. The calculation method is: Cell viability (%) = (OD 实验 - OD 空白 ) / OD 对照 × 100%.

[0087] The method for detecting the phototoxicity of TPMY nanoparticles was similar to the above. After culturing MCF-7 cells for 24 h, a medium containing TPMY nanoparticles at different concentrations (0, 1.56, 3.13, 6.25, 12.5, 25, 50, 100, 200 μg / mL) was added. After incubation for 6 h, it was irradiated with a white light xenon lamp (400 - 700 nm, 50 mW / cm 2 ) for 10 min, or irradiated with a 660 nm laser (0.5 W / cm 2 ) for 10 min, or first treated with white light for 5 min and then laser irradiated for 5 min, and then placed back in the incubator for continued culture for 18 h. Then, the cell viability was detected by the above method, and the experimental results are as Figure 4 shown. In the absence of light (Dark), the cell viability at different concentrations was above 90%, indicating that TPMY nanoparticles had almost no cytotoxicity; under white light (WL) or 660 nm laser (L) irradiation, the cell viability gradually decreased with the increase of the compound concentration. Especially when treated with white light and laser together, the cell viability could rapidly drop to <10%. The results showed that TPMY nanoparticles could efficiently kill tumor cells through the synergistic effect of photodynamic / photothermal under light irradiation conditions.

[0088] Example 11 Test on the performance of intracellular reactive oxygen species generation of TPMY nanoparticles

[0089] The experimental group and the control group were set up. MCF-7 cells in the logarithmic growth phase were taken, the MCF-7 cells were digested and counted, and 2 mL was added to a 6-well plate for a total of 5 × 10 5A cell suspension of MCF-7 cells was placed in an incubator at 37°C and 5% CO2 for 24 h. The medium was aspirated, and a medium containing TPMY nanoparticles (25 μg / mL) was added. After co-culturing for 4 h, the drug-containing medium was aspirated, and the cells were washed 3 times with PBS. A basal medium containing the reactive oxygen species indicator 2,7-dichlorofluorescein diacetate (DCFH-DA) (10 μM) was added, and the cells were incubated in the dark for 30 min and then washed 3 times with PBS. 1 mL of complete medium was added to moisten the cells. The experimental group was irradiated with white light for 10 min, while the control group was not irradiated. The control groups included the PBS group, the PBS + white light irradiation group, and the nanoparticle group; the experimental groups included the nanoparticle + white light irradiation group, the nanoparticle + 660 nm laser irradiation group, and the nanoparticle + white light + 660 nm laser irradiation group. Among them, the power of the white light xenon lamp (400 - 700 nm) was 50 mW / cm 2 , and the power of the 660 nm laser was 0.5 W / cm 2 . In the nanoparticle + white light + 660 nm laser irradiation group, the cells were first irradiated with white light for 5 min and then irradiated with a 660 nm laser for 5 min. An inverted fluorescence microscope (Axio Observer A1) was used to detect the fluorescence intensity of DCFH-DA in the cells of the control group and the experimental groups. The results are as Figure 5 shown. There was almost no green fluorescence signal in the control group, indicating that reactive oxygen species were not generated in the cells; green fluorescence appeared in the irradiated groups, and the fluorescence intensity was in the order of nanoparticle + white light + 660 nm laser irradiation group > nanoparticle + white light irradiation group > nanoparticle + 660 nm laser irradiation group, indicating that reactive oxygen species were generated in the cells, and the combined irradiation with white light and 660 nm laser had a better effect on generating reactive oxygen species.

[0090] Example 12 Evaluation of the in vivo therapeutic effect of TPMY nanoparticles

[0091] The in vivo photodynamic / photothermal synergistic therapeutic effect of TPMY nanoparticles was evaluated by establishing a tumor-bearing nude mouse model. MCF-7 cells (2×10 6 cells per nude mouse) were subcutaneously inoculated into the outer part of the right lower limb of nude mice. When the tumor volume was approximately 100 mm 3 , the nude mice were randomly divided into 5 groups for different treatments (PBS group, nanoparticle group, nanoparticle + white light irradiation group, nanoparticle + 660 nm laser irradiation group, nanoparticle + white light + 660 nm laser irradiation group). On day 0, 50 μL of normal saline or TPMY NPs solution (3 mg / mL) was injected into the tumor of each nude mouse. One irradiation was performed 24 h after the injection. The nanoparticle + white light irradiation group and the nanoparticle + 660 nm laser irradiation group were irradiated with a white light xenon lamp (400 - 700 nm, 200 mW / cm 2 ) or a 660 nm laser (0.8 W / cm 2) Irradiate for 10 min. For the nanoparticle + white light + 660 nm laser irradiation group, first irradiate with white light for 5 min, and then irradiate with laser for 5 min. Record the tumor weight and volume of nude mice in each group at 0, 3, 6, 10, and 14 days (V 肿瘤 = d 长径 × d 短径 2 / 2). After 14 days of treatment, sacrifice the nude mice, take out the tumors for weighing. The average weight and volume of tumors in each group are as Figure 6 shown. As can be seen from Figure 6 , the tumors of nude mice in the PBS group and the nanoparticle group grew rapidly, and the growth of tumors in nude mice in the irradiation groups was significantly inhibited. Among them, the nanoparticle + white light + 660 nm laser irradiation group had the best tumor inhibition effect, showing excellent photodynamic / photothermal synergistic therapy effect.

[0092] The above specifically describes the preferred embodiments of the present invention. However, the present invention is not limited to the described embodiments. Those skilled in the art can make various equivalent variations or substitutions without departing from the spirit of the present invention, and these equivalent variations or substitutions are all included within the scope defined by the claims of this application.

Claims

1. A cross-shaped near-infrared fluorescent compound with a structural general formula as shown in formula (I): Among them, R is selected from hydrogen or an alkoxy group, and Ar is selected from a phenyl group or a thiophenyl group.

2. The cross-shaped near-infrared fluorescent compound according to claim 1, characterized in that, The R is selected from one of hydrogen, a methoxy group, an ethoxy group, or a propoxy group.

3. The cross-shaped near-infrared fluorescent compound according to claim 1, characterized in that, The structural formula is as follows:

4. The preparation method of the cross-shaped near-infrared fluorescent compound according to any one of claims 1-3, characterized in that, Comprising the following steps: Using triphenylamine or 4',4'-dimethoxytriphenylamine as the parent nucleus, undergoing Suzuki coupling reaction with pinacol 4-formylphenylboronate or pinacol 5-formyl-2-thiopheneboronate, and then undergoing Knoevenagel condensation reaction with 1,3-bis(dicyanomethylene)indan to obtain the cross-shaped near-infrared fluorescent compound.

5. The preparation method according to claim 4, characterized in that, The conditions for the Suzuki coupling reaction include: being carried out in the presence of a catalyst, an inorganic base, an organic solvent, and under an inert condition; the reaction temperature is 80 - 100 °C, and the reaction time is 16 - 72 h.

6. The preparation method according to claim 5, characterized in that, The catalyst includes at least one of tetrakis(triphenylphosphine)palladium, [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium, and cuprous chloride; the inorganic base includes potassium carbonate and / or cesium carbonate; the organic solvent includes at least one of ethanol, toluene, 1,4-dioxane, and N,N-dimethylformamide.

7. The preparation method according to claim 4, characterized in that, The Knoevenagel condensation reaction is carried out in a mixed solution of 1,3-bis(dicyanomethylene)indan and acetic anhydride, the reaction temperature is 60 - 80 °C, and the reaction time is 10 - 15 h.

8. Use of the cross-shaped near-infrared fluorescent compound according to any one of claims 1 - 3 in the preparation of a photoacoustic imaging agent and a photothermal agent.

9. Use of the cross-shaped near-infrared fluorescent compound according to any one of claims 1 - 3 in the preparation of a drug for photodynamic therapy, photothermal therapy, and photodynamic / photothermal combined therapy of tumors.

10. A drug, characterized in that, Comprising the cross-shaped near-infrared fluorescent compound according to any one of claims 1 - 3.

Images