Benzopyranium fluorescent dye molecule as well as preparation method and application thereof

Abstract

The invention relates to the technical field of organic functional materials, in particular to a benzopyran onium fluorescent dye molecule, a preparation method and application, the benzopyran onium fluorescent dye molecule constructed by using an intramolecular charge transfer effect has an electron withdrawing group benzopyran onium, an electron-rich thiophene ring bridge and electron donating groups methoxy and amino, the photo-thermal conversion efficiency and the active oxygen generation capability are relatively high; the benzopyran onium fluorescent dye molecules BN-725 and BN-825 have near-infrared first region emission fluorescence, the fluorescence emission wavelength of the molecule BN-1000 can reach near-infrared second region, the stokes shift can reach 258nm to the maximum extent, and the benzopyran onium fluorescent dye has good biocompatibility, can be used for cell imaging and small animal living body imaging, and has a potential application prospect in the field of tumor treatment.

Description

Technical Field

[0001] The invention relates to the technical field of organic functional materials, and in particular to a benzopyrylium fluorescent dye molecule, a preparation method and an application thereof. Background Art

[0002] Cancer is one of the leading causes of death in humans and poses a huge threat to human health and life. Rapid diagnosis and targeted treatment of cancer are particularly important because they can significantly improve the cure rate of cancer. In traditional clinical applications, tumor diagnosis and treatment are two largely independent processes that require the use of separate diagnostic contrast agents and therapeutic agents. The extended time between the two medical procedures can easily delay the best treatment opportunity, while the negative effects of the two drug injections can cause discomfort and increase the risk to patients. To address these challenges, the integration of fluorescence imaging and photothermal treatment has become a promising approach. Since fluorescence imaging does not require ionizing radiation, it can be developed into a non-invasive or minimally invasive technology that can be conveniently used in the study of biological phenomena. At the same time, fluorescence imaging also has some key problems such as autofluorescence quenching, photobleaching, and low tissue penetration depth. The near-infrared region II (NIR-Ⅱ, 1000-1700nm) has lower autofluorescence absorption and tissue scattering. Compared with traditional visible light fluorescence imaging and near-infrared region I (NIR-I, 650-900nm) fluorescence imaging, near-infrared region II imaging has the smallest scattering and the deepest penetration depth, which can achieve high resolution and high signal-to-noise ratio, and has great application potential in molecular diagnosis and treatment. Among them, near-infrared region II dyes based on organic small molecules are a research hotspot in the field of biological imaging and chemical research due to their high biocompatibility, rapid excretion ability, and simple and fast preparation methods.

[0003] Currently, benzopyrylium is widely used in the field of biomedical imaging due to its unique D-π-A structure, which gives it a large Stokes shift and good biocompatibility. However, the emission wavelength of existing benzopyrylium fluorescent molecules is generally short, making it difficult to apply them to living organisms. Therefore, the development of a class of diagnostic and therapeutic agents based on benzopyrylium as an electron acceptor structure is of great significance in the biomedical field. Summary of the invention

[0004] In order to overcome the shortcomings of the above technical defects, the present invention provides a benzopyrylium fluorescent dye molecule, preparation method and application, and uses the intramolecular charge transfer effect to construct a series of near-infrared benzopyrylium molecules, which have the same electron-withdrawing group benzopyrylium, electron-rich thiophene ring bridge and electron-donating groups methoxy and amino. It has high photothermal conversion efficiency and active oxygen generation ability, and can be used for fluorescence imaging and photothermal therapy of organisms.

[0005] In order to achieve the above object, the technical solution of the present invention is:

[0006] In one aspect, a benzopyranium fluorescent dye molecule is provided, and its structural formula is shown in Formula I to Formula III:

[0007]

[0008]

[0009] Furthermore, the maximum emission wavelength of the fluorescent dye molecule is 700-1000 nm, and the maximum absorption wavelength is 640-750 nm.

[0010] 11. Further, the compound of formula IV reacts with the compound of formula V to obtain compound BN-725, the structure of which is shown in formula I;

[0011] Or the compound of formula IV reacts with the compound of formula VI to obtain compound BN-825, the structure of which is shown in formula II;

[0012] Or the compound of formula IV reacts with the compound of formula VII to obtain compound BN-1000, the structure of which is shown in formula III;

[0013] The structures of the compounds are as follows:

[0014]

[0015] Further, the condensation reaction conditions are: the condensation reaction conditions are: under an inert atmosphere, adding an organic solvent, reacting at 60-70° C. for 30-60 min, adding a recrystallization solvent, crystallizing and purifying to obtain;

[0016] The organic solvent is selected from one or more of anhydrous ethanol, anhydrous methanol, N,N-dimethylformamide or acetic anhydride;

[0017] The recrystallization solvent is a combination of one or more of dichloromethane, petroleum ether, ethyl ether, n-hexane and methanol.

[0018] Furthermore, the synthesis route of the compound of formula V is as follows:

[0019]

[0020] Furthermore, the synthetic route of the compound of formula VI is as follows:

[0021]

[0022] In a third aspect, a method is provided for preparing tumor photothermal therapy and / or diagnostic reagents or drugs based on the above-mentioned benzopyrylium fluorescent dye molecule and the benzopyrylium fluorescent dye molecule prepared by the above-mentioned method.

[0023] Furthermore, the tumor is breast cancer, lung cancer, liver cancer, gastric cancer, colon cancer, rectal cancer, nasopharyngeal cancer, pancreatic cancer, thyroid cancer, prostate cancer, leukemia, lymphoma, kidney tumor, sarcoma or blastoma; more preferably, the tumor is cervical cancer.

[0024] In a fourth aspect, a method for using the benzopyrylium fluorescent dye molecule and the benzopyrylium fluorescent dye molecule prepared by the above method in medical imaging is provided, wherein the medical imaging includes bioluminescence imaging and / or photoacoustic imaging.

[0025] In the fifth aspect, a pharmaceutical composition is provided, comprising the third aspect, providing a benzopyrylium fluorescent dye molecule based on the above-mentioned benzopyrylium fluorescent dye molecule, a benzopyrylium fluorescent dye molecule prepared by the above-mentioned method, or a pharmaceutically acceptable salt, solvate, enantiomer, diastereomer, or tautomer thereof.

[0026] Beneficial effects: Compared with the prior art, the benzopyrylium fluorescent dye molecules of the present invention have higher photothermal conversion efficiency and active oxygen generation capacity; the benzopyrylium fluorescent dye molecules BN-725 and BN-825 have fluorescence emission in the near-infrared region I, the fluorescence emission wavelength of the molecule BN-1000 can reach the near-infrared region II, the maximum Stokes shift can reach 258nm, and it has good biocompatibility, can be used for cell imaging and small animal living imaging, and has great potential application prospects in the field of tumor treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The benzopyrylium fluorescent dye BN-725 of the present invention 1 H NMR spectrum;

[0028] Figure 2 HRMS (ESI) spectrum of the benzopyrylium fluorescent dye BN-725 of the present invention;

[0029] Figure 3 The benzopyrylium fluorescent dye BN-825 of the present invention 1 H-NMR spectrum;

[0030] Figure 4 HRMS (ESI) spectrum of the benzopyrylium fluorescent dye BN-825 of the present invention;

[0031] Figure 5 The benzopyrylium fluorescent dye BN-1000 of the present invention 1 H NMR spectrum;

[0032] Figure 6 HRMS (ESI) spectrum of the benzopyrylium fluorescent dye BN-1000 of the present invention;

[0033] Figure 7 is the ultraviolet-visible absorption spectrum and fluorescence emission spectrum of the hemicyanine dye of the present invention;

[0034] Figure 8 The photothermal performance test of benzopyrylium fluorescent dyes of different concentrations in the present invention is as follows;

[0035] Fig. 9 The photothermal properties of the benzopyrylium fluorescent dye irradiated by lasers of different powers of the present invention;

[0036] Fig.10 is the light conversion efficiency of the benzopyrylium fluorescent dye of the present invention;

[0037] Fig.11 The photothermal stability of the benzopyrylium fluorescent dye of the present invention;

[0038] Fig.12 Determination of the active oxygen content of the benzopyrylium fluorescent dye of the present invention;

[0039] Fig.13 Fluorescence imaging of the benzopyrylium fluorescent dye of the present invention;

[0040] Fig.14 In vivo fluorescence imaging of the benzopyrylium fluorescent dye BN-1000 of the present invention;

[0041] Fig.15 Live and dead cell staining of the benzopyrylium fluorescent dye BN-1000 of the present invention;

[0042] Fig.16 This is a diagram showing the photothermal treatment effect of the hemicyanine dye BN-1000 of the present invention on tumor-bearing mice;

[0043] Among them, 16A is the in vivo photothermal imaging of benzopyrylium fluorescent dye BN-1000; 16B is the photothermal stability of benzopyrylium fluorescent dye BN-1000 in a mouse cell tumor model. DETAILED DESCRIPTION

[0044] In order to make those skilled in the art better understand the technical scheme of the present invention, the present invention is described in detail below in conjunction with specific embodiments. The experimental methods for which specific conditions are not indicated in the following examples are usually carried out under normal conditions or according to the conditions recommended by the manufacturer. The test materials used in the following examples, unless otherwise specified, are purchased from conventional biochemical reagent stores. Unless otherwise specified, percentages and parts are calculated by weight. Unless otherwise defined, all professional and scientific terms used in the text have the same meaning as those familiar to those skilled in the art. In addition, any method and material similar to or equal to the recorded content can all be applied to the present invention. The preferred implementation methods and materials described in the text are only for demonstration purposes.

[0045] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0046] The present invention aims to provide a benzopyrylium fluorescent dye molecule. Three near-infrared compounds BN-725, BN-825 and BN-1000 are obtained by modifying the benzopyrylium molecule. The series of molecules have the same electron-withdrawing group benzopyrylium, electron-rich thiophene ring bridge and electron-donating groups methoxy and amino, and have high photothermal conversion efficiency and active oxygen generation capacity. Among them, the molecules BN-725 and BN-825 have near-infrared region 1 fluorescence emission, and the molecule BN-1000 has near-infrared region 2 fluorescence emission. BN-1000 can simultaneously perform photodynamic imaging and treatment of tumor cells, and can be used for cell imaging and small animal in vivo imaging.

[0047] In some specific embodiments, a benzopyranium fluorescent dye molecule is provided, and its structural formula is shown in Formula I:

[0048]

[0049] In another specific embodiment, a preparation method based on the above-mentioned benzopyranium fluorescent dye molecule is provided, comprising the following steps:

[0050] S1. Using Michler's ketone and 4-bromobenzophenone as raw materials, adding zinc powder, TiCl4 and THF, and treating after the reaction to obtain compound 1;

[0051] S2: Compound 1 and 5-formyl-2-thiopheneboronic acid are used as raw materials, Pd(PPh3)4, K2CO3, toluene and methanol are added, and after the reaction, compound 2 is obtained by treatment, and the structural formula thereof is shown in Formula V;

[0052]

[0053] S3: Compound 2 and compound 3 (whose structural formula is shown in Formula IV) are used as raw materials, anhydrous ethanol is added, and after the reaction is completed, treatment is performed to obtain compound (BN-725),

[0054]

[0055] In some examples of this embodiment, S1 is specifically: under nitrogen protection, Michler's ketone, 4-bromobenzophenone and zinc powder are dissolved in THF, TiCl4 is quickly added at 0°C, heated to reflux, cooled to room temperature, quenched with dilute hydrochloric acid, stirred, extracted, dried, and column chromatography to obtain compound 1.

[0056] In some examples of this embodiment, S2 specifically comprises: dissolving the obtained compound 1, 5-formyl-2-thiopheneboronic acid, Pd(PPh3)4 and K2CO3 in toluene and methanol, heating, cooling to room temperature, filtering, extracting, drying and column chromatography to obtain compound 2.

[0057] In some examples of this embodiment, S3 is specifically: dissolving the obtained compound 2 and compound 3 in ethanol, heating, cooling to room temperature, adding ether, and recrystallizing to obtain compound BN-725.

[0058] In some specific embodiments, a benzopyranium fluorescent dye molecule is provided, and its structural formula is shown in Formula II:

[0059]

[0060] In another specific embodiment, a preparation method based on the above-mentioned benzopyranium fluorescent dye molecule is provided, comprising the following steps:

[0061] S1. Using 4,4-dimethoxybenzophenone and 4-bromobenzophenone as raw materials, adding zinc powder, TiCl4 and THF, and treating after the reaction to obtain compound 4;

[0062] S2: Compound 4 and 5-formyl-2-thiopheneboronic acid are used as raw materials, Pd(PPh3)4, K2CO3, toluene and methanol are added, and after the reaction, compound 5 is obtained by treatment, and the structural formula thereof is shown in Formula VI;

[0063]

[0064] S3: Compound 5 and compound 3 (whose structural formula is shown in Formula IV) are used as raw materials, anhydrous ethanol is added, and after the reaction is completed, the compound (BN-825) is obtained by treatment.

[0065]

[0066] In some examples of this implementation mode, S1 is specifically: under nitrogen protection, 4,4-dimethoxybenzophenone, 4-bromobenzophenone and zinc powder are dissolved in THF, TiCl4 is quickly added at 0°C, heated to reflux, cooled to room temperature, quenched with dilute hydrochloric acid, stirred, extracted, dried, and column chromatography to obtain compound 4.

[0067] In some examples of this embodiment, S2 specifically comprises: dissolving the obtained compound 4, 5-formyl-2-thiopheneboronic acid, Pd(PPh3)4 and K2CO3 in toluene and methanol, heating, cooling to room temperature, filtering, extracting, drying and column chromatography to obtain compound 5.

[0068] In some examples of this embodiment, S3 is specifically: dissolving the obtained compound 5 and compound 3 in ethanol, heating, cooling to room temperature, adding ether, and recrystallizing to obtain compound BN-825.

[0069] In some specific embodiments, a benzopyranium fluorescent dye molecule is provided, and its structural formula is shown in Formula III:

[0070]

[0071] In another specific embodiment, a preparation method based on the above-mentioned benzopyrylium fluorescent dye molecule is provided, comprising: using compound 3 (whose structural formula is shown in Formula IV) and aldehyde thiophene compound 6 (whose structural formula is shown in Formula VII) as raw materials, adding anhydrous ethanol, and treating after the reaction to obtain compound (BN-1000),

[0072]

[0073] In some examples of this embodiment, the preparation method is specifically: dissolving compound 3 and compound 6 in ethanol, heating, cooling to room temperature, adding ether, and recrystallizing to obtain compound BN-1000.

[0074] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments and drawings. It should be understood that the following embodiments are only used to explain the present invention and are not used to limit the present invention.

[0075] Example 1 Synthesis of fluorescent dye molecule BN-725

[0076] (1) Add Michler's ketone (2.68 g, 10 mmol), 4-bromobenzophenone (2.61 g, 10 mmol) and zinc powder (2.6 g, 40 mmol) into a 250 mL two-necked flask, add 100 mL THF, cool to 0°C, quickly add TiCl4 (3.4 mL, 30 mmol) under nitrogen protection, react for 30 min, return to room temperature naturally, and then heat under reflux for 12 h. After cooling, quench the reaction with dilute hydrochloric acid, continue stirring for 2 h, extract with dichloromethane three times, dry the organic phase with anhydrous sodium sulfate, and chromatograph on a silica gel column (eluent: petroleum ether: dichloromethane = 20:1) to obtain a light yellow solid compound 1 with a yield of 33%. 1 H NMR(500MHz, CDCl3)δ8.21(d,J=1.4Hz,2H),8.19(d,J=1.4Hz,2H),7.68–7.64(m,4H), 7.44(d,J=8.4Hz,4H),7.40(t,J=7.5Hz,4H),7.28(s,1H),6.20(s,4H),2.41(s,12H).

[0077] (2) Compound 1 (496 mg, 1 mmol), 5-formyl-2-thiopheneboronic acid (235 mg, 1.5 mmol), Pd(PPh3)4 (77 mg, 0.06 mmol) and K2CO3 (276 mg, 2 mmol) were added to a 50 mL two-necked bottle. Under nitrogen protection, 5 mL of toluene and 5 mL of methanol were added and reacted at 75°C for 18 h. After the reaction was completed, the mixture was naturally cooled to room temperature and extracted three times with dichloromethane. The organic phase was dried with anhydrous sodium sulfate to obtain a light red solid compound 2, which was directly used in the next step.

[0078] (3) Compounds 2 (528 mg, 1 mmol) and 3 (256 mg, 1 mmol) were added to a 25 mL two-necked bottle under nitrogen protection. 3 mL of ethanol was added under nitrogen protection. The mixture was reacted at 60 °C for 12 h. After cooling to room temperature, 20 mL of ether was added for recrystallization to obtain a dark black solid compound BN-725 with a yield of 60%. The compound was characterized by mass spectrometry and nuclear magnetic resonance (see Figure 1-2 ). 1H NMR (400MHz, DMSO) δ8.50(s,1H),8.32(s,1H),7.89(d,J=9.5Hz,1H),7.74(dd,J=6.6,3 .1Hz,4H),7.59(d,J=8.3Hz,1H),7.52–7.39(m,5H),7.34(t,J=7.5Hz,2H),7.27–7.22(m ,2H),7.15(dd,J=16.1,7.3Hz,2H),7.06–7.00(m,2H),6.88(dd,J=15.1,8.6Hz,2H),6.6 7(s,1H),2.91(d,J=3.4Hz,8H),2.54–2.51(m,12H),1.98(s,3H),1.28(t,J=7.0Hz,6H).

[0079] The synthetic route is as follows:

[0080]

[0081] Example 2 Synthesis of fluorescent dye molecule BN-825

[0082] (1) Compound 4,4-dimethoxybenzophenone (2.24 g, 10 mmol), compound 4-bromobenzophenone (2.61 g, 10 mmol) and zinc powder (2.6 g, 40 mmol) were added to a 250 mL two-necked bottle, 100 mL THF was added, cooled to 0°C, TiCl4 (3.4 mL, 30 mmol) was quickly added under nitrogen protection, reacted for 30 min, naturally returned to room temperature, and then heated to reflux for 12 h. After cooling, the reaction was quenched with dilute hydrochloric acid, stirred for 2 h, extracted with dichloromethane three times, the organic phase was dried with anhydrous sodium sulfate, and silica gel column chromatography (eluent: petroleum ether: dichloromethane = 20:1) was performed to obtain a light yellow solid compound 4 with a yield of 30%. 1 H NMR (500MHz, CDCl3) δ7.24 (d, J=8.4Hz, 2H), 7.11 (dd, J=13.4, 6.8Hz, 3H), 7.04–7. 00(m,2H),6.97–6.88(m,6H),6.67(dd,J=18.1,8.7Hz,4H),3.77(d,J=13.8Hz,6H).

[0083] (2) Compound 4 (472 mg, 1 mmol), 5-formyl-2-thiopheneboronic acid (235 mg, 1.5 mmol), Pd(PPh3)4 (77 mg, 0.06 mmol) and K2CO3 (276 mg, 2 mmol) were added to a 50 mL two-necked bottle. Under nitrogen protection, 5 mL of toluene and 5 mL of methanol were added and reacted at 75°C for 18 h. After the reaction was completed, it was naturally cooled to room temperature and extracted with dichloromethane three times. The organic phase was dried over anhydrous sodium sulfate and purified by silica gel chromatography (eluent: petroleum ether: dichloromethane = 10:1) to obtain a light red solid compound 5 with a yield of 53%. 1 H NMR (500MHz, CDCl3) δ9.89 (s, 1H), 7.73 (d, J = 4.0Hz, 1H), 7.44 (d, J = 8.4Hz, 2H), 7.36 (d, J = 4.0Hz, 1H), 7.14 (t, J = 7.3Hz, 3H), 7.10–7.05 (m, 4H), 6.98 (dd, J = 19.4, 8.8Hz, 4H), 6.68 (dd, J = 15.6, 8.8Hz, 5H), 3.77 (d, J = 4.6Hz, 6H).

[0084] (3) Compound 3 (176.8 mg, 0.3 mmol) and Compound 5 (100 mg, 0.19 mmol) were added to a 25 mL two-necked bottle under nitrogen protection. 3 mL of ethanol was added under nitrogen protection and reacted at 60 ° C for 12 h. After naturally returning to room temperature, 20 mL of ether was added for recrystallization to obtain a dark black solid compound BN-825 with a yield of 80%. It was characterized by mass spectrometry and nuclear magnetic resonance (see Figure 3-4 ). 1 HNMR (500MHz, CDCl3) δ8.49(s,1H),8.06(s,1H),7.89(s,1H),7.70(d,J=9.6Hz,1H),7.42(d,J=8. 3Hz,2H),7.37(s,1H),7.14(dd,J=13.6,5.9Hz,3H),7.10–7.06(m,4H),7.02(d,J=8.7Hz,2H),6.9 6(d,J=8.6Hz,2H),6.71(d,J=8.7Hz,2H),6.67(d,J=8.7Hz,2H),3.77(d,J=4.5Hz,6H),3.70(s,2H ), 3.50 (q, J = 7.0Hz, 2H), 2.93 (d, J = 31.4Hz, 5H), 2.03 (s, 3H), 1.39 (s, 6H), 1.23 (t, J = 7.0Hz, 2H).

[0085] The synthetic route is as follows:

[0086]

[0087] Example 3 Synthesis of fluorescent dye molecule BN-1000

[0088] Under nitrogen protection, compound 3 (256 mg, 1 mmol) and compound 6 (497 mg, 1 mmol) were added to a 25 mL two-necked bottle, and 3 mL of ethanol was added under nitrogen protection. The reaction was carried out at 60 ° C for 12 h. After cooling to room temperature, 20 mL of ether was added for recrystallization to obtain a dark black solid compound BN-1000 with a yield of 65%. It was characterized by mass spectrometry and nuclear magnetic resonance (see Figure 5-6 ). 1 H NMR (400MHz, CDCl3) δ8.53 (s, 1H), 8.08 (s, 1H), 7.91 (d, J = 4.3Hz, 1H), 7.72 (d, J=9.1Hz,1H),7.44(d,J=8.5Hz,2H),7.31(s,3H),7.18(d,J=3.8Hz,1H),7.13( d,J=8.9Hz,5H),6.95(d,J=8.5Hz,2H),6.90(d,J=8.9Hz,4H),3.85(s,6H),3.7 3(d,J=7.5Hz,4H), 2.96(d,J=21.7Hz,4H), 2.07(s,2H), 1.41(t,J=7.2Hz,6H).

[0089] The synthetic route is as follows:

[0090]

[0091] Example 4 Investigation of the UV Fluorescence Properties of Benzopyranium Fluorescent Dyes

[0092] The molecules synthesized in Examples 1-3 were tested for UV and fluorescence properties in DMSO. The spectra are shown in FIG. Figure 7 The maximum UV absorption peaks of BN-725, BN-825 and BN-1000 are 640nm, 640nm and 742nm, respectively, and the maximum fluorescence emission peaks are 725nm, 825nm and 1000nm, respectively, indicating that the addition of electron-rich bridged thiophene rings is beneficial to the movement of UV absorption and fluorescence emission to the long-wave direction.

[0093] Example 5 Investigation of the Photothermal Properties of Benzopyrylium Fluorescent Dyes

[0094] like Figure 8 As shown, at a fixed laser power (635nm, 1.0W / cm 2), the photothermal temperature of pure PBS did not change much, but with the increase of photosensitizer concentration, the temperature of photosensitizer BN-725, BN-825, and BN-1000 solutions also increased.

[0095] like Fig. 9 As shown, as the power density increases from 0.6W / cm 2 Increase to 1.0W / cm 2 , the temperature of photosensitizers BN-725, BN-825, and BN-1000 also increased, indicating that the temperature is positively correlated with the laser power intensity irradiating the photosensitizer.

[0096] At room temperature (22°C), using 635nm (1.0W / cm 2 ) irradiated PBS solutions (100μM) of dyes BN-725 and BN-825. The results showed that the temperature of BN-825 solution reached a maximum value of 42°C after irradiation with 635nm for 5min, and its photothermal conversion efficiency was 32.43%. Under the same conditions, BN-725 could reach 59°C, with a photothermal conversion efficiency of 43.25%. At room temperature 22°C, using 808nm (1.5W / cm 2 ) irradiated the PBS solution (100 μM) of the dye BN-1000. The results showed that the temperature of the BN-1000 solution increased significantly after being irradiated at 808 nm for 5 minutes, and the maximum temperature could reach 67°C. Its photothermal conversion efficiency was 54.71%. Figure 8-10 After 6 cycles of light heating and cooling, the highest temperatures that BN-725, BN-825 and BN-1000 can reach are almost unchanged, indicating that their light-heating stability is strong and the light-heating effect is almost unchanged. Fig.11 Compared with BN-725 and BN-825, BN-1000 has better optical and photothermal properties and has greater potential application prospects.

[0097] Example 6 Investigation of the Active Oxygen Generation Capacity of Benzopyrylium Fluorescent Dyes

[0098] In dark conditions, a 635 nm laser (0.6 W / cm 2 ) irradiated DCFH-DA, BN-725+DCFH-DA and BN-825+DCFH-DA solutions or used 808 nm laser (1 W / cm 2 ) irradiated DCFH-DA and BN-1000+DCFH-DA solutions for 300s. The results showed that BN-725, BN-825, and BN-1000 could effectively generate reactive oxygen species, among which BN-1000 had the strongest reactive oxygen species generation ability. Fig.12In order to more intuitively show the ROS production ability of BN-1000, we used the ROS probe DCFH to stain the cells. Hela cells were incubated with PBS, Rosup, and BN-1000 (20 μM) in a dark environment for 0.5 h, and an 808 nm laser (1.5 W / cm 2 ) and irradiated the cells for 5 minutes. After irradiation, the cells were immediately stained with the commercial reactive oxygen species probe DCFH for 30 minutes. The DCFH probe can react with reactive oxygen species in cells to produce green fluorescence signals, which were imaged and observed using a Leica live cell workstation. The results showed that BN-1000 can effectively produce reactive oxygen species under 808nm laser irradiation, such as Fig.13 shown.

[0099] Example 7 Investigation of in vitro fluorescence imaging performance of benzopyrylium fluorescent dyes

[0100] Using 750nm and 808nm lasers (2W / cm 2 ) irradiated BN-725, BN-825, and BN-1000 (25 μM), using the LP880 filter to obtain the best signal contrast and image quality. The results showed that BN-1000 had the best in vitro fluorescence imaging effect, while most of the energy absorbed by BN-825 was likely released in the form of intersystem crossing, resulting in a lower fluorescence quantum yield, such as Fig.14 shown.

[0101] Example 8 Investigation of the Cytotoxicity of Benzopyranium Fluorescent Dye BN-1000

[0102] To more intuitively display the phototherapy performance of BN-1000, we used calcein AM (green) and propidium iodide (red) dyes for live and dead cell staining. Green fluorescence indicates live cells, and red indicates dead cells. The 808 nm laser (2 W / cm 2 ) were irradiated with PBS, PBS+laser, BN-1000, and BN-1000+laser solutions for 10 min. Under dark conditions, no cell death was observed in the BN-1000 group, indicating that the cytotoxicity of BN-1000 was low and it had good biocompatibility. After laser irradiation, HeLa cells treated with BN-1000 showed obvious red fluorescence, indicating that most of the HeLa cells died, such as Fig.15 The results show that at a power intensity (2W / cm 2 ) laser irradiation, BN-1000 can effectively kill cancer cells and has a good phototherapy effect, which has great potential application prospects in the field of tumor treatment.

[0103] Example 9 In vivo photothermal imaging of benzopyranium fluorescent dye BN-1000

[0104] In vivo photothermal imaging experiments: Fluorophore (200 μL, 100 μM, PBS solvent) was injected subcutaneously into the thigh of BABL / C nude mice or mice, and the fluorescence was detected at 808 nm, 1 W / cm 2 The laser was irradiated for 10 minutes, and the temperature changes of the tumor were recorded every 2 minutes using an infrared thermal imager during the irradiation process.

[0105] PBS and fluorescent dye BN-1000 (200 μL, 100 μM) were injected into the subcutaneous tumor site of the thigh root of BABL / C nude mice or mice, and then 808 nm laser (1.0 W / cm 2 ) irradiation for 10 minutes, and the temperature changes of the tumor during irradiation were recorded with an infrared thermal imager every 2 minutes. Fig.16 As shown. The results showed that the local temperature of mice injected with PBS solution at the thigh root did not increase significantly under laser irradiation, while the local temperature of mice injected with BN-1000 solution increased significantly under laser irradiation, with the highest temperature reaching 57°C. The results showed that the benzopyranium fluorescent dye BN-1000 has a good photothermal imaging effect. This has potential application prospects in the field of photothermal treatment of tumors.

[0106] Finally, it should be noted that the above description is only a preferred embodiment of the present invention. Under the guidance of the present invention, those skilled in the art can make a variety of similar expressions without violating the purpose and claims of the present invention, and such changes all fall within the protection scope of the present invention.

Claims

1. A benzopyrylium fluorescent dye molecule, characterized in that: Its structural formula is shown in Formula I-Formula III:

2. The benzopyrylium fluorescent dye molecule according to claim 1, characterized in that: The maximum emission wavelength of the fluorescent dye molecule is 700-1000 nm, and the maximum absorption wavelength is 640-750 nm.

3. The method for preparing a benzopyrylium fluorescent dye molecule according to claim 1, characterized in that: The compound of formula IV undergoes a condensation reaction with the compound of formula V to obtain compound BN-725, the structure of which is shown in formula I; Or the compound of formula IV reacts with the compound of formula VI to obtain compound BN-825, the structure of which is shown in formula II; Or the compound of formula IV reacts with the compound of formula VII to obtain compound BN-1000, the structure of which is shown in formula III; The structures of the compounds are as follows:

4. The method according to claim 2, characterized in that: The condensation reaction conditions are: adding an organic solvent under an inert atmosphere, reacting at 60-70° C. for 30-60 minutes, adding a recrystallization solvent, crystallizing and purifying to obtain; The organic solvent is selected from one or more of anhydrous ethanol, anhydrous methanol, N,N-dimethylformamide or acetic anhydride; The recrystallization solvent is a combination of one or more of dichloromethane, petroleum ether, ethyl ether, n-hexane and methanol.

5. The method according to claim 2, characterized in that: The synthetic route of the compound of formula V is as follows:

6. The method according to claim 2, characterized in that The synthetic route of the compound of formula VI is as follows:

7. Use of the benzopyrylium fluorescent dye molecule according to claim 1 or 2, or the benzopyrylium fluorescent dye molecule obtained by the method according to any one of claims 3 to 6 in the preparation of tumor photothermal therapy and / or diagnostic agents or drugs.

8. The use according to claim 7, characterized in that: The tumor is breast cancer, lung cancer, liver cancer, gastric cancer, colon cancer, rectal cancer, nasopharyngeal cancer, pancreatic cancer, thyroid cancer, prostate cancer, leukemia, lymphoma, kidney tumor, sarcoma or blastoma; more preferably, the tumor is cervical cancer.

9. Use of the benzopyrylium fluorescent dye molecule according to claim 1 or the benzopyrylium fluorescent dye molecule obtained by the method according to any one of claims 3 to 6 in medical imaging, wherein the medical imaging includes bioluminescence imaging and / or photoacoustic imaging.

10. A pharmaceutical composition, characterized in that Contains the benzopyrylium fluorescent dye molecule according to claim 1 or 2, the benzopyrylium fluorescent dye molecule prepared by the method according to any one of claims 3 to 6, or a pharmaceutically acceptable salt, solvate, enantiomer, diastereomer, or tautomer thereof.

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