A benzopyrylium fluorescent dye molecule, preparation method and application

Abstract

The present invention relates to the technical field of organic functional materials, and particularly relates to a benzopyrylium fluorescent dye molecule, a preparation method and an application thereof. The benzopyrylium fluorescent dye molecule constructed by using the intramolecular charge transfer effect has an electron-withdrawing group benzopyrylium, an electron-rich thiophene ring bridge and electron-donating groups methoxy and amino, and has a high photothermal conversion efficiency and reactive oxygen generation ability; the benzopyrylium fluorescent dye molecules BN-725 and BN-825 emit fluorescence in the first near-infrared region, the fluorescence emission wavelength of the molecule BN-1000 can reach the second near-infrared region, the Stokes shift can reach up to 258 nm at most, and it has good biocompatibility and can be used for cell imaging and small animal in vivo imaging, and has great potential application prospects in the field of tumor treatment.

Description

Technical Field

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

[0002] Cancer is one of the main causes of human death and poses a great threat to human health and life. The rapid diagnosis and targeted therapy 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, and separate diagnostic contrast agents and therapeutic reagents need to be used. The extended time between the two medical procedures easily delays the optimal treatment opportunity, and the negative effects of two drug injections can cause discomfort and increase the risk to patients. To address these challenges, the integration of fluorescence imaging and photothermal therapy has become a promising method. Since fluorescence imaging does not require ionizing radiation, it can be developed into a non-invasive or minimally invasive technique and is conveniently used for 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. Near-infrared II (NIR-II, 1000-1700 nm) luminescence has lower autofluorescence absorption and tissue scattering. Compared with traditional visible light fluorescence imaging and near-infrared I (NIR-I, 650-900 nm) fluorescence imaging, near-infrared II imaging has the least scattering and the deepest penetration depth, enabling high resolution and high signal-to-noise ratio, and has great application potential in molecular diagnosis and treatment. Among them, near-infrared II dyes based on organic small molecules are a research hotspot in the current fields of biological imaging and chemical research due to their high biocompatibility, rapid excretion ability, and simple and fast preparation methods.

[0003] Currently, benzopyrylium has a large Stokes shift and good biocompatibility due to its unique D-π-A structure, etc., and is widely used in the field of biomedical imaging. However, the emission wavelengths of existing benzopyrylium fluorescent molecules are generally short, making it difficult to perform good in vivo applications. Therefore, it is of great significance to develop a class of diagnostic and therapeutic reagents based on benzopyrylium as the electron acceptor structure in the biomedical field. Summary of the Invention

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

[0005] To achieve the above object, the technical solution of the present invention is as follows:

[0006] On the one hand, a benzopyrylium fluorescent dye molecule is provided, and its structural formula is shown in Formula I - 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] Furthermore, when the compound of Formula IV reacts with the compound of Formula V through a condensation reaction, the compound BN - 725 is obtained, and its structure is shown in Formula I;

[0011] Or when the compound of Formula IV reacts with the compound of Formula VI through a condensation reaction, the compound BN - 825 is obtained, and its structure is shown in Formula II;

[0012] Or when the compound of Formula IV reacts with the compound of Formula VII through a condensation reaction, the compound BN - 1000 is obtained, and its structure is shown in Formula III;

[0013] Among them, the structures of each compound are as follows:

[0014]

[0015] Furthermore, the conditions for the condensation reaction are as follows: in an inert atmosphere, an organic solvent is added, and after reacting at 60 - 70 °C for 30 - 60 min, a recrystallization solvent is added, and crystallization and purification are carried out to obtain the product;

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

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

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

[0019]

[0020] Furthermore, the synthesis route of the compound of Formula VI is as follows:

[0021]

[0022] In a third aspect, there is provided an application of the above-mentioned benzopyrylium fluorescent dye molecule and the benzopyrylium fluorescent dye molecule prepared by the above method in the preparation of tumor photothermal therapy and / or diagnostic reagents or drugs.

[0023] Further, 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, there is provided an application of the above-mentioned benzopyrylium fluorescent dye molecule and the benzopyrylium fluorescent dye molecule prepared by the above method in medical imaging, and the medical imaging includes biofluorescence imaging and / or photoacoustic imaging.

[0025] In a fifth aspect, there is provided a pharmaceutical composition containing the benzopyrylium fluorescent dye molecule in the third aspect, the benzopyrylium fluorescent dye molecule prepared by the above method or a pharmaceutically acceptable salt, solvate, enantiomer, diastereomer, tautomer thereof.

[0026] Beneficial effects: Compared with the prior art, the benzopyrylium fluorescent dye molecule of the present invention has a high photothermal conversion efficiency and reactive oxygen species generation ability; the benzopyrylium fluorescent dye molecules BN-725 and BN-825 emit fluorescence in the first near-infrared region, and the fluorescence emission wavelength of the molecule BN-1000 can reach the second near-infrared region, and the Stokes shift can reach up to 258 nm, and it has good biocompatibility and can be used for cell imaging and small animal in vivo imaging, and has great potential application prospects in the field of tumor treatment. Description of the Drawings

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

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

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

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

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

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

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

[0034] Figure 8 This is the photothermal performance test of benzopyrylium fluorescent dyes with different concentrations of the present invention;

[0035] Figure 9 This is the photothermal performance of benzopyrylium fluorescent dyes irradiated by lasers with different powers of the present invention;

[0036] Figure 10 This is the photoconversion efficiency of the benzopyrylium fluorescent dye of the present invention;

[0037] Figure 11 This is the photothermal stability of the benzopyrylium fluorescent dye of the present invention;

[0038] Figure 12 This is the determination of the reactive oxygen content of the benzopyrylium fluorescent dye of the present invention;

[0039] Figure 13 This is the fluorescence imaging of the benzopyrylium fluorescent dye of the present invention;

[0040] Figure 14 This is the in vivo fluorescence imaging of the benzopyrylium fluorescent dye BN-1000 of the present invention;

[0041] Figure 15 This is the live-dead cell staining of the benzopyrylium fluorescent dye BN-1000 of the present invention;

[0042] Figure 16 This is the photothermal therapy effect diagram of the hemicyanine dye BN-1000 in tumor-bearing mice of the present invention;

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

[0044] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be described in detail below in conjunction with specific embodiments. For the experimental methods without specific conditions noted in the following examples, they are generally carried out under conventional conditions or according to the conditions recommended by the manufacturer. The test materials used in the following examples are all obtained from regular biochemical reagent stores without special instructions. Unless otherwise stated, percentages and parts are calculated by weight. Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to the described content can be applied to the present invention. The preferred implementation methods and materials described herein are for illustrative purposes only.

[0045] In the ranges disclosed herein, the endpoints and any values are not limited to the exact ranges or values. These ranges or values should be understood to include values close to these ranges or values. For numerical ranges, between the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values, they can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[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. This series of molecules has the same electron-withdrawing group benzopyrylium, electron-rich thiophene ring bridge, and electron-donating groups methoxy and amino, and has high photothermal conversion efficiency and reactive oxygen generation ability. Among them, molecules BN-725 and BN-825 emit fluorescence in the near-infrared region I, molecule BN-1000 emits fluorescence in the near-infrared region II, and BN-1000 can simultaneously perform photodynamic imaging and treatment on tumor cells and can be used for cell imaging and small animal in vivo imaging.

[0047] In some specific embodiments, a benzopyrylium 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 benzopyrylium fluorescent dye molecule is provided, including the following steps:

[0050] S1. Using Michler's ketone and 4-bromobenzophenone as raw materials, adding zinc powder, TiCl 4 and THF, and after the reaction is completed, the compound 1 is obtained through post-treatment;

[0051] S2: Using compound 1 and 5-formyl-2-thiopheneboronic acid as raw materials, adding Pd(PPh 3 ) 4 、K2 CO 3 , toluene and methanol, and after the reaction is completed, compound 2 is obtained through post-treatment, and its structural formula is as shown in Formula V;

[0052]

[0053] S3: Using compound 2 and compound 3 (whose structural formula is as shown in Formula IV) as raw materials, adding absolute ethanol, and after the reaction is completed, compound (BN-725) is obtained through post-treatment,

[0054]

[0055] In some embodiments of this embodiment, S1 is specifically: Under nitrogen protection, dissolve Michler's ketone, 4-bromobenzophenone and zinc powder in THF, quickly add TiCl at 0 °C 4 , heat under reflux, cool to room temperature, quench with dilute hydrochloric acid, stir, extract, dry, and perform column chromatography to obtain compound 1.

[0056] In some embodiments of this embodiment, S2 is specifically: Dissolve the obtained compound 1, 5-formyl-2-thiopheneboronic acid, Pd(PPh 3 ) 4 and K 2 CO 3 in toluene and methanol, heat, cool to room temperature, filter, extract, dry, and perform column chromatography to obtain compound 2.

[0057] In some embodiments of this embodiment, S3 is specifically: Dissolve the obtained compound 2 and compound 3 in ethanol, heat, cool to room temperature, add ether, and perform recrystallization to obtain compound BN-725.

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

[0059]

[0060] In another specific embodiment, a preparation method based on the above benzopyrylium fluorescent dye molecule is provided, including the following steps:

[0061] S1. Using 4,4-dimethoxybenzophenone and 4-bromobenzophenone as raw materials, adding zinc powder, TiCl 4 and THF, and after the reaction is completed, compound 4 is obtained through post-treatment;

[0062] S2: Using compound 4 and 5-formyl-2-thiopheneboronic acid as raw materials, adding Pd(PPh 3 ) 4 , K 2 CO 3, toluene and methanol, and after the reaction is completed, compound 5 is obtained through post-treatment, and its structural formula is as shown in Formula VI;

[0063]

[0064] S3: Using compound 5 and compound 3 (whose structural formula is as shown in Formula IV) as raw materials, adding absolute ethanol, and after the reaction is completed, compound (BN-825) is obtained through post-treatment,

[0065]

[0066] In some embodiments of this embodiment, S1 is specifically: Under nitrogen protection, dissolve 4,4-dimethoxybenzophenone, 4-bromobenzophenone and zinc powder in THF, and quickly add TiCl at 0 °C 4 , heat under reflux, cool to room temperature, quench with dilute hydrochloric acid, stir, extract, dry, and perform column chromatography to obtain compound 4.

[0067] In some embodiments of this embodiment, S2 is specifically: Dissolve the obtained compound 4, 5-formyl-2-thiopheneboronic acid, Pd(PPh 3 ) 4 with K 2 CO 3 in toluene and methanol, heat, cool to room temperature, filter, extract, dry, and perform column chromatography to obtain compound 5.

[0068] In some embodiments of this embodiment, S3 is specifically: Dissolve the obtained compound 5 and compound 3 in ethanol, heat, cool to room temperature, add ether, and perform recrystallization to obtain compound BN-825.

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

[0070]

[0071] In another specific embodiment, a preparation method based on the above benzopyrylium fluorescent dye molecule is provided, including: Using compound 3 (whose structural formula is as shown in Formula IV) and aldehyde group thiophene compound 6 (whose structural formula is as shown in Formula VII) as raw materials, adding absolute ethanol, and after the reaction is completed, compound (BN-1000) is obtained through post-treatment,

[0072]

[0073] In some embodiments of this embodiment, this preparation method is specifically: Dissolve compound 3 and compound 6 in ethanol, heat, cool to room temperature, add ether, and perform recrystallization to obtain compound BN-1000.

[0074] The technical solution of the present invention will be further described in detail below in conjunction with specific embodiments and the accompanying 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) to a 250 mL two-necked flask, add 100 mL of THF, cool to 0 °C, and quickly add TiCl 4 (3.4 mL, 30 mmol) under nitrogen protection and react for 30 min. Let it return to room temperature naturally, then heat to reflux for 12 h. After cooling, quench the reaction with dilute hydrochloric acid, continue stirring for 2 h, extract three times with dichloromethane, dry the organic phase with anhydrous sodium sulfate, and perform silica gel column chromatography (eluent: petroleum ether:dichloromethane = 20:1) to obtain a pale yellow solid compound 1 with a yield of 33%. 1 H NMR (500 MHz, CDCl 3 ) δ 8.21 (d, J = 1.4 Hz, 2H), 8.19 (d, J = 1.4 Hz, 2H), 7.68–7.64 (m, 4H), 7.44 (d, J = 8.4 Hz, 4H), 7.40 (t, J = 7.5 Hz, 4H), 7.28 (s, 1H), 6.20 (s, 4H), 2.41 (s, 12H).

[0077] (2) Add compound 1 (496 mg, 1 mmol), 5-formyl-2-thiopheneboronic acid (235 mg, 1.5 mmol), Pd(PPh 3 ) 4 (77 mg, 0.06 mmol) and K 2 CO 3 (276 mg, 2 mmol) to a 50 mL two-necked flask. Under nitrogen protection, add 5 mL of toluene and 5 mL of methanol, and react at 75 °C for 18 h. After the reaction is completed, let it cool to room temperature naturally, extract three times with dichloromethane, and dry the organic phase with anhydrous sodium sulfate to obtain a light red solid compound 2, which is directly put into the next step.

[0078] (3) Under nitrogen protection, add compound 2 (528 mg, 1 mmol) and 3 (256 mg, 1 mmol) to a 25 mL two-necked flask. Under nitrogen protection, add 3 mL of ethanol, react at 60 °C for 12 h. After cooling to room temperature, add 20 mL of ether for recrystallization to obtain a dark black solid compound BN-725 with a yield of 60%. Perform mass spectrometry and nuclear magnetic resonance characterization on it (see Figure 1-2 ). 11H NMR (400 MHz, DMSO) δ 8.50 (s, 1H), 8.32 (s, 1H), 7.89 (d, J = 9.5 Hz, 1H), 7.74 (dd, J = 6.6, 3.1 Hz, 4H), 7.59 (d, J = 8.3 Hz, 1H), 7.52–7.39 (m, 5H), 7.34 (t, J = 7.5 Hz, 2H), 7.27–7.22 (m, 2H), 7.15 (dd, J = 16.1, 7.3 Hz, 2H), 7.06–7.00 (m, 2H), 6.88 (dd, J = 15.1, 8.6 Hz, 2H), 6.67 (s, 1H), 2.91 (d, J = 3.4 Hz, 8H), 2.54–2.51 (m, 12H), 1.98 (s, 3H), 1.28 (t, J = 7.0 Hz, 6H).

[0079] The synthetic route is as follows:

[0080]

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

[0082] (1) Add 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) to a 250 mL two-necked flask, add 100 mL of THF, cool to 0 °C, and quickly add TiCl 4 (3.4 mL, 30 mmol) and react for 30 min. Naturally restore to room temperature, then heat under reflux for 12 h. After cooling, quench the reaction with dilute hydrochloric acid, continue stirring for 2 h, extract three times with dichloromethane, dry the organic phase with anhydrous sodium sulfate, and perform silica gel column chromatography (eluent: petroleum ether:dichloromethane = 20:1) to obtain the pale yellow solid compound 4 with a yield of 30%. 1 1H NMR (500 MHz, CDCl 3 ) δ 7.24 (d, J = 8.4 Hz, 2H), 7.11 (dd, J = 13.4, 6.8 Hz, 3H), 7.04–7.00 (m, 2H), 6.97–6.88 (m, 6H), 6.67 (dd, J = 18.1, 8.7 Hz, 4H), 3.77 (d, J = 13.8 Hz, 6H).

[0083] (2) Add compound 4 (472 mg, 1 mmol), 5-formyl-2-thiopheneboronic acid (235 mg, 1.5 mmol), Pd(PPh 3 ) 4 (77 mg, 0.06 mmol) and K 2 CO3 (276 mg, 2 mmol) was added to a 50 mL two-necked flask. Under nitrogen protection, 5 mL of toluene and 5 mL of methanol were added, and the reaction was carried out at 75 °C for 18 h. After the reaction was completed, it was naturally cooled to room temperature, extracted three times with dichloromethane, the organic phase was dried over anhydrous sodium sulfate, and purified by silica gel column chromatography (eluent: petroleum ether:dichloromethane = 10:1) to obtain a light red solid compound 5 with a yield of 53%. 1 H NMR (500 MHz, CDCl 3 ) δ 9.89 (s, 1H), 7.73 (d, J = 4.0 Hz, 1H), 7.44 (d, J = 8.4 Hz, 2H), 7.36 (d, J = 4.0 Hz, 1H), 7.14 (t, J = 7.3 Hz, 3H), 7.10–7.05 (m, 4H), 6.98 (dd, J = 19.4, 8.8 Hz, 4H), 6.68 (dd, J = 15.6, 8.8 Hz, 5H), 3.77 (d, J = 4.6 Hz, 6H).

[0084] (3) Under nitrogen protection, compound 3 (176.8 mg, 0.3 mmol) and 5 (100 mg, 0.19 mmol) were added to a 25 mL two-necked flask. Under nitrogen protection, 3 mL of ethanol was added, and the reaction was carried out 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 (500 MHz, CDCl 3 ) δ 8.49 (s, 1H), 8.06 (s, 1H), 7.89 (s, 1H), 7.70 (d, J = 9.6 Hz, 1H), 7.42 (d, J = 8.3 Hz, 2H), 7.37 (s, 1H), 7.14 (dd, J = 13.6, 5.9 Hz, 3H), 7.10–7.06 (m, 4H), 7.02 (d, J = 8.7 Hz, 2H), 6.96 (d, J = 8.6 Hz, 2H), 6.71 (d, J = 8.7 Hz, 2H), 6.67 (d, J = 8.7 Hz, 2H), 3.77 (d, J = 4.5 Hz, 6H), 3.70 (s, 2H), 3.50 (q, J = 7.0 Hz, 2H), 2.93 (d, J = 31.4 Hz, 5H), 2.03 (s, 3H), 1.39 (s, 6H), 1.23 (t, J = 7.0 Hz, 2H).

[0085] The synthetic route is as follows:

[0086]

[0087] Synthesis of Fluorescent Dye Molecule BN-1000 in Example 3

[0088] Under nitrogen protection, compound 3 (256 mg, 1 mmol) and 6 (497 mg, 1 mmol) were added to a 25 mL two-necked flask. Under nitrogen protection, 3 mL of ethanol was added, and 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 (400 MHz, CDCl 3 ) δ 8.53 (s, 1H), 8.08 (s, 1H), 7.91 (d, J = 4.3 Hz, 1H), 7.72 (d, J = 9.1 Hz, 1H), 7.44 (d, J = 8.5 Hz, 2H), 7.31 (s, 3H), 7.18 (d, J = 3.8 Hz, 1H), 7.13 (d, J = 8.9 Hz, 5H), 6.95 (d, J = 8.5 Hz, 2H), 6.90 (d, J = 8.9 Hz, 4H), 3.85 (s, 6H), 3.73 (d, J = 7.5 Hz, 4H), 2.96 (d, J = 21.7 Hz, 4H), 2.07 (s, 2H), 1.41 (t, J = 7.2 Hz, 6H).

[0089] The synthesis route is as follows:

[0090]

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

[0092] The UV and fluorescence properties of the molecules synthesized in Examples 1-3 were tested in DMSO, and the spectrograms are as Figure 7 shown. The maximum UV absorption peak wavelengths of BN-725, BN-825, and BN-1000 were 640 nm, 640 nm, and 742 nm respectively, and the maximum fluorescence emission peak wavelengths were 725 nm, 825 nm, and 1000 nm respectively, indicating that increasing the electron-rich bridging thiophene ring is beneficial to the red shift of UV absorption and fluorescence emission.

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

[0094] As Figure 8 shown, under the irradiation of a fixed laser power (635 nm, 1.0 W / cm 2 ), the photothermal temperature change of pure PBS was small, while with the increase of the photosensitizer concentration, the temperatures of the photosensitizer BN-725, BN-825, and BN-1000 solutions also increased.

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

[0096] At room temperature of 22 °C, using 635 nm (1.0 W / cm 2 ) to irradiate the PBS solutions (100 μM) of dyes BN-725 and BN-825, the results show that the temperature of the BN-825 solution reaches the highest value of 42 °C after being irradiated by 635 nm for 5 min, and its photothermal conversion efficiency is 32.43%. While BN-725 can reach 59 °C under the same conditions, and the photothermal conversion efficiency is 43.25%; at room temperature of 22 °C, using 808 nm (1.5 W / cm 2 ) to irradiate the PBS solution (100 μM) of dye BN-1000, the results show that the temperature of the BN-1000 solution will increase significantly after being irradiated by 808 nm for 5 min, and the highest temperature can reach 67 °C, and its photothermal conversion efficiency is 54.71%, as Figure 8-10 shown. After 6 cycles of cyclic light irradiation heating and cooling, the highest temperatures that BN-725, BN-825, and BN-1000 can reach hardly decrease, indicating that their photothermal stability is strong and the photothermal effect hardly decreases, as Figure 11 shown. Compared with BN-725 and BN-825, BN-1000 has better optical and photothermal properties and has great potential application prospects.

[0097] Example 6 examines the ability of benzopyrylium fluorescent dyes to generate reactive oxygen species

[0098] Under dark conditions, use a 635 nm laser (0.6 W / cm 2 ) to irradiate DCFH-DA, BN-725+DCFH-DA, and BN-825+DCFH-DA solutions or use an 808 nm laser (1 W / cm 2 ) to irradiate DCFH-DA and BN-1000+DCFH-DA solutions for 300 s. The results show that both BN-725 and BN-825, and BN-1000 can effectively generate reactive oxygen species, among which BN-1000 has the strongest ability to generate reactive oxygen species, as Figure 12 shown. To more intuitively show the ability of BN-1000 to generate reactive oxygen species, we use the reactive oxygen species probe DCFH to stain cells. Incubate Hela cells with PBS, Rosup, BN-1000 (20 μM) in the dark environment for 0.5 h, and use an 808 nm laser (1.5 W / cm 2)Irradiate the cells for 5 min. Immediately after the irradiation is completed, stain the cells with the commercial reactive oxygen species probe DCFH for 30 min. The DCFH probe can react with the reactive oxygen species in the cells to produce a green fluorescence signal, and an imaging observation is carried out using a Leica live cell workstation. The results show that BN-1000 can effectively generate reactive oxygen species under 808 nm laser irradiation, as Figure 13 shown.

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

[0100] Irradiate BN-725, BN-825, and BN-1000 (25 μM) with 750 nm and 808 nm lasers (2 W / cm 2 ), and use an LP880 filter to obtain the best signal contrast and image quality. The results show that BN-1000 has the best in vitro fluorescence imaging effect, while most of the energy absorbed by BN-825 may be released in the form of intersystem crossing, resulting in a low fluorescence quantum yield, as Figure 14 shown.

[0101] Example 8 examines the cytotoxicity of benzopyrylium fluorescent dye BN-1000

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

[0103] Example 9 examines the in vivo photothermal imaging of benzopyrylium fluorescent dye BN-1000

[0104] In vivo photothermal imaging experiment: Subcutaneously inject the fluorophore (200 μL, 100 μM, solvent is PBS) into the thigh root of BABL / C nude mice or mice, and at 808 nm, 1 W / cm 2Irradiate with a laser for 10 min, and use an infrared thermal imager to record the temperature change of the tumor every 2 min during the irradiation process.

[0105] Inject PBS and the fluorescent dye BN-1000 (200 μL, 100 μM) in situ at the subcutaneous tumor site in the thigh root of BABL / C nude mice or mice respectively, and then use an 808 nm laser (1.0 W / cm 2 ) to irradiate for 10 min, and use an infrared thermal imager to record the temperature change of the tumor every 2 min during the irradiation process, as Figure 16 shown. The results show that the local temperature of the mice injected with PBS solution at the thigh root did not increase significantly under laser irradiation, while the local temperature of the mice injected with BN-1000 solution increased significantly under laser irradiation, and the highest temperature reached 57 °C. The results indicate that the benzopyrylium fluorescent dye BN-1000 has good photothermal imaging effect. This has potential application prospects in the field of photothermal therapy for tumors.

[0106] Finally, it should be noted that the above description is only the preferred embodiment of the present invention. Under the inspiration of the present invention, those skilled in the art can make various similar representations without violating the purpose and claims of the present invention. Such transformations 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 3, 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 3, characterized in that: The synthetic route of the compound of formula V is as follows:

6. The method according to claim 3, 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 in the preparation of tumor photothermal therapy and / or diagnostic reagents or drugs, wherein: The tumor is Hela cells.

8. Use of the benzopyrylium fluorescent dye molecule prepared by the method according to any one of claims 3 to 6 in the preparation of tumor photothermal therapy and / or diagnostic reagents or drugs, wherein: The tumor is Hela cells.

9. Use of the benzopyrylium fluorescent dye molecule according to claim 1 or 2 in the preparation of a medical imaging preparation, wherein the medical imaging is bioluminescence imaging.

10. Use of the benzopyrylium fluorescent dye molecule prepared by the method according to any one of claims 3 to 6 in the preparation of medical imaging preparations, wherein the medical imaging is bioluminescent imaging.

11. A pharmaceutical composition, characterized in that Contains the benzopyrylium fluorescent dye molecule according to claim 1 or 2 or a pharmaceutically acceptable salt thereof.

12. A pharmaceutical composition, characterized in that Contains a benzopyrylium fluorescent dye molecule or a pharmaceutically acceptable salt thereof prepared by the method described in any one of claims 3 to 6.

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