Preparation method and application of a benzothiadiazole derivative near-infrared fluorescent probe molecule

Abstract

The application discloses a preparation method and application of a benzothiadiazole derivative near-infrared fluorescent probe molecule, and belongs to the technical field of fluorescent sensors. The preparation method comprises the following steps: performing Suzuki coupling reaction on a boronic acid or a boronic ester derivative containing an electron donor unit and 7-bromobenzo[c][1,2,5]thiadiazole-4-formaldehyde to obtain an intermediate compound containing an aldehyde group; performing Knoevenagel condensation reaction on the intermediate compound containing the aldehyde group and 3-ethyl-1,1,2-trimethyl-1H-benzo[e]indol-3-oxonium iodide in a solvent, and after the reaction is completed, the fluorescent probe is obtained through separation and purification. The overall synthesis route of the application is simple, the structure is strong in controllability, the conversion rate is high, by-products are few, and the purification process is simple.

Description

Technical Field

[0001] This invention relates to the field of fluorescence sensor technology, and in particular to a method for preparing and applying a benzothiadiazole derivative near-infrared fluorescent probe molecule. Background Technology

[0002] Cancer has become a major threat to human life and health, and developing new strategies for cancer diagnosis and treatment has been an extremely important research area in recent years. Precise targeting strategies have provided new ideas for the study of cancer-related mechanisms and the development of new diagnostic and therapeutic approaches. In recent years, organelle targeting strategies have shown great potential in cancer imaging and treatment. Among them, mitochondria exhibit significant differences between normal cells and cancer cells; for example, the mitochondrial membrane potential of cancer cells is significantly higher than that of normal cells. These differences make mitochondria an important target for cancer-related research. Mitochondria are the main energy supply site within cells; precise targeting and destruction of mitochondria in cancer cells can achieve precise imaging and treatment of tumor lesions.

[0003] Among existing cancer detection methods, MRI has low resolution and is time-consuming, CT and PET carry radiation risks, and ultrasound is easily interfered with by factors such as cavitation. There is a need to develop new, safe, and efficient diagnostic solutions to meet diverse safety and detection requirements. Fluorescence imaging technology has advantages such as rapid response and non-invasiveness, and has been widely used in clinical applications. In particular, near-infrared fluorescence imaging can significantly reduce background fluorescence interference in biological organisms and has high tissue penetration depth, showing great application potential. On the other hand, cancer treatment options also have various limitations. For example, chemotherapy causes systemic damage, radiotherapy causes radiation damage, and targeted therapy and immunotherapy are prone to drug resistance and are expensive. Therefore, there is a need to develop more safe and effective cancer treatment strategies. Photothermal therapy uses photosensitizers to convert light energy into heat energy, rapidly generating local high temperatures to kill cancer cells. It has advantages such as precision, controllability, safety, and efficiency, and has great application potential in precision cancer treatment. Furthermore, currently, cancer diagnosis and treatment are mostly conducted independently. Achieving integrated diagnosis and treatment is of positive significance for reducing toxic side effects, simplifying the treatment process, and reducing the physical and mental burden on patients. Integrating fluorescence imaging and photothermal therapy functions into the same photosensitizer enables integrated diagnosis and treatment, making it easier to achieve precise diagnosis and treatment of tumors.

[0004] Currently, in the face of the increasing demand for cancer diagnosis and treatment, there is a need to develop more new integrated diagnosis and treatment solutions that can precisely target tumors. This invention discloses a mitochondrial-targeting near-infrared fluorescent probe with photothermal properties, its preparation method and application, especially the application of this probe in cancer imaging and photothermal therapy has broad prospects. Summary of the Invention

[0005] One of the objectives of this invention is to provide a benzothiadiazole derivative near-infrared fluorescent probe molecule to solve the problem that existing technologies cannot integrate fluorescence imaging and photothermal therapy functions in the same photosensitizer.

[0006] This invention is achieved through the following technical solution: a benzothiadiazole derivative near-infrared fluorescent probe molecule, the probe molecule having the structure shown in the following formula:

[0007] Its structural formula is: an R1 group is attached to the 4 position of the benzothiadiazole core, and a vinylidene is attached to the 7 position. The vinylidene is further attached to an N-ethyl-1,1-dimethyl-1H-benzo[e]indolium salt, wherein R1 is selected from substituted or unsubstituted nitrogen-containing heterocyclic groups or substituted or unsubstituted aromatic amino groups.

[0008] Further, R1 is selected from one of the following structures: 1,2,3,4,4a,5,6,7-octahydroquinolinyl; bis(4-methoxyphenyl)amino.

[0009] Furthermore, the molecule is one of the following compounds: compound PI-DHN, whose structure is shown in the following formula:

[0010] ,

[0011] The compound PI-TPN has the following structure:

[0012] .

[0013] Another aspect of the present invention provides a method for preparing a benzothiadiazole derivative near-infrared fluorescent probe molecule. The benzothiadiazole derivative near-infrared fluorescent probe molecule as described above is prepared according to the following steps: a boric acid or borate ester derivative containing an electron donor unit is subjected to a Suzuki coupling reaction with 7-bromobenzo[c][1,2,5]thiadiazole-4-carboxaldehyde to obtain an aldehyde-containing intermediate compound; the aldehyde-containing intermediate compound is subjected to a Knoevenagel condensation reaction with 3-ethyl-1,1,2-trimethyl-1H-benzo[e]indole-3-onium iodide in a solvent; after the reaction is completed, the fluorescent probe is obtained by separation and purification.

[0014] Furthermore, when preparing the intermediate of compound PI-DHN, the derivative of the electron donor unit is (1,2,3,5,6,7-hexahydropyridino[3,2,1-ij]quinoline-9-yl)boronic acid.

[0015] Furthermore, when preparing the intermediate of compound PI-TPN, the derivative of the electron donor unit is (4-(bis(4-methoxyphenyl)amino)phenyl)boronic acid.

[0016] Furthermore, the solvent is anhydrous ethanol, the reaction temperature is 75–85°C, and the reaction time is 8–12 hours.

[0017] Furthermore, the Suzuki coupling reaction uses tetra(triphenylphosphine)palladium as the catalyst, potassium carbonate as the reaction base, and a mixture of tetrahydrofuran and water as the reaction solvent. The reaction is carried out under reflux under nitrogen protection.

[0018] In another aspect, the present invention also provides an application of a benzothiadiazole derivative near-infrared fluorescent probe molecule. The fluorescent probe molecule prepared according to the above-mentioned benzothiadiazole derivative near-infrared fluorescent probe molecule or according to the above-mentioned method for preparing a benzothiadiazole derivative near-infrared fluorescent probe molecule has the following applications: (1) application in the preparation of bioimaging reagents; (2) application in the preparation of tumor photothermal therapy drugs.

[0019] Furthermore, the bioimaging reagent is used for targeted near-infrared fluorescence imaging of tumor cell mitochondria.

[0020] Furthermore, the tumor photothermal therapy drug specifically includes: mitochondrial-targeted fluorescence imaging: utilizing the benzo[e]indolium cation in the probe molecule to specifically target the mitochondria of tumor cells and generate a fluorescence signal in the near-infrared region (>650 nm) for imaging; photothermal therapy: utilizing the photothermal conversion properties of the probe under 660 nm laser irradiation to convert light energy into heat energy to kill tumor cells.

[0021] Furthermore, photothermal therapy is based on a photothermal effect that is both probe concentration-dependent and laser power density-dependent, wherein the probe, when irradiated by a 660 nm laser at 0.5 W / cm², can generate high temperatures at the tumor site sufficient to kill cancer cells.

[0022] In another aspect, the present invention provides a mitochondrial-targeted photothermal therapy formulation comprising a benzothiadiazole derivative near-infrared fluorescent probe molecule as described above, and a pharmaceutically acceptable carrier or excipient.

[0023] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0024] 1. This invention constructs a DA-π-A' conjugated system with ultralong electron cloud delocalization capability by introducing a strong electron-donating group (R, such as octahydroquinolinyl or di(4-methoxyphenyl)amino) at one end of the benzothiadiazole core and bridging the other end with a strong electron-withdrawing benzoindolium cation via a vinylene bridge. This structure significantly reduces the band gap of the molecule, redshifting the fluorescence emission wavelength to the near-infrared region I (840 nm). This wavelength falls within the transparent window of biological tissues, significantly reducing background interference from autofluorescence and greatly improving tissue penetration depth, thus providing the possibility for precise imaging of deep tumors. Through a strong intramolecular charge transfer (ICT) effect, the probe, when irradiated by a 660 nm laser, can convert absorbed light energy into heat energy through an efficient non-radiative transition pathway. Combined with experimental data, this is achieved at extremely low concentrations (100 μM) and low power densities (0.5 μM). At a temperature of W / cm², the temperature rise can reach over 48.8℃, which is sufficient to kill tumor cells, thus achieving highly efficient photothermal therapy (PTT) under imaging guidance.

[0025] 2. This invention utilizes the inherent positive charge of the benzoindoline group and cleverly takes advantage of the biological characteristic that the mitochondrial membrane potential of tumor cells is significantly higher than that of normal cells to achieve an active targeting mechanism. The probe molecules can penetrate multiple biological membranes through electrostatic attraction and actively accumulate in the mitochondria of tumor cells. Experiments have confirmed that the Pearson correlation coefficient between the probe of this invention and commercial mitochondrial dyes is as high as 0.89, while the localization correlation with lipid droplets and cell nuclei is extremely low (0.19 and 0.01, respectively). This extremely high subcellular localization accuracy is not only beneficial for monitoring mitochondrial-related metabolic changes, but also can destroy cancer cells from the inside by generating heat in situ, significantly improving treatment efficiency.

[0026] 3. The probe of this invention exhibits extremely significant environmentally sensitive fluorescence characteristics, which have important application value in complex organisms: background suppression, the probe has extremely weak fluorescence (in a quenched state) in aqueous physiological environments such as PBS, but the fluorescence intensity increases dramatically after entering the intracellular lipid environment or high-viscosity microenvironment (such as glycerol environment); intelligent response: this spot-lighting imaging feature effectively avoids ineffective luminescence during the circulation process, significantly improves the signal-to-noise ratio of imaging, and can accurately delineate the boundaries of tumor lesions.

[0027] 4. The probe of this invention, while possessing strong photothermal killing properties, exhibits extremely high biocompatibility under light-free conditions: at the effective working concentration, the survival rate of tumor cells remains above 90%, demonstrating excellent cell compatibility; this controllable characteristic of being non-toxic in the absence of light but highly toxic when photosensitive can significantly reduce the systemic toxic side effects of traditional chemotherapy drugs, alleviate the physical and mental burden on patients, and has good prospects for clinical translation; moreover, the overall synthetic route is simple and the structure is highly tunable. Through the two-step reaction of Suzuki coupling and aldehyde-amine condensation, the conversion rate is high, the byproducts are few, and the purification process is simple (high-purity products can be obtained by column chromatography). By changing the R group in the starting material, the absorption curve and photothermal conversion efficiency of the molecule can be sensitively adjusted, providing great chemical flexibility for customizing personalized probes for different types of cancer. Attached Figure Description

[0028] The accompanying drawings, which are included to provide a further understanding of embodiments of the invention and form part of this application, do not constitute a limitation thereof. In the drawings:

[0029] Figure 1 This is a reference diagram of the reaction route provided in Embodiment 1 of the present invention.

[0030] Figure 2 Compound 3a provided in Example 1 of this invention 1 HNMR analysis diagram.

[0031] Figure 3 The PI-DHN provided in Embodiment 1 of the present invention 1 HNMR analysis diagram.

[0032] Figure 4 Compound 3b provided in Example 2 of this invention 1 HNMR analysis diagram.

[0033] Figure 5 The PI-TPN provided in Embodiment 2 of the present invention 1 HNMR analysis diagram.

[0034] Figure 6 The absorption spectrum of the probe PI-DHN provided in Embodiment 3 of the present invention is shown.

[0035] Figure 7 The absorption spectrum of the probe PI-TPN provided in Embodiment 3 of the present invention is shown.

[0036] Figure 8 The fluorescence emission spectrum of the probe PI-DHN provided in Embodiment 3 of the present invention is shown.

[0037] Figure 9The fluorescence emission spectrum of the probe PI-TPN provided in Embodiment 3 of the present invention is shown.

[0038] Figure 10 The image shows the photothermal effect detection results of the probe PI-DHN provided in Embodiment 4 of the present invention.

[0039] Figure 11 The image shows the photothermal effect detection results of the probe PI-DHN provided in Embodiment 4 of the present invention under different laser powers.

[0040] Figure 12 The image shows the photothermal effect detection results of the probe PI-TPN provided in Embodiment 4 of the present invention.

[0041] Figure 13 The image shows the photothermal effect detection results of PI-TPN under different laser powers provided in Embodiment 4 of the present invention.

[0042] Figure 14 This is a diagram showing the toxicity effect of the probe PI-TPN provided in Example 5 of the present invention on tumor cells.

[0043] Figure 15 This is a co-localization map of organelles after co-incubation with tumor cells using the probe PI-DHN and different commercially available organelle dyes provided in Example 6 of the present invention. Detailed Implementation

[0044] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are some embodiments of the present invention, but not all embodiments.

[0045] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated herein by reference to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail. The terms “comprising,” “including,” “having,” “containing,” etc., as used herein are open-ended terms, meaning that they include but are not limited to. Unless the context clearly indicates otherwise, the expressions “a” and “an” as used herein include plural references. It should be noted that “first,” “second,” etc., are used merely for convenience of description and distinction and should not be construed as indicating or implying relative importance. The term “about” as used herein indicates a range of ±20% of the following numerical value. In some embodiments, the term “about” indicates a range of ±10% of the following numerical value. In some embodiments, the term “about” indicates a range of ±5% of the following numerical value.

[0046] Materials and reagents used in the following examples: (1,2,3,5,6,7-hexahydropyrido[3,2,1-ij]quinoline-9-yl)boronic acid was purchased from Shanghai Aichun Biotechnology Co., Ltd. 7-Bromobenzo[c][1,2,5]thiadiazole-4-carboxaldehyde was purchased from Shanghai Dibai Biotechnology Co., Ltd. (4-(di(4-methoxyphenyl)amino)phenyl)boronic acid, etc., were purchased from Shanghai Haohong Biomedical Technology Co., Ltd. 3-Ethyl-1,1,2-trimethyl-1H-benzo[e]indole-3-onium iodide was purchased from Jilin Zhongke Research Technology Co., Ltd. Potassium carbonate, etc., was purchased from Shanghai Titan Technology Co., Ltd.

[0047] Example 1

[0048] This embodiment provides the specific steps for the synthesis of the benzothiadiazole derivative PI-DHN. Figure 1 A reaction route reference diagram is shown in this embodiment. The diagram illustrates the following steps:

[0049] Step 1: Synthesis of intermediate compound 3a. In a 50 mL two-necked flask, (1,2,3,5,6,7-hexahydropyrido[3,2,1-ij]quinoline-9-yl)boronic acid (compound 1a, 200.0 mg, 0.92 mmol), 7-bromobenzo[c][1,2,5]thiadiazole-4-carboxaldehyde (compound 2, 224.0 mg, 0.92 mmol), potassium carbonate (585.1 mg, 4.24 mmol), and tetra(triphenylphosphine)palladium (106.3 mg, 0.092 mmol) were added sequentially.

[0050] Under nitrogen protection, 10 mL of tetrahydrofuran (THF) and 5 mL of deionized water were injected, and the mixture was heated to reflux temperature and stirred for 12 hours. After the reaction was completed, the reaction solution was cooled to room temperature, and the organic solvent was removed by vacuum rotary evaporation.

[0051] The remaining solid was purified by silica gel column chromatography to prepare a purple solid compound 3a (213.9 mg), with a yield of 69.3%. NMR spectroscopy was performed on the prepared solid compound 3a to obtain the... 1 HNMR analysis graph as follows Figure 2 As shown in the figure. The representation data in the figure are:

[0052] ¹HNMR(400MHz,CDCl3)δ10.67(s,1H),8.20(d,J=7.3Hz,1H),7.75(d,J=7.4Hz,1H) ,7.60(s,2H),3.28(t,J=5.2Hz,4H),2.87(t,J=5.2Hz,4H),2.03(t,J=5.2Hz,4H).

[0053] Step 2: Synthesis of probe PI-DHN: 3-ethyl-1,1,2-trimethyl-1H-benzo[e]indole-3-onium iodide (compound 4, 108.8 mg, 0.298 mmol) and compound 3a (100 mg, 0.298 mmol) prepared in step 1 were added to a 25 mL two-necked flask.

[0054] Under nitrogen protection, 10 mL of anhydrous ethanol was added to dissolve the compound. The mixture was heated and refluxed at 80 °C with stirring for 8 hours. After the reaction was complete, the mixture was cooled to room temperature, and the solvent was removed under vacuum. The crude product was purified by silica gel column chromatography to obtain a dark green solid target compound PI-DHN (63.8 mg), with a yield of 31.4%. NMR spectroscopy was performed on the prepared target compound PI-DHN to obtain... 1 HNMR analysis graph as follows Figure 3 As shown in the figure. The representation data in the figure are:

[0055] 1HNMR(400MHz,DMSO)δ8.85(d,J=16.1Hz,1H),8.64(dd,J=15.0,12.1Hz,2H),8.48(d,J=8. 4Hz,1H),8.32(d,J=8.9Hz,1H),8.24(d,J=8.2Hz,1H),8.18(d,J=9.0Hz,1H),8.04(d,J=7 .9Hz,1H),7.83(d,J=11.1Hz,1H),7.78–7.71(m,1H),4.80(dd,J=14.4,7.1Hz,2H),3.33– 3.26(m,4H),2.82(d,J=6.1Hz,3H),2.11(s,5H),1.99–1.90(m,4H),1.63(t,J=7.2Hz,3H).

[0056] Example 2

[0057] This embodiment provides the specific synthesis steps for the target compound PI-TPN.

[0058] Step 1: Synthesis of intermediate compound 3b. In a 50 mL two-necked flask, (4-(di(4-methoxyphenyl)amino)phenyl)boronic acid (compound 1b, 100 mg, 0.286 mmol), 7-bromobenzo[c][1,2,5]thiadiazole-4-carboxaldehyde (compound 2, 76.5 mg, 0.315 mmol), potassium carbonate (395.3 mg, 2.86 mmol), and tetra(triphenylphosphine)palladium (10.7 mg, 0.009 mmol) were added.

[0059] Under nitrogen protection, 5 mL of tetrahydrofuran and 2 mL of deionized water were added. The mixture was heated under reflux and stirred for 12 hours. After the reaction was complete, the mixture was cooled to room temperature, the solvent was removed under vacuum, and the solid was separated by column chromatography to give a red solid compound 3b (91.5 mg), with a yield of 68.5%. NMR analysis of the prepared solid compound 3b yielded [data missing]. 1 HNMR analysis graph as follows Figure 4 As shown in the figure, the representative data are:

[0060] ¹HNMR(400MHz,DMSO-d6)δ10.61(s,1H),8.31(d,J=7.5Hz,1H),7.99(t,J=7.7Hz,3H ),7.15(d,J=8.9Hz,5H),6.98(d,J=8.9Hz,4H),6.87(d,J=8.8Hz,2H),3.77(s,6H).

[0061] Step 2: Synthesis of probe PI-TPN: 3-ethyl-1,1,2-trimethyl-1H-benzo[e]indole-3-onium iodide (compound 4, 88 mg, 0.243 mmol) and compound 3b obtained in step 1 (100 mg, 0.213 mmol) were added to a 25 mL reaction flask.

[0062] Under nitrogen protection, 10 mL of anhydrous ethanol was added to dissolve the compound. The mixture was heated and stirred at 80 °C for 8 hours. After the reaction was complete, the mixture was cooled to room temperature, and the solvent was removed under vacuum. The solid was separated by column chromatography to obtain a dark blue solid, PI-TPN (74.4 mg), with a yield of 42.8%. NMR spectroscopy was performed on the prepared PI-TPN compound to obtain the desired NMR concentration. 1 HNMR analysis graph as follows Figure 5 As shown in the figure. The representation data in the figure are:

[0063] ¹HNMR(400MHz,DMSO-d6)δ8.88(d,J=16.2Hz,1H),8.73–8.66(m,2H),8.50(d,J=8.4Hz,1H) ,8.34(d,J=9.0Hz,1H),8.25(d,J=8.1Hz,1H),8.21(d,J=9.1Hz,1H),8.09(d,J=8.6Hz,2H) ,7.85(t,J=7.6Hz,1H),7.80–7.72(m,1H),7.16(d,J=8.9Hz,3H),7.00(d,J=8.9Hz,3H),6. 90(d,J=8.8Hz,2H),4.84(d,J=7.4Hz,2H),3.78(s,5H),2.11(s,5H),1.64(t,J=7.2Hz,3H).

[0064] Example 3

[0065] In this embodiment, the UV-Vis absorption spectra and fluorescence emission spectra of the probes PI-DHN and PI-TPN prepared in Examples 1 and 2 in different solvents were tested respectively.

[0066] The test solvents were: phosphate buffered saline (PBS), dimethyl sulfoxide (DMSO), methanol (MeOH), N,N-dimethylformamide (DMF), acetonitrile (MeCN), dichloromethane (DCM), and glycerol (Glycerol).

[0067] Absorption spectrum:

[0068] PI-DHN: The maximum absorption wavelength range is 503–772 nm, exhibiting the largest absorption wavelength in dichloromethane (DCM). The absorption spectrum detection results of the PI-DHN probe are as follows... Figure 6 As shown in the figure, the maximum absorption wavelength of the probe PI-DHN varies greatly in different solvents, ranging from 503 to 772 nm, with the maximum absorption wavelength being the largest in DCM.

[0069] PI-TPN: The maximum absorption wavelength range is 597–683 nm, and it also exhibits the maximum absorption wavelength in dichloromethane (DCM). The absorption spectrum detection results of the PI-TPN probe are as follows... Figure 7 As shown in the figure, the maximum absorption wavelength of the probe PI-TPN varies greatly in different solvents, ranging from 597 to 683 nm. Similar to PI-DHN, the maximum absorption wavelength is the largest in DCM.

[0070] Conclusion: Both probes exhibited significant solvation colorimetric effects.

[0071] Fluorescence emission spectrum:

[0072] PI-DHN: No significant fluorescence emission was observed near 840 nm in low-viscosity solvents such as PBS, DMSO, and MeOH. Only in glycerol did it exhibit significant strong fluorescence emission at 840 nm. The fluorescence emission spectrum of the PI-DHN probe is shown below. Figure 8 As shown in the figure, only glycerol exhibited significant fluorescence emission at 840 nm among the different solvents examined. This emission wavelength falls within the near-infrared region I, which is advantageous for the application of the probe in in vivo fluorescence imaging. The other solvents did not show significant fluorescence emission near 840 nm.

[0073] PI-TPN: Similar to PI-DHN, it exhibits no significant fluorescence in low-viscosity solvents, showing fluorescence emission only in glycerol, but this emission is weaker than that of PI-DHN. The fluorescence emission spectrum detection results of the PI-TPN probe are as follows... Figure 9 As shown in the figure, unlike PI-DHN, the probe PI-TPN did not show obvious fluorescence emission peaks in different solvents under the laser wavelength investigated.

[0074] As can be seen from this embodiment, the probe has typical intramolecular charge transfer (ICT) characteristics and environmental viscosity sensitivity (TICT mechanism), and its emission wavelength is located in the near-infrared region I (NIR-I), which is beneficial for imaging deep tissues in vivo.

[0075] Example 4

[0076] This embodiment is used to evaluate the probe's ability to convert light energy into heat energy. The specific test method is as follows:

[0077] The probe PI-DHN prepared in Example 1 or the PI-TPN prepared in Example 2 was dissolved in DMSO at a certain concentration. The solution was irradiated with a 660nm laser at different powers, and the temperature change of the solution was monitored in real time with a thermal imager, and recorded every 30 seconds.

[0078] The photothermal effect detection results of probes PI-DHN at different concentrations are shown in the figure. Figure 10 As the irradiation time increases, the solution temperature gradually rises. Increasing the concentration of the probe can continuously increase the temperature difference of the solution. At a power of 0.5 W / cm2, a probe solution with a concentration of 100 μM can achieve a temperature difference of 48.8 °C.

[0079] The photothermal effect detection results of the PI-DHN probe at different laser powers are as follows: Figure 11 As shown in the figure, the temperature of the 50 μM probe solution increases with increasing irradiation time. Increasing the laser irradiation power can continuously increase the temperature difference of the solution; at a power of 0.5 W / cm², the probe solution can achieve a temperature difference of 29.8 °C.

[0080] The photothermal effect detection results of probe PI-TPN at different concentrations are as follows: Figure 12 As shown in the figure, the solution temperature gradually increases with the increase of irradiation time. Increasing the concentration of the probe can continuously increase the temperature difference of the solution. At a power of 0.5 W / cm2, a probe solution with a concentration of 100 μM can achieve a temperature difference of 49.4 °C.

[0081] The photothermal effect detection results of the PI-TPN probe at different laser powers are shown in the figure. Figure 13 The temperature of the 50 μM probe solution increased with increasing irradiation time. Increasing the laser irradiation power could continuously increase the temperature difference of the solution; at a power of 0.5 W / cm², the probe solution could achieve a temperature difference of 37.7 °C.

[0082] In summary, the probes PI-DHN and PI-TPN exhibit strong photothermal properties and have potential application value in tumor photothermal therapy.

[0083] Example 5

[0084] This example is used to evaluate the safety of the probe prepared in Example 2 in vivo. The specific experimental methods are as follows:

[0085] 1. Select KHOS-240S cells and seed them in 96-well plates and culture for 24 hours.

[0086] 2. Add PI-TPN solution diluted with culture medium (prepared by the method in Example 2) to make final concentrations of 0, 0.25, 0.5, 1 and 2 μM.

[0087] 3. Incubate at 37°C for 6 hours in a cell culture incubator.

[0088] 4. Carefully aspirate the culture medium and wash twice with PBS.

[0089] 5. Add fresh culture medium containing CCK-8 reagent and continue incubation for 2 hours.

[0090] 6. Use an ELISA reader to measure the absorbance of each well and calculate the cell viability.

[0091] Figure 14 The cytotoxicity of the probe PI-TPN against tumor cells is shown in the figure. As can be seen, the survival rate of KHOS-240S cells did not decrease significantly with increasing PI-TPN concentration; even at the highest tested concentration of 2 μM, cell survival remained above 90%. This indicates that this type of probe has good biocompatibility and low cytotoxicity, making it suitable for bioimaging and therapeutic applications.

[0092] Example 6

[0093] This embodiment is used to verify the specific targeting ability of the probe prepared in Example 1 to tumor cell mitochondria. The specific verification method is as follows:

[0094] 1. Seed 4T1 cells into a confocal culture dish and culture for 24 hours.

[0095] 2. Add PI-DHN molecules diluted in culture medium and incubate at 37°C for 30 minutes in the dark.

[0096] 3. Add commercially available dye diluted in the culture medium: Mito-TrackerGreen (mitochondrial green fluorescent probe) Or BODIPY493 / 503 (lipid drop dye) Or Hoechst33342 (nuclear dye)

[0097] 4. Continue incubation in the dark for another 30 minutes.

[0098] 5. After washing with PBS, images were taken using a confocal laser scanning microscope, and the Pearson correlation coefficient was calculated.

[0099] Figure 15 The figure shows colocalization maps of organelles after co-incubation with tumor cells using the probe PI-DHN and various commercially available organelle dyes. The calculated Pearson correlation coefficients are as follows:

[0100] R values ​​were 0.19 (BODIPY 493 / 503), 0.01 (Hoechest 33342), and 0.89 (Mito-TrackerGreen). The probe showed a high Pearson correlation coefficient with commercial mitochondrial probes, demonstrating its excellent targeting performance to mitochondria. The PI-DHN probe utilizes its intramolecular benzoindoline cation charge to specifically target the mitochondria of tumor cells, consistent with its differential response mechanism of mitochondrial membrane potential between normal and cancer cells, achieving precise subcellular organelle localization imaging. This indicates the probe has potential application value in mitochondrial-related tumor imaging and tumor therapy.

[0101] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A near-infrared fluorescent probe molecule of a benzothiadiazole derivative, characterized in that, The probe molecule has the structure shown in the following formula: ， Its structural formula is: an R1 group is attached to the 4-position of the benzothiadiazole core, and a vinylidene is attached to the 7-position. The vinylidene is further attached to an N-ethyl-1,1-dimethyl-1H-benzo[e]indolium salt. R1 is selected from substituted or unsubstituted nitrogen-containing heterocyclic groups or substituted or unsubstituted aromatic amino groups.

2. The benzothiadiazole derivative near-infrared fluorescent probe molecule according to claim 1, characterized in that, R1 is selected from one of the following structures: 1,2,3,4,4a,5,6,7-octahydroquinolinyl; Di(4-methoxyphenyl)amino.

3. The benzothiadiazole derivative near-infrared fluorescent probe molecule according to claim 1, characterized in that, The molecule is one of the following compounds: The compound PI-DHN has the following structure: The compound PI-TPN has the following structure: 。 4. A method for preparing a near-infrared fluorescent probe molecule of a benzothiadiazole derivative, characterized in that, The benzothiadiazole derivative near-infrared fluorescent probe molecule according to any one of claims 1 to 3 is prepared according to the following steps: A boric acid or borate ester derivative containing an electron donor unit is subjected to a Suzuki coupling reaction with 7-bromobenzo[c][1,2,5]thiadiazole-4-carboxaldehyde to obtain an intermediate compound containing an aldehyde group. The aldehyde-containing intermediate compound was subjected to a Knoevenagel condensation reaction with 3-ethyl-1,1,2-trimethyl-1H-benzo[e]indole-3-onium iodide in a solvent. After the reaction was completed, the fluorescent probe was obtained by separation and purification.

5. The method for preparing the benzothiadiazole derivative near-infrared fluorescent probe molecule according to claim 4, characterized in that, When preparing the intermediate of compound PI-DHN, the derivative of the electron donor unit is (1,2,3,5,6,7-hexahydropyridino[3,2,1-ij]quinoline-9-yl)boronic acid; When preparing the intermediate of compound PI-TPN, the derivative of the electron donor unit is (4-(bis(4-methoxyphenyl)amino)phenyl)boronic acid.

6. The method for preparing the benzothiadiazole derivative near-infrared fluorescent probe molecule according to claim 4, characterized in that, The solvent is anhydrous ethanol, the reaction temperature is 75-85℃, and the reaction time is 8-12 hours.

7. The method for preparing the benzothiadiazole derivative near-infrared fluorescent probe molecule according to claim 4, characterized in that, The catalyst used in the Suzuki coupling reaction is tetra(triphenylphosphine)palladium. The reaction base is potassium carbonate, and the reaction solvent is a mixture of tetrahydrofuran and water. The reaction is carried out under reflux under nitrogen protection.

8. The application of a benzothiadiazole derivative near-infrared fluorescent probe molecule, characterized in that, The benzothiadiazole derivative near-infrared fluorescent probe molecule has the molecular structure described in any one of claims 1 to 3, or the infrared fluorescent probe molecule prepared according to the preparation method described in any one of claims 4 to 7. Including the following applications: (1) Application in the preparation of biological imaging reagents; (2) Application in the preparation of tumor photothermal therapy drugs.

9. The application of the benzothiadiazole derivative near-infrared fluorescent probe molecule according to claim 8, characterized in that, The bioimaging reagent is for targeted near-infrared fluorescence imaging of tumor cell mitochondria; The tumor photothermal therapy drugs specifically include: Mitochondrial targeted fluorescence imaging: The probe molecule specifically targets the mitochondria of tumor cells using the benzo[e]indolium cation, and generates a fluorescence signal in the near-infrared region (>650 nm) for imaging. Photothermal therapy: Utilizing the photothermal conversion properties of the probe under 660 nm laser irradiation, light energy is converted into heat energy to kill tumor cells. The photothermal therapy is based on a probe concentration-dependent and laser power density-dependent photothermal effect, wherein the probe, under irradiation with a 660 nm laser at 0.5 W / cm², can generate high temperatures at the tumor site sufficient to kill cancer cells.

10. A mitochondrial-targeted photothermal therapy formulation, characterized in that, The present invention comprises an effective dose of the benzothiadiazole derivative near-infrared fluorescent probe molecule as described in claim 1, and a pharmaceutically acceptable carrier or excipient.

Images