2,1,3-benzothiadiazole and spiropyran conjugated fluorescent compounds and their applications in lipid droplet imaging

Abstract

The application belongs to the field of organic photo fluorescent dyes, and relates to a preparation method of a 2,1,3-benzothiadiazole-spiropyran conjugated fluorescent compound, photophysical property research and application of the 2,1,3-benzothiadiazole-spiropyran conjugated fluorescent compound in lipid droplet imaging. The chemical structure is shown in formula I: wherein R1 is selected from C1-C6 linear or branched alkyl, ether group or C1-C6 alkyl substituted acyl; and R2 is hydrogen, methyl, substituted alkyl, fluorine, chlorine, bromine, iodine, cyano, trihalomethyl, nitro or dimethylamino. The 2,1,3-benzothiadiazole-spiropyran conjugated fluorescent compound provided by the application not only has excellent reversible photochromic characteristics, but also has acid-induced color change and strong solution and solid-state fluorescence. Importantly, the probe has low toxicity, good biocompatibility, can accurately target cell lipid droplets and realize high-contrast fluorescence imaging, which has important significance for further applying the probe to research diseases caused by lipid disorders such as obesity, atheromatous arteriosclerosis and the like.

Description

Technical Field

[0001] This invention belongs to the field of organic photofluorescent dyes and relates to fluorescent compounds conjugated with 2,1,3-benzothiadiazole and spiropyran and their application in lipid droplet imaging. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Currently, spiropyrans, as promising photochromic dyes, have wide applications in anti-counterfeiting smart materials and biosensing. When stimulated by external environments (such as ultraviolet-visible light, acid / base, polarity, etc.), these molecules transform from a lipophilic spiropyran ring-closed structure to an open-ring structure with a positively charged π extension. More importantly, these two isomers typically exhibit drastically different physicochemical properties. Therefore, researchers have conducted extensive studies on their ring-switching mechanism and properties over the years. However, to date, fluorescent probes designed for the spiropyran skeleton are still relatively few, and some significant drawbacks exist. For example, modifications to spiropyran derivatives are mostly located at the sites of benzopyran and indole nitrogen atoms, while modifications to the right-hand indole benzene ring are less common. In addition, the low solid-state fluorescence quantum yield of spiropyran derivatives greatly limits their applications in organic light-emitting diodes, bioimaging, and other fields.

[0004] Fluorescent probes can be used for lipid droplet imaging and dynamic tracking. However, most current lipid fluorescent probes suffer from insufficient imaging contrast, limiting their ability to target not only lipid droplets but also other intracellular lipid structures, such as the endoplasmic reticulum. Furthermore, some key imaging parameters of current lipid droplet fluorescent probes, such as fluorescence quantum yield and imaging contrast, still require further improvement and refinement. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides fluorescent compounds conjugated with 2,1,3-benzothiadiazole and spiropyran, and their application in lipid droplet imaging. Due to their rigid donor-acceptor structure, these fluorescent dyes exhibit strong solution and solid-state dual-state fluorescence, along with advantages such as high permeability and low toxicity, enabling specific and high-contrast visualization imaging of intracellular lipid droplets.

[0006] In a first aspect, the present invention provides a fluorescent compound conjugated with 2,1,3-benzothiadiazole and spiropyran, the chemical structure of which is shown in Formula I:

[0007]

[0008] Wherein, R1 is selected from C1-C6 straight-chain or branched hydrocarbon groups, ether groups, or C1-C6 alkyl-substituted acyl groups; R2 is hydrogen, methyl, substituted alkyl, fluorine, chlorine, bromine, iodine, cyano, trihalomethyl, nitro, or dimethylamino.

[0009] In a second aspect, a method for synthesizing the above-mentioned fluorescent compound conjugated with 2,1,3-benzothiadiazole and spiropyran is provided, comprising the step of obtaining the compound shown in Formula I according to the following reaction route;

[0010]

[0011] Compound 1 is converted into an indole salt via a simple alkylation reaction, and then reacted with compound 3 via a Knoevenagel condensation reaction to obtain spiropyran derivative 4. Finally, compound 4 and 5 are coupled via a simple Suzuki coupling reaction to obtain the final compound I. In this reaction, R1 is selected from C1-C6 straight-chain or branched hydrocarbon groups, ether groups, or C1-C6 alkyl-substituted acyl groups; R2 is hydrogen, methyl, substituted alkyl, fluorine, chlorine, bromine, iodine, cyano, trihalomethyl, nitro, or dimethylamino.

[0012] This invention is based on commercially available materials and can synthesize spiropyran derivatives through a simple three-step reaction. The process is simple and the reaction conditions are not required.

[0013] Thirdly, the application of a fluorescent compound conjugated with the above-mentioned 2,1,3-benzothiadiazole and spiropyran in a fluorescent probe.

[0014] The fluorescent compound provided by this invention, which is conjugated with 2,1,3-benzothiadiazole and spiropyran, exhibits strong bistate fluorescence in nonpolar solvents and in solids; at the same time, it also has photochromic properties.

[0015] Fourthly, a composition comprising the above-described fluorescent compound conjugated with 2,1,3-benzothiadiazole and spiropyran, or a pharmaceutically acceptable salt thereof.

[0016] Fifthly, a formulation comprising an active ingredient and a pharmaceutical carrier, said active ingredient being a fluorescent compound or composition conjugated with the above-mentioned 2,1,3-benzothiadiazole and spiropyran.

[0017] Sixthly, the application of a fluorescent compound, composition, or formulation of the above-mentioned 2,1,3-benzothiadiazole and spiropyran conjugated together in lipid droplet-specific fluorescence imaging.

[0018] In a seventh aspect, a lipid droplet detection kit includes the above-mentioned fluorescent compound, composition or formulation conjugated with 2,1,3-benzothiadiazole and spiropyran, as well as a solvent or diluent.

[0019] The beneficial results of this invention are:

[0020] This invention introduces a derivative of 2,1,3-benzothiadiazole, a strong acceptor, into the spiropyran backbone via conjugated coupling. This derivative retains excellent reversible photochromic properties while also exhibiting acid-induced color change and strong solution and solid-state fluorescence. Importantly, this probe exhibits low toxicity and good biocompatibility, enabling precise targeting of cellular lipid droplets and achieving high-contrast fluorescence imaging. This is significant for its further application in researching lipid-related diseases such as obesity and atherosclerosis. Attached Figure Description

[0021] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0022] Figure 1 To observe the single-crystal structures of BT-SP-NO2 and BT-SP-NMe2 from different directions. C: gray; H: white; N: blue; O: red and S: yellow.

[0023] Figure 2 A represents the normalized absorption spectra of BT-SP-NO2 and BT-SP-NMe2 in n-hexane solution. Figure 2 B and Figure 2 C represents the fluorescence emission spectra of BT-SP-NO2 and BT-SP-NMe2 in different solvents. Figure 2 D represents the fluorescence quantum yield of BT-SP-NO2 and BT-SP-NMe2 in different solvents. Concentration: 10 μM.

[0024] Figure 3 A and Figure 3 B represents the fluorescence emission spectra of BT-SP-NO2 and BT-SP-NMe2 in different DMSO / water mixtures, showing different f values. w value. Figure 3 C is a graph showing the relationship between fluorescence intensity I / I0 and the composition of the DMSO / water mixture. Figure 3 D represents the normalized fluorescence spectra of BT-SP-NO2 and BT-SP-NMe2 in the solid state. Inset: Solid-state fluorescence images of BT-SP-NO2 and BT-SP-NMe2 taken under a 365 nm UV lamp, along with their solid-state fluorescence quantum yields (QY). Concentration: 10 μM.

[0025] Figure 4 A and Figure 4 B represents the dynamic light scattering (DLS) data of BT-SP-NO2 and BT-SP-NMe2 in a DMSO / water mixture containing 30% DMSO.

[0026] Figure 5 A and Figure 5 B represents the response spectra of BT-SP-NO2 and BT-SP-NMe2 to trifluoroacetic acid (TFA) in THF. Concentration: 10 μM.

[0027] Figure 6 A and Figure 6 B represents the normalized absorption spectra of BT-SP-NO2 and BT-SP-NMe2 in tetrahydrofuran solution after different UV irradiations. Figure 6 C and Figure 6 D represents the fluorescence spectra obtained by exciting BT-SP-NO2 in tetrahydrofuran solution with different excitation lights after the same irradiation time. Figure 6 E is a schematic diagram of the isomerization transition between BT-SP-NO2 and BT-MC-NO2. Concentration: 10 μM.

[0028] Figure 7 The fluorescence intensity values ​​of BT-SP-NO2 in tetrahydrofuran solution after 6 cycles of alternating UV-Vis irradiation. UV: 30s; Vis: 3min; Concentration: 10μM.

[0029] Figure 8 A represents the spatial electron distribution of HOMO and LUMO in the optimized ground state of BT-SP-NO2 and BT-SP-NMe2. Figure 8 B represents the molecular packing of BT-SP-NO2 and BT-SP-NMe2 crystals in different directions.

[0030] Figure 9 A and Figure 9 B represents the intramolecular dihedral angles of BT-SP-NO2 and BT-SP-NMe2 molecules, respectively.

[0031] Figure 10 A and Figure 10 B represents the distance between the spirocarbon atom on one molecule and the spirocarbon atoms on the other three molecules within the unit cell of BT-SP-NO2 and BT-SP-NMe2, respectively. (Dashed line unit:)

[0032] Figure 11 The in situ spectra of BT-SP-NO2 and BT-SP-NMe2 in HeLa cells are shown.

[0033] Figure 12CLSM images of HeLa cells incubated with BT-SP-NO2 or BT-SP-NMe2 and Lipi-Deep Red. Scale bar: 10 μm. Probe concentrations: BT-SP-NO2 (500 nM), BT-SP-NMe2 (1 μM), Lipi-Deep Red (200 nM).

[0034] Figure 13 The cytotoxicity of BT-SP-NO2 and BT-SP-NMe2 at different concentrations in HeLa cells was investigated.

[0035] Figure 14 The hydrogen spectrum of compound 2 prepared in Example 1 of this invention in deuterated DMSO.

[0036] Figure 15 The hydrogen spectrum of compound 4 prepared in Example 2 of this invention in deuterated DMSO.

[0037] Figure 16 The hydrogen spectrum of compound 7 prepared in Example 2 of this invention in deuterated DMSO.

[0038] Figure 17 The hydrogen spectrum of BT-SP-NO2 prepared in Example 3 of this invention in deuterated DMSO.

[0039] Figure 18 The hydrogen spectrum of BT-SP-NMe2 prepared in deuterated chloroform in Example 3 of this invention.

[0040] Figure 19 The carbon spectrum of BT-SP-NO2 prepared in deuterated chloroform in Example 3 of this invention.

[0041] Figure 20 The carbon spectrum of BT-SP-NMe2 prepared in deuterated chloroform in Example 3 of this invention.

[0042] Figure 21 The high-resolution mass spectrometer of BT-SP-NO2 prepared in Example 3 of this invention.

[0043] Figure 22 The high-resolution mass spectrometer of BT-SP-NMe2 prepared in Example 3 of this invention. Detailed Implementation

[0044] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0045] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0046] Given the limitations of existing spiropyran derivative probe design strategies and the limited quantum yield of solid-state fluorescence, this invention proposes a fluorescent compound conjugated with 2,1,3-benzothiadiazole and spiropyran and its application in lipid droplet imaging.

[0047] A typical embodiment of the present invention provides a fluorescent compound conjugated with 2,1,3-benzothiadiazole and spiropyran, the chemical structure of which is shown in Formula I:

[0048]

[0049] Wherein, R1 is selected from C1-C6 straight-chain or branched hydrocarbon groups, ether groups, or C1-C6 alkyl-substituted acyl groups; R2 is hydrogen, methyl, substituted alkyl, fluorine, chlorine, bromine, iodine, cyano, trihalomethyl, nitro, or dimethylamino.

[0050] In some embodiments, the compounds are selected from the following:

[0051]

[0052] A second embodiment of the present invention provides a method for synthesizing the above-mentioned fluorescent compound conjugated with 2,1,3-benzothiadiazole and spiropyran, comprising the step of obtaining the compound shown in Formula I according to the following reaction route;

[0053]

[0054] Compound 1 is alkylated to generate an indole salt (compound 2), which is then reacted with compound 3 via a Knoevenagel condensation reaction to give spiropyran derivative 4. Finally, compound 4 and 5 are coupled via a simple Suzuki coupling reaction to obtain the final compound I. In this reaction, R1 is selected from C1-C6 straight-chain or branched hydrocarbon groups, ether groups, or C1-C6 alkyl-substituted acyl groups; R2 is hydrogen, methyl, substituted alkyl, fluorine, chlorine, bromine, iodine, cyano, trihalomethyl, nitro, or dimethylamino.

[0055] In some embodiments, the reaction conditions for preparing compound 2 from compound 1 are: a temperature of 60–80°C and a reaction time of 5–7 h. Acetonitrile is used as the solvent in the reaction system.

[0056] In some embodiments, the reaction conditions for Knoevenagel condensation are: a temperature of 60–80°C and a reaction time of 4–6 h. Specifically, piperidine is added as a catalyst during the reaction. Specifically, ethanol is used as the solvent in the reaction system.

[0057] In some embodiments, the reaction conditions for the Suzuki coupling reaction are: a temperature of 95–120°C and a reaction time of 23–25 h. Specifically, tetrakis(triphenylphosphine)palladium is added as a catalyst during the reaction. Specifically, toluene and water are used as solvents in the reaction system.

[0058] A third embodiment of the present invention provides the application of the above-mentioned fluorescent compound conjugated with 2,1,3-benzothiadiazole and spiropyran in a fluorescent probe.

[0059] A fourth embodiment of the present invention provides a composition comprising the above-described fluorescent compound conjugated with 2,1,3-benzothiadiazole and spiropyran, or a pharmaceutically acceptable salt thereof.

[0060] The pharmaceutically acceptable salts described in this invention include hydrochloride, sulfate, acetate, oxalate, citrate, etc.

[0061] A fifth embodiment of the present invention provides a formulation comprising an active ingredient and a pharmaceutical carrier, wherein the active ingredient is a fluorescent compound or composition conjugated with the above-mentioned 2,1,3-benzothiadiazole and spiropyran.

[0062] The pharmaceutical carriers described in this invention include physiological saline, buffer solutions, etc.

[0063] The sixth embodiment of the present invention provides the application of the above-mentioned fluorescent compound, composition or formulation of 2,1,3-benzothiadiazole and spiropyran conjugated in lipid droplet-specific fluorescence imaging.

[0064] Specifically, a lipid droplet-specific fluorescent imaging reagent is prepared by conjugating fluorescent compounds, compositions, or formulations of 2,1,3-benzothiadiazole and spiropyran, and then lipid droplet-specific fluorescent imaging is performed.

[0065] A seventh embodiment of the present invention provides a lipid droplet detection kit comprising the above-mentioned fluorescent compound, composition or formulation of 2,1,3-benzothiadiazole and spiropyran conjugated, and a solvent or diluent.

[0066] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments and comparative examples.

[0067] In the following examples, unless otherwise stated, all chemicals were used directly as is, without any purification. Anhydrous solvents were used in the fluorescence property studies. Deionized water was used throughout the studies. The commercial lipid droplet probe Lipi-Deep Red was purchased from Dojindo Molecular Technologies, Inc. 1 H NMR (400MHz) and 13 C10 NMR (100 MHz) spectra were recorded on a Bruker AVANCE III spectrometer using tetramethylsilane as an internal reference. High-resolution mass spectrometry (HRMS) was performed on an Agilent Technologies 6510 Q-TOF LC / MS instrument operating in ESI mode. UV-Vis absorption and fluorescence spectra were obtained using a Hitachi U-4100 spectrophotometer and a Horiba FluoroMax-4 fluorometer, respectively. Absolute fluorescence quantum yield was determined using an integrating sphere.

[0068] Example 1: Synthesis of Compound 2

[0069]

[0070] Compound 1 (2.4 g, 10 mmol) was added sequentially to a Schlenk tube containing 15 mL of anhydrous acetonitrile. Then, CH3I (0.6 mL, 10 mmol) was added to the solution, and the mixture was refluxed at 80 °C for 6 hours. After cooling to room temperature, the precipitate was collected and washed with acetonitrile, and then dried under vacuum to give compound 2 as a white solid (2.76 g, 73%).

[0071] 1 ¹H NMR (400MHz, DMSO-d⁶), δ (ppm): 8.17 (s, 1H), 7.87 (t, J = 8.8Hz, 2H), 3.94 (s, 3H), 2.74 (s, 3H), 1.53 (s, 6H). Figure 14 As shown.

[0072] Example 2: Synthesis of Compound 4

[0073]

[0074] Compound 2 (379 mg, 1 mmol), compound 3 (167 mg, 1 mmol), and piperidine (200 μL, 2 mmol) were dissolved in EtOH (10 mL) and refluxed at 80 °C for 4.5 h under nitrogen protection. After cooling to room temperature, the precipitate was collected and washed with EtOH, then dried under vacuum to give compound 4 as a milky white solid (220 mg, 55%).

[0075] 1 H NMR (400MHz, DMSO-d6), δ (ppm): 8.23 ​​(d, J = 2.76Hz, 1H), 8.01 (dd, J = 8.96, 2.80Hz, 1H), 7.33-7.27 (m, 2H), 7.24 (d, J = 10. 4Hz, 1H), 6.91 (d, J = 9.0Hz, 1H), 6.60 (d, J = 8.2Hz, 1H), 5.99 (d, J = 10.36Hz, 1H), 2.66 (s, 3H), 1.21 (s, 3H), 1.12 (s, 3H). Such as Figure 15 As shown.

[0076] Example 3: Synthesis of Compound 7

[0077]

[0078] Compound 2 (379 mg, 1 mmol), compound 6 (165 mg, 1 mmol), and piperidine (200 μL, 2 mmol) were dissolved in EtOH (10 mL) and refluxed at 80 °C for 4.5 h under nitrogen protection. After cooling to room temperature, the precipitate was collected and washed with EtOH, then dried under vacuum to give compound 7 as a brown solid (238 mg, 60%).

[0079] 1 H NMR (400MHz, DMSO-d6), δ (ppm): 7.26-7.21 (m,, 2H), 6.97 (m, J = 10.2Hz, 1H), 6.63-6.60 (m, 1H), 6.56 (d, J = 1. 52Hz,2H),6.52-6.48(m,1H),5.71(d,J=10.16Hz,1H),2.78(s,6H),2.61(s,3H),1.19(s,3H),1.08(s,3H). Such as Figure 16 As shown.

[0080] Example 4: Synthesis of compound BT-SP-NO2

[0081]

[0082] Compound 5 (400 mg, 1 mmol), compound 7 (262.09 mg, 1 mmol), Pd(PPh3)4 (57.78 mg, 0.05 mmol), and K2CO3 (414.63 mg, 3 mmol) were mixed in a toluene / H2O (8 mL / 2 mL) solution and refluxed at 100 °C for 24 hours under nitrogen protection. After cooling to room temperature, the solvent was removed by evaporation under reduced pressure. The residue was purified by silica gel column chromatography using a hexane / dichloromethane (from 15:1 to 5:1, v / v) mixture as eluent to give an orange solid BT-SP-NO2 (136 mg, 30%). 1 HNMR (400MHz, DMSO-d6), δ (ppm): 8.25 (d, J=2.80Hz, 1H), 8.04-7.99 (m, 2H), 7.87 (dd, J=8.12, 1.8Hz, 1H), 7.83-7.75 (m, 3H), 7.27 ( d,J=10.36Hz,1H),6.97(d,J=9.04Hz,1H),6.81(d,J=8.20Hz,1H),6.06(d,J=10.36Hz,1H),2.77(s,3H),1.30(s,3H),1.21(s,3H). 13 C NMR (100MHz, CDCl3), δ (ppm): 159.80, 155.88, 153.81, 148.22, 141.15, 136.71, 135.07, 129.91, 129.57, 129.17, 128.54 ,126.65,126.06,122.85,121.51,119.53,118.74,115.64,107.16,106.52,,52.49,29.07,26.17,20.16.HRMS(ESI)m / z calcd for[C 25 H 21 N4O3S + ]457.1329(([M+H] + )), found 457.1300. (e.g.) Figure 17 , 19 As shown in Figure 21. The single-crystal structure is as follows: Figure 1 As shown, the cell parameters and refinement data are shown in Table 1.

[0083] Example 5: Synthesis of compound BT-SP-NMe2

[0084]

[0085] Compound 7 (398.10 mg, 1 mmol), compound 5 (262.09 mg, 1 mmol), Pd(PPh3)4 (57.78 mg, 0.05 mmol), and K2CO3 (414.63 mg, 3 mmol) were mixed in a toluene / H2O (8 mL / 2 mL) solution and refluxed at 100 °C for 24 hours under nitrogen protection. After cooling to room temperature, the solvent was removed by evaporation under reduced pressure. The residue was purified by silica gel column chromatography using a mixture of n-hexane / ethyl acetate (from 60:1 to 20:1, v / v) as eluent to give a yellow solid BT-SP-NMe2 (204 mg, 45%). 1 HNMR (400MHz, CDCl3), δ (ppm): 7.90 (dt, J = 9.8, 3.64Hz, 1H), 7.85 (dd, J = 8.08, 1.8Hz, 1H), 7.67-7.62 (m, 3H), 6.85 (d, J = 10.16 Hz,1H),6.70-6.60(m,3H),6.52(d,J=2.8Hz,1H),5.72(d,J=10.12Hz,1H),2.86(s,6H),2.80(s,3H),1.39(s,3H),1.25(s,3H). 13 C NMR (100MHz, CDCl3), δ (ppm): 155.98, 153.95, 148.94, 146.96, 145.44, 137.61, 135.50, 130.08, 129.94, 129.38, 128.24, 126.37, 1 22.79,119.78,119.16,118.97,115.81,115.44,112.06,106.73,103.89,51.70,41.98,29.12,26.18,20.56,20.56.HRMS(ESI)m / z calcdfor[C 27 H 27 N4OS + ]455.1900(([M+H] + )), found 455.1871. (e.g.) Figure 18 , 20 As shown in Figure 22. The single-crystal structure is as follows: Figure 1 The cell parameters and refinement data are shown in Table 2.

[0086] Table 1. Cell parameters and refinement data of BT-SP-NO2

[0087]

[0088] Table 2. Cell parameters and refinement data of BT-SP-NMe2

[0089]

[0090] Example 5: Characterization of basic photophysical properties

[0091] First, the photophysical properties of BT-SP-NO2 and BT-SP-NMe2 in different organic solvents were investigated. From... Figure 2 As shown in Figure A, in n-hexane, BT-SP-NO2 exhibits maximum absorption at 400 nm, while BT-SP-NMe2 shows a redshift at 420 nm compared to the former. Furthermore, according to... Figure 2 B, 2C, and 2D all exhibit excellent luminescence in nonpolar environments, and the fluorescence emission peak intensity decreases significantly and redshifts slightly with increasing solvent polarity, demonstrating typical TICT properties.

[0092] Secondly, their properties at different water fractions (f) were studied. w Emission properties of DMSO / water mixtures. Figure 3 As can be seen, both exhibit a slight decrease in fluorescence intensity at water contents ranging from 0% to 30%, accompanied by a slight red shift in the fluorescence spectrum. With further increases in water content, BT-SP-NO2 and BT-SP-NMe2 show a decrease in fluorescence intensity at f... w The maximum fluorescence intensity is observed when the concentration is 70%, which is λ. em =613nm and λ em =571nm. The decrease in fluorescence intensity under high water content may be related to their different aggregation sizes and morphologies. Furthermore, such as Figure 4 As shown, dynamic light scattering was used to demonstrate their polymer formation under high water content. The above data confirm that they are all AIE dyes. Most importantly, unlike traditional SPs, BT-SP-NMe2 exhibits a significantly higher fluorescence quantum yield in the solid state, approximately 59.0%, far exceeding that of BT-SP-NO2, which may be related to the different molecular packing arrangements.

[0093] Then, the response of the two compounds to trifluoroacetic acid (TFA) was tested. The two compounds were dispersed in a tetrahydrofuran solution and titrated. Figure 5 As shown in Figures A and B, the emission peaks of both exhibit varying degrees of redshift with increasing TFA concentration. However, BT-SP-NMe2 shows an increase in fluorescence intensity, while BT-SP-NO2 shows a different trend. This phenomenon indicates that introducing electron-donating groups into the spiropyran backbone facilitates a proton response.

[0094] Finally, the responses of the two compounds to UV-Vis light were tested. Molecules dispersed in a tetrahydrofuran (THF) solution were alternately irradiated with a handheld UV lamp and a fluorescent lamp, and the spectral changes at different irradiation times were recorded using absorption and emission spectra. Figure 6 As shown in Figure A, BT-SP-NO2 exhibits a sensitive response to ultraviolet light, displaying a new absorption peak after 5 seconds of irradiation and reaching saturation within 50 seconds. Furthermore, this molecule maintains good isomerization efficiency even after six rounds of alternating irradiation. Figure 7 Conversely, no similar situation was observed with BT-SP-NMe2 dispersed in THF. Figure 6 B). This phenomenon demonstrates that introducing electron-withdrawing groups into the spiropyran skeleton makes the molecule more responsive to light.

[0095] Example 6: Density Functional Theory Calculation (DFT) and Molecular Packing Analysis

[0096] The HOMO and LUMO electron cloud distributions of BT-SP-NO2 and BT-SP-NMe2 were calculated using density functional theory (DFT). Figure 8 A) In the LUMO, electrons are primarily delocalized within the benzothiadiazole moiety, while the electron cloud distribution of the HOMO orbitals shows a significant difference between the two. For BT-SP-NO2, NO2 reduces electron repulsion through its strong electron attraction, thus lowering the HOMO energy level. Conversely, NMe2 raises the HOMO energy level through its strong electron-donating ability, with its electron cloud mainly concentrated on the left-hand benzopyran moiety. The spatial electron distributions of HOMO and LUMO indicate that these two AIE groups exhibit significant electron separation due to a strong intramolecular charge transfer (ICT) effect.

[0097] To elucidate the interaction and stacking of BT-SP-NO2 and BT-SP-NMe2 in the crystal state, and to explain their significant differences in solid-state fluorescence quantum yield ( Figure 8 (B) In this embodiment, their crystal structures were also analyzed. Both molecules employ an alternating antiparallel packing configuration in the crystal lattice. NO2 and NMe2 significantly affect the crystal structures of both molecules, leading to marked differences in molecular packing, which may explain their different fluorescence quantum yields in the solid state. Compared to the NO2 substituent, the presence of NMe2 reduces the dihedral angle between the benzopyran and indole moieties by approximately 10°. Figure 9 This leads to π-π stacking interactions at the BTD site of BT-SP-NMe2, while such interactions are not observed at the BTD site of BT-SP-NO2. Figure 8B). Furthermore, four SP molecules within the crystal cell are selected, and their four central helical carbon atoms are connected to form two parallel spatial quadrilateral planes (B). Figure 10 Comparing the length, width, and diagonal length of these planes, it is noted that the NMe2 substituent leads to a decrease in width and an increase in length. This indicates a more compact molecular arrangement, effectively restricting molecular motion. This may explain the higher fluorescence quantum yield observed in the solid state of BT-SP-NMe2 compared to BT-SP-NO2.

[0098] Example 7: Culture and Imaging of HeLa Cells

[0099] Cervical cancer (HeLa) cells were passaged in high glucose (H-DMEM) medium containing 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin, in an incubator at 37°C with 5% CO2.

[0100] Before imaging, HeLa cells at an appropriate concentration were seeded in confocal culture dishes, and an appropriate amount of pre-prepared culture medium was added. The dishes were then cultured in an incubator for 1–2 days until the cells adhered to the culture dish before confocal imaging experiments could be performed. The synthesized dye was dissolved in dimethyl sulfoxide (DMSO) to prepare a stock solution (1 mM) of an appropriate concentration for subsequent cell staining.

[0101] For staining, add fresh culture medium and 0.5 μL of BT-SP-NO2 or 1 μL of BT-SP-NMe2 stock solution to the centrifuge tube and vortex until homogeneous. Before imaging, replace the original culture medium in the confocal culture dish with the above mixed solution and incubate at 37°C in a 5% CO2 incubator for 30 min. After staining, rinse the culture dish 2–3 times with phosphate-buffered saline (PBS), add 1 mL of fresh culture medium to maintain cell viability, and then directly image under a confocal laser scanning microscope (CLSM).

[0102] Co-staining with Lipi-Deep Red: Incubate with BT-SP-NO2 (0.5 μM) or BT-SP-NMe2 (1 μM) and 200 nM Lipi-Deep Red for 30 min. After staining, wash away residual dye with PBS, add 1 mL of fresh culture medium to maintain cell viability, and then directly image under a confocal laser scanning microscope (CLSM). The in situ fluorescence spectrum is similar to that of low-polarity solvents ( Figure 11 The fluorescence channels of BT-SP-NO2 or BT-SP-NMe2 showed good overlap with those of Lipi-Deep Red, and the corresponding Pearson coefficients were calculated to be 0.84 and 0.89, respectively. Figure 12These data confirm that the BT-SP-NO2 and BT-SP-NMe2 probes in HeLa cells exhibit high signal-to-noise ratios and selectively stain lipid droplets without washing.

[0103] Example 7: Toxicity testing of BT-SP-NO2 and BT-SP-NMe2

[0104] To test the effect of the dye on HeLa cell viability, the standard thiazolyl blue (MTT) method was used for evaluation. The main principle of this method is that succinate dehydrogenase in the mitochondria of cells can reduce exogenous MTT to water-insoluble blue-violet formazan crystals, which are then deposited in the cells. Dead cells do not have this function.

[0105] First, HeLa cells were seeded in 96-well plates and cultured at 37°C for 24 hours. Then, the medium was replaced with a mixture of fresh medium and different concentrations of dye (0, 2, 4, 6, 8, 10 μM), and the cells were cultured for another 24 hours. Next, the medium was replaced again with a freshly prepared mixture of MTT (5 mg / mL) and medium (MTT / DMEM = 1:5), and the cells were cultured for another 3 hours. Afterward, the medium in each well was replaced with DMSO, and the cells were agitated thoroughly. The absorbance at 490 nm was then measured using a microplate reader. Cell viability was calculated using the following formula:

[0106] Cell viability=(A1-A0) / (A2-A0).

[0107] A0 is the absorbance of the blank in a 96-well plate without cell inoculation, A1 is the absorbance of the experimental group after 24 hours of dye treatment, and A2 is the absorbance of the control group that was cultured in culture medium only and was not treated with dye.

[0108] See results Figure 13 The cell survival rate of HeLa cells after incubation with different concentrations of the compound for 24 hours was determined. The results showed that after incubation with the 10 μM probe for 24 hours, the cell survival rate was still as high as 85%, indicating that the probe has high biocompatibility.

[0109] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. 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 fluorescent compound conjugated with 2,1,3-benzothiadiazole and spiropyran, characterized in that, Its chemical structure is shown in Formula I: Wherein, R1 is methyl; R2 is nitro or dimethylamino.

2. A method for preparing the fluorescent compound of 2,1,3-benzothiadiazole and spiropyran conjugated as described in claim 1, characterized in that, Includes the following steps: Compound 1 is alkylated to generate compound 2. Then, compound 2 and compound 3 undergo a Knoevenagel condensation reaction to give spiropyran derivative 4. Finally, compound 4 and 5 undergo a Suzuki coupling reaction to obtain the final compound I. The reaction route is as follows: 。 3. The method for preparing the fluorescent compound conjugated with 2,1,3-benzothiadiazole and spiropyran as described in claim 2, characterized in that, The reaction conditions for preparing compound 2 from compound 1 were: temperature 60-80 °C and reaction time 5-7 h.

4. The method for preparing the fluorescent compound conjugated with 2,1,3-benzothiadiazole and spiropyran as described in claim 2, characterized in that, The reaction conditions for preparing compound 2 from compound 1 were: acetonitrile was used as the solvent in the reaction system.

5. The method for preparing the fluorescent compound conjugated with 2,1,3-benzothiadiazole and spiropyran as described in claim 2, characterized in that, The reaction conditions for Knoevenagel condensation are: temperature 60~80 ℃, reaction time 4~6 h.

6. The method for preparing the fluorescent compound conjugated with 2,1,3-benzothiadiazole and spiropyran as described in claim 2, characterized in that, The reaction conditions for Knoevenagel condensation are: piperidine is added as a catalyst during the reaction.

7. The method for preparing the fluorescent compound conjugated with 2,1,3-benzothiadiazole and spiropyran as described in claim 2, characterized in that, The reaction conditions for Knoevenagel condensation are: ethanol is used as the solvent in the reaction system.

8. The method for preparing the fluorescent compound conjugated with 2,1,3-benzothiadiazole and spiropyran as described in claim 2, characterized in that, The reaction conditions for the Suzuki coupling reaction are: temperature 95~120 ℃, reaction time 23~25 h.

9. The method for preparing the fluorescent compound conjugated with 2,1,3-benzothiadiazole and spiropyran as described in claim 2, characterized in that, The reaction conditions for the Suzuki coupling reaction are: tetra-triphenylphosphine palladium is added as a catalyst during the reaction.

10. The method for preparing the fluorescent compound conjugated with 2,1,3-benzothiadiazole and spiropyran as described in claim 2, characterized in that, The reaction conditions for the Suzuki coupling reaction are: toluene and water are used as solvents in the reaction system.

11. The use of the fluorescent compound of claim 1, which is conjugated with 2,1,3-benzothiadiazole and spiropyran, in the preparation of a fluorescent probe for selectively staining lipid droplets in HeLa cells.

12. A composition characterized in that, Fluorescent compounds comprising 2,1,3-benzothiadiazole and spiropyran conjugated as described in claim 1, or pharmaceutically acceptable salts thereof.

13. A formulation comprising an active ingredient and a pharmaceutical carrier, characterized in that, The active ingredient is the fluorescent compound of 2,1,3-benzothiadiazole and spiropyran conjugated as described in claim 1, or a pharmaceutically acceptable salt thereof.

14. The use of a fluorescent compound of claim 1, 2,1,3-benzothiadiazole and spiropyran conjugated, the composition of claim 12, or the formulation of claim 13 in the preparation of a HeLa cell lipid droplet-specific fluorescent imaging agent.

15. A lipid droplet detection kit, characterized in that, It includes the fluorescent compound of 2,1,3-benzothiadiazole and spiropyran conjugated as described in claim 1, the composition of claim 12, or the formulation of claim 13, as well as a solvent or diluent.

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