A fluorescent probe based on camphor derivatives, its preparation method and application

Abstract

The present invention discloses a fluorescent probe based on the structure of camphor derivatives, with camphor derivatives as the fluorescent group and 2,4-dinitrophenol group as the recognition group, and the structural formula is as shown in (I): The fluorescent probe of the present invention can be used for the detection of benzenethiol accumulated in water samples and biological samples. The fluorescent probe of the present invention has high sensitivity, good selectivity and anti-interference performance, can quickly identify benzenethiol, shorten the response time, and realize the real-time and effective detection of benzenethiol. The probe of the present invention can achieve benzenethiol imaging in cells and in vivo, is expected to play a role in industrial production and clinical medical detection, and has broad application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of fluorescent probes, and particularly relates to a fluorescent probe using a camphor derivative as a fluorescent group and 2,4-dinitrophenol group as a recognition group, and its application in the detection of benzenethiol. The present invention also provides a preparation method of the fluorescent probe. Background Art

[0002] Benzenethiol and its derivatives are commonly used synthetic materials, and are widely used in the manufacture of pesticides, drug production, and the production of various industrial products. In addition, benzenethiol is a class of volatile sulfur-containing aromatic compounds, and has an odor similar to meaty, grilled, and sausage flavors, so it is commonly used as a food additive to improve the taste of meat products and seasonings. For example, o-methylbenzenethiol, 2,6-dimethylbenzenethiol, benzenethiol, and 2-ethylbenzenethiol are clearly listed as safe and legal benzenethiol spices in the "National Standard for Food Additives" (GB2760-2014) in China. However, despite the wide application of benzenethiol, the toxicity caused by its excessive use to animals and humans is a problem that cannot be ignored. Benzenethiol in soil and water will damage aquatic organisms and natural habitats. Benzenethiol is toxic to fish and mice, and its median lethal dose (LC50) values are determined to be 0.01 - 0.4 mM and 46.2 mg / kg, respectively. In addition, long-term exposure to benzenethiol liquid or vapor may cause serious health problems, such as damage to the central nervous system, muscle weakness, vomiting, coma, and even death. Considering its disadvantages, benzenethiol has been listed as a priority pollutant by the United States Environmental Protection Agency (USEPA).

[0003] So far, there are many methods for detecting benzenethiol, such as gas chromatography, high performance liquid chromatography, and nanomaterial-based sensor methods, etc. These methods can meet the needs of benzenethiol detection, but there are still some problems, such as complex sample processing methods, expensive experimental instruments, and the need for professional analysts, etc. In recent years, the fluorescence detection technology based on small molecule fluorescent probes has been recognized as one of the most attractive molecular detection technologies, and has the advantages of simplicity, convenience, high sensitivity, etc., and is widely used in the visual detection of target analytes in chemistry, biology, and the environment.

[0004] In recent years, many reported benzenethiol fluorescent probes are designed based on the strong nucleophilicity of benzenethiol. A strongly electron-withdrawing 2,4-dinitrobenzenesulfonyl group or 2,4-dinitrophenol group is coupled with a fluorescent dye, and these nitrobenzene groups quench the fluorescence of the dye through intramolecular charge transfer (ICT) or photoinduced electron transfer (PET) mechanisms. When the probe reacts with benzenethiol, the nitrobenzene group is easily subjected to nucleophilic aromatic substitution (S N(Ar), releasing the fluorescent dye results in enhanced fluorescence. However, there are few reports on coupling the 2,4-dinitrophenol group as a group to inhibit twisted intramolecular charge transfer (TICT) with a fluorescent dye to enhance the fluorescence performance of the dye.

[0005] Many fluorescent probes for detecting benzenethiol can be classified into rhodamine, BODIPY, hemicyanine, fluorescein, coumarin, etc. according to the structure of the fluorescent group. However, there are very few reports on constructing a fluorescent group by structurally modifying renewable natural camphor. In addition, most of the reported benzenethiol probes are interfered by aliphatic thiols such as glutathione (GSH), cysteine (Cys), and homocysteine (Hcy), because aliphatic thiols have similar chemical structures or nucleophilicities to benzenethiol. For example, the literature "A rapid and visible colorimetric fluorescent probe for benzenethiol flavor detection" discloses a fluorescent probe with (4-nitrophenyl)-3-oxoprop-1-enyl)naphthalene as the fluorescent group and 2,4-dinitrophenol group as the recognition group (Food Chemistry, 2019, 286: 322-328.). When this probe is applied to the detection of benzenethiol, aliphatic thiols can all cause a considerable degree of fluorescence enhancement. Similarly, there is also a probe based on squaric acid structure disclosed in the literature "Highly Sensitive Squaraine-Based Water-Soluble Far-Red / Near-Infrared Chromofluorogenic Thiophenol Probe" (ACS Sensors 2017, 2, 599-605.). In addition, most of the reported probes are only applied to the imaging of benzenethiol in water samples and living cells, and rarely applied to the imaging of benzenethiol accumulated in vivo.

[0006] As an important source of natural terpenoid compounds, camphor is widely distributed in Lauraceae plants and can be used as an ideal starting material for preparing various compounds, such as anti-tumor drugs, anesthetics, antibacterial agents, and catalysts. In addition, camphor derivatives have good biocompatibility and low cytotoxicity. However, there are few reports on fluorescent materials based on camphor derivatives. The rigid structure of the camphor molecule can effectively limit the intermolecular interaction and reduce the energy consumption during the collision process, which is beneficial to the optical properties of camphor derivatives. Therefore, based on the natural camphor structure, designing a fluorescent probe that can distinguish aliphatic thiols and aromatic benzenethiol and applying it to the imaging of benzenethiol accumulated in vivo has important research value and development prospects. Summary of the Invention

[0007] To solve the deficiencies of the prior art, the technical problems to be solved by the present invention are to provide a fluorescence probe based on a camphor derivative, which can specifically react with benzenethiol, can be used for qualitative and quantitative analysis of benzenethiol, and is not interfered by aliphatic thiols in the sample. Another technical problem to be solved by the present invention is to provide a preparation method of the fluorescence probe. Another technical problem further solved by the present invention is to provide the application of the fluorescence probe in imaging benzenethiol in living cells and in vivo.

[0008] To achieve the above objectives, the present invention adopts the following technical solutions:

[0009] A fluorescence probe based on a camphor derivative, with 2,4-dinitrophenol group as the recognition group.

[0010] In a specific example of the present invention, the structural formula of the fluorescence probe is shown as formula (Ⅰ):

[0011] 。

[0012] The present invention also discloses the application of the above fluorescence probe in the preparation of an indicator for detecting thiol compounds. Preferably, the thiol compound is a benzenethiol compound. More preferably, the benzenethiol compounds include benzenethiol, 2,6-dimethylbenzenethiol, 2-ethylbenzenethiol, 2-methylbenzenethiol, 4-trifluoromethylbenzenethiol, 4-fluorobenzenethiol, 4-methylbenzenethiol, 4-aminobenzenethiol, 4-methoxybenzenethiol.

[0013] The fluorescence probe of the present invention can be used for qualitative or quantitative detection of benzenethiol compounds.

[0014] A specific example of the above application is liquid, cell or organism (in vivo) fluorescence imaging.

[0015] The fluorescence probe of the present invention is used as a diagnostic reagent in the diagnosis of diseases related to the excessive accumulation of benzenethiol. The diseases related to the excessive accumulation of benzenethiol are selected from liver damage, skin damage, central nervous system damage, muscle weakness, vomiting, coma. The fluorescence probe of the present invention is also used as a detection reagent to deeply study the kinetic mechanism of the processes such as the transport and accumulation of benzenethiol compounds in the environment and in organisms.

[0016] A specific example, the application of the fluorescence probe (Ⅰ) described in the present invention includes the following steps:

[0017] (1) Prepare a fluorescence probe system;

[0018] (2) Prepare a solution of benzenethiol compounds;

[0019] (3) Add the solution of the phenylthiol compound to the fluorescence probe system to prepare a mixed solution with different concentrations of the phenylthiol compound;

[0020] (4) Measure the fluorescence intensity of the mixed solution at 560 nm and plot a standard curve according to the relationship between the fluorescence intensity and the concentration of the phenylthiol compound;

[0021] (5) Quantitatively detect the content of the phenylthiol compound in the test solution according to the standard curve.

[0022] The steps for preparing the fluorescence probe system described above include:

[0023] (1) Dissolve the fluorescence probe (Ⅰ) in an organic solvent to obtain a mother liquor, and make the concentration of the probe in the mother liquor 5×10 -7 ~ 5×10 -3 mol / L;

[0024] (2) Dilute the concentration of the mother liquor with a buffer solution to obtain a fluorescence probe system;

[0025] (3) The organic solvent for preparing the fluorescence probe mother liquor is one or more of methanol, ethanol, acetonitrile, tetrahydrofuran, acetone, acetic acid, DMF, DMA, DMSO;

[0026] (4) The buffer solution for diluting the mother liquor is one or more of PBS buffer solution, HEPES buffer solution, Tris buffer solution.

[0027] Another object of the present invention is to provide a preparation method of the fluorescence probe (Ⅰ) of the present invention, including the following steps:

[0028] S1: React camphorquinone with 4,7-dibromobenzo[c][1,2,5]thiadiazole-5,6-diamine in an organic solvent to prepare compound (Ⅱ),

[0029] .

[0030] The above-mentioned organic solvent is selected from one or more of formic acid, acetic acid, propionic acid, acetonitrile, THF, N,N dimethylformamide, N,N dimethylacetamide, DMSO, toluene, xylene, 1,4-dioxane, 2-methylpyrrolidone.

[0031] Preferably, the reaction temperature is 0 °C to 200 °C.

[0032] S2: Dissolve compound (Ⅱ) and 4-hydroxyphenylboronic acid pinacol ester in an organic solvent, and under nitrogen protection, react with a catalyst and a base to obtain compound (Ⅲ),

[0033] .

[0034] The above-mentioned organic solvent is selected from one or more of acetonitrile, THF, N,N -dimethylformamide, N,N -dimethylacetamide, DMSO, toluene, xylene, 1,4-dioxane, 2-methylpyrrolidone, ethanol, methanol, ethylene glycol dimethyl ether, and water.

[0035] The selected catalyst is selected from one or more of Pd(PPh3)4, Pd(PPh3)2Cl2, Pd(dppf)Cl2, Pd(dba)2-PCy3, PdCl2, Pd(OAc)2, Pd / C, and NiCl2(dppf).

[0036] The selected base is selected from one or more of K2CO3, K3PO4, Na2CO3, CsF, Cs2CO3, t- BuONa, t -BuOK, Ba(OH)2, NaOH, NaHCO3, triethylamine, N,N- diisopropylethylamine, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU).

[0037] The preferred reaction temperature is 0 °C to 200 °C.

[0038] S3: Compound (III) and 2,4-dinitrofluorobenzene are dissolved in an organic solvent, and a base is added for reaction to obtain the fluorescent probe (I).

[0039] .

[0040] The above-mentioned organic solvent is selected from one or more of dichloromethane, chloroform, acetone, acetonitrile, THF, N,N -dimethylformamide, N,N -dimethylacetamide, DMSO, toluene, xylene, 1,4-dioxane, 2-methylpyrrolidone, ethanol, methanol, ethylene glycol dimethyl ether.

[0041] The selected base is selected from one or more of K2CO3, K3PO4, Na2CO3, CsF, Cs2CO3, t- BuONa, t -BuOK, Ba(OH)2, NaOH, NaHCO3, sodium acetate, potassium acetate, triethylamine, N,N- diisopropylethylamine, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU).

[0042] The preferred reaction temperature is 0 °C to 200 °C.

[0043] A specific preparation method of the present invention is as follows:

[0044] S1: Dissolve camphorquinone and 4,7-dibromo-benzo[c][1,2,5]thiadiazole-5,6-diamine in a reaction solvent, and heat with stirring. After the reaction is completed, cool the reaction to room temperature and filter to obtain the crude product. Separate the product (Ⅱ) by column chromatography.

[0045] S2: Dissolve compound (Ⅱ), 4-hydroxybenzeneboronic acid pinacol ester, a base, and a catalyst in a reaction solvent, and heat with stirring in a nitrogen atmosphere. After the reaction is completed, cool the reaction to room temperature, rotary evaporate to remove most of the solvent, and then extract with ethyl acetate. Wash the combined organic layers with saturated brine, add anhydrous Na2SO4 for drying. Separate the product (Ⅲ) by column chromatography.

[0046] S3: Dissolve compound (Ⅲ), 2,4-dinitrofluorobenzene, and a base in a reaction solvent, and heat with stirring. After the reaction is completed, cool the reaction to room temperature, rotary evaporate to remove most of the reaction solvent, and then add water to the reaction system and extract with ethyl acetate. Wash the combined organic layers with saturated brine, add anhydrous Na2SO4 for drying. Separate the fluorescent probe (Ⅰ) by column chromatography.

[0047] The above specific reaction conditions can be referred to the examples.

[0048] The beneficial effects of the present invention are as follows:

[0049] (1) The fluorescent probe provided by the present invention introduces a natural camphor molecule into the dye as a fluorescent matrix, and selects 2,4-dinitrophenol group which is easily subjected to nucleophilic aromatic substitution (SNAr) as the recognition group, which can respond to benzenethiol compounds with high selectivity and high sensitivity. In the presence of benzenethiol compounds, the fluorescence intensity decreases significantly, and it can be used for qualitative and quantitative detection of benzenethiol compounds, and will not be interfered by aliphatic thiols such as glutathione (GSH), cysteine (Cys), and homocysteine (Hcy), and has a fast detection speed and high detection accuracy.

[0050] (2) The fluorescent probe provided by the present invention has low toxicity to cells and good biocompatibility. It can not only detect benzenethiol in solution, but also detect benzenethiol in cells and in zebrafish. It can be used to deeply study the kinetic mechanism of the transport and accumulation of benzenethiol in the environment and organisms, and is also helpful to clarify the biological role and mechanism of benzenethiol in the pathogenic process. Brief Description of the Drawings

[0051] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other related drawings can also be obtained based on these drawings.

[0052] Figure 1 are the changes in the ultraviolet spectrum (a), fluorescence spectrum (b), and solution color (inset) after adding benzenethiol (700 μM) to the fluorescent probe (Ⅰ) (10 μM). The detection system is tetrahydrofuran-PBS (pH = 7.4, v / v = 40 / 60), and the temperature during detection is 37 °C. The excitation wavelength of the fluorescence spectrum is 450 nm, and the slit is 5 / 5 nm.

[0053] Figure 2 are the changes in the fluorescence spectrum of the fluorescent probe (Ⅰ) (10 μM) in tetrahydrofuran-PBS (pH = 7.4) mixed solutions with different water contents (a) and the changes in the fluorescence intensity of the solution at 560 nm before and after adding benzenethiol (700 μM) (b). Inset: The color change of the solution under a 365 nm ultraviolet lamp after adding benzenethiol (700 μM) to tetrahydrofuran-PBS (pH = 7.4) mixed solutions with different water contents. The temperature during detection is 37 °C, the excitation wavelength of the fluorescence spectrum is 450 nm, and the slit is 5 / 5 nm.

[0054] Figure 3 is the change in the fluorescence intensity at 560 nm over time within 5 minutes after adding benzenethiol (350, 500, 700 μM) to the fluorescent probe (Ⅰ) (10 μM). The detection system is tetrahydrofuran-PBS (pH = 7.4, v / v = 40 / 60), the temperature during detection is 37 °C, the excitation wavelength is 450 nm, and the slit is 5 / 5 nm.

[0055] Figure 4 are the changes in the fluorescence spectrum (a) of the fluorescent probe (Ⅰ) (10 μM) and the fluorescence intensity (b) at 560 nm with the change in the concentration of benzenethiol (0 - 600 μM) after adding different concentrations of benzenethiol. Inset: The linear fitting graph of fluorescence intensity and concentration in the range of 0 - 30 μM. The detection system is tetrahydrofuran-PBS (pH = 7.4, v / v = 40 / 60), the temperature during detection is 37 °C, incubated for 60 minutes, the excitation wavelength is 450 nm, and the slit is 5 / 5 nm.

[0056] Figure 5It is the change in the fluorescence intensity at 560 nm of the fluorescence spectrum after adding 700 μM thiophenol to the fluorescent probe (I) (10 μM) under different pH conditions. The detection system is tetrahydrofuran-PBS (v / v = 40 / 60), the temperature during detection is 37 °C, incubated for 30 minutes, the excitation wavelength is 450 nm, and the slit is 5 / 5 nm.

[0057] Figure 6 It is the change in the fluorescence spectrum (a) and the fluorescence intensity at 560 nm (b) before and after adding 700 μM of different kinds of thiophenols (2,6-dimethylthiophenol, 2-ethylthiophenol, 2-methylthiophenol, 4-trifluoromethylthiophenol, 4-fluorothiophenol, 4-methylthiophenol, 4-aminothiophenol, 4-methoxythiophenol) to the fluorescent probe (I) (10 μM). The detection system is tetrahydrofuran-PBS (pH = 7.4, v / v = 40 / 60), the temperature during detection is 37 °C, incubated for 30 minutes, the excitation wavelength is 450 nm, and the slit is 5 / 5 nm.

[0058] Figure 7 It is the color change of the solution before and after adding 700 μM of different kinds of thiophenols (2,6-dimethylthiophenol, 2-ethylthiophenol, 2-methylthiophenol, 4-trifluoromethylthiophenol, 4-fluorothiophenol, 4-methylthiophenol, 4-aminothiophenol, 4-methoxythiophenol) to the fluorescent probe (I) (10 μM) under visible light (a) and under a 365 nm ultraviolet lamp (b).

[0059] Figure 8 It is the change in the fluorescence spectrum (a) and the fluorescence intensity at 560 nm (b) before and after adding 700 μM of thiophenol or 700 μM of various analytes (Cys, GSH, Hcy, Na2S2O3, Na2SO4, NaHSO3, 3-mercaptopropionic acid, His, Leu, Ser, Tyr, Arg, Asp, aniline, phenol, CaCl2, CuSO4, K2CO3, KI, KBr, KNO3, MgSO4, NaNO2, and ZnCl2) to the fluorescent probe (I) (10 μM). The detection system is tetrahydrofuran-PBS (pH = 7.4, v / v = 40 / 60), the temperature during detection is 37 °C, incubated for 30 minutes, the excitation wavelength is 450 nm, and the slit is 5 / 5 nm.

[0060] Figure 9The color of the solution under visible light after adding 700 μM of benzenethiol or 700 μM of various analytes (Cys, GSH, Hcy, Na2S2O3, Na2SO4, NaHSO3, 3-mercaptopropionic acid, His, Leu, Ser, Tyr, Arg, Asp, aniline, phenol, CaCl2, CuSO4, K2CO3, KI, KBr, KNO3, MgSO4, NaNO2, and ZnCl2) to the fluorescent probe (Ⅰ) (10 μM).

[0061] Figure 10 The color of the solution under 365 nm ultraviolet light after adding 700 μM of benzenethiol or 700 μM of various analytes (Cys, GSH, Hcy, Na2S2O3, Na2SO4, NaHSO3, 3-mercaptopropionic acid, His, Leu, Ser, Tyr, Arg, Asp, aniline, phenol, CaCl2, CuSO4, K2CO3, KI, KBr, KNO3, MgSO4, NaNO2, and ZnCl2) to the fluorescent probe (Ⅰ) (10 μM).

[0062] Figure 11 The change in fluorescence intensity at 560 nm of the fluorescence spectrum after adding 700 μM of benzenethiol and 700 μM of various analytes (Cys, GSH, Hcy, Na2S2O3, Na2SO4, NaHSO3, 3-mercaptopropionic acid, His, Leu, Ser, Tyr, Arg, Asp, aniline, phenol, CaCl2, CuSO4, K2CO3, KI, KBr, KNO3, MgSO4, NaNO2, and ZnCl2) to the fluorescent probe (Ⅰ) (10 μM) simultaneously. The detection system is tetrahydrofuran-PBS (pH = 7.4, v / v = 40 / 60), the temperature during detection is 37 °C, incubated for 30 minutes, excitation wavelength 450 nm, slit 5 / 5 nm.

[0063] Figure 12 The color of the solution under visible light after adding 700 μM of benzenethiol and 700 μM of various analytes (Cys, GSH, Hcy, Na2S2O3, Na2SO4, NaHSO3, 3-mercaptopropionic acid, His, Leu, Ser, Tyr, Arg, Asp, aniline, phenol, CaCl2, CuSO4, K2CO3, KI, KBr, KNO3, MgSO4, NaNO2, and ZnCl2) to the fluorescent probe (Ⅰ) (10 μM) simultaneously.

[0064] Figure 13The color of the solution under a 365 nm ultraviolet lamp after adding 700 μM of benzenethiol and 700 μM of various analytes (Cys, GSH, Hcy, Na2S2O3, Na2SO4, NaHSO3, 3-mercaptopropionic acid, His, Leu, Ser, Tyr, Arg, Asp, aniline, phenol, CaCl2, CuSO4, K2CO3, KI, KBr, KNO3, MgSO4, NaNO2, and ZnCl2) to the fluorescent probe (Ⅰ) (10 μM).

[0065] Figure 14 Laser confocal microscopy images of the fluorescent probe (Ⅰ) (10 μM) for detecting the accumulation of benzenethiol in HeLa cells.

[0066] Figure 15 Fluorescence microscopy images of the fluorescent probe (Ⅰ) (10 μM) for detecting the accumulation of benzenethiol in zebrafish. Detailed implementation manners

[0067] Next, the technical solutions in the embodiments of the present invention will be described clearly and completely in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention and should not be used to limit the protection scope of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts belong to the protection scope of the present invention.

[0068] Example 1: Preparation of the fluorescent probe

[0069]

[0070] S1: Preparation of 4,11-dibromo-6,12,12-trimethyl-6,7,8,9-tetrahydro-6,9-methano[1,2,5]thiadiazolo[3,4-b]phenazine (Ⅱ)

[0071] Dissolve camphorquinone (1.66 g, 10 mmol) and 4,7-dibromobenzo[c][1,2,5]thiadiazole-5,6-diamine (3.24 g, 10 mmol) in the reaction solvent (120 mL), and stir at 120 °C for 12 h. After the reaction is completed (monitored by TLC), cool the mixture to room temperature, evaporate the solvent under reduced pressure, dissolve the residue in dichloromethane (300 mL), wash it three times with saturated NaHCO3 solution (100 mL), wash the organic layer with saturated brine, and dry it with anhydrous Na2SO4. Separate the product by column chromatography (eluent ethyl acetate: petroleum ether = 1:100 to elute the target compound). Obtain 2.41 g of yellow solid (Ⅱ) with a yield of 53%.

[0072] Structure determination:

[0073] 1 H NMR (600 MHz, CDCl3) δ 3.26 (d, J J = 4.6 Hz, 1H), 2.44 – 2.33 (m, 1H), 2.15 (dd, J J = 16.8, 5.4 Hz, 1H), 1.59 (t, J J = 8.5 Hz, 2H), 1.50 (s, 3H), 1.20 (s, 3H), 0.73 (s, 3H); 13 C NMR (150 MHz, CDCl3) δ 169.4, 167.7, 151.6, 151.5, 139.7, 139.6, 114.0, 113.4, 54.4, 53.2, 52.6, 31.8, 24.7, 20.9, 18.2, 9.9; HRMS (ESI + ) calcd for C 16 H 14 Br2N4S [M+H] + 452.9379, found 452.9378.

[0074] S2: Preparation of 4,4'-(6,12,12-trimethyl-6,7,8,9-tetrahydro-6,9-methano[1,2,5]thiadiazolo[3,4-b]phenazine-4,11-diyl)diphenol (Ⅲ)

[0075] 4,11-Dibromo-6,12,12-trimethyl-6,7,8,9-tetrahydro-6,9-methano[1,2,5]thiadiazolo[3,4-b]phenazine (Ⅱ, 454 mg, 1.0 mmol), 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol (880 mg, 4.0 mmol), and Na2CO3 (420 mg, 4.0 mmol) were dissolved in a mixed solvent of toluene / ethanol / H2O (10 / 6 / 5 mL). PdCl2(dppf) (74 mg, 0.1 mmol) was added to the reaction system, and the system was heated to 70 °C and stirred for 6 h under a nitrogen atmosphere. After the reaction was completed (monitored by TLC), the reaction mixture was cooled to room temperature, the solvent was removed by rotary evaporation, and then extracted with dichloromethane (3 × 15 ml). The combined organic layers were washed with saturated brine and dried over anhydrous Na2SO4. The product was separated by column chromatography (eluent ethyl acetate: petroleum ether = 1:5 to elute the target compound). A red solid (Ⅴ) of 306 mg was obtained, with a yield of 63.6%.

[0076] Structure determination:

[0077] 1 H NMR (600 MHz, DMSO) δ 9.66 (d, J J = 8.1 Hz, 2H), 7.70 (d, J J = 7.9 Hz,2H), 7.58 (d, J J = 7.8 Hz, 2H), 6.94 (d, J J = 7.5 Hz, 4H), 2.95 (s, 1H), 2.27 (s,1H), 2.04 (t, J J = 10.1 Hz, 1H), 1.41 (t, J J = 10.5 Hz, 2H), 1.23 (s, 3H), 1.08 (s,3H), 0.63 (s, 3H); 13 C NMR (150 MHz, DMSO) δ 166.3, 164.8, 157.7, 152.7, 152.4,137.6, 137.2, 134.5, 134.1, 129.2, 129.0, 126.3, 126.0, 114.8, 114.6, 53.8,52.8, 52.5, 31.9, 31.4, 24.8, 22.5, 20.7, 18.3, 14.4, 10.1; HRMS (ESI + ) calcdfor C 28 H 24 N4O2S [M+H] + 481.1693, found 481.1697.

[0078] S3: Preparation of Fluorescent Probe (Ⅰ)

[0079] Dissolve 4,4'-(6,12,12-trimethyl-6,7,8,9-tetrahydro-6,9-methano[1,2,5]thiadiazolo[3,4-b]phenazine-4,11-diyl)diphenol (Ⅲ, 48 mg, 0.1 mmol), 2,4-dinitrofluorobenzene (56 mg, 0.3 mmol) in dichloromethane (8 mL). After adding triethylamine (100 μL) to the reaction system, stir at room temperature for 12 h. After the reaction is completed (monitored by TLC), rotary evaporate to remove the solvent, then add 50 mL of water, and extract with dichloromethane (3×15 mL). Wash the combined organic layers with saturated brine, and dry over anhydrous Na2SO4. Separate the product by column chromatography (eluent ethyl acetate: petroleum ether = 1:12 to elute the target compound). Obtain 70 mg of yellow solid (Ⅰ), with a yield of 86%.

[0080] Structure determination:

[0081] 1 H NMR (600 MHz, CDCl3) δ 8.98 – 8.83 (m, 2H), 8.41 (dd, J = 9.2, 2.7Hz, 2H), 8.06 (d, J = 8.5 Hz, 2H), 7.99 (d, J = 7.8 Hz, 2H), 7.48 – 7.31 (m, 6H),3.07 (s, 1H), 2.34 (s, 1H), 2.09 (t, J = 9.9 Hz, 1H), 1.54-1.48 (m, 2H), 1.33(s, 3H), 1.15 (s, 3H), 0.73 (s, 3H); 13 C NMR (151 MHz, CDCl3) δ 167.4, 165.8,156.1, 155.9, 153.6, 153.4, 152.5, 152.2, 141.7, 141.6, 139.9, 139.8, 138.0,137.9, 135.1, 134.9, 133.3, 133.2, 128.9, 128.6, 128.5, 122.2, 122.1, 119.7,119.4, 119.1, 54.1, 53.2, 52.5, 31.9, 26.9, 24.9, 20.8, 18.2, 9.7; HRMS (ESI + ) calcd for C 40 H 28 N8O10 S [M+H] + 813.1722, found 813.1723.

[0082] Example 2: Spectral Response of Fluorescent Probe (Ⅰ) to Benzenethiol

[0083] Dissolve the fluorescent probe (Ⅰ) in dimethyl sulfoxide (DMSO) to prepare a probe stock solution with a concentration of 10 mM, and store it in a drug cool cabinet at 4 °C. During the test, dilute the probe stock solution with DMSO to a concentration of 1 mM as the stock solution for spectral testing to explore the changes in the ultraviolet and fluorescence spectral properties after the probe responds to benzenethiol. Prepare a benzenethiol solution with a concentration of 210 mM using tetrahydrofuran (THF) as the solvent. To simulate physiological experiments, all experiments were carried out at pH = 7.4 (THF-PBS, pH = 7.4, v / v = 40 / 60) and 37 °C. Mix the benzenethiol solution with the probe solution, with the final concentration of the probe being 10 μM and the concentration of benzenethiol being 700 μM. Shake the solution well and equilibrate for 30 min, then measure the ultraviolet spectrum and fluorescence spectrum. Among them, the excitation wavelength of the fluorescence spectrum is 450 nm, and the slit width is 5 nm / 5 nm.

[0084] The changes in the ultraviolet absorption spectrum of probe (Ⅰ) before and after adding benzenethiol are as shown in Figure 1 (a) in it. As can be seen from Figure 1 (a) in it: After adding benzenethiol, the ultraviolet absorption of the detection solution is enhanced, and the maximum absorption wavelength redshifts from 450 nm to 500 nm. Under visible light, the color of the solution changes from yellow to red (the inset of (a) in Figure 1 (a)).

[0085] The changes in the fluorescence spectrum of probe (Ⅰ) before and after adding benzenethiol are as shown in Figure 1 (b) in it. As can be seen from Figure 1 (b) in it: Before adding benzenethiol, the fluorescence of the detection solution is very strong, and the maximum emission wavelength is 560 nm; after adding benzenethiol, the fluorescence intensity of the solution at 560 nm is reduced by 145 times. Under a 365 nm ultraviolet lamp, the color of the solution changes from yellow to purple (the inset of (b) in Figure 1 (b)).

[0086] The above research results show that through the changes in ultraviolet and fluorescence spectra and visual observation, the fluorescent probe (Ⅰ) has a good fluorescence response to benzenethiol.

[0087] Example 3: Influence of Moisture on the Detection Performance of Fluorescent Probe (Ⅰ)

[0088] A mixed solution composed of tetrahydrofuran and PBS buffer solution in different ratios (tetrahydrofuran - PBS, pH = 7.4) was used as the reaction system, and a reaction mixed solution of 10 μM probe (Ⅰ) and 700 μM benzenethiol was prepared. The prepared reaction mixed solution was incubated at 37 °C for 30 min, and then the fluorescence spectrum was measured. Among them, the excitation wavelength of the fluorescence spectrum was 450 nm, the maximum emission wavelength was 560 nm, and the slit width was 5 nm / 5 nm.

[0089] The fluorescence spectra of probe (Ⅰ) in mixed solutions composed of tetrahydrofuran and PBS buffer solution in different ratios and the changes in fluorescence intensity at 560 nm are as Figure 2 shown. As can be seen from Figure 2 it: as the water content increases, f water increases from 0 to 70%, and the fluorescence intensity of probe (Ⅰ) at 560 nm continuously decreases, which may be attributed to the intramolecular twisted charge transfer (TICT) effect caused by the increase in solvent polarity; when the water content continues to increase, f water increases from 70% to 90%, and the fluorescence intensity increases instead. This may be because the further increase in water will lead to the formation of aggregates, resulting in a great hindrance to the free rotation of 2,4 - dinitro - 1 - phenoxyphenyl, thereby enhancing the fluorescence intensity. This compound has the characteristic of aggregation - induced emission (AIE).

[0090] Benzenethiol was added to the probe (Ⅰ) solutions with different water contents described above, and the concentration of benzenethiol was made to reach 700 μM, and the changes in fluorescence intensity of the solution at 560 nm were studied. As shown in (b) of Figure 2 : only in the solution with a water content of 30 - 70%, the addition of benzenethiol will cause a sharp decrease in fluorescence intensity, showing good detection effect; Figure 2 The inset in (b) of

[0091] also well proves this point. Only in the solution with a water content of 30 - 70%, the color of the solution will change from yellow to purple.

[0092] Example 4: Response time of fluorescence probe (Ⅰ) to benzenethiol

[0093] A reaction mixture solution of 10 μM probe (Ⅰ) and 350, 500, 700 μM benzenethiol was prepared using a mixed solution composed of tetrahydrofuran and PBS buffer solution (tetrahydrofuran - PBS, pH = 7.4, v / v = 40 / 60) as the reaction system. The prepared reaction mixture solution was measured for fluorescence spectra at different time points (0 - 300 s) at 37°C. Among them, the excitation wavelength of the fluorescence spectrum was 450 nm, the maximum emission wavelength was 560 nm, and the slit width was 5 nm / 5 nm.

[0094] The change in fluorescence intensity of probe (Ⅰ) at 560 nm after adding benzenethiol is as Figure 3 shown. From Figure 3 it can be seen that: after adding 35, 50, 70 equivalents of benzenethiol, the fluorescence intensity at 560 nm of the solution decreased rapidly and reached the lowest point and plateau within 5 minutes. Therefore, probe (Ⅰ) can be used for the rapid detection of benzenethiol ( Figure 3 ).

[0095] Example 5: Detection limit of fluorescence probe (Ⅰ) for benzenethiol

[0096] A reaction mixture solution of 10 μM probe (Ⅰ) and benzenethiol at different concentrations was prepared using a mixed solution composed of tetrahydrofuran and PBS buffer solution (tetrahydrofuran - PBS, pH = 7.4, v / v = 40 / 60) as the reaction system. The prepared reaction mixture solution was incubated at 37°C for 1 h and then measured for fluorescence spectra. Among them, the excitation wavelength of the fluorescence spectrum was 450 nm, the maximum emission wavelength was 560 nm, and the slit width was 5 nm / 5 nm. The detection limit of probe (Ⅰ) for benzenethiol was calculated according to the 3σ / k method, where σ represents the standard deviation of the measured emission intensity values of ten blank samples, and k represents the slope of the fitted straight line between the fluorescence intensity and the concentration of benzenethiol.

[0097] The fluorescence spectra (a) of probe (Ⅰ) after adding benzenethiol at different concentrations (0 - 600 μM) and the change in fluorescence intensity at 560 nm (b) are as Figure 4 shown. Before adding benzenethiol, the fluorescence intensity of probe (Ⅰ) was very strong. As the concentration of benzenethiol increased, the fluorescence at 560 nm of the solution decreased rapidly. The fluorescence intensity was linearly related to benzenethiol in the range of 0 - 30 μM, and the linear regression equation was y = -124.90x + 5686.8, R 2 = 0.9916 ( Figure 4 inset of (b) in

[0098] Example 6: Effect of pH on the Detection of Benzenethiol by Fluorescent Probe (I)

[0099] A mixed solution composed of tetrahydrofuran and PBS buffer solution (tetrahydrofuran-PBS, v / v = 40 / 60) was used as the reaction system, and the pH of the mixed solution was adjusted with 3 mol / L hydrochloric acid and 3 mol / L NaOH solution. Subsequently, reaction mixed solutions of 10 μM probe (I) and 700 μM benzenethiol were prepared using these reaction systems with different pH values. After incubating the prepared reaction mixed solutions at 37 °C for 30 min, the fluorescence spectra were measured. Among them, the excitation wavelength of the fluorescence spectrum was 450 nm, the maximum emission wavelength was 560 nm, and the slit width was 5 nm / 5 nm.

[0100] By testing the change in fluorescence intensity at 560 nm of the detection system with a fluorescence spectrometer, the stability of probe (I) under different pH conditions and the detection ability for benzenethiol are as Figure 5 shown. It can be clearly seen from the figure that the fluorescent probe has good stability in a relatively long pH range (2 - 10). When pH > 6, the probe has good response to benzenethiol. Therefore, the fluorescent probe (I) has the potential value for further detecting benzenethiol accumulated in cells and animals.

[0101] Example 7: Recognition Performance of Fluorescent Probe (I) for Different Kinds of Benzenethiols

[0102] Benzenethiol compounds are widely used in fields such as food and chemistry. The substituents on the benzene ring of benzenethiol will affect the nucleophilicity of the mercapto group. Therefore, a fluorescent probe with excellent performance needs to respond to different kinds of benzenethiols and is not affected by the substituents on the benzene ring of benzenethiol. According to Example 1, various benzenethiol solutions (2,6-dimethylbenzenethiol, 2-ethylbenzenethiol, 2-methylbenzenethiol, 4-trifluoromethylbenzenethiol, 4-fluorobenzenethiol, 4-methylbenzenethiol, 4-aminobenzenethiol, 4-methoxybenzenethiol) with a concentration of 210 mM were prepared using tetrahydrofuran as the solvent. To study the recognition ability of probe (I) for different kinds of benzenethiols, reaction mixed solutions of 10 μM probe (I) and 700 μM various benzenethiols were prepared using a mixed solution composed of tetrahydrofuran and PBS buffer solution (tetrahydrofuran-PBS, pH = 7.4, v / v = 40 / 60) as the reaction system. After incubating the prepared reaction mixed solutions at 37 °C for 0.5 h, the fluorescence spectra were measured. Among them, the excitation wavelength of the fluorescence spectrum was 450 nm, the maximum emission wavelength was 560 nm, and the slit width was 5 nm / 5 nm.

[0103] The fluorescence spectra (a) of probe (I) after adding 700 μM of various benzenethiols (2,6-dimethylbenzenethiol, 2-ethylbenzenethiol, 2-methylbenzenethiol, 4-trifluoromethylbenzenethiol, 4-fluorobenzenethiol, 4-methylbenzenethiol, 4-aminobenzenethiol, 4-methoxybenzenethiol) and the changes in fluorescence intensity (b) at 560 nm are as Figure 6 shown. All benzenethiols can cause changes in the fluorescence spectra of the probe, indicating that the response of probe (I) to benzenethiols is not affected by the substituents on the benzene ring (whether electron-withdrawing or electron-donating).

[0104] The selectivity of probe (I) can also be demonstrated by observing the change in the color of the solution with the naked eye. As Figure 7 shown, after adding different types of benzenethiols, the probe solution changes from yellow to light red under visible light; and from yellow to purple under 365 nm ultraviolet light. Therefore, probe (I) can be used as a general probe for detecting benzenethiols, and is not affected by the substituents on the benzene ring of benzenethiols.

[0105] Example 8: Selectivity of Fluorescent Probe (I) for the Recognition of Benzenethiol

[0106] Generally speaking, high selectivity for the target analyte is the core requirement of all detection methods. According to Example 1, various solutions of biological thiols, sulfur-containing salts, amino acids, metal salts, aniline, and phenol (Cys, GSH, Hcy, Na2S2O3, Na2SO4, NaHSO3, 3-mercaptopropionic acid, His, Leu, Ser, Tyr, Arg, Asp, aniline, phenol, CaCl2, CuSO4, K2CO3, KI, KBr, KNO3, MgSO4, NaNO2, and ZnCl2) with a concentration of 210 mM were prepared using deionized water as the solvent. To study the selectivity of probe (I) for benzenethiol, a reaction mixture solution of 10 μM probe (I) and 700 μM benzenethiol or 700 μM of various solutions of biological thiols, sulfur-containing salts, amino acids, metal salts, aniline, and phenol was prepared using a mixed solution composed of tetrahydrofuran and PBS buffer solution (tetrahydrofuran-PBS, pH = 7.4, v / v = 40 / 60) as the reaction system. The prepared reaction mixture solution was incubated at 37 °C for 0.5 h and then the fluorescence spectrum was measured. Among them, the excitation wavelength of the fluorescence spectrum was 450 nm, the maximum emission wavelength was 560 nm, and the slit width was 5 nm / 5 nm.

[0107] The fluorescence spectra (a) of probe (I) after adding 700 μM benzenethiol or 700 μM of various biological thiols, sulfur-containing salts, amino acids, metal salts, aniline, and phenol and the changes in fluorescence intensity (b) at 560 nm are as Figure 8As shown, only benzenethiol can cause a significant change in the fluorescence spectrum of the probe, while the remaining analytes do not cause a significant change in the fluorescence spectrum of the probe, indicating that the probe (I) has high selectivity for benzenethiol.

[0108] The selectivity of the probe (I) can also be illustrated by observing the change in the color of the solution with the naked eye. As Figure 9 shown, under visible light, when other analytes are added, the color of the probe solution remains yellow; when benzenethiol is added, the color of the solution quickly turns light red. Similarly, under a 365 nm ultraviolet lamp, when other analytes are added, the color of the probe solution remains yellow; when benzenethiol is added, the color of the solution quickly turns purple ( Figure 10 ). The above results demonstrate the high selectivity of the probe for benzenethiol.

[0109] Example 9: Competitive experiment of the fluorescent probe (I)

[0110] To prove that the fluorescent probe has good application potential, it is necessary to test its anti-interference performance. Analytes that may interfere with the detection of the fluorescent probe (I) are added to the detection system to examine whether the ability of the fluorescent probe (I) to detect benzenethiol in the presence of these analytes will be interfered.

[0111] A reaction mixture solution containing 10 μM probe (I), 700 μM benzenethiol, and 700 μM of biological thiols, sulfur-containing salts, amino acids, metal salts, aniline, and phenol solutions (Cys, GSH, Hcy, Na2S2O3, Na2SO4, NaHSO3, 3-mercaptopropionic acid, His, Leu, Ser, Tyr, Arg, Asp, aniline, phenol, CaCl2, CuSO4, K2CO3, KI, KBr, KNO3, MgSO4, NaNO2, and ZnCl2) is prepared using a mixed solution of tetrahydrofuran and PBS buffer solution (tetrahydrofuran-PBS, pH = 7.4, v / v = 40 / 60) as the reaction system. The prepared reaction mixture solution is incubated at 37 °C for 0.5 h and then the fluorescence spectrum is measured. Among them, the excitation wavelength of the fluorescence spectrum is 450 nm, the maximum emission wavelength is 560 nm, and the slit width is 5 nm / 5 nm. As Figure 11 shown, the remaining analytes do not interfere with the detection of benzenethiol by the probe, indicating that the probe (I) has good anti-interference performance.

[0112] The anti-interference ability of the probe (I) can also be illustrated by observing the change in the color of the solution with the naked eye. As Figure 12As shown, in visible light, when benzenethiol coexists with other analytes (Cys, GSH, Hcy, Na2S2O3, Na2SO4, NaHSO3, 3-mercaptopropionic acid, His, Leu, Ser, Tyr, Arg, Asp, aniline, phenol, CaCl2, CuSO4, K2CO3, KI, KBr, KNO3, MgSO4, NaNO2, and ZnCl2), the color of the probe solution remains light red. Similarly, under ultraviolet light at 365 nm, when benzenethiol coexists with other analytes, the color of the probe solution remains purple ( Figure 13 ). The above results indicate that the presence of other analytes does not affect the detection of benzenethiol by the probe.

[0113] Example 10: Detection of intracellularly accumulated benzenethiol using fluorescent probe (I)

[0114] Pre-culture HeLa cells in a confocal dish for 24 h to allow cell attachment, and perform laser confocal imaging under a laser confocal microscope (excitation wavelength: 450 nm, fluorescence collection range: 500 nm - 700 nm). Add 10 μM probe (I) to the first group of HeLa cells and incubate for 30 min, then perform laser confocal imaging under a laser confocal microscope. The imaging results are as shown in the upper part of Figure 14 ; Add 10 μM probe (I) to the second group of HeLa cells and incubate for 30 min, then add 100 μM benzenethiol and continue to incubate for 30 min, and perform laser confocal imaging under a laser confocal microscope. The imaging results are as shown in the lower part of Figure 14 .

[0115] Comparing Figure 14 the upper and lower parts in it, it can be seen that the fluorescent probe (I) shows green fluorescence in cells, and the fluorescence quenches once benzenethiol is added, which is in line with the experimental expectation. Therefore, it can be proved that the fluorescent probe (I) can be used to detect the toxic substance benzenethiol accumulated in cells.

[0116] Example 11: Detection of intracellularly accumulated benzenethiol in zebrafish using fluorescent probe (I)

[0117] Image zebrafish under a fluorescence microscope (excitation wavelength: 450 nm, fluorescence collection range: 500 nm - 700 nm). The imaging results are as shown in A of Figure 15 : Add probe (10 μM) to the first group of zebrafish and incubate for 30 min, remove the culture medium, and wash 3 times with fish-raising water, then collect the fluorescence in the green channel; Add probe (10 μM) to the second and third groups of zebrafish and incubate for 30 min, remove the culture medium and wash 3 times with fish-raising water, then add 50 and 100 μM benzenethiol and incubate for 30 min, and collect the fluorescence in the green channel. The fluorescence intensity change of each group is asFigure 15 as shown in B in

[0118] Comparison Figure 15 It can be seen that: before adding benzenethiol, the fluorescence intensity of probe (I) in zebrafish is very strong; after adding benzenethiol, the fluorescence intensity of probe (I) decreases rapidly. Therefore, it can be proved that the fluorescent probe (I) can be used for the detection of benzenethiol accumulated in zebrafish.

[0119] It should be noted that the above embodiments do not limit the present invention in any form. Any technical solutions obtained by means of equivalent substitution or equivalent transformation fall within the protection scope of the present invention.

Claims

1. A fluorescent probe based on camphor derivatives, characterized in that: The structural formula is as shown in formula (I): 。 2. Use of the fluorescent probe according to claim 1 in the preparation of an indicator for detecting thiol compounds, wherein the thiol compound is a benzenethiol compound.

3. The application according to claim 2, wherein The benzenethiol compounds are selected from benzenethiol, 2,6-dimethylbenzenethiol, 2-ethylbenzenethiol, 2-methylbenzenethiol, 4-trifluoromethylbenzenethiol, 4-fluorobenzenethiol, 4-methylbenzenethiol, 4-aminobenzenethiol, 4-methoxybenzenethiol.

4. The application according to claim 2, wherein The detection is qualitative or quantitative detection.

5. The application according to claim 3, characterized in that The application is fluorescence imaging in solution, cells or organisms.

6. Use of the fluorescent probe according to claim 1 in the preparation of a diagnostic reagent for diseases caused by the accumulation of benzenethiol.

7. The application according to claim 6, wherein The diseases caused by the abnormal accumulation of benzenethiol are selected from liver injury, skin injury, slow growth, Alzheimer's disease, depression, cardiovascular disease, neural tube defect, inflammatory bowel disease, osteoporosis.

8. The preparation method of the fluorescent probe according to claim 1, wherein Comprising the following steps: S1: React camphorquinone with 4,7-dibromo-1,2,5-benzothiadiazole-5,6-diamine to prepare compound (II), ； S2: React the compound (II) prepared in S1 with 4-hydroxybenzeneboronic acid pinacol ester to prepare compound (III), ； S3: Dissolve the compound (III) prepared in S2 and 2,4-dinitrofluorobenzene in an organic solvent, add a base and react to obtain the fluorescent probe (I), 。 9. The preparation method of the fluorescence probe for detecting benzenethiol according to claim 8, wherein: Comprising the following steps: S1: Dissolve camphorquinone and 4,7-dibromo-benzo[c][1,2,5]thiadiazole-5,6-diamine in an organic solvent and heat to obtain compound (II). The organic solvent is selected from one or more of formic acid, acetic acid, propionic acid, acetonitrile, THF, N,N -dimethylformamide, N,N -dimethylacetamide, DMSO, toluene, xylene, 1,4-dioxane, 2-methylpyrrolidone, and the reaction temperature is 0 °C to 200 °C; S2: Compound (II) and 4-hydroxyphenylboronic acid pinacol ester are dissolved in an organic solvent. Under nitrogen protection, they react with a catalyst and a base to obtain compound (III). The organic solvent is selected from one or more of acetonitrile, THF, N,N -dimethylformamide, N,N -dimethylacetamide, DMSO, toluene, xylene, 1,4-dioxane, 2-methylpyrrolidone, ethanol, methanol, ethylene glycol dimethyl ether, water. The catalyst is selected from one or more of Pd(PPh3)4, Pd(PPh3)2Cl2, Pd(dppf)Cl2, Pd(dba)2-PCy3, PdCl2, Pd(OAc)2, Pd / C, NiCl2(dppf). The selected base is selected from one or more of K2CO3, K3PO4, Na2CO3, CsF, Cs2CO3, t- BuONa, t -BuOK, Ba(OH)2, NaOH, NaHCO3, triethylamine, N,N -diisopropylethylamine, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU). The reaction temperature is 0 °C to 200 °C; S3: Compound (III) and 2,4-dinitrofluorobenzene are dissolved in an organic solvent, and a base is added for reaction to obtain the fluorescent probe (I). The solvent is selected from one or more of dichloromethane, chloroform, acetone, acetonitrile, THF, N,N -dimethylformamide, N,N -dimethylacetamide, DMSO, toluene, xylene, 1,4-dioxane, 2-methylpyrrolidone, ethanol, methanol, ethylene glycol dimethyl ether. The base is selected from one or more of K2CO3, K3PO4, Na2CO3, CsF, Cs2CO3, t -BuONa, t -BuOK, Ba(OH)2, NaOH, NaHCO3, sodium acetate, potassium acetate, triethylamine, N,N- diisopropylethylamine, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU). The reaction temperature is 0 °C to 200 °C.

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