A type of cycloheptatriene pyrrole salt fluorescent dye and preparation method thereof

Abstract

The present invention provides a class of cycloheptatrienolpyrrole fluorescent dyes and a preparation method thereof, which overcome a series of defects of existing hemicyanine fluorescent dyes. The cycloheptatrienolpyrrole fluorescent dyes of the present invention have a certain red shift in maximum absorption wavelength and emission wavelength compared with traditional hemicyanine dyes. Compounds C1 and C2 prepared in the examples of the present application are verified by spectral testing to have fluorescence emission wavelengths between 1020 nm and 1050 nm, and are well suited for long-wavelength fluorescence detection scenarios. The cycloheptatrienolpyrrole fluorescent dyes have readily available raw materials, low preparation cost, simple operation, high yield, mild reaction conditions, and simple synthesis steps, and have high commercial application value.

Description

Technical Field

[0001] The invention belongs to the technical field of organic synthesis, and in particular relates to a cycloheptatriene pyrrolidine salt fluorescent dye and a preparation method thereof. Background Art

[0002] Fluorescence imaging technology is widely used in the identification of various biomolecules and the study of their biological effects due to its non-destructive, highly sensitive, and real-time spatial imaging characteristics. Fluorescent dyes, as the cornerstone of fluorescence imaging technology, have also received extensive attention and research from researchers.

[0003] Hemicyanine fluorophores consist of a nitrogen heterocyclic electron acceptor, a conjugated linker, and an aromatic donor with a functional terminus, forming a unique D-π-A structure. However, xanthene-type hemicyanine dyes, in particular, exhibit absorption and emission in the NIR region, flexible and tunable spectral properties, and excellent stability. Based on hemicyanines, researchers can develop multifunctional fluorescent probes that can monitor important biomolecules or enzymes in living systems through changes in fluorescence and photoacoustic signals, and use chemotherapy, photothermal therapy, photodynamic therapy, or combined therapies for diagnosis and treatment.

[0004] Currently, hemicyanine fluorescent dyes are typically prepared using quaternary salts with an active (acidic) methyl group at the 2- or 4-position, such as quaternary salts of 2-methylpyridine (α-pyridine / 2-pyridine), 2-methylquinoline (quinoline), 4-methylpyridine (γ-pyridine / 4-pyridine), and 4-methylquinoline. Most of these quaternary salts are primarily based on five- or six-membered rings, whereas the present invention introduces a seven-membered heterocyclic pyrrole quaternary salt to construct a hemicyanine fluorescent dye. Existing hemicyanine fluorescent molecules typically have short emission wavelengths, making them susceptible to interference from biological background fluorescence during imaging and analysis. However, the fluorescence emission wavelength of the cycloheptatrienolpyrrole salt and its fluorescent dye of the present invention can reach the near-infrared region II. Summary of the Invention

[0005] In order to overcome a series of defects of existing fluorescent dyes, the present invention provides a class of cycloheptatriene pyrrole salt fluorescent dyes and preparation methods thereof. The present invention is a hemicyanine fluorescent dye of a seven-membered heterocyclic pyrrole salt, which is not a traditional five-membered ring or six-membered ring pyrrole or pyridinium salt category. The cycloheptatriene pyrrole salt fluorescent dye of the present invention has a certain red shift compared to the maximum absorption wavelength and emission wavelength of traditional hemicyanine dyes. Compound C1 and compound C2 prepared in the examples of the present application were verified by spectral testing, and their fluorescence emission wavelengths were between 1020nm-1050nm, which can be well applied to the scene of long-wavelength fluorescence detection. This class of cycloheptatriene pyrrole salts and their fluorescent dye raw materials are easy to obtain and low in cost, easy to operate, high in yield, mild in reaction conditions and simple in synthesis steps, and have high commercial application value.

[0006] In order to achieve the above technical objectives, the technical solution adopted by the present invention is:

[0007] A type of cycloheptatriene pyrrole salt fluorescent dye, the general structural formula is as follows:

[0008]

[0009] R1 is selected from hydrogen, alkyl with 1-18 carbon atoms; R2 is selected from hydrogen, alkyl with 1-18 carbon atoms, aryl, benzyl; R3 is selected from hydrogen, hydroxyl, methoxy; Y is selected from halogen ion, ClO4 - 、BF4 - 、CH3COO - CF3COO - or OTs - .

[0010] A method for preparing a cycloheptatriene pyrrole salt fluorescent dye comprises:

[0011] Step S1: Preparation of intermediate compound A-1

[0012] 2,5-hexanedione, an amine containing a substituent R1, and aminosulfonic acid are stirred at room temperature, and after extraction, compound A-1 containing a substituent R1 is obtained;

[0013] Step S2: Preparation of intermediate compound A-2

[0014] Dissolve compound A-1 in DMF, add POCl3, and stir at room temperature. After the reaction is complete, pour the solution into a mixture of sodium acetate and ice water and stir. After forming a solid suspension, filter, wash, and air-dry to obtain intermediate compound A-2.

[0015] Step S3: Preparation of intermediate compound A-3

[0016] Compound A-2 is dissolved in ethanol, a ketone containing a substituent R2 is added, and then an aqueous sodium hydroxide solution is added; the mixture is stirred at room temperature, and then extracted, concentrated, and purified to obtain an intermediate compound A-3 containing R1 and R2 substitutions;

[0017] Step S4: Preparation of intermediate compound A-4

[0018] Compound A-3 is dissolved in THF under anhydrous and oxygen-free conditions, CH3MgCl is added, and then an inorganic acid is added. After extraction, concentration, and purification, an intermediate compound A-4 containing R1 and R2 substituents is obtained;

[0019] Step S5: Preparation of intermediate compound B-1

[0020] PBr3, DMF and CHCl3 were added to cyclohexanone and stirred at room temperature for neutralization reaction; then compound B-1 was obtained by extraction, washing and drying;

[0021] Step S6: Preparation of intermediate compound B-2

[0022] Compound B-1, salicylaldehyde containing R3 substituent, and Cs2CO3 are dissolved in DMF and stirred at room temperature, and then extracted, concentrated, and purified to obtain intermediate compound B-2 containing R3 substituent;

[0023] Step S7: Preparation of Compound C

[0024] Compound B-2 and compound A-4 were dissolved in acetic anhydride, triethylamine was added, and the mixture was heated, condensed and refluxed. Compound C was obtained after extraction, concentration and purification;

[0025] The reaction process is as follows:

[0026]

[0027] R1 is selected from hydrogen, alkyl with 1-18 carbon atoms; R2 is selected from hydrogen, alkyl with 1-18 carbon atoms, aryl, benzyl; R3 is selected from hydrogen, hydroxyl, methoxy; Y is selected from halogen ion, ClO4 - 、BF4 - 、CH3COO - CF3COO - or OTs - .

[0028] Furthermore, in step S1, the prepared compound A-1 is extracted with ether, then extracted with dichloromethane and water, the organic layer is dried over Na2SO4, filtered, and concentrated in vacuo to obtain compound A-1; in step S1, the molar ratio of 2,5-hexanedione and the amine containing the substituent R1 is 1:1.1-1.5 equivalents, and aminosulfonic acid is a catalytic amount.

[0029] Furthermore, in step S2, the mass ratio of sodium acetate to ice in the sodium acetate-ice-water mixture is 1:6; the mass ratio of compound A-1 to sodium acetate is 12.24:100; and the washing solvent is distilled water.

[0030] Furthermore, in step S3, the molar ratio of compound A-2 to the ketone containing the substituent R2 is 1:1.1; the mass concentration of the sodium hydroxide aqueous solution is 10%; extraction is performed with distilled water and dichloromethane; the concentration method is reduced pressure distillation; and the crude product is purified by column chromatography, and the column chromatography eluent is petroleum ether and ethyl acetate.

[0031] Furthermore, in step S4, extraction is performed using distilled water and dichloromethane; the concentration method is vacuum distillation; and the crude product is purified by recrystallization.

[0032] Furthermore, in step S5, the molar ratio of PBr3 to cyclohexanone is 130.5:48.4; the reaction is neutralized with a saturated sodium bicarbonate solution, and extracted with dichloromethane and distilled water.

[0033] Furthermore, in step S6, the molar ratio of compound B-1, salicylaldehyde containing an R3 substituent, and Cs2CO3 is 5.4:4.5:13.5; the product is washed with H2O; the organic layer is dried over Na2SO4, filtered, concentrated in vacuo, and purified by silica gel column chromatography.

[0034] Furthermore, in step S7, the molar ratio of compound B-2 to compound A-4 is 1:1.2; after extraction with dichloromethane solvent and water, the solvent is evaporated and concentrated, purified by silica gel column chromatography, and then recrystallized from dichloromethane and ethyl acetate to obtain compound C.

[0035] The fluorescent dye is used in the context of long-wavelength fluorescence detection.

[0036] Beneficial effects

[0037] Compared with the prior art, the present invention has the following advantages:

[0038] 1. The fluorescent dye raw materials of the cycloheptatrienolpyrrole salt of the present invention are easily available, easy to operate, high in yield and simple in synthesis steps, and have high commercial application value.

[0039] 2. The cycloheptatrienolpyrrole fluorescent dyes described herein exhibit a red-shift in both maximum absorption and emission wavelengths compared to conventional hemicyanine dyes. Spectral measurements of Compounds C1 and C2, prepared in the Examples of this application, confirmed that their fluorescence emission wavelengths range from 1020 nm to 1050 nm, making them well-suited for long-wavelength fluorescence detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 The general synthetic route of the cycloheptatrienolpyrrole fluorescent dye in the embodiment of the present invention is shown in FIG.

[0041] Figure 2 This is a synthetic route for compound C1 in Example 1 of the present invention;

[0042] Figure 3 is the H NMR spectrum of compound C1 prepared in Example 1 of the present invention;

[0043] Figure 4 is the C NMR spectrum of compound C1 prepared in Example 1 of the present invention;

[0044] Figure 5 is the mass spectrum of compound C1 prepared in Example 1 of the present invention;

[0045] Figure 6 This is the fluorescence emission spectrum of compound C1 prepared in Example 1 of the present invention at an excitation wavelength of 808 nm;

[0046] Figure 7 This is a synthetic route for compound C2 in Example 2 of the present invention;

[0047] Figure 8 is the H NMR spectrum of compound C2 prepared in Example 2 of the present invention;

[0048] Figure 9 is the C NMR spectrum of compound C2 prepared in Example 2 of the present invention;

[0049] Figure 10 is the mass spectrum of compound C2 prepared in Example 2 of the present invention;

[0050] Figure 11 This is the fluorescence emission spectrum of compound C2 prepared in Example 1 of the present invention at an excitation wavelength of 808 nm. DETAILED DESCRIPTION

[0051] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0052] Example 1:

[0053] Figure 1 The following is a general synthetic route for the cycloheptatrienolpyrrole fluorescent dyes according to the present invention; the synthesis of compound C1 is performed as an example:

[0054] The synthetic route of compound C1 is as follows Figure 2 As shown, compound C1 was synthesized according to the synthetic route:

[0055] 1. Preparation of Compound A-1

[0056] 2,5-Hexanedione (5.70 g, 0.05 mmol) and n-butylamine (4.38 g, 0.06 mmol) were placed in a 100 mL round-bottom flask equipped with a magnetic stirrer. 10 mol% aminosulfonic acid was added. The mixture was stirred at room temperature for 12 h. After the reaction, the mixture was extracted with ether (2 × 10 mL), followed by dichloromethane and water. The organic layer was dried over Na2SO4, filtered, and concentrated in vacuo to yield compound A-1 (4.3 g, Y = 57.0%).

[0057] 2. Preparation of Compound A-2

[0058] To a 250 mL round-bottom flask equipped with a magnetic stir bar and thermometer, add 12.24 g of Compound A-1, followed by 60 mL of DMF. The flask was cooled in an ice bath, and the mixture was treated with 19 mL of POCl₃ in 1 mL increments. For the first 8 mL, each 1 mL produced a strong exotherm, causing the solution to warm from 10°C to 20°C. After the initial 8 mL, the addition of phosphorus oxychloride produced almost no temperature increase. After the addition of POCl₃, the ice bath was removed, and the flask containing the light orange solution was placed in an oil bath, which was then heated to 100°C. After heating for two hours, the oil bath was removed, and the solution was allowed to cool for 15 minutes. The solution was then poured into a mixture of 600 g of ice and 100 g of sodium acetate and stirred. After 25 minutes, a gray solid suspension was formed. The mixture was filtered, and the solid was washed with 2×100 ml of water and air-dried to obtain compound A-2 (10.24 g, Y=60.99%).

[0059] 3. Preparation of Compound A-3

[0060] Compound A-2 (1.03 g, 5 mmol) was dissolved in ethanol at room temperature. 5.5 mmol of 3-pentanone in 3 ml of ethanol was added very slowly in droplets to the solution (over 3 hours). Then, 4 ml of a mixture of water and sodium hydroxide solution (10% mass concentration) was added. The mixture was stirred at room temperature, and then 10 ml of water was added. The aqueous phase was extracted five times, each time with 15 ml of chloroform. The organic phase was dried over anhydrous MgSO4 and then evaporated. The residue was chromatographed on an aluminum column using thioether / chloroform = 75 / 25 as the eluent. Compound A-3 (0.37 g, Y = 28.9%) was collected.

[0061] 4. Preparation of Compound A-4

[0062] Compound A-3 (2.58 g, 10 mmol) was added to anhydrous diethyl ether (15 mL) and added dropwise to a Grignard reagent prepared by conventional methods by adding magnesium (1.03 g, 43.5 mmol) and methyl iodide (6.2 g, 174 mmol) to anhydrous reagent-grade diethyl ether (50 mL) at room temperature under argon. The solution partially discolored due to the precipitation of a yellow solid. The resulting mixture was stirred at 40°C, and the solvent was evaporated in vacuo. A mixture of acetic acid (20 mL) and HBF4 (50%, 15 mL) was then added. The resulting blue solution was poured into water (250 mL). After 12 hours, the solution was extracted with dichloromethane (3 x 50 mL) and dried over Na2SO4. After evaporation of the solvent, the solid was fermented with diethyl ether and recrystallized from isopropanol to yield compound A-4 (2.37 g, Y = 75.2%).

[0063] 5. Preparation of Compound B-1

[0064] PBr3 (12.4 mL, 130.5 mmol) was added dropwise to a mixture of DMF (11.2 mL) and chloroform (50 mL) at 0°C. After stirring for 45 minutes, cyclohexanone (5 mL, 48.4 mmol) was added. The resulting solution was stirred at room temperature for 16 hours, then poured into 50 mL of ice water, neutralized with solid NaHCO3, and extracted with dichloromethane. The layers were separated, and the aqueous layer was extracted with CH2Cl2 (100 mL). The organic layer was dried over Na2SO4, filtered, and concentrated in vacuo to yield the target compound B-1 (7.2 g, 38.52 mmol, Y = 75.7%).

[0065] 6. Preparation of Compound B-2

[0066] At room temperature, compound B-1 (1.02 g, 5.4 mmol) was dissolved in DMF (30 mL), and 2-hydroxy-4-methoxybenzaldehyde (685 mg, 4.5 mmol) and Cs2CO3 (4.4 g, 13.5 mmol) were added. The mixture was stirred at 25°C for 16 h. A strong yellow spot appeared on a TLC plate (hexane:AcOEt = 8:2). The insoluble material was filtered through a silica gel pad, and the filtrate was concentrated. The resulting residue was diluted with dichloromethane (150 mL) and washed with H2O (10 x 15 mL). The organic layer was dried over Na2SO4, filtered, and concentrated in vacuo. Purification by silica gel column chromatography (CH2Cl2:AcOEt = 20:1) gave compound B-2 as a dark yellow solid (0.72 g, Y = 65.8%).

[0067] 7. Preparation of Compound C1

[0068] At 100°C, compound B-2 (484 mg, 2 mmol, 1 eq) and compound A-4 (857 mg, 2.4 mmol, 1.2 eq) were dissolved in 30 mL of acetic anhydride, and 2 mL of triethylamine was added to catalyze the reaction. The reaction progress was monitored by TLC. The reaction was stopped after stirring for 1-24 h. The mixture was extracted with dichloromethane and water, and the solvent was evaporated. The mixture was purified by silica gel column chromatography (CH2Cl2:MeOH=20:1), and then recrystallized from dichloromethane and ethyl acetate to obtain compound C1 (403 mg, 0.84 mmol, Y=42.0%).

[0069] Figure 3 is the H NMR spectrum of compound C1 prepared in Example 1 of the present invention; Figure 4 is the C NMR spectrum of compound C1 prepared in Example 1 of the present invention; Figure 5 This is the mass spectrum of compound C1 prepared in Example 1 of the present invention.

[0070] 1 H NMR (600MHz, CDCl3) δ = 8.20 (s, 2H), 7.93 (d, J = 15.0Hz 1H),7.14(d,J=8.5Hz,1H),6.82(s,1H),6.77(s,1H),6.65-6.70(m,2H),4.29(t,J=7.2Hz,2H),3.88(s,3H),2.72(s,6H),2.71(s, 6H),2.64(t,J=5.4Hz,2H),2.59(t,J=6.0Hz,2H),1.87-1.90(m,2H),1.84-1.86(m,2H),1.40-1.47(m,2H),0.98(t,J=7.8Hz,3H).

[0071] 13 C NMR (100MHz, CDCl3) δ = 168.5, 162.9, 156.3, 154.3, 141.9, 140.4, 134.7, 130.8, 128.9, 127.7, 127.2, 124.8, 124.0, 116. 0,115.0,113.5,112.4,101.6,100.4,68.0,56.1,46.3,41.0,32.3,29.7,29.4,29.0,25.6,24.1,21.1,20.1,13.7,10.1.

[0072] ESI-MS (m / z): calculated value: 480.29, spectrum display: 480.3.

[0073] Example 2:

[0074] Figure 1 The general synthetic route of the cycloheptatrienolpyrrole fluorescent dye in the embodiment of the present invention is shown; the synthesis of compound C2 is taken as an example:

[0075] Synthesis route diagram Figure 7 As shown, compound C2 was synthesized according to the synthetic route:

[0076] 1. Preparation of Compound B-3

[0077] A mixed solution of BBr (1.5 mL) and CHCl (3 mL) was added to a solution of compound B-2 (2.42 g, 10 mmol) in anhydrous CHCl (25 mL) at 0°C. The resulting mixture was stirred at 0°C overnight and then quenched with water. After filtration, the reaction was chromatographed on silica gel using CHCl:CHOH (20:1) as the eluent to obtain compound B-3 (1.45 g, Y = 63.5%).

[0078] 2. Preparation of Compound C2

[0079] At 100°C, compound B-3 (338 mg, 1.5 mmol, 1 eq) and compound A-4 (589 mg, 1.65 mmol, 1.1 eq) were dissolved in 20 mL of acetic anhydride, and 1.0-2.0 mL of triethylamine was added to catalyze the reaction. The reaction progress was monitored by TLC. The reaction was stopped after stirring for 1-24 h. The mixture was extracted with dichloromethane and water, and the solvent was evaporated. The mixture was purified by silica gel column chromatography (CH2Cl2:MeOH=20:1), and then recrystallized from dichloromethane and ethyl acetate to obtain compound 2 (324 mg, 0.73 mmol, Y=48.4%).

[0080] Figure 8 is the H NMR spectrum of compound C2 prepared in Example 2 of the present invention; Figure 9 is the C NMR spectrum of compound C2 prepared in Example 2 of the present invention; Figure 10 is the mass spectrum of compound C2 prepared in Example 2 of the present invention;

[0081] 1H NMR (400MHz, CDCl3) δ = 8.49 (s, 2H), 7.54 (d, J = 15.9Hz, 1H), 7.11 (d, J = 8.0Hz, 1H),6.86(s,1H),6.76-6.79(m,1H),6.70(d,J=16.0Hz,1H),6.54(s,1H),4.45 (t,J=8.0Hz,2H),2.84(s,6H),2.74(s,6H),2.57-2.65(m,2H),2.30(s,2H),1 .85-1.93(m,2H),1.76-1.84(m,2H),1.44-1.54(m,2H),1.02(t,J=7.2Hz,3H).

[0082] 13 C NMR (100MHz, CDCl3) δ = 169.1, 168.0, 153.1, 151.5, 144.9 (3C), 136.2 (2C), 132.1 (2C), 130.1 (2C), 126.7, 126. 3,125.5,124.0,119.8,116.9,112.1,109.1,47.3,32.2,29.7,28.9,24.4,21.1,20.7,20.1,18.39,13.6,10.2.

[0083] ESI-MS (m / z): [M+MeCN] + Calculated value: 508.27, spectrum display: 508.3.

[0084] Example 3:

[0085] Fluorescence spectrum test of compound C1 and compound C2:

[0086] like Figure 6 As shown, 10.0 μM compound C1 was placed in dichloromethane, dimethyl sulfoxide, and PBS buffer, and the fluorescence emission spectra of the different solutions were measured under 808 nm laser excitation. Compound C1 exhibited strong fluorescence in the NIR-II region, with the maximum emission peak between 1000 and 1200 nm.

[0087] like Figure 11 As shown, 10.0 μM compound C2 was placed in dichloromethane, dimethyl sulfoxide, and PBS buffer, and the fluorescence emission spectra of the different solutions were measured under 808 nm laser excitation. Compound C2 exhibited strong fluorescence in the NIR-II region, with the maximum emission peak between 1000 and 1200 nm.

[0088] In summary, the present invention discloses a method for preparing a cycloheptatriene pyrrole salt fluorescent dye. The cycloheptatriene pyrrole salt and its fluorescent dye of the present invention have a certain red shift compared to the maximum absorption wavelength and emission wavelength of traditional hemicyanine dyes. Compound C1 and compound C2 prepared in the embodiment of the present application are verified by spectral testing, and their fluorescence emission wavelength is between 1020nm-1050nm, which can be well applied to the scene of long-wavelength fluorescence detection. Such cycloheptatriene pyrrole salt and its fluorescent dye raw material are easy to obtain and low cost, easy to operate, high yield, mild reaction conditions and simple synthesis steps, with high commercial application value.

[0089] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a list of elements includes not only those elements, but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0090] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0091] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. A cycloheptatriene pyrrolidine salt fluorescent compound, characterized in that: The structural formulas of the cycloheptatriene pyrrolidine salt fluorescent compounds C1 and C2 are: ； 。 2. The method for preparing a cycloheptatriene pyrrolidine salt fluorescent compound as claimed in claim 1, wherein: The preparation method of the cycloheptatriene pyrrolidine salt fluorescent compounds C1 and C2 comprises: Step S1: Preparation of intermediate compound A-1 2,5-Hexanedione, n-butylamine, and aminosulfonic acid were stirred at room temperature and extracted to obtain compound A-1; Step S2: Preparation of intermediate compound A-2 Dissolve compound A-1 in DMF, add POCl3, and stir at room temperature. After the reaction is complete, pour the solution into a mixture of sodium acetate and ice water and stir. After forming a solid suspension, filter, wash, and air-dry to obtain intermediate compound A-2. Step S3: Preparation of intermediate compound A-3 Compound A-2 was dissolved in ethanol, 3-pentanone was added, and then an aqueous sodium hydroxide solution was added; the mixture was stirred at room temperature, and then extracted, concentrated, and purified to obtain an intermediate compound A-3; Step S4: Preparation of intermediate compound A-4 Compound A-3 was dissolved in THF under anhydrous and oxygen-free conditions, CH3MgCl was added, and then an inorganic acid was added. After extraction, concentration, and purification, the intermediate compound A-4 was obtained. Step S5: Preparation of intermediate compound B-1 PBr3, DMF and CHCl3 were added to cyclohexanone and stirred at room temperature for neutralization reaction; then compound B-1 was obtained by extraction, washing and drying; Step S6: Preparation of intermediate compound B-2: Compound B-1, 2-hydroxy-4-methoxybenzaldehyde, and Cs2CO3 were dissolved in DMF and stirred at room temperature, and then extracted, concentrated, and purified to obtain the intermediate compound B-2; Preparation of intermediate compound B-3: A mixed solution of BBr3 and CH2Cl2 was added to an anhydrous CH2Cl2 solution of the intermediate compound B-2; the resulting mixture was stirred overnight and then quenched with water; after filtration, silica gel chromatography was performed to obtain compound B-3; Step S7: Preparation of compound C1: Compound B-2 and compound A-4 were dissolved in acetic anhydride, triethylamine was added to catalyze the reaction, and the mixture was heated, condensed and refluxed. After extraction, concentration and purification, compound C1 was obtained; Preparation of compound C2: Compound B-3 and compound A-4 were dissolved in acetic anhydride, triethylamine was added, and the reaction was stopped after stirring. The solvent was evaporated after extraction with dichloromethane solvent and water, and the mixture was purified by silica gel column chromatography and then recrystallized from dichloromethane and ethyl acetate to obtain compound C2.

3. The method for preparing a cycloheptatriene pyrrolidine salt fluorescent compound as claimed in claim 2, wherein: In the step S1, the prepared compound A-1 is extracted with ether, then extracted with dichloromethane and water, the organic layer is dried over Na2SO4, filtered, and concentrated in vacuo to obtain compound A-1; in the step S1, the molar ratio of 2,5-hexanedione and n-butylamine is 1:1.1-1.5 equivalents, and aminosulfonic acid is a catalytic amount.

4. The method for preparing a cycloheptatriene pyrrolidine salt fluorescent compound as claimed in claim 2, wherein: In step S2, the mass ratio of sodium acetate to ice in the sodium acetate-ice-water mixture is 1:6; the mass ratio of compound A-1 to sodium acetate is 12.24:100; and the washing solvent is distilled water.

5. The method for preparing a cycloheptatriene pyrrolidine salt fluorescent compound as claimed in claim 2, wherein: In step S3, the molar ratio of compound A-2 to 3-pentanone is 1:1.1; the mass concentration of the sodium hydroxide aqueous solution is 10%; extraction is performed with distilled water and dichloromethane; the concentration method is reduced pressure distillation; and the crude product is purified by column chromatography, and the column chromatography eluent is petroleum ether and ethyl acetate.

6. The method for preparing a cycloheptatriene pyrrolidine salt fluorescent compound as claimed in claim 2, wherein: In the step S4, extraction is performed with distilled water and dichloromethane; the concentration method is vacuum distillation; and the crude product is purified by recrystallization.

7. The method for preparing a cycloheptatriene pyrrolidine salt fluorescent compound as claimed in claim 2, wherein: In step S5, the molar ratio of PBr3 to cyclohexanone is 130.5:48.4; the reaction is neutralized with a saturated sodium bicarbonate solution, and extracted with dichloromethane and distilled water.

8. The method for preparing a cycloheptatriene pyrrolidine salt fluorescent compound as claimed in claim 2, wherein: In step S6, in the preparation of intermediate compound B-2, the molar ratio of compound B-1, 2-hydroxy-4-methoxybenzaldehyde, and Cs2CO3 is 5.4:4.5:13.5; the mixture is washed with H2O; the organic layer is dried over Na2SO4, filtered, concentrated in vacuo, and purified by silica gel column chromatography; in the preparation of intermediate compound B-3, the ratio of BBr3, CH2Cl2, and compound B-2 is 1.5 mL:3 mL:10 mmol, and the reaction temperature is 0°C.

9. The method for preparing a cycloheptatriene pyrrolidine salt fluorescent compound as claimed in claim 2, wherein: In step S7, in the preparation of compound C1, the molar ratio of compound B-2 to compound A-4 is 1:1.2; after extraction with dichloromethane solvent and water, the solvent is evaporated and concentrated, purified by silica gel column chromatography, and then recrystallized from dichloromethane and ethyl acetate to obtain compound C1; in the preparation of compound C2, the molar ratio of compound B-3 to compound A-4 is 1:1.1, the reaction temperature is 100°C, and the reaction time is 1-24h.

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