Urea compound for inducing antigen-specific reaction, fluorescent marker thereof, preparation method and use thereof

Abstract

The present invention discloses a urea compound, 1-(4-hydroxycyclohexyl)-3-(3-(2-oxoxazolidin-3-yl)phenyl)urea, for inducing an antigen-specific reaction, as shown in Formula 1; a fluorescent marker thereof, and a preparation method and use thereof. The invention relates to the fields of medicinal chemistry and pharmacotherapy. The preparation method comprises the following steps: dissolving 2-oxazolidinone, N,N-dimethylglycine, cuprous iodide, and potassium carbonate in a solvent, and reacting them with 3-bromoaniline to produce intermediate I; dissolving intermediate I, and reacting it with triethylamine and phenyl chloroformate to produce intermediate II; and reacting intermediate II with 4-aminocyclohexane-1-ol hydrochloride and N,N-diisopropylethylamine (DIPEA) to produce a product. The aforementioned small molecule compound has the characteristics of small molecular weight, easy binding to CD91, and rapid entry into DC cells. #imgabs0#

Description

Technical Field

[0001] The present invention relates to the fields of pharmaceutical chemistry and pharmacotherapy, and in particular to urea compounds, BODIPY fluorescent markers thereof, and preparation methods and uses thereof. Background Art

[0002] Cellular immunotherapy is gaining increasing attention for its promising therapeutic effects in the treatment of infectious diseases and cancer. This approach uses antigen-presenting cells (also known as dendritic cells (DCs)) to effectively present and stimulate immune T cells to kill viruses.

[0003] Complexes of the heat shock protein glycoprotein 96 (gp96) and antigenic peptides are taken up by antigen-presenting cells and presented by major histocompatibility complex (MHC) class I molecules. To explain the extraordinary efficiency of this process, gp96 uptake is thought to occur through a receptor, the low-density lipoprotein receptor-related protein (CD91). Peptide complexes of heat shock protein 90 (hsp90), calreticulin, and heat shock protein 70 (hsp70) are also taken up by macrophages and dendritic cells (DCs) and re-presented by MHC class I molecules. All heat shock proteins utilize the CD91 receptor, even though some of these proteins share no homology with each other.

[0004] Heat shock protein (HSP)-stimulated activated proteoglycan cells (APCs) can activate NF-κB, release pro-inflammatory cytokines, and increase the expression of CD40, B7, and MHC II (classic markers of DC maturation). CD91 is a key receptor for HSP-mediated endocytosis and cross-presentation.

[0005] In the existing technology, CD91 is bound to protein macromolecules, which have a large molecular weight, large volume, inconvenient storage, and relatively high cost. Therefore, designing a small molecule compound that can bind to CD91 to replace the protein macromolecule to carry the antigen into DC cells is an urgent problem that needs to be solved. Summary of the Invention

[0006] The purpose of the present invention is to provide a small molecule 1-(4-hydroxycyclohexyl)-3-(3-(2-oxooxazolidin-3-yl)phenyl)urea that can activate CD91 on DC cells, and through a fluorescent labeling method, it is confirmed that the aforementioned compound can effectively bind to and activate CD91 molecules on DC cells, quickly enter DC cells, and upregulate the expression of MHC-I molecules that present antigens.

[0007] To achieve the above objectives, the present invention provides the following technical solutions:

[0008] A urea compound for inducing an antigen-specific reaction, having a structure as shown in Formula 1:

[0009]

[0010] A method for preparing the compound comprises the following steps:

[0011] ① Dissolve 2-oxazolidinone, N,N-dimethylglycine, cuprous iodide, and potassium carbonate in N,N-dimethylformamide (DMF) and add 3-bromoaniline dropwise to generate Intermediate I. ② Dissolve Intermediate I in dichloromethane, add triethylamine, cool, and add phenyl chloroformate dropwise in an ice bath to generate Intermediate II.

[0012] ③ Intermediate II and 4-aminocyclohexane-1-ol hydrochloride are dissolved in a solvent and reacted with N,N-diisopropylethylamine (DIPEA) to obtain 1-(4-hydroxycyclohexyl)-3-(3-(2-oxooxazolidin-3-yl)phenyl)urea.

[0013] Furthermore, in step ①, the reaction temperature is 100-120° C., and the reaction is continued for 18-24 hours to obtain the intermediate I, which is 3-(3-aminophenyl)oxazolidin-2-one.

[0014] Furthermore, in step ③, intermediate II and 4-aminocyclohexane-1-ol hydrochloride are dissolved in ethyl acetate, and DIPEA is added, and the mixture is reacted at room temperature for 9-12 hours.

[0015] Furthermore, the compound 1-(4-hydroxycyclohexyl)-3-(3-(2-oxooxazolidin-3-yl)phenyl)urea is used in cell immunotherapy. The compound has been shown to be able to activate CD91 molecules to enter DC cells through fluorescent labeling.

[0016] Furthermore, a BODIPY fluorescent label of 1-(4-hydroxycyclohexyl)-3-(3-(2-oxooxazolidin-3-yl)phenyl)urea as shown in Formula 6.

[0017] Furthermore, a method for preparing the BODIPY fluorescent marker as described in Formula 6 is characterized in that the method comprises the following steps:

[0018] ① Dissolve intermediate II in a solvent and react with N-Boc-1,4-butanediamine to obtain intermediate III;

[0019] ② Dissolve intermediate III in a solvent and react with trifluoroacetic acid (TFA) to obtain intermediate IV;

[0020] ③ Dissolve intermediate IV, BODIPY-CO2H and 1-hydroxybenzotriazole (BtOH) in a solvent, add EDCI and triethylamine under ice bath conditions to react and obtain the BODIPY fluorescent marker of the small molecule compound.

[0021] Furthermore, in step ①, intermediate II is dissolved in chloroform and reacted with N-Boc-1,4-butanediamine at room temperature for 10-12 hours to obtain intermediate III which is tert-butyl (4-(3-(3-(2-oxoxazolin-3-yl)phenyl)ureido)carbamate.

[0022] Furthermore, in step ②, intermediate III is dissolved in dichloromethane and reacted with trifluoroacetic acid at room temperature to obtain intermediate IV, which is 1-(4-aminobutyl)-3-(3-(2-oxooxazolidin-3-yl)phenyl)urea hydrochloride.

[0023] Furthermore, the solvent in step ③ is tetrahydrofuran (THF); ice bath cooling is performed, EDCI and triethylamine are added, the mixture is stirred while maintaining the temperature, and then the mixture is transferred to room temperature for reaction for 10-14 hours to obtain the product.

[0024] As the present invention adopts the above technical solution, compared with the prior art, for example, the small molecule in the present invention has the characteristics of small molecular weight, easy binding to CD91 and rapid entry into DC cells.

[0025]

[0026] BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the NMR spectrum of intermediate I.

[0028] Figure 2 This is the NMR spectrum of intermediate II.

[0029] Figure 3 is the NMR spectrum of formula 1.

[0030] Figure 4 This is the NMR spectrum of intermediate III.

[0031] Figure 5 This is the NMR spectrum of formula 6.

[0032] Figure 6 qPCR analysis of the differences in the interference of three sequences with CD91 RNA.

[0033] Figure 7 Flow cytometry analysis was used to confirm the interference of pAVE3578 sequence on CD91 RNA.

[0034] Figure 8 This is a fluorescence photograph of DC cells treated with the compounds provided in the examples of the present invention.

[0035] Figure 9 Changes in fluorescence intensity were analyzed for flow cytometry.

[0036] Figure 10 MHC expression was detected by qPCR. DETAILED DESCRIPTION

[0037] The compounds disclosed in the present invention are described in further detail below in conjunction with the accompanying drawings and specific examples. It should be noted that the technical features or the combination of technical features described in the following examples should not be considered to be isolated, and they can be combined with each other to achieve better technical effects. In the accompanying drawings of the following examples, the same reference numerals appearing in each accompanying drawing represent the same features or parts, which can be applied in different embodiments. Therefore, once a certain item is defined in an accompanying drawing, it does not need to be further discussed in the subsequent accompanying drawings.

[0038] Example 1

[0039] Preparation of 3-(3-aminophenyl)oxazolidin-2-one (Intermediate I)

[0040]

[0041] 2-oxazolidinone, N,N-dimethylglycine, cuprous iodide and potassium carbonate were added to a dry 10 mL round-bottom flask. N,N-dimethylformamide (DMF) was added to dissolve the above compounds. 3-bromoaniline was added dropwise and the mixture was reacted at 120°C for 24 h.

[0042] The reaction was monitored by thin-layer chromatography until 3-bromoaniline was completely consumed. Developing solvent: PE:EA = 1:2. The product was visible under UV light and developed with alkaline potassium permanganate solution and phosphomolybdic acid.

[0043] The reaction solution was rotary evaporated to remove the solvent, and the residue was separated by silica gel column chromatography (PE:EA=1:2) to obtain Intermediate I (white solid) with a yield of 92%.

[0044] See also Figure 1 Shown is the NMR spectrum of intermediate I.

[0045] Nuclear magnetic spectrum: 1H NMR (600 MHz, CDCl3) δ7.17-7.11 (m, 2H), 6.70 (dd, J = 7.8, 1.8 Hz, 1H), 6.47 (dd, J = 7.8, 1.8 Hz, 1H), 4.48-4.44 (m, 2H), 4.05-4.00 (m, 2H).

[0046] Example 2

[0047] Preparation of (3-(2-oxooxazolidin-3-yl)phenyl)carbamate (Intermediate II)

[0048]

[0049] Intermediate I was added to a dry 10-mL round-bottom flask, dissolved in dichloromethane, and triethylamine was added. The mixture was cooled to 0-5°C in an ice bath. Phenyl chloroformate was added dropwise in an ice bath. The mixture was stirred for 30 minutes and refluxed at 40°C for 12 hours.

[0050] The reaction was monitored by thin layer chromatography until the intermediate I was completely consumed. Developing solvent: PE:EA=2:1.

[0051] The reaction solution was rotary evaporated to remove the solvent, and the product was purified by silica gel column chromatography (PE:EA=2:1) ​​to obtain Intermediate II (white solid) with a yield of 95%.

[0052] See also Figure 2 Shown is the NMR spectrum of intermediate II.

[0053] NMR spectrum: 1 H NMR(600MHz, CDCl3)δ7.78(bs,1H),7.42-7.37(m,2H),7.34-7.30(m,2H),7 .26-7.22(m,3H),7.20-7.16(m,2H),4.48-4.43(m,2H),4.06-4.01(m,2H).

[0054] Example 3

[0055] Preparation of 1-(4-hydroxycyclohexyl)-3-(3-(2-oxoxazolidin-3-yl)phenyl)urea

[0056]

[0057] 1-(4-Hydroxycyclohexyl)-3-(3-(2-oxooxazolidin-3-yl)phenyl)urea

[0058] Intermediate II and 4-aminocyclohexane-1-ol hydrochloride were added to a dry 50-mL round-bottom flask, dissolved with ethyl acetate, and N,N-diisopropylethylamine (DIPEA) was added, and the mixture was reacted at room temperature for 10 h.

[0059] The reaction was monitored by thin layer chromatography until the conversion of Intermediate II was complete. Developing solvent: DCM:MeOH=10:1.

[0060] The reaction mixture was rotary evaporated to remove the solvent, and the product was purified by silica gel column chromatography (DCM:MeOH=10:1) to obtain 1-(4-hydroxycyclohexyl)-3-(3-(2-oxooxazolidin-3-yl)phenyl)urea as a white solid in a yield of 91%.

[0061] See also Figure 3 Shown is the NMR spectrum of formula 1.

[0062] Nuclear magnetic spectrum: 1H NMR (600MHz, DMSO-d6) δ8.45 (s, 1H), 7.65 (s, 1H), 7.23-7.17 (m, 2H), 7.05-7.02 (m, 1H), 5.99 (d, J = 7.8Hz, 1H), 4.56 (d, J = 4.2Hz, 1H), 4.44-4.39 (m, 2H), 4.03-3.98 (m, 2H), 3.44-3.34 (m, 1H), 1.86-1.86 (m, 4H), 1.27-1.11 (m, 4H).

[0063] Also included is the preparation of boron dipyrrole dye (BODIPY) labels of 1-(4-hydroxycyclohexyl)-3-(3-(2-oxooxazolidin-3-yl)phenyl)urea.

[0064] Example 4

[0065] Preparation of tert-butyl (4-(3-(3-(2-oxoxazolin-3-yl)phenyl)ureido)carbamate (Intermediate III)

[0066]

[0067] Intermediate II was added to a dry 10-mL round-bottom flask, dissolved in chloroform, and N-Boc-1,4-butanediamine was added, and the mixture was reacted at room temperature for 12 h.

[0068] The reaction was monitored by thin layer chromatography until the substrate 4 was completely consumed. Developing solvent: DCM:MeOH = 30:1. The product was visualized with alkaline potassium permanganate solution and ninhydrin.

[0069] The reaction solution was rotary evaporated to remove the solvent, and the residue was purified by silica gel column chromatography (DCM:MeOH=30:1) to obtain Intermediate III as a white solid with a yield of 96%.

[0070] See also Figure 4 Shown is the NMR spectrum of intermediate III.

[0071] NMR spectrum: 1 H NMR (600MHz, CDCl3) δ7.85(bs,1H),7.67(bs,1H),7.28-7.25(m,1H),7.23-7.18(m,1H),6.99(bd,J=7.2Hz,1H),5.78(bs,1H ),4.87(bs,1H),4.46-4.40(m,2H),4.02-3.96(m,2H),3.23-3.16(m,2H),3.13-3.06(m,2H),1.51-1.45(m,4H),1.43(s,9H).

[0072] Example 5

[0073] Preparation of 1-(4-aminobutyl)-3-(3-(2-oxooxazolidin-3-yl)phenyl)urea hydrochloride (Intermediate IV)

[0074]

[0075] Intermediate III was added to a dry 5 ml round-bottom flask, dissolved in dichloromethane, and trifluoroacetic acid (TFA) was added, followed by stirring at room temperature.

[0076] The reaction was monitored by thin-layer chromatography (TLC) until the intermediate III was completely consumed. Developing solvent: DCM:MeOH = 30:1. The product was visualized with alkaline potassium permanganate solution and ninhydrin.

[0077] The reaction solution was rotary evaporated to remove the solvent to obtain a crude intermediate IV as a white solid with a yield of 99%, which was used directly in the next step without purification.

[0078] Example 6

[0079] Preparation of BODIPY fluorescent label of 1-(4-hydroxycyclohexyl)-3-(3-(2-oxooxazolidin-3-yl)phenyl)urea.

[0080]

[0081] Intermediate IV, BODIPY-CO2H and 1-hydroxybenzotriazole (BtOH) were added to a dry 5-mL round-bottom flask, dissolved with THF, cooled to 0-5°C in an ice bath, EDCI and triethylamine were added, stirred at this temperature for 30 minutes, and then moved to room temperature for reaction for 12 hours.

[0082] The reaction was monitored by thin-layer chromatography until compound 8 was completely consumed. Developing solvent: PE:EA = 1:3. The product had strong fluorescence (Rf = 0.1).

[0083] The reaction solution was rotary evaporated to remove the solvent, and the product was separated by silica gel column chromatography (PE:EA=1:3) to obtain the fluorescent labeled product with a yield of 58%.

[0084] See also Figure 5 Shown is the NMR spectrum of formula 6.

[0085] Nuclear magnetic spectrum: 1H NMR (600MHz,CDCl3)δ8.19(bs,1H),(7.88,7.73)(d,J=8.4Hz,1H),7.68(s,1H),7.46-7.42(m,1H),7.41-7.37(m,1H),7.22-7.19(m,1H),7.13(bs,1H),7.09(s,1H),6.88(bd,J=3.6Hz ,1H),6.54(bs,1H),6.26(bd,J=3.6Hz,1H),6.11(s,1H),4.47-4.40(m,2H),4.07-3.99(m, 2H),3.28-3.17(m,6H),2.65(t,J=7.2Hz,2H),2.53(s,3H),2.24(s,3H),1.51-1.40(m,4H).

[0086] Example 7

[0087] Cell culture

[0088] DC cells were cultured normally in 10% FBS + RPMI-1640 medium and passaged when they reached over 80% confluence. Discard the cell culture supernatant, wash the cells with 2 mL of PBS, and discard. Add 700 μL of 0.25% trypsin and place in a CO2 incubator for approximately 1.5 minutes. Observe under a microscope for rounded cells. Gently tap the culture flask to detach the cells.

[0089] Add 2 mL of 10% FBS + RPMI-1640 culture medium to completely detach the cells. Pipette the cell mixture into a sterile 15 mL centrifuge tube and centrifuge at 1000 rpm for 3 minutes at room temperature. Discard the supernatant and resuspend the cells in 1 mL of complete culture medium. Take 10 μL of the cell suspension and add 1 μL of trypan blue, mix thoroughly, and count the cells on a cell counting plate.

[0090] Example 8

[0091] qPCR was used to analyze the effect of CD91 RNA interference on DC cells.

[0092] On the basis of Example 7, the cell viability was ensured to be above 90%, and 6-well plates were plated, with 6×10 5After the cells adhered, the supernatant was discarded and replaced with complete culture medium. Adenoviruses packaged with the following three CD91 interference sequences, pAVE-3576, pAVE-3577, and pAVE-3578, and adenovirus supernatant packaged with the control plasmid, pAVE-Control, were added. After 24 hours of culture, samples were collected, total RNA was extracted, and cDNA was reverse transcribed. qPCR analysis was performed using the CD91 primer sequences shown in the table below and the GAPDH internal control primer sequence to verify the interference effects of the three CD91 interference sequences on CD91.

[0093] Table 1 Three CD91 interference sequences are as follows

[0094]

[0095] Table 2 The primer sequences of each gene are as follows

[0096]

[0097]

[0098] See also Figure 6 qPCR analysis of the differences in CD91 RNAi activity among the three sequences is shown. The experiment demonstrates the CD91 RNA transcription levels in each group. The results indicate that the third sequence, pAVE3578, has the best interference effect and can be used in subsequent experiments.

[0099] Example 9

[0100] Flow cytometry analysis confirmed the interference effect of pAVE3578 plasmid on CD91 RNA.

[0101] Based on Example 7, after culturing for 24 hours, samples were collected from the pAVE3578 group with the best interference effect, and the expression level of CD91 was detected by flow cytometry:

[0102] Experimental steps:

[0103] 1. Harvest the cells, centrifuge at 1000 rpm for 5 minutes at 4°C, and discard the supernatant.

[0104] 2. Add 50 mL of staining buffer to resuspend the cells and count them. Use Trypan Blue to detect cell viability.

[0105] 3. Centrifuge the cell suspension and discard the supernatant; resuspend the cells in Staining Buffer and adjust the cell concentration to 2 × 107 cells / mL.

[0106] 4. Add 50 μL of diluted primary antibody (antibody diluted to an appropriate concentration with Staining Buffer) to each flow cytometry tube; add 50 μL of Staining Buffer to a blank tube.

[0107] 5. Add 50 μL of cell suspension to each tube and mix gently.

[0108] 6. Incubate at 4°C in the dark for 20 minutes.

[0109] 7. After incubation, centrifuge at 1000 rpm for 5 minutes at 4°C and discard the supernatant.

[0110] 8. Repeat washing three times with 100 μL Staining Buffer.

[0111] 9. Resuspend the cells in 100 μL Staining Buffer and analyze on flow cytometer.

[0112] Data Analysis:

[0113] Data were analyzed using GraphPad Prism 8, and statistical analysis was performed using Bonferroni's multiple comparisons test with alpha = 5.000%. Figure 7 Flow cytometry analysis confirms the interference of pAVE3578 on CD91 RNA. As can be seen from the figure, after DC cells were treated with the interfering adenovirus, the expression of CD91 in the pAVE3578 group was significantly reduced compared to the control group.

[0114] Example 10

[0115] Total RNA extraction

[0116] Experimental steps:

[0117] Aspirate the culture medium in the 6-well plate, add 1 mL of Trizol Reagent to each well, and use a pipette to blow to completely lyse the cells.

[0118] The lysate was transferred to a 1.5 mL EP tube and placed at room temperature for 10 minutes.

[0119] Add 200 μL of chloroform, shake vigorously to mix, and let it stand at room temperature for 10 minutes.

[0120] Centrifuge at 12,000 rpm at 4°C for 10 minutes, aspirate the supernatant into a new centrifuge tube, add an equal volume of isopropanol, and allow to precipitate at room temperature for 10 minutes. Centrifuge at 12,000 rpm at 4°C for 15 minutes, and discard the supernatant.

[0121] Wash the pellet once with 500 μL of 75% ethanol. Centrifuge at 12,000 rpm at 4°C for 5 minutes, recover the pellet, and discard the supernatant. Invert and air-dry at room temperature for 10 minutes.

[0122] Dissolve the precipitate in 20 μL DEPC-H2O, measure OD260 and OD280, and calculate the RNA concentration. Check the integrity of the RNA by agarose gel electrophoresis.

[0123] Example 11

[0124] The primer design table for real-time PCR analysis is shown in Table 2.

[0125] Reagents and instruments

[0126] Real-time fluorescence quantitative universal reagent (Shanghai Biotechnology Co., Ltd.)

[0127] Real-time fluorescence quantitative PCR instrument (Applied Biosystems, 7900, US)

[0128] The experimental method is as follows:

[0129] Reverse transcription reaction system

[0130]

[0131]

[0132] Reverse transcription program: 42℃ 30min; 85℃ 10min.

[0133] Real-time fluorescence quantitative reaction system

[0134]

[0135] Quantitative PCR reaction procedure

[0136] 95℃, 5 minutes denaturation

[0137] 95℃, 12 seconds; 60℃, 40 seconds. 40 cycles

[0138] 95°C, 10 seconds; 60°C, 10 seconds

[0139] 40℃, 30 seconds

[0140] Example 12

[0141] Treatment of DC cells with fluorescently labeled small molecule compounds

[0142] Ensure that the cell viability is above 90%, and plate 6-well plates with 6×10 5After the cells adhered, the supernatant was discarded and replaced with complete culture medium. The supernatant of the control plasmid pAVE-Control and the supernatant of the third sequence pAVE3578 adenovirus were added and mixed. After 24 hours of culture, the cells were treated with fluorescently labeled compounds (40 μM) and photographed at 3, 6, 12, 24, and 48 hours.

[0143] See also Figure 8 Shown are fluorescent images of DC cells treated with compounds provided in Examples of the present invention. The images show images of the same field of view at each time point, both under white light and fluorescence. The most pronounced differences were observed between 24 and 48 hours after treatment with the compounds.

[0144] Example 13

[0145] Flow cytometry for cell fluorescence

[0146] DC cell samples were collected at various time points and the cell fluorescence intensity was detected using flow cytometry. The steps are as follows:

[0147] 1. Harvest the cells, centrifuge at 1000 rpm for 5 minutes at 4°C, and discard the supernatant.

[0148] 2. Add 100 μL PBS to resuspend the cells and count them. Use Trypan Blue to detect cell activity.

[0149] 3. Centrifuge the cell suspension and discard the supernatant; resuspend the cells with PBS to adjust the cell concentration to 2×10 7 pieces / mL.

[0150] 4. Place the cells on a flow cytometer for detection and perform significance analysis at α = 0.05.

[0151] See also Figure 9 The figure shows the changes in fluorescence intensity analyzed by flow cytometry. As can be seen from the figure, after DC cells were treated with small molecule compounds for 24 and 48 hours and CD91 RNA interference was performed (pAVE-3587 group), the fluorescence intensity was significantly lower than that of the CD91 RNA non-interference group (pAVE-Control group) (p<0.05). There was no significant difference in fluorescence intensity between the experimental and control groups at other time points (p>0.05).

[0152] Example 14

[0153] Detection of MHC expression MHC (major histocompatibility complex) is a tool for DC cells to present antigens; upregulation of MHC expression in DC cells is an important sign of enhanced antigen presenting ability.

[0154] DC cells were treated with fluorescently labeled compounds, and DC cell samples were collected at 6, 16, and 24 hours. Total RNA was extracted according to the method described in Example 6, and Real-time PCR analysis was performed according to the method described in Example 11. The MHC-I primer sequences are shown in Table 2.

[0155] See also Figure 10 The figure shows the expression of MHC detected by qPCR. As shown in the figure, the expression of MHC on DC cells was detected at 6, 16, and 24 hours. It was found that after the application of small molecules, the expression of MHC on DC cells was upregulated at 16 hours.

[0156] Although example aspects of the present disclosure have been described for illustrative purposes, those skilled in the art will appreciate that the foregoing description is merely a description of preferred embodiments of the present invention and does not limit the scope of the present invention in any way. The scope of the preferred embodiments of the present invention includes alternative implementations in which functions may be performed out of the order in which they appear or are discussed. Any changes or modifications made by those skilled in the art based on the foregoing disclosure are intended to fall within the scope of the claims.

Claims

1. A BODIPY fluorescent marker as shown in Formula 6 2. A method for preparing the BODIPY fluorescent marker according to claim 1, characterized in that: The method comprises the following steps: ① Dissolve intermediate II in a solvent and react with N-Boc-1,4-butanediamine to obtain intermediate III ② Dissolve intermediate III in a solvent and react with trifluoroacetic acid (TFA) to obtain intermediate IV, i.e. ③ Dissolve intermediate IV, BODIPY-CO2H, and 1-hydroxybenzotriazole (BtOH) in a solvent, add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) and triethylamine under ice bath conditions to react and obtain the BODIPY fluorescent marker of the small molecule compound.

3. The method according to claim 2, wherein: In step ①, intermediate II is dissolved in chloroform and reacted with N-Boc-1,4-butanediamine at room temperature for 10-12 hours to obtain intermediate III.

4. The method according to claim 2, wherein: In step ②, intermediate III is dissolved in dichloromethane and reacted with trifluoroacetic acid at room temperature to obtain intermediate IV.

5. The method according to claim 2, wherein: The solvent in step ③ is tetrahydrofuran (THF); ice-cool the mixture, add EDCI and triethylamine, maintain the temperature and stir, then transfer the mixture to room temperature for 10-14 hours to obtain the product.

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