A frequency up-conversion xanthene fluorescent dye and its preparation method and application in biological fluorescence labeling

Abstract

The present invention provides a frequency up-conversion xanthene fluorescent dye and a preparation method thereof and an application in biological fluorescence labeling, belonging to the field of biological optical labeling technology. The frequency up-conversion xanthene fluorescent dye provided by the present invention uses xanthene as a parent, and the xanthene structure is connected to the nitrogen heterocycle of indole, has frequency up-conversion luminescence performance, can not only realize near-infrared emission, but also realize short-wavelength luminescence ability under anti-Stokes shift long wavelength (730nm) excitation, can enter cells in a short time to realize fluorescent staining, and can realize schistosome fluorescent labeling imaging. At the same time, the excitation conditions of the up-conversion luminescence of the frequency up-conversion xanthene fluorescent dye provided by the present invention are mild, which can reduce the thermal effect or light damage caused by the excitation light, thereby avoiding the problems of short wavelength and high power excitation existing in traditional organic fluorescent dyes.

Description

Technical Field

[0001] The present invention relates to the technical field of bio-optical labeling, and in particular to a frequency up-conversion xanthene fluorescent dye, a preparation method thereof, and an application thereof in bio-fluorescence labeling. Background Art

[0002] Schistosomiasis is an infectious disease prevalent in tropical and subtropical regions, which poses a serious threat to people's health and affects social and economic development. It is listed by the World Health Organization (WHO) as a highly reproducible infectious disease. Adult schistosomiasis can cause great harm to the human body, causing hepatosplenomegaly, cirrhosis, and irreversible liver fibrosis, and in severe cases in the late stage can cause death. Praziquantel is the drug of choice for killing adult schistosomiasis, but due to the long-term, widespread and large-scale use of anti-schistosomiasis drugs, schistosomiasis has developed resistance to it. At the same time, the actual mechanism of action of this type of drug in killing schistosomiasis is still unclear, so in-depth research on anti-schistosomiasis drugs is still needed. Fluorescence imaging can intuitively and in situ visualize and monitor biological molecules and biological processes in living cells and living bodies, with high sensitivity and selectivity, so it is widely used in the study of drug action mechanisms. However, the following disadvantages exist when using traditional organic fluorescent dyes for schistosome imaging: (1) short-wavelength excitation and emission are interfered by spontaneous biological background fluorescence; (2) most organic small molecule fluorescent dyes have poor hydrophilicity, which limits their biological applications; (3) low tissue penetration ability and large tissue damage are not conducive to fluorescence imaging.

[0003] Frequency up-conversion luminescence is a single-photon tropical absorption mechanism. It requires low excitation energy, has a large absorption cross section, and has mild excitation conditions, which makes it potential for application in bioluminescence detection and fluorescence imaging. Currently, the types of fluorophores with frequency up-conversion luminescence properties that have been reported mainly include xanthenes, BODPIYs, and cyanines. However, not all dyes with the above-mentioned types of fluorophores have good frequency up-conversion luminescence properties. Therefore, the selection of fluorophore types and the design of molecular structures become difficulties. Summary of the invention

[0004] The purpose of the present invention is to provide a frequency up-conversion xanthene fluorescent dye and a preparation method thereof and an application in biological fluorescence labeling. The xanthene fluorescent dye provided by the present invention has frequency up-conversion luminescence performance, and the excitation conditions of the up-conversion luminescence are mild, which can reduce the thermal effect or light damage caused by the excitation light, and has the characteristics of small background interference of biological tissue and less damage to biological tissue during imaging, thereby avoiding the problems of short wavelength and high power excitation existing in traditional organic fluorescent dyes.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a frequency up-conversion xanthene fluorescent dye having a structure shown in Formula 1, Formula 2 or Formula 3:

[0007]

[0008] The present invention provides a method for preparing the frequency up-conversion xanthene fluorescent dye described in the above technical solution, comprising the following steps:

[0009] Compound A and compound B are subjected to an addition elimination reaction to obtain a frequency up-conversion xanthene fluorescent dye having a structure shown in Formula 1, Formula 2 or Formula 3;

[0010] The compound A has a structure shown in formula A, and the compound B has a structure shown in formula B:

[0011]

[0012] In the formula B, R is

[0013] Preferably, the molar ratio of compound A to compound B is (1-1.2):1.

[0014] Preferably, the addition elimination reaction is carried out in a protective atmosphere and in the presence of an acid binding agent and an organic solvent.

[0015] Preferably, the acid binding agent includes potassium acetate or triethylamine, and the organic solvent includes acetic anhydride or acetic acid.

[0016] Preferably, the temperature of the addition elimination reaction is 75-90° C. and the time is 2-5 hours.

[0017] The present invention provides the use of the frequency up-conversion xanthene fluorescent dye described in the above technical solution in biological fluorescence labeling.

[0018] Preferably, the biological fluorescent labeling objects suitable for the frequency up-conversion xanthene fluorescent dye include cells or schistosomes.

[0019] Preferably, the schistosome is an adult schistosome.

[0020] Preferably, when performing the biofluorescence labeling, the excitation wavelength is 635 nm, and the fluorescence signal of 650 to 750 nm is collected.

[0021] The present invention provides a frequency up-conversion xanthene fluorescent dye (wherein, the frequency up-conversion xanthene fluorescent dyes having structures shown in Formula 1, Formula 2, and Formula 3 are respectively recorded as fluorescent dyes SUPC-1, SUPC-2, and SUPC-3). The frequency up-conversion xanthene fluorescent dye provided by the present invention uses xanthene as a parent, and the xanthene structure is connected to the nitrogen heterocycle of indole, and has frequency up-conversion luminescence performance, which can not only realize near-infrared emission (such as the maximum emission peaks of fluorescent dyes SUPC-1, SUPC-2, and SUPC-3 are respectively at 698nm, 687nm, and 674nm), but also realize short-wavelength luminescence ability under anti-Stokes shift long wavelength (730nm) excitation, can enter cells in a short time to realize fluorescent staining, and can realize schistosome fluorescent labeling imaging; while traditional organic dyes generally have low tissue penetration ability due to the excitation and emission wavelengths in the ultraviolet to visible region, and due to poor hydrophilicity, they cannot enter living cells and schistosomes well, and cannot obtain fluorescent signals. The frequency up-conversion xanthene fluorescent dye provided by the present invention has excellent fluorescent labeling effect in cells and schistosomes, provides a new means for exploring the fluorescent labeling of schistosomes, and can be used as a fluorescent labeling agent for schistosomes. At the same time, the excitation conditions of the up-conversion luminescence of the frequency up-conversion xanthene fluorescent dye provided by the present invention are mild, and the excitation wavelength is 730nm (near infrared light 650-900nm), which is longer than the wavelength of ultraviolet visible light excitation used by traditional fluorescent dyes, and the excitation energy is lower, which can reduce the thermal effect or light damage caused by the excitation light, and has the characteristics of small background interference of biological tissues and small damage to biological tissues during imaging, thereby avoiding the problems of short wavelength and high power excitation of traditional organic fluorescent dyes, and providing a reference for the use of frequency up-conversion fluorescent dyes in the field of parasites. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Absorption spectra and fluorescence emission spectra of fluorescent dyes SUPC-1, SUPC-2 and SUPC-3;

[0023] Figure 2 Frequency up-conversion fluorescence spectra of the fluorescent dyes SUPC-1, SUPC-2, and SUPC-3;

[0024] Figure 3 This is the laser confocal fluorescence imaging of cells using fluorescent dyes SUPC-1, SUPC-2 and SUPC-3;

[0025] Figure 4 Fluorescence imaging of adult schistosomes labeled with fluorescent dyes SUPC-2 and SUPC-3. DETAILED DESCRIPTION

[0026] The present invention provides a frequency up-conversion xanthene fluorescent dye having a structure shown in Formula 1, Formula 2 or Formula 3:

[0027]

[0028] The present invention provides a method for preparing the frequency up-conversion xanthene fluorescent dye described in the above technical solution, comprising the following steps:

[0029] Compound A and compound B are subjected to an addition elimination reaction to obtain a frequency up-conversion xanthene fluorescent dye having a structure shown in Formula 1, Formula 2 or Formula 3;

[0030] The compound A has a structure shown in formula A, and the compound B has a structure shown in formula B:

[0031]

[0032] In the formula B, R is

[0033] In the present invention, unless otherwise specified, the raw materials used are all known compounds, which can be obtained from commercial channels or synthesized according to methods designed in relevant literature.

[0034] In the present invention, the preparation method of the compound A preferably comprises the following steps: at 0°C, 3.0-3.5 mL of cyclohexanone is added dropwise at a rate of 2-3 mL / min to 40 mL of concentrated sulfuric acid with a concentration of 96-98 wt%, and then 15-16.5 mmol of 4-diethylamino ketoacid is added in small amounts and multiple times while stirring, and the 4-diethylamino ketoacid addition process lasts for 1.5-2.5 h. After the addition is completed, the resulting reaction system is transferred to a nitrogen atmosphere and subjected to a nucleophilic addition reaction at 85-95°C for 2-3 h; after the reaction is completed, the resulting product system is added to a beaker filled with ice at a rate of 2-3 mL / min, and then 1.25-1.75 mL of a 70 wt% perchloric acid solution is added to the beaker, and after the solid is completely precipitated, it is filtered under reduced pressure, and a brown solid is obtained after being washed with distilled water for multiple times to obtain a crude product of compound A, which is directly used in the next step of the reaction. In the present invention, the role of perchloric acid in the perchloric acid solution is to salt the product and precipitate a solid; that is, the negative charge in the compound A is provided by the perchlorate ion.

[0035] After obtaining compound A, the present invention conducts an addition elimination reaction between compound A and compound B to obtain a frequency up-conversion xanthene fluorescent dye having a structure shown in Formula 1, Formula 2 or Formula 3. In the present invention, the molar ratio of compound A to compound B is preferably (1-1.2):1. In the present invention, the addition elimination reaction is preferably carried out under a protective atmosphere and in the presence of an acid binding agent and an organic solvent. In the present invention, the acid binding agent preferably includes potassium acetate or triethylamine; the amount ratio of potassium acetate to compound A is preferably (130-150) mg:0.51 mmol, more preferably 140 mg:0.51 mmol; the amount ratio of triethylamine to compound A is preferably (1.5-2.5) mL:0.51 mmol, more preferably 2 mL:0.51 mmol. In the present invention, the organic solvent preferably includes acetic anhydride or acetic acid; the ratio of the acetic anhydride to the compound A is preferably (3-3.5) mL:0.51 mmol; the ratio of the acetic acid to the compound A is preferably (5-8) mL:0.51 mmol, more preferably (6-7) mL:0.51 mmol. In the present invention, the temperature of the addition elimination reaction is preferably 75-90°C, more preferably 85-90°C; the time is preferably 2-5 h, more preferably 3-4 h.

[0036] After the addition elimination reaction is completed, the present invention preferably cools the obtained product system to room temperature, adds it dropwise into distilled water, then adds sodium bicarbonate powder until no bubbles are generated, separates the obtained material into solid and liquid, and dries the obtained filter cake to obtain a crude product; the crude product is subjected to column chromatography separation to obtain a frequency up-conversion oxygen anthene fluorescent dye having a structure shown in Formula 1, Formula 2 or Formula 3. In the present invention, the drying method is preferably natural drying; the reagents used for the column chromatography separation are preferably CH2Cl2 and CH3OH, and the volume ratio of CH2Cl2 and CH3OH is preferably 15:1. In the present invention, the frequency up-conversion oxygen anthene fluorescent dye having a structure shown in Formula 1, Formula 2 or Formula 3 is specifically provided with negative charge by perchlorate.

[0037] The present invention provides the use of the frequency up-conversion xanthene fluorescent dye described in the above technical solution in biological fluorescence labeling. In the present invention, the biological fluorescence labeling objects applicable to the frequency up-conversion xanthene fluorescent dye preferably include cells or schistosomes; the schistosomes are preferably adult schistosomes. In the present invention, when performing the biological fluorescence labeling, the excitation wavelength is specifically 635nm, and the fluorescence signal of 650-750nm is collected.

[0038] The technical solutions in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0039] In the following examples, the physical and chemical parameters involved were measured by the following instruments: 1 HNMR spectra were measured on a Bruker MercuryPlus at 400 MHz with TMS as the internal standard. Mass spectrometry data were obtained using an AB SCIEX 5800 mass spectrometer (MALDI-TOF-MS). UV-visible absorption spectra were obtained using a Shimadzu UV-2700 UV-visible absorption spectrometer. Fluorescence emission spectra and frequency up-conversion emission spectra were measured using an Edinburgh FS5 fluorescence spectrometer. Fluorescence imaging of adult Schistosoma japonicum was collected using an OLYMPUS FV1000 laser confocal fluorescence microscope, with the laser providing an excitation light source of 635 nm and collecting emitted fluorescence signals in the range of 650 to 750 nm.

[0040] Example 1

[0041] At 0°C, cyclohexanone (3.3 mL, 32 mmol) was added dropwise to 40 mL of concentrated sulfuric acid (98 wt%) at a rate of 2.5 mL / min, and then 4-diethylaminoketoacid (5.01 g, 16 mmol) was added in small amounts and multiple times while stirring. The addition of 4-diethylaminoketoacid lasted for 2 h. After the addition was completed, the obtained reaction system was transferred to a nitrogen atmosphere and subjected to a nucleophilic addition reaction at 90°C for 2 h. After the reaction was completed, the obtained product system was added to a beaker filled with ice at a rate of 2.5 mL / min, and then a perchloric acid solution (70 wt%, 1.5 mL) was added to the beaker. After the solid was completely precipitated, it was filtered under reduced pressure and washed with distilled water for multiple times to obtain a brown solid, thereby obtaining a crude product of compound A, which was directly used in the next step of the reaction.

[0042] 0.51 mmol of compound A, 0.5 mmol of 5-methoxy-1-methyl-indole-3-carboxaldehyde and 140 mg of potassium acetate were added to a round-bottom flask, and then 3 mL of acetic anhydride was added, and an addition elimination reaction was carried out at 90° C. for 3 hours under nitrogen protection; then the obtained product system was cooled to room temperature, added dropwise into distilled water, and then sodium bicarbonate powder was added until no bubbles were generated, the obtained material was solid-liquid separated, and the obtained filter cake was naturally dried to obtain a crude product; the crude product was separated by column chromatography (the reagent used was CH2Cl2:CH3OH=15:1 by volume ratio) to obtain the fluorescent dye SUPC-1.

[0043] NMR and mass spectrometry characterization data of fluorescent dye SUPC-1: 1 H NMR (400MHz, CD3OD) δ8.10(d,J=7.6Hz,1H),7.83(s,1H),7.58(dtd,J=27.5,7.5,1.2Hz,2H),7.38(d,J=1.9Hz ,2H),7.26(s,1H),7.15(d,J=7.5Hz,1H),7.00(dd,J=8.8,2.4Hz,1H),6.71(d,J=9.1Hz,1H),6.63(d,J=2.5Hz, 1H),6.53(dd,J=9.1,2.6Hz,1H),3.97(s,3H),3.84(s,3H),3.47(q,J=7.1Hz,4H),2.89–2.68(m,2H),2.24(dd d,J=15.9,7.0,4.5Hz,1H),1.89(ddd,J=15.9,10.1,4.8Hz,1H),1.81–1.68(m,2H),1.25(t,J=7.1Hz,6H).HRMS Chemical Formula:C 35 H 35 N2O4 + ,m / z:547.2591,found:547.2608.

[0044] Example 2

[0045] The fluorescent dye SUPC-2 was prepared according to the method of Example 1, except that 5-methoxy-1-methyl-indole-3-carboxaldehyde was replaced by 1-methylindole-3-carboxaldehyde.

[0046] NMR and mass spectrometry characterization data of fluorescent dye SUPC-2: 1H NMR (400MHz, CD3OD) δ8.63(s,1H),8.17(dd,J=7.0,1.7Hz,1H),8.12(dd,J=6.0,3.1Hz,1H),7.97(s,1H ),7.66–7.60(m,2H),7.55(dd,J=7.3,1.5Hz,1H),7.38(pd,J=7.1,1.3Hz,2H),7.27(d,J=2.4Hz,1H),7 .18(t,J=2.7Hz,1H),7.16(d,J=3.7Hz,1H),7.10(dd,J=9.5,2.4Hz,1H),3.98(s,3H),3.68(dd,J=14.9 ,7.8Hz,4H),2.96(s,2H),2.64–2.45(m,2H),1.96(dt,J=13.6,7.0Hz,2H),1.34(t,J=7.1Hz,6H).HRMS Chemical Formula:C 34 H 33 N2O3 + ,m / z:518.2564,found:518.2536.

[0047] Example 3

[0048] The fluorescent dye SUPC-3 was prepared according to the method of Example 1, except that 5-methoxy-1-methyl-indole-3-carboxaldehyde was replaced by indole-3-carboxaldehyde.

[0049] NMR and mass spectrometry characterization data of fluorescent dye SUPC-3: 1 H NMR (400MHz, CD3OD) δ8.49–8.42(m,1H),8.27(s,1H),8.15–8.09(m,1H),8.06(dd,J=6. 3,2.8Hz,1H),8.01(s,1H),7.66–7.60(m,2H),7.47–7.42(m,2H),7.39(d,J=2.4Hz,1H) ,7.27(d,J=1.8Hz,1H),7.24–7.17(m,2H),7.16–7.10(m,2H),3.70(q,J=7.1Hz,4H),3. 03(d,J=7.4Hz,2H),2.49(dt,J=43.8,6.8Hz,2H),1.92(s,2H),1.35–1.32(m,6H).HRMS Chemical Formula:C 33 H 31 N2O3 + ,m / z:503.2329,found:503.2345.

[0050] Test Example 1

[0051] The absorption and fluorescence spectra of fluorescent dyes SUPC-1, SUPC-2 and SUPC-3 are tested in the following steps:

[0052] The fluorescent dyes SUPC-1, SUPC-2 and SUPC-3 were prepared with dimethyl sulfoxide (DMSO) to 10 mmol·L -1 The mother solution was then diluted with anhydrous ethanol to a concentration of 5 μmol·L -1 The ultraviolet spectrum was measured with a scanning range of 400-800 nm, and its ultraviolet absorption spectrum characteristics were recorded; and then the fluorescence spectrum was measured.

[0053] Figure 1 is the absorption spectrum and fluorescence emission spectrum of the fluorescent dyes SUPC-1, SUPC-2 and SUPC-3, Figure 1 It can be seen that the maximum absorption peaks of the fluorescent dyes SUPC-1, SUPC-2, and SUPC-3 are located at 637nm, 614nm, and 632nm, respectively, and the maximum emission peaks are located at 698nm, 687nm, and 674nm, respectively. The emission of these three fluorescent dyes under traditional excitation is located in the near-infrared region (650-900nm).

[0054] Test Example 2

[0055] Frequency up-conversion fluorescence test of fluorescent dyes SUPC-1, SUPC-2, and SUPC-3. The specific steps are as follows:

[0056] The fluorescent dyes SUPC-1, SUPC-2 and SUPC-3 were prepared with dimethyl sulfoxide (DMSO) to 10 mmol·L -1 The mother solution was then diluted with anhydrous ethanol to a concentration of 5 μmol·L -1 A frequency up-conversion fluorescence test was performed, wherein the excitation light source used a 730 nm laser, a 700 nm short-pass filter, and the spectrum from 550 to 800 nm was collected.

[0057] Figure 2 The frequency up-conversion fluorescence spectra of the fluorescent dyes SUPC-1, SUPC-2 and SUPC-3 are shown in Figure 2. Figure 2 It can be seen that the upconversion fluorescence intensity of the fluorescent dyes SUPC-1, SUPC-2 and SUPC-3 has a linear relationship with the frequency, that is, these three fluorescent dyes all have upconversion luminescence effects.

[0058] Test Example 3

[0059] Cell fluorescence imaging of fluorescent dyes SUPC-1, SUPC-2 and SUPC-3:

[0060] (1) Reagents and materials

[0061] Dimethyl sulfoxide (AR) was purchased from Aladdin Chemical Reagent Co., Ltd., RPMI 1640 culture medium was purchased from Thermo Fisher Scientific, and human breast cancer cells MCF-7 were purchased from the Institute of Biochemistry and Cell Biology (Chinese Academy of Sciences).

[0062] (2) Cell fluorescence imaging

[0063] Human breast cancer cells MCF-7 were cultured in RPMI 1640 medium containing 10% fetal bovine serum and grown in an incubator at 37°C and 5% CO2. Fluorescent dyes SUPC-1, SUPC-2 and SUPC-3 were prepared into 1mmol / L stock solution with dimethyl sulfoxide, and then diluted into 5μmol / L solution with RPMI 1640 medium, and then incubated with MCF-7 cells for different time (5min, 15min, 30min), washed three times with PBS, and then placed on OLYMPUS FV 1000 laser confocal fluorescence microscope for testing, with an excitation wavelength of 635nm, and collecting fluorescence signals of 650-750nm for fluorescence imaging.

[0064] Figure 3 This is the laser confocal fluorescence imaging of cells with fluorescent dyes SUPC-1, SUPC-2 and SUPC-3. Figure 3 It can be seen that the fluorescent dyes SUPC-1, SUPC-2 and SUPC-3 can quickly enter the cells within 30 minutes and have a fluorescent labeling effect on the cells.

[0065] Test Example 4

[0066] Fluorescent dyes SUPC-2 and SUPC-3 for fluorescent labeling and imaging of schistosomes:

[0067] (1) Reagents and materials

[0068] Dimethyl sulfoxide (AR) was purchased from Aladdin Chemical Reagent Co., Ltd., RPMI 1640 culture medium was purchased from Thermo Fisher Scientific, and phosphate buffer (PBS, pH=7.4) was purchased from Jiangsu Biyuntian Biological Reagent Co., Ltd.; adult Schistosoma japonicum was provided by the Jiangxi Provincial Key Laboratory of Organic Pharmaceutical Chemistry, Gannan Normal University.

[0069] (2) Fluorescence labeling imaging of adult Schistosoma japonicum

[0070] Fluorescent dyes SUPC-2 and SUPC-3 are respectively dissolved in dimethyl sulfoxide (DMSO) to accurately prepare a 1mmol / L mother solution, and then diluted with PBS buffer to a 5μmol / L fluorescent dye dilution solution; 200μL of the fluorescent dye dilution solution is transferred to a culture dish, adult schistosomes are picked up with tweezers and added to the fluorescent dye dilution solution, and incubated for 5min to 3h at 37°C and 5% CO2 air atmosphere, and then the adult schistosomes are transferred to a special container for fluorescence imaging, and the fluorescent labeling of the adults is observed using a laser confocal fluorescence microscope under 635nm excitation conditions, and the fluorescence signals of 650 to 750nm are collected for fluorescence imaging.

[0071] Figure 4 This is a fluorescence image of adult Schistosoma japonicum labeled with fluorescent dyes SUPC-2 and SUPC-3. Figure 4 It can be seen that the fluorescent dyes SUPC-2 and SUPC-3 can be used for fluorescent labeling and imaging of adult Schistosoma japonicum.

[0072] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An application of a frequency up-conversion xanthene fluorescent dye in biological fluorescence labeling for the purpose of non-disease diagnosis, wherein the excitation wavelength is 635 nm when the biological fluorescence labeling is performed, and the fluorescence signal of 650-750 nm is collected. The biological fluorescence labeling object applicable to the frequency up-conversion xanthene fluorescent dye is cells or schistosomes. The frequency up-conversion xanthene fluorescent dye has a structure shown in Formula 2, and the negative charge in the frequency up-conversion xanthene fluorescent dye is provided by perchlorate: Formula 2.

2. The use according to claim 1, It is characterized in that The method for preparing the frequency up-conversion xanthene fluorescent dye comprises the following steps: Compound A and compound B are subjected to an addition elimination reaction to obtain a frequency up-conversion xanthene fluorescent dye having a structure shown in Formula 2; The compound A has a structure shown in formula A, and the negative charge in the compound A is provided by perchlorate; the compound B has a structure shown in formula B: Formula A; Formula B; In the formula B, R is .

3. The use according to claim 2, It is characterized in that The molar ratio of compound A to compound B is (1-1.2):

1.

4. The use according to claim 2, It is characterized in that The addition elimination reaction is carried out in a protective atmosphere and in the presence of an acid-binding agent and an organic solvent.

5. The use according to claim 4, It is characterized in that The acid binding agent includes potassium acetate or triethylamine, and the organic solvent includes acetic anhydride or acetic acid.

6. The use according to claim 2, It is characterized in that The temperature of the addition elimination reaction is 75-90°C and the time is 2-5 h.

7. The use according to claim 1, It is characterized in that The schistosomes are adult schistosomes.

Images