CN109350742B

CN109350742B - A kind of bipolar photosensitizer and preparation method thereof

Info

Publication number: CN109350742B
Application number: CN201811413898.XA
Authority: CN
Inventors: 康健; 陈依慧; 欧好; 张薇薇; 周大维; 王臻; 陈静; 黄进华; 鲁建云; 郭爱元; 谢媛媛; 马思祺
Original assignee: Third Xiangya Hospital of Central South University
Current assignee: Third Xiangya Hospital of Central South University
Priority date: 2018-11-26
Filing date: 2018-11-26
Publication date: 2021-04-23
Anticipated expiration: 2038-11-26
Also published as: CN109350742A

Abstract

The invention discloses a bipolar photosensitizer and a preparation method thereof. The bipolar photosensitizer also comprises glyceride acyl residues, wherein the glyceride acyl residues are used as hydrophobic groups and are combined with the EtNBSe photosensitizer through covalent bonds to form a structure with one hydrophilic end and one hydrophobic end, the structure is similar to phosphatidylcholine which is an essential component for forming a biological membrane, and due to the principle of similar intermiscibility, the bipolar photosensitizer is easier to enter cancer cells, so that the uptake rate of the photosensitizer is greatly increased, the uptake rate is improved by about 46.1%, and the weight percentage of the photosensitizer in a medicament can be properly reduced on the basis of the uptake rate, so that the treatment effect of the bipolar photosensitizer is greatly improved while the side effect of the medicament is reduced.

Description

Bipolar photosensitizer and preparation method thereof

Technical Field

The invention relates to a bipolar photosensitizer and a preparation method thereof, in particular to an EtNBSe derivative used as the photosensitizer kills specific tumor cells by utilizing photodynamic effect and improves the killing effect by combining an AMPK signal channel activator, belonging to the field of biological medicine.

Background

In recent years, the prevalence of skin cancer, such as squamous cell carcinoma, has increased in people due to environmental factors, poor lifestyle, and the like. Squamous cell carcinoma is one of the high-grade cancers of the skin, can develop and spread to deep tissues when serious, thereby being life-threatening, and has become a common disease which jeopardizes the life health of human beings. So that quick and effective intervention treatment is important.

Photodynamic therapy is a novel technology for treating cancer in recent years, wherein laser with specific wavelength is used for irradiating tumor tissues, photosensitizer absorbed by tumor cells transfers energy to surrounding oxygen under the excitation of light to generate singlet oxygen with strong activity, and the singlet oxygen and adjacent biological macromolecules generate oxidation reaction to generate cytotoxicity effect, so that the tumor cells are damaged or even die. The photodynamic therapy is particularly effective on squamous cell carcinoma, and compared with other common treatment schemes such as chemotherapy, surgical excision, electrocautery, liquid nitrogen freezing and the like, the photodynamic therapy has small side effect, and patients do not need to suffer pain and have no great stimulation response in the treatment process; no scar is left after treatment; the doctor is easy to operate; the treatment is more accurate, and the radical treatment rate is higher; has great advantages in clinical treatment. However, photodynamic therapy still has its drawbacks.

In general, the performance of the photosensitizer largely determines the efficacy of photodynamic therapy. The first and second photosensitizers, such as hematoporphyrin derivatives, have complex components and smaller wavelength of maximum absorption light wave, and the depth of light wave with smaller wavelength penetrating through human tissues is limited, so that deeper tissue cells cannot be treated. For example, in the invention patent of a preparation method of hypocrellin B as a novel photosensitizer (application No. 200910154644.5, publication No. CN 102070432A), the hypocrellin B has short main absorption wavelength and weak absorption capability at near infrared light wavelength, and is difficult to treat deep tissues. Secondly, the rate of entering tumor cells is low, which is not beneficial to the aggregation of the photosensitizer in the tumor cells. For example, in the invention patent of composition of 5-aminolevulinic acid and derivatives thereof and application thereof (application number: 200810044109.X, publication number: CN 101745102A), the hydrophilicity of the composition of 5-aminolevulinic acid and derivatives thereof influences the efficiency of the photosensitizer for entering cells through cell membranes, and has low targeting selectivity, so that the photosensitizer is difficult to be accumulated in a large amount in tumor cells to achieve ideal treatment effect.

Disclosure of Invention

In view of the above-mentioned problems associated with the prior art, it is an object of the present invention to provide a coupled drug of a bipolar highly permeable photosensitizer and an AMPK signal pathway activator with little side effects, which greatly enhances the therapeutic effect of photodynamic therapy, and the following is a specific technical solution of the present invention.

The technical scheme of the invention is to provide a bipolar photosensitizer, wherein the bipolar photosensitizer is ammonium salt, and cations in the ammonium salt are as follows:

preferably, wherein the anion in the ammonium salt is chloride.

The invention also provides a preparation method of the bipolar photosensitizer, which comprises the following steps:

s1: dissolving a selenium-containing benzophenoxazine photosensitizer (EtNBSe photosensitizer for short) in anhydrous glycerin, then dropwise adding concentrated sulfuric acid with the mass fraction of 98%, and stirring to generate an intermediate product A, wherein the reaction formula is as follows:

s2: adding sodium hydroxide solution into the reaction solution obtained in the step S1, pressurizing to the relative pressure of 4-7MPa, heating, and reacting to generate an intermediate product B, wherein the reaction formula is as follows:

when the reaction liquid is cooled (natural cooling is selected) to be below 80 ℃, decompressing and exhausting, and then adding acetic anhydride to generate an intermediate product C, wherein the reaction formula is as follows:

s3: adding concentrated nitric acid into the reaction solution obtained in the step S2, then supplementing concentrated sulfuric acid, and heating to generate a nitro compound, wherein the reaction formula is as follows:

then adding Fe, continuously introducing hydrogen, and reducing the nitro compound into an intermediate product D, wherein the reaction formula is as follows:

s4: adding a sodium nitrite solution into the reaction liquid obtained in the step S3, and then introducing hydrogen fluoride gas into the reaction liquid to generate an intermediate product E, wherein the reaction formula is as follows:

s5: adding catalyst anhydrous aluminum chloride into the reaction liquid obtained in the step S4, introducing HCl gas, and then adding cell signal pathway activator Acadesine into the reaction liquid to obtain an intermediate product F;

s6, dissolving phosphatidylcholine in cyclohexane, adding water, introducing excessive HBr, and oscillating to generate an intermediate product G; the chemical structural formula of the intermediate product G is as follows:

intermediate F is mixed with intermediate G and K is added₂CO₃Heating, and carrying out Williamson reaction to generate an intermediate product H, wherein the reaction formula is as follows:

s7: and (4) evaporating and concentrating the reaction solution obtained in the step S6, washing with deionized water, collecting washing liquor, adjusting to be alkaline, extracting, adding deionized water into the extract liquor, and then adding acid to adjust the pH of the solution to be acidic, so as to generate the bipolar photosensitizer.

Preferably, in step S7, hydrochloric acid is added to adjust the pH of the solution to acidity, resulting in a bipolar photosensitizer.

Preferably, in the step S1 of synthesizing the intermediate product A, 5 parts of selenium-containing benzophenoxazine photosensitizer and 0.5 part of 98% concentrated sulfuric acid by mass are added, and the mixture is reacted for 10-15min at 120-128 ℃.

Preferably, in the step S2 of synthesizing the intermediate product B, 3 parts of sodium hydroxide solution by mass are reacted for 30min, wherein the concentration is 1.0mol/L, the temperature is controlled at 297-302 ℃; in the reaction for synthesizing the intermediate product C in the step S2, 3 parts of acetic anhydride with the concentration of 2.5mol/L react for 5 min.

Preferably, in the step S3 of synthesizing the nitro compound, 1 part by mass of concentrated nitric acid is 68%; 1 part of concentrated sulfuric acid, the mass fraction of which is 98 percent, and the reaction temperature is 145-150 ℃.

Preferably, according to the mass parts, in the reaction for synthesizing the intermediate product D in the step S3, the amount of Fe is 0.5mol per liter of reaction liquid, the hydrogen flow rate is 1.5-2L/min, and the reaction is continuously conducted for 5 min; in the reaction of synthesizing the intermediate product E in the step S4, 3 parts of sodium nitrite solution with the concentration of 3.2mol/L is reacted for 8min at the temperature of 10-13 ℃ while the flow rate of hydrogen fluoride gas is controlled to be 0.3-0.8L/min.

Preferably, in the step S5 of synthesizing the intermediate product F, anhydrous aluminum chloride is added in an amount of 0.3-0.6mol per liter of reaction solution, and Acadesine is added in an amount of 3 parts by mass, the flow rate of HCl is controlled to be 1-2L/min, and the pH is maintained to be 0.5.

Preferably, in the step S6 of synthesizing the intermediate product G, 4 parts of phosphatidylcholine are used, and the concentration of phosphatidylcholine is 0.5-1.0 mol/L; in the reaction for synthesizing the intermediate product H in the step S6, the pH of the reaction solution is maintained between 10 and 13, and the reaction temperature is maintained between 75 and 77 ℃.

The "ambipolar" of the ambipolar photosensitizers and the "EtNBSe" of the EtNBSe photosensitizers in the present invention are only used to distinguish the two photosensitizers.

The method is used for keeping the polarity of ammonium salt amino nitrogen, the concentration of the obtained product is diluted to be within the range of 100-800 mu mol/L, the mixed solution is used as photosensitizer mixed solution, and the photosensitizer mixed solution and auxiliary components are mixed and prepared into corresponding medicine forms to obtain the applicable medicine.

The pharmaceutical auxiliary component of the bipolar photosensitizer of the present invention includes, but is not limited to, one or more of glycerin, microcrystalline cellulose, sucrose, starch, mucilage, dextrin, magnesium stearate, and water for injection. Specifically, the bipolar photosensitizer adopts different auxiliary components according to different pharmaceutical modes.

The intensity of the irradiating light is required to activate the photosensitizer and can not damage normal tissues of a human body, the intensity range of the optional light is 10-80 mJ/cm2, and the light intensity range is preferably 20-30 mJ/cm2 in consideration of various factors such as the activation effect of the photosensitizer and the adaptation condition of the human body. The irradiation time of the light is 5-15 min, the specific irradiation time is adapted to the light intensity, the irradiation time needs to be prolonged when the light intensity is small, and the irradiation time can be properly reduced when the light intensity is relatively large.

The AMPK cell signal pathway activator can activate an AMPK cell signal pathway, thereby promoting apoptosis of tumor cells, enhancing targeted killing of the tumor cells of the pharmaceutical composition, and improving treatment effect.

The photosensitizer is 100 to 255 mug/kg by weight, and preferably 150 to 200 mug/kg by weight in view of the magnitude of side effects, therapeutic effects, and the like.

Further, the weight percentage of the AMPK cell signal pathway activator in the bipolar photosensitizer is 135-180 mug/kg, and considering various factors such as the activation effect of the AMPK cell signal pathway, the full utilization of the activator and the like, the weight percentage is preferably 140-155 mug/kg.

The bipolar photosensitizer also comprises glyceride acyl residues, wherein the glyceride acyl residues are combined with the EtNBSe photosensitizer as hydrophobic groups through covalent bonds to form a structure with one hydrophilic end and one hydrophobic end, the structure is similar to phosphatidylcholine which is an essential component for forming a biological membrane, and the bipolar photosensitizer is easier to enter cancer cells due to the principle of similar intermiscibility, so that the uptake rate of the photosensitizer is greatly increased, and is improved by about 46.1%, and the weight percentage of the bipolar photosensitizer in a medicament can be properly reduced on the basis, so that the treatment effect of the bipolar photosensitizer is greatly improved while the side effect of the medicament is reduced.

The bipolar photosensitizer also comprises F atoms, wherein the F atoms are directly connected with aromatic rings, and the strong electron-withdrawing induction effect can reduce the aromaticity of the aromatic rings, so that the absorption spectrum of the photosensitizer shifts towards the infrared direction (red shift), the utilization rate of laser light energy is increased, the intensity of irradiated light can be reduced, the duration of light irradiation can be shortened, the negative influence of the light irradiation on normal tissues of patients can be avoided, the time cost is saved, and the treatment is facilitated.

Drawings

FIG. 1 shows a hydrogen spectrum of a coupled drug of a bipolar photosensitizer and an AMPK signaling pathway activator.

FIG. 2 shows a carbon spectrum of a coupled drug of a bipolar photosensitizer and an AMPK signaling pathway activator.

FIG. 3 is a graph showing the changes of tumors in the left and right sides of a nude mouse with time.

FIG. 4 is a graph showing the relative expression of p-AMPK in A-431 cells after gradient dose bipolar photosensitizer-PDT treatment.

FIG. 5 is a bar graph of the relative expression of p-AMPK in A-431 cells versus the dose of bipolar photosensitizer.

FIG. 6 is a graph showing the relative expression of p-AMPK in A-431 cells at different time periods after 400nM bipolar photosensitizer-PDT treatment.

FIG. 7 is a bar graph of the relative expression of p-AMPK in A-431 cells versus the time elapsed after bipolar photosensitizer-PDT treatment.

FIG. 8 is a graph showing the activity of A-431 cells in MTT assay after 400nmol of EtNBSe and 400nmol of the bipolar photosensitizer were separately exposed to light.

FIG. 9 shows the chemical structure of the cation of the bipolar photosensitizer of the present invention.

Detailed Description

The present invention will be further described with reference to the following examples.

Example 1: preparation of bipolar photosensitizer

S1 mixing five parts of photosensitizer containing selenium benzophenoxazine

Dissolving in anhydrous glycerin, slowly dropwise adding 0.5 part of 98% concentrated sulfuric acid, stirring the mixed solution by using a magnetic stirrer, heating to 120-128 ℃, and reacting for 13min to generate an intermediate product A:

s2: adding three parts of 1.0mol/L sodium hydroxide solution into the final reaction solution in the step S1, pressurizing to 5MPa by using a pressure-resistant kettle, raising the temperature to 297-302 ℃, and co-melting with sodium hydroxide for 30min to generate an intermediate product B:

when the reaction liquid is cooled to below 80 ℃, decompressing and exhausting gas to open the kettle, then adding three parts of 2.5mol/L acetic anhydride, and reacting for 5min to generate an intermediate product C:

s3: adding 1 part of 68% concentrated nitric acid into the S2 reaction solution, then slowly stirring and replenishing 1 part of concentrated sulfuric acid, and reheating the reaction solution to 145-150 ℃ to generate a nitro compound:

and then adding 0.5mol of Fe into each liter of reaction solution, and then smoothly and continuously introducing hydrogen for 5min at the speed of 1.5-2L/min to reduce the nitro compound into an intermediate product D:

s4: adding 3 parts of 3.2mol/L sodium nitrite solution into the reaction solution obtained in S3, slowly and constantly introducing hydrogen fluoride gas into the reaction solution, controlling the flow rate of the hydrogen fluoride gas to be 0.3-0.8L/min, controlling the temperature of the diazo compound solution to be below 40 ℃, preferably 10-13 ℃, reacting for 8min, and generating an intermediate product E, wherein the yield is 79.2 percent:

s5: adding 0.3-0.6mol of catalyst anhydrous aluminum chloride into each liter of reaction solution obtained in the step S4, stably and smoothly introducing HCl gas at the speed of 1-2L/mol, keeping the pH value at 0.5, and then adding 3 parts of AMPK cell signal pathway activator Acadesine:

under the catalysis of anhydrous aluminum chloride strong Lewis acid, the Acadesine attacks the EtNBSe photosensitizer by using keto positive ions, so that an intermediate product F:

s6, dissolving 4 parts of 0.5-1.0 mol/L phosphatidylcholine in cyclohexane, adding a small amount of water, introducing excessive HBr in a large amount, violently oscillating, removing phosphorylcholine, and substituting with Br to generate an intermediate product G:

mixing the above reaction solution with the reaction solution obtained in S5, and adding appropriate amount of K₂CO₃And (3) heating the reaction solution to 75-77 ℃ to ensure that the pH value is 10-13, and carrying out Williamson reaction to generate an intermediate product H:

s7: evaporating and concentrating the product S6 at low temperature, washing the solution with deionized water for 6 times, collecting washing liquor, adjusting to alkalescence, carrying out methylation, extracting with cyclopentane, extracting cyclopentane with glycerol for two times, adding a small amount of deionized water, and then adding dilute hydrochloric acid to adjust the pH of the solution to acidity, thus generating the final product of ammonium salt as follows:

(FIG. 9); the anion of the ammonium salt is chloride.

Application example 1

EtNBSe-PDT example for treatment of cutaneous squamous cell carcinoma

(1) Animal experiments

Mouse squamous cell tumor model, selecting nude mouse, male, 18-20 g. In the experiment, A-431 cells with good growth are taken, trypsinized, diluted by sterile normal saline according to the proportion of 1:10 to prepare tumor cell suspension, 500uM EtNBSe medicament is injected into the left tumor of a nude mouse by subcutaneous injection, the right tumor is not treated as a control, and 20J/cm is used²Red light for 15min, and tumor status was observed at 2 days, 4 days, 7 days, 12 days, 14 days, and 17 days.

(2) Results of the experiment

Changes in tumors were observed at different times after EtNBSe-PDT treatment, and the results showed significant necrosis and shrinkage of tumor masses after EtNBSe-PDT treatment, indicating that EtNBSe-PDT was able to significantly treat squamous cell tumors (FIG. 3).

Application example 2

Example of EtNBSe-PDT activation of squamous cell AMPK Signaling

(1) Experimental methods

The squamous carcinoma cell A-431 is cultured in DMEM culture solution of 10% calf serum, 100IU/L penicillin and 100mg/L streptomycin are added, the DMEM culture solution is placed in a constant-temperature incubator at 37 ℃ and 5% CO2 for conventional culture, after the cell grows to the bottom of a bottle, the culture solution is discarded, PBS is washed twice, then 0.25% pancreatin and 0.02% EDTA are used for digestion for 5 minutes, the bottle culture is carried out again, the cell is subcultured for one time after 2 to 3 days, and the cell in the logarithmic growth phase is taken for experiment.

Pancreatin digestion of A-431 cells in logarithmic growth phase, inoculating to six-well culture plate, culturing at 37 deg.c in 5% CO2 culture box, adding EtNBSe agent to the wells to final concentration of 0nM, 100nM, 200nM, 400nM and 800nM after incubation for 1 hr, and adding to the culture plate 20J/cm²Irradiating for 15min with red light, collecting cells after half an hour, extracting total protein, and detecting the expression of p-AMPK in the cells by using a Western Blot method.

Pancreatin digestion of A-431 cells in logarithmic growth phase, inoculating to culture dish, culturing at 37 deg.c in 5% CO2 culture box, adding 400nM EtNBSe agent to each culture dish for 1 hr after growth to 80-90% fusion state, and incubating with 20J/cm²Irradiating with red light for 15min, collecting cells at 0.5h, 1h, 2h, 8h and 16h after EtNBSe-PDT treatment, extracting total protein, and detecting the expression of p-AMPK in the cells by using a Western Blot method.

(2) Results of the experiment

Western Blot analysis of p-AMPK expression levels of A-431 cells treated by EtNBSe-PDT at gradient doses revealed that the expression levels of p-AMPK were significantly reduced with the increase of the EtNBSe dose, and that the EtNBSe-PDT was able to significantly inhibit p-AMPK signals (FIG. 4 and FIG. 5).

Western Blot analysis of p-AMPK expression levels by collecting proteins at gradient time after EtNBSe-PDT treatment of A-431 cells revealed that p-AMPK expression levels were minimized at 0.5h after EtNBSe-PDT treatment of A-431 cells, and then gradually returned to normal levels, even with a slight increase in p-AMPK expression levels at 16h (FIG. 6, FIG. 7).

Comparative example: contrast of Bipolar photosensitizer with EtNBSe photosensitizer for inhibiting A-431 cell Activity

(1) Experimental methods

In a culture solution containing 10% fetal calf serum, a squamous carcinoma cell A-431 cell line is prepared into a single cell suspension, and the cell suspension is inoculated into three 96-well plates according to the standard of 1000-10000 cells per well, wherein the volume of each well is 200 ul. Then, physiological saline, EtNBSe 400nmol and a bipolar photosensitizer 400nmol were added to each of the three well plates, and after laser irradiation for 1min, the A-431 cell line was routinely cultured in a 5% CO2 incubator at 37 ℃ for 3 days.

After the incubation, 20ul of 5mg/ml MTT solution in PBS was added to each well of the well plate and incubated for 4 hours at 37 ℃ in a 5% CO2 incubator. Subsequently, the culture supernatant in the well was slowly aspirated, and after centrifugation, the culture supernatant in the well was again aspirated. Subsequently, 150ul DMSO was added to each well of the well plate to dissolve the crystals sufficiently, and after 10 minutes of shaking, monochromatic light with a wavelength of 490nm was selected, and the absorbance of the liquid in each well was measured in an enzyme-linked immunosorbent instrument, and the results were recorded.

(2) Results of the experiment

From the MTT analysis results of EtNBSe or the effect of the coupled drug alone on the activity of a-431 cells, it can be seen that under the laser irradiation condition, the activity of a-431 cells treated with EtNBSe and the bipolar photosensitizer is very low compared with the control group, however, compared with the control group, the bipolar photosensitizer has higher cell lethality, indicating that the bipolar photosensitizer has higher stability and cytotoxicity than EtNBSe photosensitizer in the a-431 cell line (fig. 8).

Claims

1. a bipolar photosensitizer, is characterized in that, described bipolar photosensitizer is ammonium salt, and the cation in described ammonium salt is:

2 . The bipolar photosensitizer according to claim 1 , wherein the anion in the ammonium salt is chloride ion. 3 .

3. a preparation method of bipolar photosensitizer as claimed in claim 1, is characterized in that, comprises the following steps:

S1: Dissolve the selenium-containing benzophenoxazine photosensitizer in anhydrous glycerol, then add dropwise concentrated sulfuric acid with a mass fraction of 98%, stir to generate an intermediate product A, and its reaction formula is:

S2: add sodium hydroxide solution to the reaction solution obtained in step S1, and pressurize it to a relative pressure of 4-7MPa, heat up, and react to generate intermediate product B, and its reaction formula is:

When the reaction solution is cooled to below 80°C, decompress and exhaust, and then add acetic anhydride to generate intermediate product C, whose reaction formula is:

S3: add concentrated nitric acid to the reaction solution obtained in step S2, then add concentrated sulfuric acid, heat, generate nitro compounds, and its reaction formula is:

Add Fe again, continuously feed hydrogen, and reduce described nitro compound into intermediate product D, and its reaction formula is:

S4: add sodium nitrite solution to the reaction solution obtained in the step S3, then feed hydrogen fluoride gas into the above-mentioned reaction solution to generate intermediate product E, and its reaction formula is:

S5: adding catalyst anhydrous aluminum chloride to the reaction solution obtained in step S4, and feeding HCl gas, and then adding cell signaling pathway activator Acadesine to the reaction solution to obtain intermediate product F;

S6: phosphatidylcholine is dissolved in cyclohexane, water is added, excess HBr is passed through, and vibration is generated to generate intermediate product G; the chemical structural formula of intermediate product G is:

The intermediate product F and the intermediate product G are mixed, K ₂ CO ₃ is added, heated, and the Williamson reaction is carried out to generate the intermediate product H. The reaction formula is:

S7: Evaporate and concentrate the reaction solution obtained in step S6, wash with deionized water, collect the washing solution, adjust to alkalinity, perform methylation, and then extract, add deionized water to the extract, and then add acid to adjust the pH of the solution to acidity, resulting in bipolar photosensitizers.

4. The preparation method according to claim 3, characterized in that, in step S7, hydrochloric acid is added to adjust the pH of the solution to be acidic to generate a bipolar photosensitizer.

5. preparation method as claimed in claim 3 is characterized in that, by mass, in the reaction of step S1 synthesizing intermediate product A, add 5 parts of selenium-containing benzophenoxazine photosensitizer, mass fraction 98% concentrated sulfuric acid 0.5 parts, and react at 120-128°C for 10-15min.

6. preparation method as claimed in claim 3 is characterized in that, by mass, in the reaction of step S2 synthesizing intermediate product B, 3 parts of sodium hydroxide solution, concentration is 1.0mol/L, and temperature is controlled at 297-302 ℃, the reaction is carried out for 30 min; in the reaction of synthesizing the intermediate product C in step S2, 3 parts of acetic anhydride, the concentration is 2.5 mol/L, the reaction is carried out for 5 min.

7. preparation method as claimed in claim 3 is characterized in that, by mass, in the reaction of step S3 synthesizing nitro compound, 1 part of concentrated nitric acid, its mass fraction is 68%; 1 part of concentrated sulfuric acid, its mass fraction was 98%, and the reaction temperature was 145-150 °C.

8. preparation method as claimed in claim 3 is characterized in that, by mass parts, in the reaction of step S3 synthesis intermediate product D, the amount that adds Fe is every liter of reaction solution 0.5mol, and hydrogen flow rate is 1.5-2L/min , continue to feed 5min; in the reaction of synthesizing intermediate product E in step S4, 3 parts of sodium nitrite solution, its concentration is 3.2mol/L, control hydrogen fluoride gas flow rate 0.3-0.8L/min, 10-13 ℃ of reaction 8min.

9. preparation method as claimed in claim 3 is characterized in that, by mass, in the reaction of step S5 synthesizing intermediate product F, the amount of adding anhydrous aluminum chloride is 0.3-0.6mol per liter of reaction solution, Acadesine 3 The flow rate of HCl was controlled to be 1-2 L/min, and the pH was maintained at 0.5.

10. The preparation method according to claim 3, characterized in that, in parts by mass, in the reaction of synthesizing intermediate product G in step S6, 4 parts of phosphatidylcholine, the concentration of which is 0.5～1.0mol/L; step S6 synthesis In the reaction of the intermediate product H, the pH of the reaction solution is maintained between 10-13, and the reaction temperature is 75-77°C.