Preparation method of regadenoson

Abstract

The invention discloses a preparation method of regadenoson and a preparation and detection method of impurities of regadenoson, the regadenoson is prepared by the following steps: (1) 2-chloroadenosine reacts with hydrazine hydrate and acetone in sequence to prepare a compound (1); (2) reacting the compound (1) with 2-formyl-3-oxo ethyl propionate to obtain a compound (2); and (3) reacting the compound (2) with a methylamine aqueous solution to prepare regadenoson. The regadenoson preparation method provided by the invention is short in reaction route, easy in reaction and capable of realizing large-scale preparation, meanwhile, the impurity content in regadenoson is greatly reduced, the purity and the yield of the prepared product are remarkably improved, and the regadenoson preparation method can be widely applied to the field of regadenoson preparation. Meanwhile, the invention also provides a preparation and detection method of the regadenoson impurity, the content of the impurity can be effectively detected, and the quality control of the product is further improved.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical preparation synthesis, and specifically to a method for preparing Reganoxan. Background Technology

[0002] Reganoxon is a novel A2A adenosine receptor agonist developed by CV Therapeutics in the United States. It was approved by the FDA in April 2008 for myocardial perfusion imaging (MPI). Compared to the classic drug adenosine, this product does not cause bronchoconstriction or atrioventricular block, and has significant effects with fewer adverse reactions.

[0003] Currently reported synthetic routes for reganosine primarily use 2-chloroadenosine or 2-chloropurine as starting materials, followed by substitution, condensation, and ammonolysis to obtain the reganosine product. US Patent 6403567 first disclosed this compound and a method for its preparation.

[0004] US Patent 7732595 provides a detailed description of another method for preparing Reganoxane. Using 2-chloroadenosine as a substrate, it is reacted with hydrazine hydrate at 40-45°C to generate 2-hydrazyladenosine. 2-hydrazyladenosine reacts with ethyl 2-formyl-3-oxopropionate to generate ethyl 1-{9[4S,2R,3R,5R]-3,4-dihydroxy-5-(hydroxymethyl)oxacyclopentan-2-yl]-6-aminopurine-2-yl}pyrazole-4-carboxylate. The nucleoside analogue of this ethyl formate reacts with an aqueous solution of methylamine in a pressure reactor at 50-70°C to generate compound (I)(1-{9[4S,2R,3R,5R]-3,4-dihydroxy-5-(hydroxymethyl)oxacyclopentan-2-yl]-6-aminopurine-2-yl}pyrazol-4-yl)-N-methylformamide, namely Reganoxon.

[0005] Liu Wei et al. used the hydroxyl group of 2-chloroadenosine as a raw material, protected with tert-butyldimethylsilane, and reacted with hydrazine hydrate to obtain 2',3',5'-tritert-butyldimethylsiloxy-2-hydrazinoside. After cyclization with ethyl 2-formyl-3-oxopropionate, it was methylated to obtain the protected reganosine. Finally, the protecting group was removed to obtain reganosine.

[0006] Luo Hairong et al. used 2-chloroadenine as the starting material to obtain 2-hydrazinoadenine via hydrazine hydrate reaction; then reacted with ethyl 2-formyl-3-oxopropionate to generate ethyl 1-(6-amino-9H-purin-2-yl)-1H-pyrazole-4-carboxylate; after ammonolysis with methylamine, 1-(6-amino-9H-purin-2-yl)-1H-pyrazole-4-formylmethylamine was obtained; and after condensation with tetraacetylribose and hydrolysis to remove the acetyl group, Reganosin was obtained.

[0007] All of the above routes use ethyl 2-formyl-3-oxopropionate, which generates impurities M and N during its preparation. In view of this, Chinese patent CN202010620425 protects the amino group by using an acetyl group. However, the acetyl group needs to be removed in a high-pressure vessel in the later stage, which is cumbersome, increases production costs, and is not conducive to commercial production.

[0008] Therefore, there is an urgent need for a method that has a short reaction route, is easy to react, has simple post-processing conditions, and can be used to prepare high-purity reganoxan on a large scale. Summary of the Invention

[0009] Purpose of the invention: In view of the shortcomings of the prior art, the purpose of this invention is to provide a method for preparing high-purity reganorogen with a shorter reaction route, easier reaction, simpler post-processing conditions, and the ability to prepare it on a large scale, as well as to provide a reganorogen impurity and its preparation method.

[0010] Technical solution: This invention provides a method for preparing Reganoxan, comprising the following steps: (1) Compound (1) was prepared by reacting 2-chloroadenosine with hydrazine hydrate and acetone in sequence. (2) Compound (1) was reacted with ethyl 2-formyl-3-oxopropionate to prepare compound (2); (3) Reganoxane was prepared by reacting compound (2) with aqueous methylamine.

[0011] Further, step (1) specifically involves: adding 2-chloroadenosine to a hydrazine hydrate solution and heating it to 60-70°C. After reacting for 3-4 hours, the reaction solution is cooled to 20-30°C, and acetone is added dropwise. After the addition is complete, the mixture is stirred to crystallize for 2.5-3 hours. The mixture is then filtered, washed, and dried to obtain compound (1).

[0012] Further, step (2) specifically involves adding compound (1) and ethyl 2-formyl-3-oxopropionate to anhydrous ethanol, heating to 75-85°C, reacting for 3-4 hours, cooling to 0-10°C, crystallizing for 2-3 hours, filtering and drying to obtain compound (2).

[0013] Further, step (3) specifically involves: adding compound (2) to a 30-50 wt% aqueous solution of methylamine, heating to 40-50°C, reacting for 2-3 hours, cooling the reaction solution to 0-10°C, stirring for 2-2.5 hours, vacuum filtering, washing and drying to obtain the Reganosen.

[0014] Further, in step (1), the mass ratio of 2-chloroadenosine, hydrazine hydrate and acetone is 1:(5-8):(4-8).

[0015] Furthermore, in step (2), the mass ratio of compound (1) to ethyl 2-formyl-3-oxopropionate is 1:(0.5-1); in step (3), the mass ratio of compound (2) to aqueous methylamine is 1:(8-12); and the concentration of aqueous methylamine is 30-50 wt%.

[0016] This invention also provides a method for preparing reganosin, wherein the preparation method is achieved by reducing or inhibiting the formation of impurities M and N, wherein, The impurity M has the structure shown in the following formula M: ; The impurity N has the structure shown in the following formula: Furthermore, the preparation method is achieved by introducing acetone to protect the amino group in 2-chloroadenosine, thereby reducing or inhibiting the formation of impurities M and N; Furthermore, the generation mechanism of the impurity M is as follows: The generation mechanism of impurity N is as follows: .

[0017] Beneficial effects (1) The method for preparing Reganorogen provided by the present invention has a short reaction route, is easy to react and can be prepared on a large scale. At the same time, it greatly reduces the impurity content in Reganorogen, and the purity and yield of the product are significantly improved. It can be widely used in the field of Reganorogen preparation. (2) The preparation method of Reganoxane provided by the present invention is that the reaction system in step (1) is in a dissolved state, acetone is the crystallization solvent, and acetone reacts with amino groups to form an imine structure, which protects the amino group and avoids the generation of impurities M and N in step (2), which can significantly improve the purity and yield of Reganoxane. (3) The preparation method of Reganoxane provided by the present invention has a lower stability of imine structure compared with amide structure in intermediate compound (1) and compound (2). The protection can be completely removed in the aqueous methylamine solution in step (3) at 40-50°C. No closed high-pressure equipment is required. Compared with the preparation method in the prior art, it has a shorter reaction route and simpler reaction conditions, and can be applied to the large-scale production of Reganoxane. (4) This invention also provides the structure, preparation method and detection method of impurities M and N in Reganorsen, providing new reference standards for the detection of impurities in Reganorsen, which is more conducive to the impurity control and quality research of Reganorsen; by analyzing the pathway of the impurity generation, the synthesis process design of Reganorsen can be guided to prevent the generation of impurities; the development of the related substances HPLC (high performance liquid chromatography) method further improves the quality requirements of Reganorsen products. Attached Figure Description

[0018] Figure 1 The HPLC chromatogram and data of compound (1) in Example 1 are shown below; Figure 2 The HPLC chromatogram and data of compound (1) in Example 2 are shown below. Figure 3 The HPLC chromatogram and data of compound (1) in Example 3; Figure 4 The HPLC chromatogram and data of compound (2) in Example 1; Figure 5 The HPLC chromatogram and data of the product of Reganol in Example 1 are shown below. Figure 6 The mass spectrum of the product of regano in Example 1; Figure 7 The 1H NMR spectrum of the product of Reganol in Example 1 is shown. Detailed Implementation

[0019] This invention provides a method for preparing reganosin, which is achieved by reducing or inhibiting the formation of impurities M and N, wherein... The impurity M has the structure shown in the following formula M: ; The impurity N has the structure shown in the following formula: .

[0020] The principles and processes of impurity M and N generation are as follows: (1) Generation route of impurity M: Impurity M preparation method 1: A. Compound A reacts with ethyl 3-oxopropionate or ethyl 3-ethoxy-2-(ethoxymethyl)propionate to generate compound AM; BA-M reacts with aqueous methylamine to obtain the impurity M.

[0021] Method 2 for preparing impurity M: A. Compound B reacts with ethyl 3-oxopropionate or ethyl 3-ethoxy-2-(ethoxymethyl)propionate to generate compound B-M'; BB-M' reacts with aqueous methylamine to generate B-M''; CB-M'' condenses with tetraacetylribose to obtain the impurity M.

[0022] The route of impurity N generation: Method 1 for preparing impurity N: A. Compound A reacts with ethyl 2-formyl-3-oxopropionate to generate compound AN; B. Compound AN reacts with aqueous methylamine to obtain the impurity N.

[0023] Method 2 for preparing impurity N: A. Compound B-N' is prepared by reacting compound B with ethyl 2-formyl-3-oxopropionate; B. (2) Compound B-N' is prepared by reacting compound B-N' with aqueous methylamine solution; C. Impurity N is prepared by condensing compound B-N'' with tetraacetylribose.

[0024] Based on the above principles, the present invention provides a method for preparing Reganoxan, and the specific synthetic route is as follows: The present invention will be described below with reference to specific embodiments. It should be noted that the following embodiments are examples of the present invention and are used only to illustrate the invention, not to limit it. Other combinations and various modifications within the scope of the present invention can be made without departing from its spirit or scope.

[0025] The reagents and equipment used in this invention are conventional reagents and equipment in this technical field.

[0026] Example 1 Reganoxone was prepared using the following steps: (1) Preparation of compound (1): 300 ml of hydrazine hydrate was added to a 1 L flask, and 50 g of 2-chloroadenosine was slowly added while stirring. After the addition was completed, the reaction solution was heated to 70 °C and reacted for 4 h. After the reaction was completed, the reaction solution was cooled to 20 °C, and 300 ml of weighed acetone was added dropwise. After the addition was completed, the mixture was stirred to crystallize for 3 h. The mixture was then vacuum filtered, and the filter cake was washed with a small amount of acetone. The mixture was then vacuum dried at 50 °C for 4 h to obtain 51.6 g of compound (1) with a purity of 99.22% (e.g., ...). Figure 1 As shown in the figure, the yield was 92.3%.

[0027] (2) Preparation of compound (2): 500 ml of anhydrous ethanol was added to a 1 L flask, followed by stirring and the addition of 50 g of compound (1) and 32 g of ethyl 2-formyl-3-oxopropionic acid. After the addition was complete, the reaction solution was heated to 80 °C and reacted for 4 h. After the reaction was completed, the temperature was lowered to 0 °C and stirred to crystallize for 2 h. The mixture was then vacuum filtered, and the filter cake was washed with a small amount of methyl tert-butyl ether. The mixture was then dried under vacuum at 50 °C for 4 h to obtain 56.2 g of compound (2); purity 99.81%. Figure 4 The yield was 85.1%, and the HPLC chromatogram and data of compound (2) are as follows: Figure 4 As shown; (3) Preparation of Reganoxane: 500 ml of 40 wt% methylamine aqueous solution was added to a 1 L flask, and 50 g of compound (2) was added with stirring. After the addition was completed, the reaction solution was heated to 50 °C and stirred for 3 h. The reaction was stopped, the temperature of the reaction solution was lowered to 0 °C and stirred for 2 h. Vacuum filtration was performed, and the filter cake was washed with purified water and anhydrous ethanol respectively. The solution was dried under vacuum at 60 °C to obtain 40.7 g of Reganoxane product. The purity was 99.88% and the yield was 88.7%. The HPLC chromatogram and data of Reganoxane product are as follows. Figure 5 As shown.

[0028] The mass spectra of the product of Regano are as follows. Figure 6 As shown, the proton NMR spectrum is as follows: Figure 7 As shown, its 1H NMR data are: 1H NMR (400 MHz, DMSO) δ 8.97 (s, H8), 8.44 (s, H21), 8.40 (d, H26), 8.09 (s, H23), 7.80 (s, H15), 5.97 (d, H10), 5.55 (d, H16), 5.28 (d, H17), 5.08 (t, H19), 4.66 (d, 14), 4.21 (m, H13), 3.99 (m., H12), 3.70 (m, H18), 2.78 (d, H28).

[0029] Example 2 Basically the same as Example 1, except that in step (1) acetone is replaced with 200 mL. The HPLC chromatogram and data of compound (1) are as follows. Figure 2 As shown; in step (2), ethyl 2-formyl-3-oxopropionate is changed to 20g; in step (3), methylamine aqueous solution is changed to 400mL.

[0030] Example 3 Basically the same as Example 1, except that in step (1) acetone is replaced with 400 mL. The HPLC chromatogram and data of compound (1) are as follows: Figure 3As shown; in step (2), ethyl 2-formyl-3-oxopropionate is changed to 50g; in step (3), methylamine aqueous solution is changed to 600mL.

[0031] Example 4 (1) Impurity M was prepared by the following method 20 ml of DMF was added to a 50 ml three-necked flask. Compound (1) (1.0 g, 8.6 mmol), ethyl 3-oxopropionate (2.0 g, 17.2 mmol), and 3 g of anhydrous magnesium sulfate were added with stirring. After the addition was complete, the mixture was heated to reflux and stirred for 14 h. After the reaction was complete, the mixture was cooled to room temperature, filtered to remove the anhydrous magnesium sulfate, and the filtrate was concentrated to dryness under reduced pressure to obtain compound (2). Compound (2) was added to a 30% aqueous methylamine solution and heated to 40 °C with stirring for 6 h. After the reaction was complete, the mixture was cooled to room temperature, filtered, and the filter cake was washed with methanol to obtain crude product containing impurity M.

[0032] Silica gel column chromatography purification: mobile phase: methanol: dichloromethane = 1:6, the target fraction was collected and concentrated to dryness under pressure to obtain impurity M 0.73g, with a purity of 95.2%.

[0033] Impurity N was prepared by the following method

[0034] 20 ml of DMF was added to a 50 ml three-necked flask. Compound (1) (1.0 g, 8.6 mmol), ethyl 2-formyl-3-oxopropionate (2.5 g, 17.2 mmol), and 3 g of anhydrous magnesium sulfate were added while stirring. After the addition was complete, the mixture was heated to reflux and stirred for 12 h. After the reaction was complete, the mixture was cooled to room temperature, filtered to remove the anhydrous magnesium sulfate, and the filtrate was concentrated to dryness under reduced pressure to obtain compound (2). Compound (2) was added to a 30% aqueous methylamine solution and heated to 40 °C, stirring for 4 h. After the reaction was complete, the mixture was cooled to room temperature, filtered, and the filter cake was washed with methanol to obtain crude product containing impurity N.

[0035] Silica gel column chromatography purification: mobile phase: methanol: dichloromethane = 1:8, the target fraction was collected and concentrated to dryness under pressure to obtain impurity N 0.73g, purity 98.8%.

[0036] Comparative Example 1 (Preparation of Reganoxane by Liu Wei et al.) used tert-butyldimethylsilyl as the glycoside protecting group of 2-chloroadenosine. The synthesis route is as follows: (1) 2-Chloroadenosine (50.0 g, 0.17 mol) was dissolved in 500 ml of pyridine, and tert-butyldimethylchlorosilane (210 g, 1.40 mol) and imidazole (90.0 g, 1.32 mol) were added with stirring. The reaction was carried out at room temperature for 24 h. The pyridine was removed by concentration under reduced pressure, and 500 ml of dichloromethane was added to dissolve the remaining solid. Then, the solid was washed twice with saturated sodium bicarbonate solution and three times with saturated sodium chloride solution. The dichloromethane phase was dried with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give compound 2 (91.8 g, yield 86%), with a purity of 92%. (2) Compound 2 (50.0 g, 0.078 mol) was dissolved in 200 ml of anhydrous ethanol and stirred at 50 °C. Under nitrogen protection, 80% hydrazine monohydrate (80.0 g, 1.28 mol) was added, and the temperature was raised to 70 °C for 20 h. The reaction solution was slowly poured into 2 L of ice water, stirred for 1 h, and filtered. The filter cake was washed three times with purified water, filtered, and dried under reduced pressure at 55 °C to obtain compound 3 (46.7 g, yield 94.0%), purity 92%. (3) Compound 3 (50.0 g, 0.078 mol) was dissolved in 500 ml of isopropanol under nitrogen protection, heated to 80 °C, and ethyl 2-formyl-3-oxopropionate (13.0 g, 0.090 mol) was added. The reaction was carried out for 2 h. The reaction solution was poured into 4 L of ice-cold purified water, stirred for 30 min, filtered, and the filter cake was washed twice with 100 ml of ice-cold isopropanol-water (1:6) mixture. The filter cake was dried under vacuum at 55 °C to obtain compound 4 (52.6 g, yield 90.0%) with a purity of 88%. (4) Under normal pressure, compound 4 (25.0 g, 0.033 mol) was dissolved in 500 g of methanol solution (40%) containing methylamine, and the temperature was raised to 65 °C for 24 h. Methylamine was removed under reduced pressure at 30 °C. At room temperature, tetrabutylammonium fluoride (25.0 g, 0.096 mol) was added directly to the reaction solution, and the reaction was carried out for 12 h. A solid precipitated out, was filtered, and the filter cake was added to 200 ml of a mixture of ethanol and water (1:5), stirred for 2 h, filtered, and the filter cake was washed with 30 ml of purified water and dried under reduced pressure at 55 °C to obtain a white solid (7.16 g, yield 64.9%) with a purity of 99.0%.

[0037] Comparative Example 2 (Preparation of Reganoxan by Luo Hairong et al.) Unlike Example 1, 2-chloroadenine was selected as the starting material, and then 1,2,3,5-tetra-O-acetyl-β-D-ribose was reacted with sodium hydroxide to remove the acetyl group, yielding Reganoxan. The specific synthetic route is as follows: (1) 2-Chloroadenine (50 g, 0.29 mol) was mixed with 250 mL of hydrazine hydrate (4.85 mol), heated to 120 °C, reacted under nitrogen protection for 2 h, cooled to room temperature, filtered, the filter cake was washed with water, and dried under reduced pressure at 60-70 °C to obtain white compound 2 (43.8 g, yield 90%), with a purity of 98.16%; (2) Add 2 (40 g, 0.24 mol) to 800 ml of anhydrous ethanol, add ethyl 2-formyl-3-oxopropionate (34.9 g, 0.26 mol), and heat under reflux for 2 h. Cool to room temperature, filter, and dry under reduced pressure at 60–70 °C to obtain yellow solid 3 (52.9 g, 80%) with a purity of 97.8%. (3) Add 3 (50 g, 0.18 mol) to 30% methylamine solution (500 ml), react at room temperature for 3 h, remove the solvent under reduced pressure, add water (500 mL) to the residue, stir at room temperature for 6 h, filter, and dry under reduced pressure at 60-70 °C to obtain light brown solid 4 (37.8 g, 80%) with a purity of 97.2%; (4) Under nitrogen protection, 4 (35 g, 0.14 mol) was added to a mixture of acetonitrile (400 ml) and N,O-bis(trimethylsilyl)acetamide (200 ml), heated to 55-60 °C, and after complete dissolution, 5 (51.8 g, 0.16 mol) of acetonitrile solution (200 ml) and TMSOTf (60.2 g, 0.27 mol) of acetonitrile solution (200 ml) were added dropwise. After the addition was complete, the mixture was reacted at 50-60 °C for 3 h. After cooling to room temperature, 5 mol / L sodium hydroxide solution (200 L) was added, and the mixture was reacted at room temperature for 3 h. After standing and separating into layers, deionized water (400 ml) was added to the organic phase, and the mixture was heated to 50-55 °C to precipitate a solid. After 45 min, add deionized water (400 ml), cool to 10°C, continue stirring for 45 min, filter, wash the filter cake with water (100 ml), and dry under reduced pressure at 55-60°C to obtain a white solid 1 (39.7 g, 75.0%) with a purity of 98.6%.

[0038] Purity testing The following steps were used to perform HPLC quantitative detection of Reganoxan and its impurities M and N in Examples 1-3 and Comparative Examples 1-2: Column conditions: A Welch Xtimate C18 column, 4.6 mm × 250 mm, 5 μm, was used; gradient elution was performed using 5 mmol / L diammonium hydrogen phosphate solution-methanol (95:5) as mobile phase A and 5 mmol / L diammonium hydrogen phosphate solution-methanol (5:95) as mobile phase B; the flow rate was 1.0 mL / min; the detection wavelength was 247 nm; the column temperature was 40 °C; the injection volume was 50 μL; and the elution program was set as shown in the table below. Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 95 5 40 65 35 50 65 35 50.1 95 5 60 95 5 Preparation of reference solution: Accurately weigh 10.0 mg of Reganoxen reference standard and place it in a 100 mL volumetric flask. Add an appropriate amount of methanol to dissolve it, sonicate for 5 minutes to aid dissolution, cool, and then dilute to the mark with methanol. Shake well to obtain the reference standard stock solution (100 μg / mL). Accurately measure 1.0 mL of the reference standard stock solution and place it in a 50 mL volumetric flask. Dilute to the mark with methanol and shake well to obtain the reference standard working solution (2 μg / mL) (prepare immediately before use). (3) Preparation of test solution: Accurately weigh 0.5 g of Reganoxane raw material prepared in Example 1 and Comparative Examples 1 and 2, place it in a 50 mL volumetric flask, add 30 mL of methanol, sonicate for 10 minutes to completely dissolve, cool and then dilute to the mark with methanol and shake well; take 5 mL of the above solution, place it in a 10 mL volumetric flask, dilute to the mark with methanol, shake well, filter through a 0.45 μm organic phase filter membrane, and take the filtrate as the test solution; (5) Detection: Inject 20 μL of blank solvent (methanol), 20 μL of reference working solution, and 20 μL of test solution in sequence, and record the chromatograms respectively. Then, calculate the content of each impurity in Reganoxon injection according to the external standard method.

[0039] Performance testing Based on the spectra, the purity and yield of compounds (1), (2), and reganosin in Examples 1-3 and Comparative Examples 1-2 were detected and calculated respectively. The results are shown in Tables 1 and 2: Table 1. Purity and yield data of intermediate products and reganosin in Examples 1-3. Compound (1) purity / % Compound (1) yield / % Purity of compound (2) / % Compound (2) yield / % Riganosheng purity / % Regano's yield / % Example 1 99.22 92.3 99.81 85.1 99.88 88.7 Example 2 99.25 89.9 99.83 85.3 99.88 88.9 Example 3 99.28 94.8 99.81 85.4 99.87 89.0 Table 2. Purity and yield data of intermediates and reganosin in Comparative Examples 1 and 2. Purity of compound (2) / % Compound (2) yield / % Purity of compound (3) / % Compound (3) yield / % Compound (4) purity / % Compound (4) yield / % Riganosheng purity / % Regano's yield / % Comparative Example 1 92 86 92 94 88 90% 99.0% 64.9% Comparative Example 2 98.16 90 97.8 80 97.2 80 98.6% 75.0% Based on the comparison of the test results of Examples 1-3 and Comparative Examples 1-2, it can be seen that by changing the preparation method of Reganoxane, the present invention can effectively avoid the generation of impurities, improve the purity and yield of the product, and at the same time avoid the use of closed high-pressure equipment, reduce production costs, and can be applied on a large scale in the preparation of Reganoxane.

[0040] The above embodiments are only for illustrating the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it. They should not be used to limit the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A method for preparing Reganoxan, characterized in that, Includes the following steps: (1) Compound (1) was prepared by reacting 2-chloroadenosine with hydrazine hydrate and acetone in sequence. (2) Compound (1) was reacted with ethyl 2-formyl-3-oxopropionate to prepare compound (2); (3) Reganoxane was prepared by reacting compound (2) with aqueous methylamine.

2. The method for preparing Reganoxan according to claim 1, characterized in that, The specific steps (1) are as follows: 2-chloroadenosine is added to hydrazine hydrate solution and heated to 60-70℃. After reacting for 3-4 hours, the reaction solution is cooled to 20-30℃ and acetone is added dropwise. After the addition is completed, the mixture is stirred to crystallize for 2.5-3 hours. After filtration, washing, and drying, compound (1) is obtained.

3. The method for preparing Reganoxan according to claim 1, characterized in that, The specific steps (2) are as follows: Compound (1) and ethyl 2-formyl-3-oxopropionate are added to anhydrous ethanol, the temperature is raised to 75-85℃, the reaction is carried out for 3-4 hours, the temperature is lowered to 0-10℃, crystallization is carried out for 2-3 hours, and then the mixture is filtered and dried to obtain compound (2).

4. The method for preparing Reganoxan according to claim 1, characterized in that, The specific steps (3) are as follows: add compound (2) to a 30-50 wt% aqueous solution of methylamine, heat to 40-50℃, react for 2-3 hours, cool the reaction solution to 0-10℃, stir for 2-2.5 hours, vacuum filter, wash and dry to obtain the Reganosen.

5. The method for preparing Reganoxan according to claim 1 or 2, characterized in that, In step (1), the mass ratio of 2-chloroadenosine, hydrazine hydrate and acetone is 1:(5-8):(4-8).

6. The method for preparing Reganoxan according to claim 1 or 3, characterized in that, In step (2), the mass ratio of compound (1) to ethyl 2-formyl-3-oxopropionate is 1:(0.5-1).

7. The method for preparing Reganoxan according to claim 1 or 4, characterized in that, In step (3), the mass ratio of compound (2) to the aqueous methylamine solution is 1:(8-12), and the concentration of the aqueous methylamine solution is 30-50 wt%.

8. A method for preparing Reganoxan, characterized in that, The preparation method is achieved by reducing or suppressing the formation of impurities M and N, wherein, The impurity M has the structure shown in the following formula M: ； The impurity N has the structure shown in the following formula: 。 9. The method for preparing Reganoxan according to claim 8, characterized in that, The preparation method is achieved by introducing acetone to protect the amino group in 2-chloroadenosine, thereby reducing or inhibiting the formation of impurities M and N.

10. The method for preparing Reganoxan according to claim 8, characterized in that, The generation mechanism of the impurity M is as follows: The generation mechanism of impurity N is as follows: 。

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