A method for preparing a high-purity peramivir intermediate

Abstract

The present invention relates to a method for preparing a high-purity peramivir intermediate. The method comprises the following steps: Step 1: adding compound II to a reaction kettle, and dropwise adding a sodium borohydride methanol solution; after the dropwise addition is completed, the reaction is stirred at a controlled temperature; purified water and a sodium nitrite solution are added, and the reaction is stirred to prepare compound (III); Step 2: adding acetic anhydride dropwise to compound (III), and the reaction is stirred at a controlled temperature; Step 3: adding a sodium carbonate aqueous solution, stirring, separating the liquids, collecting the organic phase, concentrating under reduced pressure, adding a solvent, stirring and crystallizing, and centrifuging until no obvious liquid flows out; vacuum drying the filter cake to obtain an off-white crude peramivir acetylate (IV); Step 4: refining to obtain an off-white solid peramivir acetylate (IV). #imgabs0#

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceuticals and medicines, and discloses a method for preparing a key intermediate of peramivir, (1S,2S,3R,4R)-3-[(1S)-1-acetylamino-2-ethylbutyl]-4[[(1,1-dimethylethoxy)carbonyl]amino]-2-hydroxycyclopentanoic acid methyl ester. Background Art

[0002] Peramivir, developed by Biocrystal Pharmaceuticals, Inc. in the United States, can effectively inhibit the replication and transmission of multiple influenza virus strains. It has the advantages of good tolerance and low toxicity. It is used to treat and prevent influenza A and B in adults and children. The chemical name of peramivir is (1S,2S,3R,4R)-3-[(1S)-1-(acetylamino)-2-ethylbutyl]-4-guanidino-2-hydroxycyclopentanecarboxylic acid trihydrate, and its structure is as shown in Formula I:

[0003]

[0004] In October 2009, the US FDA granted emergency approval for Compound I for the treatment of H1N1 influenza; in January 2010, peramivir was launched in Japan. In 2012, the China Food and Drug Administration approved the marketing of peramivir sodium chloride injection. On December 19, 2014, the FDA approved peramivir for the treatment of influenza infections in adults.

[0005] Currently, the patents for representative synthesis methods of peramivir are WO9933781A1, J.Med.Chem.2000,43,3482-3486, ZL200710143607.5, ZL201180070519.1, ZL201510461348.5 and literature J.Med.Chem,2001,44,4379-4392, etc. The general synthesis method is to use vins lactam as the raw material, and finally obtain peramivir through amide alcoholysis resolution, amino / carboxyl protection, 1,3-dipolar cycloaddition, reductive ring opening, acetylation, deprotection, hydrolysis and guanidination.

[0006] Patent WO9933781A1 and literature J.Med.Chem.2000,43,3482-3486, J.Med.Chem,2001,44,4379-4392 report that the key intermediate (1S,2S,3R,4R)-3-[(1S)-1-acetylamino-2-ethylbutyl]-4[[(1,1-dimethylethoxy)carbonyl]amino]-2-hydroxycyclopentanoic acid methyl ester (II) is prepared by reacting (1S,4R)-4-((tert-butoxycarbonyl)amino)cyclopent-2-ene-1-carboxylic acid methyl ester with 3-(nitromethyl)pentane and phenyl isocyanate. The preparation process uses highly toxic reagents, which is not conducive to safe production and waste liquid treatment. Chinese Journal of Antibiotics, 2009, 34(8): 475-478 and J Org Chem, 2003, 68(17): 6591-6596 used different methods to prepare compound II by reacting diethylbutyraldehyde oxime or diethylbutyraldehyde chlorooxime, avoiding the use of phenyl isocyanate.

[0007] The ring-opening methods for the key intermediate (1S,2S,3R,4R)-3-[(1S)-1-acetylamino-2-ethylbutyl]-4[[(1,1-dimethylethoxy)carbonyl]amino]-2-hydroxycyclopentanoic acid methyl ester (II) generally include PtO2-H2 reduction (WO9933781A1, J.Med.Chem. 2000, 43, 3482-3486, J.Med.Chem, 2001, 44, 4379-4392) or NaBH4 / NiCl2 reduction (ZL200710143607.5, etc.). Both methods have their advantages and disadvantages. PtO2-H2 reduction is expensive, requires specialized reaction equipment, and requires the addition of a Pt removal unit. NaBH4 / NiCl2 reduction is rapid and inexpensive, but also requires the treatment of nickel-containing wastewater.

[0008] The above preparation methods all involve the key intermediate (1S,2S,3S,4R)-3-[(1S)-1-acetylamino-2-ethylbutyl]-4-(Boc)-amino-2-ylcyclopentane-1-acid ester, specifically as shown in Formula II:

[0009]

[0010] Earlier literature reported that Compound II was ring-opened, aminoacetylated, and then guanidinium-grouped with N,N'-di-Boc-S-methylisothiourea / HgCl2. This reaction used highly toxic HgCl2 and produced methyl mercaptan as a byproduct. Domestic patents such as ZL200710143607.5 also describe the ring-opening, aminoacetylation, and deprotection of Compound II followed by reaction with a guanidinium-grouping reagent (typically 1H-1,2,4-triazole-1-carboxamidine hydrochloride, carboxamidine hydrochloride, or pyrazolecarboxamidine hydrochloride). ZL201180070519.1 uses chiral cyclopentene as a starting material and reacts it with Boc-protected pyrazolecarboxamidine hydrochloride. After Boc protection, the key intermediate is cyclized with 2-ethyl-N-hydroxybutyliminochloride. Ring-opening with sodium borohydride / nickel chloride is followed by acetyl addition and deprotection to yield peramivir.

[0011] The route for preparing peramivir from compound II is as follows:

[0012]

[0013] The process for preparing Compound III from Compound II involves a reductive ring-opening reaction, where a chiral center (1S) is added to the 3-position of Compound III's structure after reduction. Early patents and literature reported little on the post-processing and purification of Compound III, which was directly used in the next chemical reaction without isolation. ZL200710143607.5 used methanol as the reduction solvent. After the reaction, the solid was separated by treatment with sodium nitrite, ammonium chloride, and 25% aqueous ammonia. The solid was suspended in toluene, washed with 25% ethylenediaminetetraacetic acid, complexed with sodium ethylenediaminetetraacetic acid to remove metal ions, and then cooled and crystallized to obtain Compound III. Reference Synthetic Communications, 2013, 43, 2641-2647, also used methanol as the reduction solvent. After the reaction, the solvent was concentrated and then treated with sodium nitrite, ammonium chloride, and aqueous ammonia to crystallize to obtain Compound III. ZL202010966511.4 uses dichloromethane and methanol as the reaction solvents. After the reaction is completed, it is treated with sodium ethylenediaminetetraacetic acid, sodium nitrite, ammonium chloride, and ammonia water. The separated liquids are concentrated to remove the solvent, and then recrystallized from a methanol-water system to obtain compound III (up to 97.56%). ZL202310222820.4 uses toluene as the reduction reaction solvent. After the reaction is completed, it is washed, extracted, concentrated to remove the solvent, and recrystallized from methanol to obtain compound III. However, methanol has good solubility for compound III, the yield is greatly affected by the crystallization temperature, and the reproducibility is relatively poor.

[0014] Report ZL201710002936.1 reproduces a related method to prepare Compound III, with an isomer content of up to 10%. This patent uses an iron-cobalt alloy as a catalyst, but Compound III is obtained by column chromatography, which is not suitable for commercial production. A small amount of the 3-(1R) isomer is present during the preparation of Compound III, and its generation equation is as follows:

[0015]

[0016] Therefore, it is necessary to develop a purification method that can effectively remove the reaction impurities and 3-(1R) isomer impurities in the preparation of compound III.

[0017] The structure of compound III contains both amino and hydroxyl groups. During the acetylation process to prepare compound IV, over-acetylated diacetylated impurities will be generated, as follows:

[0018]

[0019] Most literature and patents use compound IV directly in subsequent chemical reactions after preparation, or use column chromatography to purify compound IV to a higher purity. A purification method that can efficiently remove diacetylated impurities is of great significance for drug quality control.

[0020] Consistent quality control of APIs is a key focus in the drug development process. In accordance with current international and domestic drug registration requirements for drug quality by design, the purification and detection of intermediates during the preparation of technical drugs are extremely important links in the process development and research process, thereby avoiding adverse reactions caused by impurities and potential risks to the efficacy and safety of the drug. Summary of the Invention

[0021] The present invention aims to provide a method for preparing a high-purity peramivir intermediate (1S,2S,3R,4R)-3-[(1S)-1-acetylamino-2-ethylbutyl]-4[[(1,1-dimethylethoxy)carbonyl]amino]-2-hydroxycyclopentanoic acid methyl ester (peramivir acetylate IV). The conventional method for preparing peramivir is to use compound II as a starting material, perform ring-opening reduction, and then react with acetic anhydride to prepare the peramivir acetylate (Compound IV). The acetylate is then deprotected from the amino group, the ester group is hydrolyzed, and then reacted with triazole carboxamidine to produce peramivir.

[0022] The preparation method of peramivir reported in the existing literature is a one-step process for acetylation, deprotection and guanidination, and each intermediate is directly reacted without separation. The operation process is relatively simplified, but at the same time, the intermediates in each step are not separated and purified, and the process control cannot be effectively carried out, which increases the quality risk of the final product peramivir as a raw material drug. Therefore, in order to increase the quality controllability of peramivir, the present invention provides a method for preparing, separating and purifying a peramivir intermediate acetylated product. The acetylated product prepared by the method is an off-white solid with a yield of >95% and a purity of >99%. In particular, the method of the present invention can control the content of diacetylated impurities to <0.1%. This impurity is a process impurity that can be transferred backward with the reaction and is a recalcitrant impurity. Conventional drug purification methods such as recrystallization and beating have poor removal effects on this impurity and subsequent corresponding impurities.

[0023] At the same time, a method for refining compound III is invented. Toluene is used to purify compound III, and the purity thereof is above 98%, and the yield is stable at above 85%.

[0024] Compared with the prior art, the present invention offers the following beneficial technical effects: Compound III is purified with toluene and then used in an acetylation reaction to obtain Compound IV. The crude acetylated Compound IV is then crystallized and recrystallized to yield high-purity Compound IV. This method is simple to operate, precipitates the target product from the system, and exhibits excellent properties, high yield, and high quality. It is particularly effective in controlling recalcitrant hydroxyacetylated impurities.

[0025] The specific content of the invention is as follows:

[0026]

[0027] Step 1: Add Compound II, nickel chloride hexahydrate, and methanol to a reactor. Cool and add sodium borohydride methanol solution dropwise. After completion of the addition, control the temperature and stir the reaction. Add purified water and sodium nitrite solution, and stir the reaction. After completion of the reaction, filter, and wash the filter cake with ammonia and then EDTA-2Na aqueous solution to obtain crude Compound III. Refine the crude Compound III by adding it to a certain amount of solvent, heating to dissolve, cooling and stirring to crystallize, filtering, and transferring the filter cake to a vacuum drying oven to dry, yielding Compound III as an off-white solid.

[0028] Step 2: Add the solvent and the purified peramivir methyl ester (III) to the reactor, start stirring, and add acetic anhydride dropwise under temperature control. Add the mixture within a certain period of time, and then stir and react under temperature control. The reaction is considered complete when the IV material point disappears under TLC monitoring.

[0029] Step 3: After the reaction is completed, add sodium carbonate aqueous solution, stir, separate the liquids, collect the organic phase, concentrate under reduced pressure, add solvent, stir and crystallize, and centrifuge until no obvious liquid flows out; vacuum dry the filter cake to obtain an off-white crude product of peramivir acetylate (IV);

[0030] Step 4: Add the crude product of peramivir acetylate (IV) to the reactor, mix the solvent, start stirring, and heat until the system is clear; cool down, add solvent, solid precipitates from the system, continue cooling and stirring to crystallize; centrifuge until no obvious liquid flows out, and vacuum dry the filter cake to obtain an off-white solid peramivir acetylate (IV).

[0031] Wherein, the solvent for the purification of compound III described in step 1 is benzene, toluene, or xylene, preferably toluene, and the amount of toluene used is 10 to 20 times (mass ratio) of the amount of compound II fed, preferably 15 times.

[0032] The dropping temperature in step 2 is 0 to 30°C, preferably 0 to 10°C.

[0033] The reaction time in step 2 is 0 to 8 hours, preferably 2 to 3 hours.

[0034] The solvent in step 3 is an alkane solvent such as n-pentane, n-hexane, or n-heptane, preferably n-heptane.

[0035] The solvent described in step 4 is a lower ester or a mixed solvent of a lower ester and an alkane solvent, wherein the lower ester is ethyl formate, ethyl acetate, isopropyl acetate, etc., preferably ethyl acetate; the alkane solvent is n-pentane, n-hexane, n-heptane, preferably n-heptane; the mass ratio of the mixed solvent is 1:(3-10), preferably 1:4.5.

[0036] At the same time, the present invention provides a method for detecting related substances of compound III and a method for detecting related substances of compound IV.

[0037] Isomer detection method of compound III:

[0038] Octadecylsilane bonded silica gel was used as the filler, potassium dihydrogen phosphate solution was used as the mobile phase A, and acetonitrile-water was used as the mobile phase B for gradient elution.

[0039] The chromatographic column described in the detection method for Compound III is a NanoChrom ChromCore AQ C18, 4.6 mm × 250 mm, 5 μm, or a chromatographic column of equivalent performance, and a trapping column (NanoChrom Ghost-Remover, 4.6 mm × 50 mm, or a trapping column of equivalent performance) is connected after the mixer and before the injector.

[0040] The mobile phase described in the detection method of compound III is as follows: mobile phase A is 0.02 mol / L potassium dihydrogen phosphate solution (pH adjusted to 4.5 with phosphoric acid), and mobile phase B is acetonitrile-water (90:10).

[0041] The elution gradient described in the detection method of compound III is:

[0042] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 80 20 20 70 30 70 20 80 80 20 80 82 80 20 95 80 20

[0043] The flow rate described in the detection method of compound III was 1.0 mL per minute; the column temperature was 25° C., the detection wavelength was 195 nm; and the injection volume was 20 μL.

[0044] Detection method of related substances of compound IV:

[0045] Octadecylsilane bonded silica gel was used as the filler, potassium dihydrogen phosphate solution was used as the mobile phase A, and acetonitrile-water was used as the mobile phase for gradient elution.

[0046] The chromatographic column described in the detection method of compound IV is NanoChrom ChromCore AQ C18, 4.6 mm × 250 mm, 5 μm or a chromatographic column of equivalent performance, and a trapping column (NanoChromChromCore AQ C18, 4.6 mm × 250 mm, 5 μm or a chromatographic column of equivalent performance) is connected after the mixer and before the injector.

[0047] The mobile phase described in the detection method of compound IV is as follows: mobile phase A is 0.02 mol / L potassium dihydrogen phosphate solution (pH adjusted to 4.5 with phosphoric acid), mobile phase B is acetonitrile-water (90:10),

[0048] The elution gradient described in the detection method of compound IV is:

[0049] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 80 20 20 70 30 70 60 40 80 60 40 82 80 20 95 80 20

[0050] The flow rate described in the detection method of compound IV was 1.0 mL per minute; the column temperature was 25° C., the detection wavelength was 195 nm; and the injection volume was 20 μL.

[0051] The intermediate preparation method of the present invention, wherein the reaction conditions and are obtained through screening, the screening process is as follows:

[0052] The purification method of compound III in step 1 was investigated, and benzene, toluene and xylene were used as solvents to investigate the purification effect. The results showed that toluene was the preferred solvent. The amount of toluene was investigated to be 10 to 20 times the amount of compound II (mass ratio), with 15 times being the preferred amount.

[0053] The temperature of adding acetic anhydride in step 2 was investigated and the results showed that a temperature range of 0 to 30°C is feasible, preferably 0 to 10°C.

[0054] The reaction time of step 2 was investigated to be in the range of 0 to 8 h, preferably 2 to 3 h.

[0055] The crystallization solvents in step 3 were investigated: alkane solvents n-pentane, n-hexane, and n-heptane. The results showed that all of them can meet the requirements of the present invention, with n-heptane being preferred.

[0056] The recrystallization solvent in step 4 was investigated, and a mixture of lower esters or lower esters and alkane solvents was investigated, wherein the lower esters are ethyl formate, ethyl acetate, isopropyl acetate, etc., preferably ethyl acetate; the alkane solvents are n-pentane, n-hexane, n-heptane, preferably n-heptane; the mass ratio of the mixed solvent is 1:(3-10), preferably 1:4.5. The method of the present invention differs from the existing method in that:

[0057] Compared with the prior art, the present invention uses toluene to purify compound III and then uses it for acetylation reaction to obtain compound IV. The crude compound IV is crystallized and recrystallized to obtain high-purity compound IV. Compound IV is separated, solidified and purified, thereby enhancing the quality control of peramivir.

[0058] The advantages are:

[0059] The method of the invention is simple to operate, can precipitate the target product from the system, has good properties, high yield and good quality, and has a significant control effect on stubborn hydroxyacetylated impurities. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 HPLC system suitability profile of compound III

[0061] Figure 2 HPLC spectrum of compound III

[0062] Figure 3 HPLC system suitability profile of compound IV

[0063] Figure 4 HPLC spectrum of compound IV DETAILED DESCRIPTION

[0064] The specific embodiments of the present invention are further described below through examples.

[0065] Example 1

[0066] Weigh 2.00 kg of compound II, 1.42 kg of nickel chloride hexahydrate, and 6.00 kg of methanol into a 50 L reactor. Cool to below 0°C and dropwise add a methanolic sodium borohydride solution (0.85 kg of sodium borohydride, 5.00 kg of methanol). After addition, control the temperature of the reaction system at 0-10°C and stir for 2 hours. Add 10.00 kg of purified water and, controlling the temperature at 20-30°C, add the sodium nitrite solution to the reaction mixture. Continue stirring at 20-25°C for 14 hours. Filter and wash the filter cake with 25.00 kg of ammonia water and then an aqueous EDTA-2Na solution (1.30 kg of EDTA-2Na, 22.00 kg of purified water) to obtain the crude product of compound III.

[0067] The crude compound III was added to 30.00 kg of toluene and heated to 60-70°C to dissolve. The temperature was then cooled to 20-30°C with stirring for 1 hour to allow crystallization. The mixture was filtered, and the filter cake was rinsed with toluene and purified water. The filter cake was transferred to a vacuum drying oven and dried at 45°C ± 5°C to obtain 1.78 kg of an off-white solid compound III in an 89% yield (HPLC purity 98.188%, isomers 0.023%).

[0068] Example 2

[0069] Weigh 2.00 kg of compound II, 1.42 kg of nickel chloride hexahydrate, and 6.00 kg of methanol into a 50 L reactor. Cool to below 0°C and dropwise add a methanolic sodium borohydride solution (0.85 kg of sodium borohydride, 5.00 kg of methanol). After addition, control the temperature of the reaction system at 0-10°C and stir for 2 hours. Add 10.00 kg of purified water and, controlling the temperature at 20-30°C, add the sodium nitrite solution to the reaction mixture. Continue stirring at 20-25°C for 14 hours. Filter and wash the filter cake with 25.00 kg of ammonia water and then an aqueous EDTA-2Na solution (1.30 kg of EDTA-2Na, 22.00 kg of purified water) to obtain the crude product of compound III.

[0070] The crude compound III was added to 40.00 kg of toluene and heated to 60-70°C to dissolve. The temperature was then cooled to 20-30°C and stirred to allow crystallization for 1 hour. The mixture was filtered, and the filter cake was rinsed with toluene and purified water. The filter cake was transferred to a vacuum drying oven and dried at 45°C ± 5°C to obtain 1.68 kg of an off-white solid compound III in an 85% yield (HPLC purity 98.281%, isomers 0.015%).

[0071] Example 3

[0072] To a reactor, 23.50 kg of ethyl acetate and 2.90 kg of compound III were added, followed by the dropwise addition of 0.90 kg of acetic anhydride over 2-3 hours. The reaction mixture was stirred at 20-30°C. After completion of the reaction, 5.00 kg of a 10% aqueous sodium carbonate solution was added, stirred for 20 minutes, and allowed to stand for 20 minutes. The mixture was separated, and the organic phase was collected and distilled under reduced pressure (vacuum ≤ -0.08 MPa) until 80-90% of the added volume was reached. 23.65 kg of n-heptane was added, causing a large amount of solid to precipitate. The mixture was cooled to 0-10°C and stirred for 1 hour. The mixture was centrifuged, rinsed with n-heptane, and the filter cake was vacuum-dried at 45-50°C to yield 3.15 kg of crude off-white solid intermediate IV, a 97.2% yield (HPLC purity 99.552%, diacetylated impurities 0.095%).

[0073] To a reaction kettle, 3.00 kg of crude IV, 5.40 kg of ethyl acetate, and 4.00 kg of n-heptane were added. Stirring was initiated and the temperature was raised to 50-60°C, resulting in a clear solution. The temperature was lowered and 20.00 kg of n-heptane was added, causing a large amount of solid to precipitate. The temperature was further lowered to 0-10°C, followed by stirring and crystallization for 1 hour. The mixture was centrifuged, the filter cake was rinsed with n-heptane, and vacuum dried at 45-50°C to obtain 2.89 kg of an off-white solid, peramivir intermediate IV, in a 96% yield (HPLC purity 99.732%, diacetylated impurity 0.032%).

[0074] Example 4

[0075] The crude compound IV was prepared using the same method as in Example 3. 3.00 kg of crude IV, 5.40 kg of ethyl acetate, and 4.00 kg of n-heptane were added to a reaction kettle, stirring was initiated, and the temperature was raised to 50-60°C. The mixture became clear. The temperature was lowered and 34.00 kg of n-heptane was added. A large amount of solid precipitated. The temperature was further lowered to 0-10°C, stirring for 1 hour, and crystallization occurred. The mixture was centrifuged, the filter cake was rinsed with n-heptane, and vacuum dried at 45-50°C to obtain 2.70 kg of an off-white solid, peramivir intermediate IV, in a 90% yield (HPLC purity 99.580%, diacetylated impurity 0.065%).

[0076] Example 5

[0077] The crude compound IV was prepared in the same manner as in Example 3. 3.00 kg of crude IV, 6.70 kg of ethyl acetate, and 4.00 kg of n-heptane were added to a reaction kettle, stirring was initiated, and the temperature was raised to 50-60°C. The mixture became clear. The temperature was lowered and 20.00 kg of n-heptane was added. A large amount of solid precipitated. The temperature was further lowered to 0-10°C, stirring for 1 hour, and crystallization occurred. The mixture was centrifuged, the filter cake was rinsed with n-heptane, and vacuum dried at 45-50°C to obtain 2.13 kg of an off-white solid, peramivir intermediate IV, in a 71% yield (HPLC purity 99.511%, diacetylated impurity 0.152%).

[0078] Example 6

[0079] The crude compound IV was prepared using the same method as in Example 3. 3.00 kg of crude IV, 6.70 kg of ethyl acetate, and 4.00 kg of n-hexane were added to a reaction kettle, stirring was initiated, and the temperature was raised to 50-60°C. The mixture became clear. The temperature was lowered and 20.00 kg of n-hexane was added. Solids precipitated. The temperature was further lowered to 0-10°C, stirring for 1 hour, and crystallization occurred. The mixture was centrifuged, the filter cake was rinsed with n-hexane, and vacuum-dried at 45-50°C to obtain 2.64 kg of an off-white solid, peramivir intermediate IV, in an 88% yield (HPLC purity 99.322%, diacetylated impurities 0.184%).

[0080] Example 7

[0081] The (1S,2S,3R,4R)-3-[(1S)-1-acetylamino-2-ethylbutyl]-4[[(1,1-dimethylethoxy)carbonyl]amino]-2-hydroxycyclopentanoic acid methyl ester (III) obtained in Example 1 was detected by high performance liquid chromatography, wherein octadecylsilane bonded silica gel was used as a filler (octadecylsilane bonded silica gel was used as a filler (NanoChrom ChromCore AQ C18, 4.6 mm × 250 mm, 5 μm or a chromatographic column with equivalent performance), a trapping column (NanoChrom Ghost-Remover, 4.6 mm × 50 mm or equivalent trapping column); mobile phase A: 0.02 mol / L potassium dihydrogen phosphate solution (pH adjusted to 4.5 with phosphoric acid); mobile phase B: acetonitrile-water (90:10); gradient elution: according to the table below; flow rate: 1.0 mL / min; column temperature: 25°C; detection wavelength: 195 nm; injection volume: 20 μL.

[0082] In this embodiment, the specified gradient elution program is shown in the following table.

[0083] Table 1 Gradient elution program

[0084] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 80 20 20 70 30 70 20 80 80 20 80 82 80 20 95 80 20

[0085] The HPLC spectrum of the obtained compound III is as follows Figure 1 In the system suitability solution chromatogram, the isomer and III peaks appear in sequence, and the resolution between the two peaks is greater than 0.5.

[0086] The system suitability chart and HPLC chart of compound III are attached. Figure 1 、 2 .

[0087] Example 8

[0088] The (1S,2S,3R,4R)-3-[(1S)-1-acetylamino-2-ethylbutyl]-4-[[(1,1-dimethylethoxy)carbonyl]amino]-2-hydroxycyclopentane-1-carboxylic acid methyl ester (IV) obtained in Example 2 was detected by high performance liquid chromatography, wherein octadecylsilane bonded silica gel was used as a filler (NanoChrom ChromCore AQ C18, 4.6 mm × 250 mm, 5 μm or a chromatographic column of equivalent performance), a trapping column (NanoChrom Ghost-Remover, 4.6 mm × 50 mm or equivalent trapping column); mobile phase A: 0.02 mol / L potassium dihydrogen phosphate solution (pH adjusted to 4.5 with phosphoric acid); mobile phase B: acetonitrile-water (90:10); gradient elution: according to the table below; flow rate: 1.0 mL / min; column temperature: 25°C; detection wavelength: 195 nm; injection volume: 20 μL.

[0089] In this embodiment, the specified gradient elution program is shown in the following table.

[0090] Table 2 Gradient elution program

[0091] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 80 20 20 70 30 70 60 40 80 60 40 82 80 20 95 80 20

[0092] The system suitability spectrum of compound IV (diacetyl impurity RRT = 1.20) and HPLC spectrum are attached. Figure 3 、 4 .

[0093] Comparative Example

[0094] Comparative Example 1

[0095] 2.00 kg of crude compound III was added to 4.00 kg of methanol and 4.00 kg of purified water, stirred, heated to 60-70 ° C to dissolve, cooled to 20-30 ° C, stirred and crystallized for 1 hour, filtered, and the filter cake was rinsed with purified water until no liquid flowed out; the filter cake was transferred to a vacuum drying oven and dried at 45 ° C ± 5 ° C to obtain 1.05 kg of off-white solid Peramivir intermediate III, with a yield of 53% and an HPLC purity of 97.25%.

[0096] Comparative Example 2

[0097] Preparation of V: Weigh 2.00 kg of compound IV and 4.77 kg of dichloromethane, add them to the reactor, start stirring, cool down, control the temperature at 0℃~10℃, add 30% hydrochloric acid dropwise, stir at 0℃~10℃ for 1 hour after the addition is complete. Monitor by TLC until the material point of compound IV (developing solvent: V 二氯甲烷 :V 甲醇 :V 氨水=100:5:0.8) disappears, the reaction is considered complete. After the reaction is complete, the mixture is allowed to stand and the aqueous phase is separated. 2.00 kg of purified water is added to the organic phase, stirred, allowed to stand, and the aqueous phases are combined. 2.66 kg of dichloromethane is added to the aqueous phase for washing, and the aqueous phase is separated and set aside.

[0098] Preparation of VI: Cool the above aqueous phase to below 10°C, and add 30% sodium hydroxide aqueous solution dropwise under temperature control; after completion of the addition, stir at 0°C-10°C for 1 h, monitor by TLC (developing solvent: methanol), and the reaction is considered complete when the V material point disappears.

[0099] Preparation of I: Warm the aqueous solution to 20°C to 30°C, add 0.96 kg of 1H-1,2,4-triazole-1-carboximidamide hydrochloride, stir, and dropwise add 30% aqueous sodium hydroxide solution to adjust the pH to 8.2-8.5. Stir and react at 20°C to 30°C for 14 hours. After completion of the reaction, cool to -5°C to 5°C, stir and crystallize for 7 hours, then filter.

[0100] Purification: 1.50 kg of peramivir (I) filter cake, 7.50 kg of purified water, and 6.00 kg of methanol were added to a reactor, stirred, and heated to reflux. After the solution was clear, it was filtered while hot. The filtrate was cooled to 20°C-25°C, stirred, and crystallized for 2 hours, then cooled to 0°C-5°C, stirred, and crystallized for 2 hours, and filtered. The filter cake was dried under vacuum at 20°C-25°C to obtain 1.01 kg of an off-white crystalline solid with a yield of 61.6% (HPLC: 99.97%, maximum single impurity 0.015%).

[0101] The methods described herein are not limited to the specific embodiments described. The above embodiments are merely illustrative of the present invention, and the present invention may also be implemented in other specific ways or in other specific forms without departing from the gist or essential characteristics of the present invention. Therefore, the embodiments described herein are to be considered in all respects as illustrative and not restrictive. The scope of the present invention is to be determined by the appended claims, and any variations that are equivalent to the intent and scope of the claims are intended to be within the scope of the present invention.

Claims

1. A method for preparing peramivir acetylate IV, characterized in that: Described method, synthetic route is as follows: The method comprises the following steps: adding 23.50 kg of ethyl acetate and 2.90 kg of compound III to a reactor, adding 0.90 kg of acetic anhydride dropwise over 2 to 3 hours; controlling the temperature at 20 to 30° C. and stirring for reaction; after the reaction is completed, adding 5.00 kg of a 10% sodium carbonate aqueous solution, stirring for 20 minutes, and standing for 20 minutes; separating the liquids, collecting the organic phase, performing reduced pressure distillation at a vacuum degree of ≤-0.08 MPa, and concentrating the organic phase until 80 to 90% of the added amount is reached; adding 23.65 kg of n-heptane, precipitating a large amount of solid, and cooling the mixture to 0 to 10 ℃ and stirred for 1h; centrifuged, rinsed with n-heptane, and dried the filter cake in vacuum at 45-50℃ to obtain 3.15kg of off-white solid intermediate IV crude product; added 3.00kg of IV crude product, 5.40kg of ethyl acetate and 4.00kg of n-heptane to the reactor, started stirring, and heated to 50-60℃ to dissolve the system; cooled and added 20.00kg of n-heptane, a large amount of solid precipitated in the system, and continued to cool to 0-10℃ and stirred for crystallization for 1h; centrifuged, rinsed with n-heptane, and dried the filter cake in vacuum at 45-50℃ to obtain off-white solid peramivir intermediate IV.

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