A preparation method of a remdesivir isomer and its analysis method

Abstract

The present invention provides a remdesivir isomer and a preparation and analysis method thereof. The preparation method can obtain a high-purity isomer compound, and the analysis method provided by the present invention can achieve effective separation of the isomer and remdesivir. The detection method is simple, rapid, accurate, highly sensitive, repeatable, and accurate, and is suitable for industrial production.

Description

Technical Field

[0001] The present invention relates to the field of medicinal chemistry, and specifically to an isomer of remdesivir and a method for preparing and analyzing the isomer. Background Art

[0002] Remdesivir is a new experimental broad-spectrum antiviral drug developed by Gilead Sciences, Inc., USA, which is used to target Ebola virus and is believed to be effective in inhibiting SARS virus and MERS virus in respiratory epithelial cells. It is a nucleotide-like prodrug that can effectively inhibit RNA-dependent RNA synthase. Its specific structure is as follows:

[0003]

[0004] At present, the research on this drug mainly focuses on its use, clinical trial scheme, preparation method, etc., and there is little research on its isomers. This article aims to study its isomers and provide a reference for its quality control standards. Summary of the invention

[0005] The present invention discloses an isomer of remdesivir, and a preparation and analysis method thereof, and its specific structure is shown in Formula I:

[0006]

[0007] Furthermore, the compound of formula I of the present invention is prepared by compound 005-A, and the reaction scheme is as follows:

[0008]

[0009] Further, the preparation method of the compound of formula I is as follows:

[0010]

[0011] The specific reaction steps are as follows:

[0012] (b-1) adding concentrated sulfuric acid dropwise to an acetone solution of (2S,3R,4S,5R)-2-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-carbonitrile and 2,2-dimethoxypropane;

[0013] (b-2) Add (3aR,4S,6R,6aR)-4-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-6-(hydroxymethyl)-2,2-dimethyltetrahydrofuran[3,4-d][1,3]dioxazole-4-carbonitrile, ((S)-(4-nitrophenoxy)(phenoxy)phosphoryl)-L-alanine-2-ethylbutyl ester and anhydrous magnesium chloride into a two-necked flask, and then add acetonitrile and diisopropylethylamine;

[0014] (b-3) ((R)-((((3aR,4R,6S,6aR)-6-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-6-cyano-2,2-dimethyltetrahydrofuran[3,4-d][1,3]dioxazol-4-yl)methoxy)(phenoxy)phosphoryl)-L-alanine-2-ethylbutyl ester is dissolved in anhydrous tetrahydrofuran, and then 37% concentrated hydrochloric acid is added dropwise to obtain a compound of formula I.

[0015] Further, the preparation method of the compound of formula I is as follows:

[0016]

[0017] Specifically include the following reaction steps:

[0018] (a-1) adding N-iodosuccinimide to a DMF solution of 4-aminopyrrolo[2,1-f][1,2,4]-triazine;

[0019] (a-2) dissolving 7-iodopyrrolo[2,1-f][1,2,4]triazine-4-amine in tetrahydrofuran, adding dropwise trimethylsilyl chloride, phenylmagnesium chloride, isopropylmagnesium chloride-lithium chloride, and then adding a tetrahydrofuran solution of (3R,4R,5R)-3,4-bis(benzyloxy)-5-((benzyloxy)methyl)dihydrofuran-2(3H)-one;

[0020] (a) dissolving (3R,4R,5R)-2-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-3,4-bis(benzyloxy)-5-((benzyloxy)methyl)tetrahydrofuran-2-ol in anhydrous dichloromethane, and adding trimethylsilyl cyanide and trimethylsilyl trifluoromethanesulfonate dropwise;

[0021] (b) dissolving (2R,3R,4R,5R)-2-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-3,4-bis(benzyloxy)-5-((benzyloxy)methyl)tetrahydrofuran-2-carbonitrile in anhydrous dichloromethane, adding boron trichloride, adding methanol dropwise, and then adding a methanol solution of triethylamine;

[0022] (b-1) adding concentrated sulfuric acid dropwise to an acetone solution of (2S,3R,4S,5R)-2-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-carbonitrile and 2,2-dimethoxypropane;

[0023] (b-2) Add (3aR,4S,6R,6aR)-4-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-6-(hydroxymethyl)-2,2-dimethyltetrahydrofuran[3,4-d][1,3]dioxazole-4-carbonitrile, ((S)-(4-nitrophenoxy)(phenoxy)phosphoryl)-L-alanine-2-ethylbutyl ester and anhydrous magnesium chloride into a two-necked flask, and then add acetonitrile and diisopropylethylamine;

[0024] (b-3) ((R)-((((3aR,4R,6S,6aR)-6-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-6-cyano-2,2-dimethyltetrahydrofuran[3,4-d][1,3]dioxazol-4-yl)methoxy)(phenoxy)phosphoryl)-L-alanine-2-ethylbutyl ester is dissolved in anhydrous tetrahydrofuran, and then 37% concentrated hydrochloric acid is added dropwise to obtain a compound of formula I.

[0025] Furthermore, the preparation method described in step (a) is as follows:

[0026] Dissolve (3R,4R,5R)-2-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-3,4-bis(benzyloxy)-5-((benzyloxy)methyl)tetrahydrofuran-2-ol in anhydrous dichloromethane, add trimethylsilyl cyanide and trimethylsilyl trifluoromethanesulfonate dropwise; after the reaction is completed, quench with aqueous sodium bicarbonate solution, separate the organic phase, extract the aqueous phase with dichloromethane, separate through a silica gel column, and elute with a gradient of 0-5% methanol and dichloromethane.

[0027] Further, the gradient elution conditions of 0-5% methanol and dichloromethane in step (a) are as follows:

[0028] Time (min) Gradient (methanol:dichloromethane) 0-10 0:100 10-50 0:100-5:100

[0029] Furthermore, the preparation method of step (b) is as follows: (2R, 3R, 4R, 5R)-2-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-3,4-bis(benzyloxy)-5-((benzyloxy)methyl)tetrahydrofuran-2-carbonitrile is dissolved in anhydrous dichloromethane, boron trichloride is added, methanol is added dropwise, and then a methanol solution of triethylamine is added; the reaction mixture is concentrated, washed with dichloromethane, the aqueous phase is retained, and separation is performed by preparation.

[0030] Further, the conditions for the preparation and separation in step (b) are as follows:

[0031] C18 reverse phase gradient elution, mobile phase: phase A: 0.1% formic acid aqueous solution; phase B: acetonitrile; detection wavelength: 254nm; temperature: room temperature;

[0032] Further, the conditions for the preparation and separation in step (b) are as follows:

[0033] C18 reverse phase gradient elution, mobile phase: phase A: 0.1% formic acid aqueous solution; phase B: acetonitrile; detection wavelength: 254nm; temperature: room temperature;

[0034] The specific gradients are as follows:

[0035] Time (min) Gradient (B%) 0 2 10 18 10.1 95 12 95 12.1 2 14 2 .

[0036] Further, the present invention provides an intermediate OO5-A for preparing a compound of formula I,

[0037]

[0038] Furthermore, the compound 005-A is prepared by the following steps, and the specific reaction route is as follows:

[0039]

[0040] Dissolve (2R,3R,4R,5R)-2-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-3,4-bis(benzyloxy)-5-((benzyloxy)methyl)tetrahydrofuran-2-carbonitrile in anhydrous dichloromethane, add boron trichloride, add methanol dropwise, and then add a methanol solution of triethylamine; concentrate the reaction mixture, wash with dichloromethane, retain the aqueous phase, and separate by preparation.

[0041] Further, the conditions for the preparation and separation are as follows:

[0042] C18 reverse phase gradient elution, mobile phase: phase A: 0.1% formic acid aqueous solution; phase B: acetonitrile; detection wavelength: 254nm; temperature: room temperature;

[0043] Further, the conditions for the preparation and separation are as follows:

[0044] C18 reverse phase gradient elution, mobile phase: phase A: 0.1% formic acid aqueous solution; phase B: acetonitrile; detection wavelength: 254nm; temperature: room temperature;

[0045] The specific gradients are as follows:

[0046] Time (min) Gradient (B%) 0 2 10 18 10.1 95 12 95 12.1 2 14 2 .

[0047] Further, the preparation method of 005-A is as follows:

[0048]

[0049] The specific steps include:

[0050] (a) dissolving (3R,4R,5R)-2-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-3,4-bis(benzyloxy)-5-((benzyloxy)methyl)tetrahydrofuran-2-ol in anhydrous dichloromethane, and adding trimethylsilyl cyanide and trimethylsilyl trifluoromethanesulfonate dropwise;

[0051] (b) (2R,3R,4R,5R)-2-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-3,4-bis(benzyloxy)-5-((benzyloxy)methyl)tetrahydrofuran-2-carbonitrile was dissolved in anhydrous dichloromethane, boron trichloride was added, methanol was added dropwise, and then a methanol solution of triethylamine was added.

[0052] Furthermore, the preparation method described in step (a) is as follows:

[0053] Dissolve (3R,4R,5R)-2-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-3,4-bis(benzyloxy)-5-((benzyloxy)methyl)tetrahydrofuran-2-ol in anhydrous dichloromethane, add trimethylsilyl cyanide and trimethylsilyl trifluoromethanesulfonate dropwise; after the reaction is completed, quench with aqueous sodium bicarbonate solution, separate the organic phase, extract the aqueous phase with dichloromethane, separate through a silica gel column, and elute with a gradient of 0-5% methanol and dichloromethane.

[0054] Further, the gradient elution conditions of 0-5% methanol and dichloromethane in step (a) are as follows:

[0055] Time (min) Gradient (methanol:dichloromethane) 0-10 0:100 10-50 0:100-5:100 .

[0056] Further, the preparation method of 005-A is as follows:

[0057] (a) (3R,4R,5R)-2-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-3,4-bis(benzyloxy)-5-((benzyloxy)methyl)tetrahydrofuran-2-ol was dissolved in anhydrous dichloromethane, and trimethylsilyl cyanide and trimethylsilyl trifluoromethanesulfonate were added dropwise; after the reaction was completed, the reaction was quenched with aqueous sodium bicarbonate, the organic phase was separated, the aqueous phase was extracted with dichloromethane, the phase was separated by silica gel column, and the phase was eluted with a gradient of 0-5% methanol and dichloromethane, and the gradient conditions were as follows:

[0058] Time (min) Gradient (methanol:dichloromethane) 0-10 0:100 10-50 0:100-5:100 ;

[0059] (b) Dissolve (2R,3R,4R,5R)-2-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-3,4-bis(benzyloxy)-5-((benzyloxy)methyl)tetrahydrofuran-2-carbonitrile in anhydrous dichloromethane, add boron trichloride, dropwise add methanol, and then add a methanol solution of triethylamine; concentrate the reaction mixture, wash with dichloromethane, retain the aqueous phase, and perform preparative separation using C18 reverse phase gradient elution, mobile phase: phase A: 0.1% formic acid aqueous solution; phase B: acetonitrile; detection wavelength: 254 nm; temperature: room temperature; elution gradient is as follows:

[0060]

[0061]

[0062] Through the above preparation method, the intermediate 005-a with high purity can be quickly prepared, and the compound of formula I with high purity can be prepared through the above intermediate.

[0063] Furthermore, the present invention also provides a method for analyzing remdesivir isomers, and the analysis method is as follows:

[0064] Normal phase separation was performed by high performance liquid chromatography, mobile phase: n-hexane-isopropanol-triethylamine; flow rate: 1.0 ml / min; column temperature: 25° C.; detection wavelength: 245 nm; solvent: ethanol.

[0065] Furthermore, the volume ratio of the mobile phase, n-hexane-isopropanol-triethylamine is 500:500:1.

[0066] Furthermore, the chromatographic column of the analytical method is a chiral column with cellulose-tris(3,5-dichlorophenylcarbamate) covalently bonded to the surface of silica gel as a filler, preferably a CHIRALPAK IC chromatographic column, and its specific size is 4.6 mm×250 mm, 5 μm.

[0067] The above analytical method has good separation and can effectively separate the compound of formula I and remdesivir.

[0068] Furthermore, the present invention also provides the use of the compound of formula I as a remdesivir impurity reference substance.

[0069] Certain abbreviations and acronyms are used to describe the test details, although most of them will be understood by those skilled in the art and their definitions are given in the table below.

[0070] Table 1 Meaning of abbreviations and acronyms

[0071] BRIEF DESCRIPTION OF THE DRAWINGS

[0072] Figure 1 Example 4-1 Compound prepared 1 H NMR spectra;

[0073] Figure 2 Example 7 Preparation of the compound 1 H NMR spectra;

[0074] Figure 3 Mass spectrum of the compound prepared in Example 7;

[0075] Figure 4 Example 12 System Compatibility Solution Analysis Spectrum;

[0076] Figure 5 Example 12 Analysis of the test solution. Specific embodiments

[0077] The specific implementation of the concentration analysis method of the present invention is further described in detail below in conjunction with the examples. These implementations are only used to illustrate the present invention, but not to limit the present invention.

[0078] The HPLC analysis method of the compound is as follows:

[0079] High performance liquid chromatography (Chinese Pharmacopoeia 2015 edition, Part IV, General Rules 0512);

[0080] Chromatographic conditions: Mobile phase: Phase A: 0.1% formic acid aqueous solution; Phase B: acetonitrile

[0081] Chromatographic column: Kromasil, 100-5-C18, 4.6×150mm

[0082] gradient:

[0083] Time (min) Gradient (B%) 0 5 2 5 12 95 14 95 14.1 5 16 5

[0084] Detection wavelength: 254nm

[0085] Temperature: Room temperature.

[0086] Example 1 Preparation of 7-iodopyrrolo[2,1-f][1,2,4]triazine-4-amine

[0087] N-iodosuccinimide (33.72 g) was added to a cold solution of 4-aminopyrrolo[2,1-f][1,2,4]-triazine (19.5 g, 0.145 mol) in DMF (137 mL) in 5 batches while maintaining the internal temperature at about 0°C. After the reaction was completed (about 3 hours at 0°C, monitored by a spot plate), the reaction mixture was transferred to a 1M aqueous sodium hydroxide solution (522 mL), and the resulting slurry was stirred at about room temperature for 1.5 hours, then filtered and rinsed with water (100 mL). The solid was dried under vacuum at 50°C to obtain 30 g of a brown solid.

[0088] Example 2 Preparation of (3R, 4R, 5R)-2-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-3,4-bis(benzyloxy)-5-((benzyloxy)methyl)tetrahydrofuran-2-ol

[0089] Dissolve 7-iodopyrrolo[2,1-f][1,2,4]triazine-4-amine (32.2 g, 76.9 mmol) in tetrahydrofuran (480 mL), add trimethylsilyl chloride (16.87 g, 153.8 mmol, 2 eq.) dropwise under nitrogen protection, stir at room temperature for 10 minutes, and place in an ice-water bath. When cooled to about 0°C, slowly add phenylmagnesium chloride (2M, 77 mL, 2 eq.). Continue stirring for 20 minutes after the addition is complete, then slowly add isopropylmagnesium chloride-lithium chloride (1.3 M, 62 mL, 1.05 eq.), and keep the internal temperature below 5°C during the addition. After the addition was complete, the reaction mixture was cooled to about -20°C, and a solution of (3R,4R,5R)-3,4-bis(benzyloxy)-5-((benzyloxy)methyl)dihydrofuran-2(3H)-one (20 g, 76.9 mmol, 1 eq.) in tetrahydrofuran (97 ml) was added, while maintaining the internal reaction temperature at about -20°C. After the addition was complete, the reaction was continued for 1 hour, then the temperature was raised to 0°C, and then quenched with methanol (64 mL), followed by the addition of acetic acid (64 mL) and water (64 mL), the resulting mixture was raised to room temperature, and then concentrated under reduced pressure. The resulting concentrate was partitioned between ethyl acetate (800 mL) and aqueous hydrochloric acid (1 M, 800 mL). The organic layer was separated, washed with saturated aqueous sodium bicarbonate (800 mL) and saturated brine (800 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Separation by silica gel column eluting with a gradient of 0-5% methanol and dichloromethane gave 17.6 g of a light yellow solid.

[0090] Example 3 Preparation of (3R, 4R, 5R)-2-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-3,4-bis(benzyloxy)-5-((benzyloxy)methyl)tetrahydrofuran-2-carbonitrile

[0091] Example 3-1

[0092] Dissolve (3R,4R,5R)-2-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-3,4-bis(benzyloxy)-5-((benzyloxy)methyl)tetrahydrofuran-2-ol (8.0 g, 14.48 mmol) in anhydrous dichloromethane (320 mL) and stir in an ice-water bath under nitrogen protection. When the internal temperature drops to about 2°C, add trimethylsilyl cyanide (7.18 g, 72.4 mmol, 5 eq.) dropwise. Stir for 10 minutes after the addition is complete, then add trimethylsilyl trifluoromethanesulfonate (16.14 g, 72.4 mmol, 5 eq.) dropwise, and control the internal temperature below 5°C. Continue to react in an ice-water bath for 2 hours. After the reaction is completed, add 300 mL of saturated sodium bicarbonate aqueous solution to quench. After the reaction mixture was stirred for 10 minutes, the organic phase was separated, and the aqueous phase was extracted three times with dichloromethane (100 mL x 3). The organic phase was collected and dried over anhydrous sodium sulfate, and then concentrated under reduced pressure. The mixture was separated by silica gel column and eluted with a gradient of 0-5% methanol and dichloromethane. The specific gradient elution conditions were as follows:

[0093] Time (min) Gradient (methanol:dichloromethane) 0-10 0:100 10-50 0:100-5:100

[0094] Finally, 6 g of light yellow solid was obtained.

[0095] Example 4 Preparation of (2S, 3R, 4S, 5R)-2-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-carbonitrile

[0096] Example 4-1

[0097] (2R,3R,4R,5R)-2-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-3,4-bis(benzyloxy)-5-((benzyloxy)methyl)tetrahydrofuran-2-carbonitrile (6.0 g, 10.7 mmol) was dissolved in anhydrous dichloromethane (60 mL) and cooled to -78°C under nitrogen. Boron trichloride (1M, 40.6 mL, 3.8 eq.) was then added, the reaction mixture was warmed to -40°C and stirred for 2 hours. The reaction mixture was then cooled to -78°C again and methanol (12 mL) was added dropwise. A solution of triethylamine (16 mL) in methanol (24 mL) was added dropwise and the reaction mixture was finally warmed to room temperature. The mixture was concentrated and the remaining solid residue was dissolved in methanol (60 mL) and heated to 45°C. Water (60 mL) was added, and the resulting mixture was concentrated under reduced pressure at 45°C to remove volatiles, then washed three times with dichloromethane (30 mL x 3), the aqueous phase was retained, and the specific conditions for preparing the liquid phase were as follows:

[0098] Chromatographic conditions: Mobile phase: Phase A: 0.1% formic acid aqueous solution; Phase B: acetonitrile

[0099] Chromatographic column: Kromasil, 100-5-C18, 30×150mm

[0100] gradient:

[0101] Time (min) Gradient (B%) 0 2 10 18 10.1 95 12 95 12.1 2 14 2

[0102] Detection wavelength: 254nm

[0103] Temperature: Room temperature.

[0104] Finally, 362 mg of white powdery solid was obtained.

[0105] 1 H NMR(400MHz, Deuterium Oxide)δ7.89(s,1H),7.00(d,J=4.7Hz,1H),6.92(d,J=4.7Hz,1H),5.06(d,J=4.4Hz,1H),4.70(dd,J=9.0,4 .4Hz, 1H), 4.21 (ddd, J=9.0, 4.4, 2.5Hz, 1H), 4.01 (dd, J=13.0, 2.6Hz, 1H), 3.83 (dd, J=13.0, 4.5Hz, 1H) (see attachment for details Figure 1 ). 13 C NMR (101MHz, Deuterium Oxide) δ153.16,143.54,123.99,117.29,114.65,111.95,104.66,83.01,77.63,75.96,70.56,60.22.

[0106] Example 4-2 Effect of different preparation liquid phase conditions on compound 005

[0107] The separation time required for the separation products of different mobile phases and the purity of the final products were compared. The results are shown in the following table:

[0108] Mobile phase Elution time Product Purity Mobile phase: Phase A: 0.1% formic acid aqueous solution; Phase B: acetonitrile 15 minutes 98.2％ <![CDATA[Mobile phase: Phase A: 0.1% aqueous TFA solution; Phase B: acetonitrile 1 > 40 minutes 97.5％

[0109] 1 The method shown is for preparing and separating with reference to paragraph

[0847] of the specification of CN108348526

[0110] Example 5 (3aR, 4S, 6R, 6aR)-4-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-6-(hydroxymethyl)-2,2-dimethyltetrahydrofuran[3,4-d][1,3]dioxazole-4-carbonitrile

[0111] Concentrated sulfuric acid was added dropwise to a solution of (2S,3R,4S,5R)-2-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-carbonitrile (400 mg, 1.37 mmol) and 2,2-dimethoxypropane (687 mg, 6.59 mmol, 4.8 eq.) in acetone (10 mL) at room temperature. The reaction mixture was stirred at room temperature for 30 minutes, heated to 45°C, and the reaction was continued for 30 minutes. Then it was cooled to room temperature, and solid sodium bicarbonate (0.5 g) and water (0.5 mL) were added in sequence. After the mixture was stirred for 15 minutes, it was concentrated under reduced pressure. The residue was dissolved in ethyl acetate (30 mL) and water (15 mL). The organic layer was separated, and the aqueous layer was extracted with ethyl acetate (10 mL×2). The combined organic phases were dried over anhydrous sodium sulfate, and then concentrated under reduced pressure to obtain a crude product.

[0112] Example 6 ((R)-((((3aR,4R,6S,6aR)-6-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-6-cyano-2,2-dimethyltetrahydrofuran[3,4-d][1,3]dioxazol-4-yl)methoxy)(phenoxy)phosphoryl)-L-alanine-2-ethylbutyl ester

[0113] (3aR, 4S, 6R, 6aR)-4-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-6-(hydroxymethyl)-2,2-dimethyltetrahydrofuran[3,4-d][1,3]dioxazole-4-carbonitrile (390 mg, 1.18 mmol), ((S)-(4-nitrophenoxy)(phenoxy)phosphoryl)-L-alanine-2-ethylbutyl ester (640 mg, 1.42 mmol, 1.2 eq.) and anhydrous magnesium chloride (112 mg, 1.18 mmol, 1 eq.) were added to a two-necked flask, and acetonitrile (6 mL) was added under nitrogen protection. After stirring at 50°C for 10 minutes, diisopropylethylamine (0.52 mL, 2.95 mmol, 2.5 eq.) was added and the reaction was continued for 20 minutes. After the reactant was cooled to room temperature, 35 mL of ethyl acetate was added, and the mixture was washed with 5% citric acid (15 mL), saturated aqueous ammonium chloride solution (15 mL), 5% aqueous sodium carbonate solution (15 mL×2) and saturated brine. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The mixture was separated by silica gel column and eluted with a gradient of 0-5% methanol and dichloromethane to obtain 510 mg of a transparent oil.

[0114] Example 7 ((R)-((((2R,3S,4R,5S)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazine-7-yl)-5-cyano-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphoryl)-L-alanine-2-ethylbutyl ester (Compound of Formula I)

[0115] ((R)-((((3aR,4R,6S,6aR)-6-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-6-cyano-2,2-dimethyltetrahydrofuran[3,4-d][1,3]dioxazol-4-yl)methoxy)(phenoxy)phosphoryl)-L-alanine-2-ethylbutyl ester (490 mg, 0.76 mmol) was dissolved in anhydrous tetrahydrofuran (3.8 mL). Stir in an ice-water bath for 10 minutes, then add concentrated hydrochloric acid (0.76 mL) dropwise. Stir the reaction at room temperature for 5 hours. Then add 3.8 mL of water and adjust the pH to 8 with a saturated sodium carbonate aqueous solution. The mixture is extracted with ethyl acetate (5 mL × 3), the organic phase is dried over anhydrous sodium sulfate and concentrated, and separated by a silica gel column, eluted with a gradient of 0-10% methanol and dichloromethane to obtain 250 mg of a white powdery solid with a purity of 93.33%.

[0116] 1 H NMR (400MHz, DMSO-d6) δ7.91(s,3H),7.37(t,J=7.9Hz,2H),7.25(d,J=8.4Hz,2H),7.18(t,J=7.3Hz,1H ),6.91–6.87(m,1H),6.64(d,J=4.4Hz,1H),6.04(dd,J=13.0,10.1Hz,1H),4.77(d,J=4.1Hz,1H),4.36 (dt, J = 11.7, 5.8 Hz, 2H), 4.22–4.16 (m, 1H), 4.11 (dt, J = 12.4, 6.3 Hz, 1H), 4.00 (dd, J = 10.9, 5.9 Hz, 1H), 3.93 (dt, J = 10.0, 6.8 Hz, 2H), 1.51–1.41 (m, 1H), 1.38–1.21 (m, 10H), 0.82 (td, J = 7.4, 2.4 Hz, 6H) (see Appendix for details) Figure 2 ). LCMS: [M+H]+: 603.3 (see attached Figure 3 ). 13C NMR(101MHz,DMSO-d6)δ173.71,173.65,155.91,151.29,151.23,148.31,139.67,130.04,125.38,124.98,122.95,120.73,120.69,118.42,11 6.02,110.28,101.22,81.22,81.15,78.21,75.71,71.86,66.63,66.43 ,50.25,34.86,30.89,23.06,23.02,21.51,20.36,20.30,11.27,11.22.

[0117] Example 8 Remdesivir isomer analysis method

[0118] High performance liquid chromatography (Chinese Pharmacopoeia 2015 edition, Part IV, General Rules 0512):

[0119] System suitability solution preparation: Take appropriate amounts of remdesivir and enantiomers, add ethanol to dissolve by ultrasound and dilute to make a mixed solution containing approximately 0.5 mg of remdesivir and 2.5 μg of enantiomers per 1 ml;

[0120] Chromatographic column: CHIRALPAK OD-H (4.6mm×250mm, 5μm)

[0121] Mobile phase: n-hexane-isopropanol (80:20)

[0122] Flow rate: 1.0ml / min

[0123] Column temperature: 25°C

[0124] Detection wavelength: 245nm

[0125] Injection volume: 20 μl

[0126] The system suitability solution was injected into the liquid chromatography, and the result was: the retention time of the isomers overlapped with that of remdesivir and could not be separated.

[0127] Example 9 Remdesivir isomer analysis method

[0128] High performance liquid chromatography (Chinese Pharmacopoeia 2015 edition, Part IV, General Rules 0512):

[0129] System suitability solution preparation: Take appropriate amounts of remdesivir and enantiomers, add ethanol to dissolve by ultrasound and dilute to make a mixed solution containing approximately 0.5 mg of remdesivir and 2.5 μg of enantiomers per 1 ml;

[0130] Chromatographic column: CHIRALPAK IA (4.6 mm × 250 mm, 5 μm)

[0131] Mobile phase: n-hexane-isopropanol (60:40)

[0132] Flow rate: 1.0ml / min

[0133] Column temperature: 25°C

[0134] Detection wavelength: 245nm

[0135] Injection volume: 20 μl

[0136] The system suitability solution was injected into the liquid chromatography, and the results showed that the remdesivir peak had severe tailing and the isomers were on its tailing peak.

[0137] Example 10 Remdesivir isomer analysis method

[0138] High performance liquid chromatography (Chinese Pharmacopoeia 2015 edition, Part IV, General Rules 0512):

[0139] System suitability solution preparation: Take appropriate amounts of remdesivir and enantiomers, add ethanol to dissolve by ultrasound and dilute to make a mixed solution containing approximately 0.5 mg of remdesivir and 2.5 μg of enantiomers per 1 ml;

[0140] Chromatographic column: CHIRALPAK IC (4.6mm×250mm, 5μm)

[0141] Mobile phase: n-hexane-isopropanol-trifluoroacetic acid (500:500:1)

[0142] Flow rate: 1.0ml / min

[0143] Column temperature: 25°C

[0144] Detection wavelength: 245nm

[0145] Injection volume: 20 μl

[0146] The system suitability solution was injected into the liquid chromatography, and the results showed that the peak diffusion of remdesivir was severe and the sensitivity was low.

[0147] Example 11 Remdesivir isomer analysis method

[0148] High performance liquid chromatography (Chinese Pharmacopoeia 2015 edition, Part IV, General Rules 0512):

[0149] System suitability solution preparation: Take appropriate amounts of remdesivir and enantiomers, add ethanol to dissolve by ultrasound and dilute to make a mixed solution containing approximately 0.5 mg of remdesivir and 2.5 μg of enantiomers per 1 ml;

[0150] Mobile phase: n-hexane-isopropanol-triethylamine (500:500:1)

[0151] Flow rate: 1.0ml / min

[0152] Column temperature: 25°C

[0153] Detection wavelength: 245nm

[0154] Injection volume: 20 μl

[0155] The system suitability solution was injected into the liquid chromatography. The result was: separation was possible, but the peak symmetry was slightly poor.

[0156] Example 12 Remdesivir isomer analysis method

[0157] High performance liquid chromatography (Chinese Pharmacopoeia 2015 edition, Part IV, General Rules 0512):

[0158] System suitability solution preparation: Take appropriate amounts of remdesivir and enantiomers, add ethanol to dissolve by ultrasound and dilute to make a mixed solution containing approximately 0.5 mg of remdesivir and 2.5 μg of enantiomers per 1 ml;

[0159] Test solution: Take an appropriate amount of the product, add ethanol, ultrasonically dissolve and dilute to make a solution containing about 0.5 mg per 1 ml.

[0160] Mobile phase: n-hexane-isopropanol-triethylamine (500:500:2)

[0161] Flow rate: 1.0ml / min

[0162] Column temperature: 25°C

[0163] Detection wavelength: 245nm

[0164] Injection volume: 20 μl

[0165] The system adaptability solution and the test solution were injected into the chromatograph. The results showed that the separation was good and the peak shape was good. The system adaptability solution spectrum is shown in the attached Figure 4 The retention time of the compound of formula I is 5.084, and the retention time of remdesivir is 8.485. The test solution is shown in the attached Figure 5 .

Claims

1. A method for analyzing remdesivir isomers, the specific analysis method is as follows: normal phase separation is performed by high performance liquid chromatography, the mobile phase is n-hexane-isopropanol-triethylamine, the volume ratio of the three is 500:500:1 or 500:500:2; flow rate: 1.0 ml / min; column temperature: 25°C; detection wavelength: 245 nm; solvent: ethanol, the chromatographic column is a chiral column with cellulose-tris (3,5-dichlorophenylcarbamate) covalently bonded to the silica gel surface as a filler, wherein the remdesivir isomer has the following structure of Formula I:

2. A method for analyzing remdesivir isomers according to claim 1, wherein the chromatographic column of the analysis method is a CHIRALPAK IC chromatographic column, and its specific size is 4.6 mm×250 mm, 5 μm.

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