A process for the preparation of monabiravir

Abstract

The application provides a preparation process of an anti-virus drug monabiravir for treating COVID-19, which comprises adding a base, reacting 2'-3'-isopropylidene cytidine with isobutyric anhydride to obtain 5'-isobutyrate containing amide impurities; then treating the reaction mixture with p-toluenesulfonic acid monohydrate to deprotect the acetonide and obtain pure isobutyryl cytidine p-toluenesulfonic acid salt containing no amide impurities; converting the p-toluenesulfonic acid salt into a free base, and then reacting with hydroxylamine to obtain monabiravir.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical synthesis technology, specifically relating to an improved preparation process for monobirapvir (MK-4482, formerly known as EIDD-2801). Monobirapvir is an orally administered active antiviral agent, and its application in treating COVID-19 infection is currently under investigation. Background Technology

[0002] Monabiravir's chemical name is methyl [(2R,3S,4R,5R)-3,4-dihydroxy-5-[4-(hydroxyamino)-2-oxopyrimidin-1-yl]oxo-2-yl]methyl-2-methylpropionate, with the structure shown in Formula I:

[0003]

[0004] Monaviravir was discovered by Emory University, which is currently collaborating with Ridgeback Biotherapeutics and Merck & Co. on clinical development.

[0005] The first synthetic route for monobiraperivir reportedly used uridine as a starting material, which is expensive and in limited supply [WO2019113462; WO2019173602]. Furthermore, this route yielded low yields. Cytidine is less expensive than uridine, and it can be directly transaminated with hydroxylamine. Therefore, new routes based on cytidine (Schemes 1 and 2) have been reported.

[0006]

[0007] Scheme 1 discloses the selective esterification of the primary alcohol of cytidine with isobutyrylacetone oxime using Novozyme 435 (immobilized Candida antarcticis lipase B). The cytidine ester is then reacted with hydroxylamine sulfate to yield monobirapvir (Chem. Commun., 2020, Vol. 56, 13363-13364). This scheme is very costly due to the use of an immobilized enzyme. Furthermore, despite the enzyme, a certain amount of diester and triester impurities are generated in the first stage, and partial deamination is observed in the second stage. Both stages require purification by column chromatography.

[0008] Option 2 uses a chemical method to selectively esterify the primary alcohol of cytidine to replace the enzyme system.

[0009]

[0010] Cytidine reacts with acetone, sulfuric acid, and 2,2-dimethoxypropane to give 2',3'-O-isopropylidene cytidine sulfate. 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) and a catalytic amount of 4-dimethylaminopyridine (DMAP) are added, followed by reaction with isobutyric anhydride in acetonitrile. The resulting cytidine acetone ester is reacted with hydroxylamine sulfate in 70% IPA (KF titration of 24% water), followed by deprotection of the acetone ester with formic acid to give monobiravir. The final product is purified by column chromatography in 64% yield. Alternatively, hydroxylation and deprotection of the acetone ester can be achieved in one step by treating with hydroxylamine sulfate in 40% IPA (60% water) for a longer period, directly converting cytidine acetone ester to monobiravir. However, this results in the hydrolysis of a large amount of esters, forming approximately 20% N-hydroxycytidine byproducts (Synlett., 2021, Vol. 32(3), pp. 326-328).

[0011] Another major drawback of Scheme 2 is that we observed carbon monoxide being released during the deprotection of acetal using formic acid. Some literature on the deprotection of acetal using formic acid (WO2019113462; WO2019173602; WO2016 / 106050A1) also explicitly states that formic acid is a source of carbon monoxide, and that storing it for an extended period will release carbon monoxide (the container needs frequent ventilation).

[0012] Therefore, both of these cytidine-based approaches have certain drawbacks, and a safer alternative process is needed. Summary of the Invention

[0013] In studying the esterification of 2',3'-O-isopropylcytidine sulfate (II) with butyric anhydride by adding alkali, this invention found that in addition to 5'-isobutyrylcytidine acetal (Formula III), approximately 8% of amide byproducts (impurity-1) were also formed due to cross-reaction of the amino groups of cytidine (Scheme 3).

[0014] Without using column chromatography, this invention investigated the salt formation of various acids and obtained a purification method. When using p-toluenesulfonic acid (PTSA) as a monohydrate for salt formation, in addition to obtaining a pure salt free of amide impurities, deprotection of the acetal group was observed, and 5'-isobutyrylcytidine p-toluenesulfonate (Formula IV) was directly obtained (Scheme 3).

[0015] The fact that azeotropic PTSA can form toluenesulfonate but cannot hydrolyze acetone groups further demonstrates the importance of water of crystallization.

[0016] Equally surprising is that the water of crystallization present in the acid only hydrolyzes the acetone group, without affecting the isobutylene ester group present in the molecule.

[0017] Then, p-toluenesulfonate (Formula IV) is converted into the free base of 5-isobutyrylcytidine (Formula V), and then reacted with hydroxylamine to obtain monobirapvir (Formula I).

[0018]

[0019] The beneficial effects of this invention are:

[0020] The main advantage of this process is that it avoids the use of formic acid as a carbon monoxide source for acetone deprotection and can achieve purification without the use of column chromatography. Detailed Implementation

[0021] This invention provides a novel process for preparing monobirapir, comprising the following steps:

[0022] a. Add a non-nucleophilic base to react 2',3'-O-isopropylcytidine sulfate with the structure of formula II with isobutyric anhydride in the solvent at room temperature to obtain 5-isobutyrate with the structure of formula III and N-isobutyric acid amide as impurity-1.

[0023]

[0024] b. The reaction product obtained in step a is reacted with p-toluenesulfonic acid monohydrate to obtain 5'-isobutyrylcytidine p-toluenesulfonate (Formula IV), which does not contain N-isobutyric acid amide impurities.

[0025]

[0026] c. Convert 5'-isobutyryl cytidine salt (Formula IV) into its free base (Formula V), and then react the free base with hydroxylamine to obtain monobiravir (Formula I).

[0027] The required starting material, namely 2',3'-O-isopropylcytidine sulfate of formula (II), can be prepared by the method described in Synlett (2021), 32(3), 326-328.

[0028] Isobutyrate (Formula III) is obtained by esterification of 2',3'-O-isopropylcytidine sulfate (Formula II) with isobutyric anhydride with the addition of a non-nucleophilic base. This reaction can be carried out in acetonitrile at room temperature. Suitable non-nucleophilic bases include triethylamine, diisopropylethylamine (DIPEA), 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), N-methylmorpholine (NMM), and 1,4-diazabicyclo-[2.2.2]octane (DABCO). The addition of a catalytic amount of 4-dimethylaminopyridine promotes the reaction. The reaction takes approximately 18-24 hours to complete, after which the mixture is concentrated, and the residue is stirred in a mixture of water and dichloromethane for approximately 5 minutes. After separation, the organic layer is dried and concentrated to obtain a foamy solid containing approximately 90% yield (Formula III). The foamy solid also contains approximately 8% impurity-1 (an amide derivative) and approximately 0.5% unknown impurity-2, as determined by HPLC analysis.

[0029] Treatment of the above-mentioned foamy solid with p-toluenesulfonic acid monohydrate yields 5'-isobutyrylcytidine toluenesulfonate and acetone hydrolysate. This reaction can be carried out in ethyl acetate or acetonitrile. At room temperature, only toluenesulfonate is formed. However, after reacting at 75-80°C for approximately 24 hours, acetone partially hydrolyzes to form (Formula IV) with an HPLC purity of 98%. It is completely free of impurity-1, and the content of impurity-2 is reduced to approximately less than 0.05%. The acetone hydrolysis is caused by the water of crystallization in the p-toluenesulfonic acid monohydrate, which is approximately 12%. PTSA azeotropically reacted with xylene does not induce acetone hydrolysis. Drying the PTSA monohydrate in a desiccator does not reduce the water content. When acetonitrile is used as a solvent, approximately 5% ester hydrolysis is observed. The p-toluenesulfonate (Formula IV) reacts with a base to give 5'-isobutyrylcytidine (Formula V). Although various organic bases can be used, dicyclohexylamine (DCHA) yields the best results. Formula IV was treated with acetone at room temperature with 1-1.5 equivalents of DCHA for approximately 1 hour to produce a precipitate of PTSA-DCHA salt, which was then removed by filtration. The filtrate was concentrated, and the residue was treated with water and dichloromethane to remove residual PTSA-DCHA salt and remaining free DCHA.

[0030] Since 1965, it has been known that N-hydroxycytidine can be obtained by reacting cytidine derivatives with hydroxylamine (Biochemical & Biophysical Research Communications, No. 18(4), 1965, 617-622).

[0031] The free base of cytidine butyrate (V) can be converted to monobiravir by reacting with hydroxylamine. This reaction can be carried out using commercially available hydroxylamine salts, such as hydrochloric acid or sulfate. The reaction requires 3–5 molar equivalents of hydroxylamine and heating at 70–80°C for 15–20 hours.

[0032] The following describes further embodiments of the invention, which do not limit the scope of the invention in any way.

[0033] Example 1: Preparation of 5-isobutyrylcytidine p-toluenesulfonate (IV)

[0034] Cytidine (II) sulfated in acetone (22 g, 57.69 mmol), 4-dimethylaminopyridine (1.41 g, 0.2 equivalents), and 1,4-diazabicyclo[5.4.0]undec-7-ene (27.23 g, 3.1 equivalents) were dissolved in acetonitrile (220 mL), and isobutyric anhydride (14.6 g, 1.6 equivalents) was added over 15 minutes. The solution was stirred for 20 hours. The reaction mixture was concentrated, and the residue was treated with water (100 mL) and dichloromethane (100 mL). After stirring for 5 minutes, the mixture was separated into two layers. The organic layer was dried over anhydrous sodium sulfate and concentrated to give 20.9 g of foamy solid containing 89.7% III, 8.54% impurity-1, and 0.54% unknown impurity-2.

[0035] The solid was dissolved in ethyl acetate (100 mL), and a solution of p-toluenesulfonic acid monohydrate (10.98 g, 1.0 equivalent) and ethyl acetate (100 mL) was added. The reaction mixture was heated to 75-80 °C and stirred for 24 hours. After cooling to room temperature, the precipitated solid was filtered, washed with ethyl acetate (100 mL), and dried under vacuum for 4 hours to give 21.5 g (yield 76.7%) of IV. HPLC purity: 98.53%, p-toluenesulfonate of III: 0.24%, impurity-1: none, impurity-2: 0.03%.

[0036] FT-IR (KBr, cm -1 ): 3481,3415,3282,3137,2923,1724,1690,1542,1497,1453,1420,1397,1331,1280,1243,1198,1172,1138,1126,1099,1035,1010,919,875,830,814,765,683,622,585,567,527. 1H-NMR (300MHz, DMSO-d6): δ9.50(s,1H),8.44(s,1H),7.95-7.93(d,1H),7.50-7.46(d,2H,Ar-H),7.13-7.10(d,2H,Ar-H),6.12-6.09( d,1H),5.71-5.69(d,1H),4.33-4.20(m,2H),4.12-4.04(m,2H),3.93-3.89(m,1H),2.64-2.54(m,1H),2.29(s,3H),1.11-1.05(d,6H). 13 C-NMR (75MHz, DMSO-d6): δ176.40,159.64,147.65,145.05,144.87,138.82,128.76,125.93, 94.65,90.99,81.64,73.73,69.71,63.90,33.60,21.25,19.24,19.19.ESI-MS:314.18[M+H] + (Free base MW: 313.31).

[0037] Example 2: Two-step preparation of 5-isobutyrylcytidine p-toluenesulfonate (IV)

[0038] Cytidine (II) sulfated acetone (30.3 g), 4-dimethylaminopyridine (1.94 g, 0.2 equivalent), and 1,4-diazabicyclo[5.4.0]undec-7-ene (37.5 g, 3.1 equivalent) were dissolved in acetonitrile (300 mL), and isobutyric anhydride (20.1 g, 1.6 equivalent) was added dropwise. The reaction was carried out as described in Example-1 to obtain 29.1 g of foamy solid. HPLC analysis showed that it contained 88.17% (III), 7.31% impurity-1, and 0.43% unknown impurity-2.

[0039] The above solid was dissolved in ethyl acetate (150 mL), and a solution of p-toluenesulfonic acid monohydrate (17.23 g, 1.1 equivalents) and ethyl acetate (100 mL) was added. The reaction mixture was stirred at 25–30 °C for one hour, the precipitated solid was filtered, washed with ethyl acetate (100 mL), and dried under vacuum for 4 hours to give 32.8 g (yield 78.54%) of p-toluenesulfonate III. HPLC purity: 97.4%, IV: 1.98%, impurity-1: none, MR: 160–169 °C.

[0040] FT-IR (KBr, cm -1): 3256,3059,2987,2941,2786,1731,1702,1660,1543,1496,1458,1409,1388,1373,1352,1326,1265,1252,1233,1205,1191,1166,1154,1124,1111,1092,1072,1034,1009,970,899,868,817,755,681,623,593,577,564 1 H-NMR (300MHz, DMSO-d6): δ9.51(s,1H),8.42(s,1H),8.00-7.98(d,1H),7.49-7 .47(d,2H,Ar-H),7.13-7.10(d,2H,Ar-H),6.08-6.06(d,1H),5.80-5.79(d,1H), 5.07-5.04(dd,1H),4.80-4.77(dd,1H),4.36-4.32(m,1H),4.28-4.17(m,2H),2. 54-2.47(m,1H and DMSO),2.29(s,3H),1.49(s,3H),1.29(s,3H),1.08-1.05(d,6H). 13 C-NMR (75MHz, DMSO-d6): δ176.27,159.93,147.68,146.80,145.46,138.55,128.68,125.93,113.63 ,94.44,85.48,84.38,81.12,64.21,33.51,27.32,25.54,21.25,19.19,19.10.ESI-MS:354.12[M+H] + (Free base MW: 353.37).

[0041] The above solid, namely p-toluenesulfonate (III) (32.7 g), was suspended in ethyl acetate (300 mL), heated to 75-80 °C, and stirred for 20-24 hours. After cooling to room temperature, the solid was filtered, washed with ethyl acetate (100 mL) and acetone (100 mL), and dried under vacuum for 3-4 hours to obtain 21.5 g of IV. HPLC purity: 98.8%, O-isopropylidene cytidine-5-isobutyrate (III): 0.16%.

[0042] Example 3: Preparation of 5-isobutyrylcytidine p-toluenesulfonate (IV)

[0043] The experiment was conducted as described in Example-1, except that the solvent ethyl acetate was replaced with acetonitrile (yield: 80.6%; HPLC purity: 98.6%).

[0044] Example 4: Preparation of 2',3'-O-isopropylcytidine-5'-isobutyrate (III)

[0045] Cytidine acetone sulfate (II) (3 g), 4-dimethylaminopyridine (0.19 g, 0.2 equivalents), and diisopropylethylamine (DIPEA) (3.15 g, 3.1 equivalents) were dissolved in acetonitrile (30 mL), and isobutyric anhydride (3.15 g, 1.6 equivalents) was added over 15 minutes. The solution was stirred for 16 hours. The reaction mixture was concentrated, and the residue was treated with water (100 mL) and dichloromethane (100 mL); after stirring for 5 minutes, the mixture was separated into two layers. The organic layer was dried over anhydrous sodium sulfate and concentrated to give 2.6 g (yield 95%) (III). HPLC purity: 83.15%, impurity-1: 9.04%, unknown impurity-2: 0.31%.

[0046] Example 5: Preparation of the free base (V) of cytidine-5-isobutyrate

[0047] Cytidine-5'-isobutyrate p-toluenesulfonate (IV) (15 g, 0.031 mol) was suspended in acetone (300 mL) and stirred. Dicyclohexylamine (6.72 g, 0.037 mol) was added to the suspension, and stirring was continued for 2 hours; the precipitated dicyclohexylammonium toluenesulfonate was removed by filtration. The solid was washed with acetone (50 mL); the acetone filtrate was polymerized and concentrated under reduced pressure; the residue was dissolved in water (100 mL) and washed with dichloromethane (2 x 100 mL). The aqueous solution was concentrated under reduced pressure, and the resulting solid residue was azeotropically reacted with acetone to give 8.3 g (85% yield) of foamy solid (V). HPLC purity: 99.5%.

[0048] Example 6: Preparation of cytidine-5-isobutyrate free base (V)

[0049] Except for the use of 1,4-dioxane instead of acetone, the experiment was carried out as described in Example-4, yielding 7.8 g (yield 80.6%) V, HPLC purity: 98.6%.

[0050] Example 7: Preparation of Monaviravir (I)

[0051] Isobutyrylcytidine (V) (5.0 g, 0.15 mmol) was suspended in 70% isopropanol, and hydroxylamine sulfate (8.4 g, 0.51 mmol) was added. The reaction mixture was heated to 75–80 °C for 16 hours. The isopropanol layer was separated and concentrated under reduced pressure. The residue was dissolved in isopropanol (60 mL), filtered to remove insoluble substances, and the filtrate was concentrated. The residue was then dissolved again in isopropanol at 50 °C. The solution was cooled to clarify, and the solid was filtered to give 3.7 g (71% yield) of colorless solid (I), HPLC purity: 99.29%.

Claims

1. A preparation process for monobirapvir having a structure of Formula I, characterized in that: Includes the following steps: a. Add a non-nucleophilic base to react 2',3'-O-isopropylcytidine sulfate with the structure of formula II with isobutyric anhydride in the solvent at room temperature to obtain 5-isobutyrate with the structure of formula III and N-isobutyric acid amide as impurity-1. b. The reaction product obtained in step a is reacted with p-toluenesulfonic acid monohydrate to obtain 5'-isobutyrylcytidine p-toluenesulfonate of formula IV, which does not contain N-isobutyric acid amide impurity-1. c. Convert the 5'-isobutyryl cytidine salt of formula IV into its free base of formula V, and react formula V with hydroxylamine to obtain monobirapvir with the structure of formula I; The nonnucleophilic base mentioned in step a is selected from diisopropylethylamine, N-methylmorpholine, 1,4-diazabicyclo-[2.2.2]octane, 1,8-diazabicyclo[5.4.0]undec-7-ene and triethylamine.

2. The preparation process of monobirapvir having the structure of Formula I as described in claim 1, characterized in that: in, The reaction described in step b is carried out at 50-90°C.

3. The preparation process of monobirapvir having the structure of Formula I as described in claim 1, characterized in that: in, The p-toluenesulfonic acid monohydrate described in step b contains 8-15% water hydrate.

4. The preparation process of monobirapvir having the structure of Formula I as described in claim 1, characterized in that: wherein, In step c, dicyclohexylamine is used to convert the 5'-isobutyryl cytidine salt of formula IV into its free base of formula V.

5. The preparation process of monobirapvir having the structure of Formula I as described in claim 1, characterized in that: wherein, In step c, acetone or 1,4-dioxane is used as a solvent to convert the 5'-isobutyryl cytidine salt of formula IV into its free base of formula V.

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