DESC:Field of Invention:
The present invention is in the field of process for synthesis of Molnupiravir. More particularly, the present invention relates to a chemo-enzymatic process for synthesis of Molnupiravir from Cytidine.

Background of the Invention:
Molnupiravir, also known as MK-4482, EIDD-2801, was originally developed by Emory University, around 2000, and was further developed in partnership with Merck, for treatment of influenza. The Drug was first disclosed in WO2002032920 for treatment or prophylaxis of host exhibiting a Flaviviridae, Orthomyxoviridae or Paramyxoviridae viral infection or abnormal cellular proliferation. Subsequently, Molnup iravir and its pharmaceutical compositions were claimed in Indian Patent Application ?s 201717025098 and 202017019418, corresponding to pending European Patent Application ? EP3236972 and EP3706762, respectively.

Recently, it has been reported that Molnupiravir is effective in treatment of SARS CoV-2/COVID-19 virus, which has caused the current pandemic. It has been reported that Molnupiravir effectively inhibits viral replication in SARS CoV-2/Covid19 patients within 24hrs of administration. Hence, it has been repurposed to be used as a treatment of Coronavirus disease (COVID-19).

Molnupiravir is a prodrug of the synthetic nucleoside derivative N4-hydroxycytidine, and exerts its antiviral action through introduction of copying errors during viral RNA replication.

Molnupiravir has, conventionally, been produced from uridine or cytidine through a multi-step chemical or enzymatic process.

The chemical process was discussed in Indian Patent Application ? 201717025098 and 202017019418. The processes as discussed therein are chemical processes which uses uridine as starting material. Uridine is a costly raw material which has a limited availability. Further, two more chemical routes for synthesis of Molnupiravir were discussed in article published by V. Gopalsamuthiram et al. (September, 2020) and in an article published by A. Steiner et al (October, 2020).

V. Gopalsamuthiram et al. reports a 5-step process for synthesis of Molnupiravir starting from cytidine. The process as disclosed in A. Steiner et al is a six-step process for synthesis of Molnupiravir starting from uridine.

To reduce the cost of raw material, use of cytidine was proposed by N. Vasudevan et al in their article published in October 2020. This article describes the preparation of Molnupiravir consisting of an esterification and hydroxyamination of cytidine, in which Enzyme catalysed esterification of cytidine was carried out by using immobilized CALB (Candida Antarctica Lipase B). However, the disclosure of N. Vasudevan et al reports two alternative routes for converting cytidine to Molnupiravir via a two-step process. The process involves direct transformation of cytidine or N-hydroxycytidine to Molnupiravir without protecting the group. This might lead to more impurities, thereby increasing the expenses towards purification of final product.

However, the processes as described in aforementioned prior arts give a considerably low yield, which are around 10 – 30%. Further, the cost of process is also high owing to the cost involved in procuring the raw material. Hence overall, the chemical processes, as discussed in prior arts are economically unfeasible.

An enzyme-based or enzymatic process for synthesis of Molnupiravir from ribose was disclosed in an article by Tamas Benkovics et al published in December 2020. The process of Tamas Benkovics et al used Nov435, MTR Kinase; Acetate Kinase; Pyruvate Oxidase; Uridine Phosphorylase to synthesise Molnupiravir by a three-step process. The process proposed by Tamas Benkovics et al is a three-step route from ribose which uses two enzymatic steps consisting of five enzymes, thereby increasing the overall cost of the process. However, the cost of post-process effluent treatment is considerably reduced as an enzymatic process is greener as compared to pure chemical process.

Hence, it is an endeavour of current inventors to combine the advantages of chemical and enzymatic process to develop an economical and greener process for synthesis of Molnupiravir using a single enzyme.

Summary of the Invention:
In an aspect the invention provides a chemo-enzymatic process for synthesis of Molnupiravir of formula I from cytidine of Formula II using a lipase for esterification reaction.

Formula I Formula II

The process of the invention comprises of following steps:
(a) The compound of formula II is reacted with acetone in presence of sulphuric acid to obtain compound of formula III

Formula III

(b) The compound of Formula III is then enzymatically converted to compound of Formula IV.

Formula IV

(c) Formula IV is transaminated by hydroxylamine sulphate or hydroxylamine hydrochloride and subsequently deprotected to achieve the compound of Formula I

In another aspect, the current invention provides an enzymatic process of obtaining 5’-isobutylester of formula IV using IMMOBILIZED CAL B lipase enzyme expressed in Pichia pastoris.

Detailed Description of the Invention:
The present invention describes a novel process for synthesis of Molnupiravir, whereby the process uses a combination of chemical reactions and enzymatic transformation. The chemo-enzymatic reactions used in the process of the invention effectively reduces the overall cost of the process for synthesis of the Molnupiravir. Furthermore, as the use of chemicals is significantly reduced, the process is greener as compared to the processes known in the art.

The process of the invention achieves an improved yield as compared to the pure chemical process, by about 4 times, thereby further improving the economic advantage achieved by the process.

In an embodiment, the process of the invention comprises of following steps:
1. Synthesis of Cytidine Acetonide
Accordingly, in an embodiment the first step of the process of invention involves treatment of Cytidine (Formula II) with Acetone only in presence of Concentrated Sulphuric acid to obtain Cytidine Acetonide sulphate of Formula III, which can be used in further steps.

In the above embodiment, one equivalent of Cytidine is treated with in procuring 20-120 volumes of Acetone preferably 40-100 volumes more preferably 60-90 volumes of Acetone and 1-5 volumes of concentrated sulphuric acid. The reaction mass is stirred at 25-35°C for 6-30 hours preferably 12-24 hours till the analysis indicates completion of reaction. The reaction mass is further filtered and purified to isolate Cytidine Acetonide sulphate.

Alternatively, Compound of Formula II can be treated with Acetone & 2,2-Dimethoxypropane in presence of Concentrated Sulphuric acid to get Cytidine Acetonide Sulphate (Formula III) and used in further step.

In the above embodiment, one equivalent of Cytidine is treated with either 5-20 volumes of Acetone preferably 6-15 volumes more preferably 8-13 volumes of Acetone, 1-10 volumes of 2,2-Dimethoxypropane preferably 1.5-6 volumes more preferably 2-4 volumes and 0.1-1 volume concentrated sulphuric acid. The reaction mass is stirred at 25-35°C for 6-30 hours preferably 12-24 hours till the analysis indicates completion of reaction. The reaction mass is further filtered and purified to isolate Cytidine Acetonide sulphate.
In an alternative embodiment, the isolated Cytidine Acetonide Sulphate of Formula III can also be neutralised with Ammonium Hydroxide and used as Cytidine Acetonide in subsequent steps.

In accordance with above embodiment, in the next step, the Cytidine Acetonide sulphate (Formula III) is treated with Acetone isobutryl oxime ester in presence of IMMOBILIZED enzyme CAL B 1000 -12000 u/gm, preferably 3000-10000 u/gm & more preferably 5000-10000u/gm & Triethylamine in a solvent selected from Tetrahydrofuran, Methyl tetrahydrofuran, 1,4-Dioxane, Methyl-tert-butyl ether, Diethyl ether, Toluene or a mixture of Toluene & n-heptane, for 6-50 hours at 15-100°C. After purification, the isolated product 5’-Isobutyl ester of Cytidine Acetonide i.e CYTIDINE 5’-ISOBUTYRATE ACETONIDE or CYTIDINE -5’-(2-METHYLPROPANOATE) ACETONIDE is used in the next step.

The biocatalyst used in this enzymatic step is Candida antarctica lipase-B (IMMOBILIZED CAL-B) expressed in Pichia pastoris.

Finally, 5’-Isobutyl ester of Cytidine Acetonide is treated with either Hydroxylamine sulphate in Isopropanol -water mixture at 70-80°C or Hydroxylamine chloride in a Methanol- water mixture at 40°C for 14-40 hours. After completion of the reaction, the desired compound is further treated with either Sulphuric acid or Formic acid to obtain Molnupiravir of Formula I. The crude product is purified to isolate 99%+ pure Molnupiravir.

The process according to above embodiment can be repressed as shown in Reaction Scheme A, as below:

In subsequent embodiment, the invention relates to a process for obtaining acetone isobutyryl oxime ester, which functions as acyl donor for enzymatic for conversion of compound of Formula III to compound of Formula IV. Accordingly, the process comprises of reacting acetone (Formula IIIA) with hydroxylamine hydrochloride or hydroxylamine sulphate to obtain acetone oxime (Formula IIIB). The acetone oxime hence formed is then converted to acetone isobutryl oxime ester (Formula IIIC).

The above reaction can be effected by reacting compound of Formula IIIB with isobutyric anhydride in presence of pyridine and 4-dimethylaminopyridine. Alternatively, the compound of Formula IIIC can be obtained by reacting compound of Formula IIIB with isobutyric acid in presence of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine in Methylene dichloride solvent. In yet another alternative, the compound of Formula IIIC is obtained by reacting compound of Formula IIIB with isobutanoyl chloride in presence of triethylamine at 0°C. Preferably the compound of Formula IIIC is obtained by reacting compound of Formula IIIB with isobutyric acid in presence of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine in Methylene dichloride solvent, as this process is economical and gives good yield.

In a preferred embodiment, the detailed process is discussed herein below:

Step 1. 1 equivalent of Formula II is treated with either 5-20 volumes of Formula IIIA preferably 6-15 volumes more preferably 8-13 volumes of Formula IIIA, 1-10 volumes of 2,2-Dimethoxypropane preferably 1.5-6 volumes more preferably 2-4 volumes and 0.1-1 volume concentrated sulphuric acid OR with 20-120 volumes of Formula IIIA preferably 40-100 volumes more preferably 60-90 volumes of Formula IIIA and 1-5 volumes of concentrated sulphuric acid, Stirred at 25-35°C for 6-30 hours preferably 12-24 hours till the analysis indicates completion of reaction. The reaction mass is filtered and purified to isolate compound of Formula III. The isolated compound of Formula III can also be neutralised with Aqueous Ammonia and the isolated Cytidine Acetonide can be used as such in the enzymatic reaction.

Step 2. 100 gms (0.2624M) of compound of Formula III is treated with 1.1 - 5 equivalents of compound of Formula IIIC preferably 2-5 equivalents more preferably 2.2- 3.3 equivalents in 1,4-Dioxane or tetrahydrofuran or Diethyl ether or methyl tert-butyl ether or Di-isopropyl ether or Toluene or a mixture of Toluene & n-hexane or n-Heptane with 10%-300% w/w of IMMOBILIZED enzyme CAL B 1000-12000 preferably with 50- 200% w/w of the substrate and more preferably with 100-200% w/w of the substrate. A base preferably selected from Triethylamine or Diethyl amine or N, N-diisopropyl ethylamine is added to the reaction mass and the reaction is stirred between 15°C – 100°C preferably at 25-65°C & more preferably at 30-40°C for 6-50 hours. TLC analysis after 6-50 hours indicates completion of reaction. HPLC analysis indicated 90% formation of the desired product. The reaction mass is filtered, washed with the relevant solvent and the filtrate is evaporated. The residue after evaporation is purified by column chromatography to give 84 gms of compound of Formula IV. HPLC analysis indicated the purified sample to be 95%-98% pure.

Step 3. To 1 equivalent of compound of Formula IV in 20 volumes of Isopropanol water mixture (24% water content) is added 2-7 equivalents of Hydroxylamine sulphate preferably 2-5 equivalents more preferably 3-5 equivalents & the entire reaction mass was heated at 25-80°C preferably 35-80°C more preferably 40-78°C for 6-45 hours. After TLC analysis the reaction mass is worked up and the crude product is either treated with 5-25 volumes of Formic acid preferably 6-20 volumes more preferably 10-15 volumes at 0-40°C preferably at 20-40°C more preferably at 23-35°C for 3-10 hours till the TLC indicates completion of the reaction. After the reaction mass is worked up and purified by column chromatography to isolate pure 98%+ compound of Formula I.

Alternatively, to 1 equivalent of compound of Formula IV in 20 volumes of water is added 2-7 equivalents of Hydroxylamine chloride preferably 2- 5 equivalents more preferably 3-5 equivalents in 5 volumes of methanol OR 7 -11 volumes of isopropanol or ethanol & the entire reaction mass is heated at 25-80°C preferably at 35-80°C more preferably at 40-60°C for 6-45 hours preferably for 20-35 hours. After TLC analysis the reaction mass is worked up and the crude product is either treated with 5-25 volumes of Formic acid preferably 6-20 volumes more preferably 10-15 volumes at 0-40°C preferably at 20-40°C more preferably at 23-35°C for 3-10 hours till the TLC indicates completion of the reaction. The reaction mass is worked up and purified by column chromatography to isolate pure 98%+ compound of Formula I.

In an advantageous embodiment, the CAL B enzyme used in the process of invention is expressed in Pichia pastoris. Enzymes used for biocatalysis are immobilized on solid polyacrylate supports. The supports are in form of porous hydrophobic polymer beads which may or may not contain active functional groups being any of epoxy, aldehyde or ionic groups.

Biocatalyst resulting from immobilization on these polymer beads by either adsorption, ionic interaction or covalent binding exhibit varying degree of selectivity, reactivity, and recyclability in the enzymatic reactions under different reaction conditions.

A reference immobilized form is denoted as Biocatalyst CALB TA 10000 and variants represented with alphanumeric notation in bracket following it (C1, C2…) wherein difference in polymer support represented by numericals and alphabet representing enzyme variant used. It has been found that heterologous expression of CAL B in P. pastoris host, shows increased activity of lipase up to 3 to 10 times as compared to the native expression.

EXAMPLES
Example 1: Synthesis of Cytidine Acetonide Sulphate:
In a 3 neck 3L round bottom flask cytidine (100g, 0.41mol) was taken. To this anhydrous acetone (1.2L) was added, followed by 2,2-dimethoxypropane (240 ml) under nitrogen atmosphere. Neat sulfuric acid (50 ml) was added to the above suspension with vigorous stirring and stirring was continued for 10 to 15 hr. The solid obtained was filtered and was washed multiple (3 to 4) times with acetone (250mL X 4) followed by ether(250mLX2) to obtain Cytidine Acetonide Sulphate as white solid.
Dry Weight: 152 g
HPLC Purity: ~95%
Isolated yield: 97.5%

Example 2: Synthesis of Cytidine Acetonide Sulphate:
Cytidine (100g, 0.41mol) is treated with 8 litres of anhydrous acetone under nitrogen atmosphere. Neat sulfuric acid (100 ml) was added to the above suspension with vigorous stirring for 7-15 hours. After TLC analysis the reaction mass was filtered and the solid obtained was washed multiple (3 to 4) times with acetone (250mL X 4) followed by ether(250mLX2).
Dry Weight: 131 g
HPLC Purity: ~95%
Isolated yield: 83%

Example 3: Synthesis of Acetone isobutyryl oxime ester (acyl donor):
To a 2-L three-neck round bottom flask equipped with an overhead stirrer, nitrogen gas inlet was charged acetone oxime (100.0 g, 1.37 mol, 1.0 equiv.), dichloromethane (1200 mL) and EDC. HCl (314g, 1.64 mol, 1.2 equiv.) was added followed by DMAP (14g, 0.1 equiv.). The reaction mixture was stirred at room temperature for 5 hours. Reaction was monitored by GC analysis. The reaction mass was washed successively with H2O (250 mLX4) and brine solution (250 mLX2). The organic layer was dried over anhydrous Na2SO4, evaporated under vacuum in rotavapor to give desired oxime ester as light-yellow oil (195 g, quantitative yield, >98% purity). The 1HNMR, C13 NMR & the mass spectra of the isolated matched with the structure of the compound isolated by the above reaction

Example 4: Synthesis of Cytidine Acetonide 5’-Isobutyrate(Cytidine Acetonide 5’-(2-Methyl propanate):
Cytidine acetonide salt (150g, 0.39 mol) in a 5L three neck round bottom flask was added anhydrous THF (3L). Triethylamine (28.9 ml, 0.39mol, 1 equiv.) was added to the above flask upon which the suspension became a clear solution after stirring at room temperature for 5 minutes. Biocatalyst CAL B 10000 (300g, 200 wt%) (BIOCATALYST CAL B TA 10000, 10000 u/g, expressed in Pichia pastoris, (Fermenta Biotech ltd) and acetone oxime O-iso butyryl ester (169 g, 1.18 mol,3 equiv.) was then added and the resultant reaction was left stirring at room temperature for 24h. The reaction mixture was then filtered to remove enzymes and the solvent was removed by rotary evaporation, yielding the crude product as a yellow oil. The crude product was redissolved in Dichloromethane (1.5L) and washed the organic layer thoroughly with water (250ml X 4) and then with brine solution (250ml X 2), dichloromethane layer separated and dried with anhydrous Sodium sulphate. The Solvent was evaporated under vacuum yielding the crude product as yellowish oil. The crude product was purified by column chromatography (2-5 % gradient of Methanol in dichloromethane).
Dry Weight: 126 g
HPLC Purity: >98%
Isolated yield: 90%

Example 5: Synthesis of Synthesis of Cytidine Acetonide 5’-Isobutyrate(Cytidine Acetonide 5’-(2-Methyl propanate):
Cytidine acetonide salt (15g, 0.039 mol) in a 1 litre three neck round bottom flask was added anhydrous Tetrahydrofuran (300ml). Triethylamine (2.89 ml, 0.039mol, 1 equiv.) was added to the above flask upon which the suspension became a clear solution after stirring at room temperature for 5 minutes. Biocatalyst CAL B 10000 (15g,100 wt%) (BIOCATALYST CAL B TA 10000, 10000 u/g, expressed in Pichia pastoris, and acetone oxime O-iso butyryl ester (16.7 g, 0.115 mol,3 equiv.) was then added and the resultant reaction was left stirring at room temperature for 40 hours. The reaction mixture was then filtered to remove enzymes and the solvent was removed by rotary evaporation, yielding the crude product as a yellow oil. The crude product was redissolved in Dichloromethane (500ml) and washed Dichloromethane layer thoroughly with water (50ml X 4) and then with brine solution (50ml X 2), dichloromethane layer separated and dried with anhydrous sodium sulphate. Solvent was removed by rotary evaporation yielding the crude product as yellowish oil. The crude product was purified by column chromatography (2-5 % gradient of Methanol in dichloromethane).
Dry Weight: 8.2 g
HPLC Purity: >98%
Isolated yield: 60%

Example 6: Synthesis of Synthesis of Cytidine Acetonide 5’-Isobutyrate (Cytidine Acetonide 5’-(2-Methyl propanate):
Cytidine acetonide salt (15g, 0.039 mol) in a 1 litre three neck round bottom flask was added anhydrous 1,4-Dioxane (300ml). Triethylamine (2.89 ml, 0.039mol, 1 equiv.) was added to the above flask upon which the suspension became a clear solution after stirring at room temperature for 5 minutes. Biocatalyst CAL B 10000 (30g, 200 wt%) (BIOCATALYST CAL B TA 10000, 10000 u/g, expressed in Pichia pastoris, Fermenta Biotech ltd.,) and acetone oxime O-iso butyryl ester (16.7 g, 0.115 mol,3 equiv.) was then added and the resultant reaction was left stirring at room temperature for 45 hours. The reaction mixture was then filtered to remove enzymes and the solvent was removed by rotary evaporation, yielding the crude product as a yellow oil. The crude product was redissolved in dichloromethane (500ml) and washed dichloromethane layer thoroughly with water (50ml X 4) and then with brine solution (50ml X 2), DICHLOROMETHANE layer separated and dried with anhydrous sodium sulphate. Solvent was removed by rotary evaporation yielding the crude product as yellowish oil. The crude product was purified by column chromatography (2-5 % gradient of Methanol in dichloromethane).
Dry Weight: 9.2 g
HPLC Purity: >98%
Isolated yield: 66%

Example 7: Synthesis of Synthesis of Cytidine Acetonide 5’-Isobutyrate(Cytidine Acetonide 5’-(2-Methyl propanate):
Cytidine acetonide salt (15g, 0.039 mol) in a 1 litre three neck round bottom flask was added anhydrous 1,4-Dioxane (300ml). Triethylamine (2.89 ml, 0.039mol, 1 equiv.) was added to the above flask upon which the suspension became a clear solution after stirring at room temperature for 5 minutes. Biocatalyst CAL B 10000 (30g, 200 wt%) (BIOCATALYST CAL B TA 10000, 10000 u/g, expressed in Pichia pastoris, (Fermenta Biotech ltd) and acetone oxime O-iso butyryl ester (16.7 g, 0.115 mol,3 equiv.) was then added and the resultant reaction was left stirring at 50-55°C for 24-29 hours. The reaction mixture was then filtered to remove enzymes and the solvent was removed by rotary evaporation, yielding the crude product as a yellow oil. The crude product was redissolved in dichloromethane (500ml) and washed dichloromethane layer thoroughly with water (50ml X 4) and then with brine solution (50ml X 2), dichloromethane layer separated and dried with anhydrous sodium sulphate. Solvent was removed by rotary evaporation yielding the crude product as yellowish oil. The crude product was purified by column chromatography (2-5 % gradient of Methanol in dichloromethane).
Dry Weight: 11 g
HPLC Purity: >98%
Isolated yield: 79%

Example 8: Synthesis of Molnupiravir:
To 5’-Isobutyl Cytidine acetonide ester [Cytidine Acetonide 5’-Isobutyrate(Cytidine Acetonide 5’-(2-Methyl propanate)] (100 g, 0.28 mol) in a 2L three neck round bottom flask was added hydroxylamine sulphate (68.0g, 0.98mol 3.5 equiv.) followed by 70 % IPA (By Karl fisher titration, 24 % water content, 2L). The reaction mixture was heated to 78°C for 20 hr upon which time HPLC showed the almost consumption of starting material and formation of product. Solvent was removed on a rotary evaporator and acetonitrile (1L) was then added and the resulting slurry was stirred for 1hr and the insoluble residue was then filtered, and the filtrate concentrated under reduced pressure to afford crude material. Toluene (500 ml) was added, and concentration done under reduced pressure to remove water azeotropically to give a colourless semi solid.

Further to the semisolid mass of N-Hydroxy cytidine acetonide ester formic acid (1.5L) was added and stirred at room temperature for 5hrs. Solvent was removed under reduced pressure and EtOH(1L) was added. The resultant solution was again concentrated under vacuum to afford an oil. To the oily residue Methyl tert-butyl ether and Isopropanol (500ml each) were successively added and concentrated to give crude product which was purified by silica gel column chromatography in 10% Methanol/ DICHLOROMETHANE
Dry Weight: 60 g
HPLC Purity: >99.8%
Isolated yield: 65%

Analytical data:
1H NMR (400 MHz, DMSO): d 6.93 (d, 1H), 5.84 (d, 1H), 5.64 (d, 1H), 4.31 (d, 2H), 4.16 (t, 1H), 4.11 (p,2H), 2.64 (p, 1H), 1.20 (d, 6H);
13C NMR (151 MHz, DMSO): d 178.3, 151.5, 146.1, 131.7, 99.6,90.4, 82.6, 74.4, 71.5, 64.9, 35.1, 19.4, 19.3 ppm
Mass Spectra: (M+): 330
(M-): 328
Purity: 99.8% (assessed by HPLC).
M.pt: 155-158°C

Example 9: Synthesis of Molnupiravir:
10 gms (0.02832M) of 5’-Isobutyl ester of Cytidine acetonide [Cytidine Acetonide 5’-Isobutyrate(Cytidine Acetonide 5’-(2-Methyl propanate)] is suspended in 50 ml water at 25-30°C.In another flask 6.4 gms (0.0920M, 3.25 equivalents) of Hydroxylamine hydrochloride is dissolved in 55 ml methanol at 25-30°C. 5.1gms (0.09107M) of Potassium hydroxide in 20 ml Methanol is added to the above Hydroxylamine hydrochloride solution, stirred for 30 minutes. The solids are filtered, washed with 17 ml Methanol and the entire filtrate is cooled to 10-15°C. Subsequently 5.48 gms (0.09133M) of Acetic acid is added dropwise in a duration of 4-10 minutes at 10-15°C. To this solution the previously prepared 10 gms of Cytidine-5’-Isobutyrate acetonide in 50 ml water is added dropwise at 10-15°C. After complete addition, 150 ml water is added and the reaction mixture is stirred at 40°C for 22-34 hours till analysis indicates the completion of reaction. After 34 hours the solvent and water are evaporated under vacuum and the residue is stripped of the residual water by Toluene. The crude dry residue is treated with Formic acid and heated at 23-33°C for 3-5 hours till the TLC analysis indicated the completion of the reaction. After the completion of reaction, the solvent is evaporated under vacuum and stripped with Methanol, isopropanol & methyl tert-butyl ether repeatedly and the crude residue is purified by column chromatography using Ethyl Acetate: Methanol (0-15% gradient respectively).

After evaporation 6.5 gms of the white solid is isolated. The HPLC analysis of the isolated compound indicates 98%+ purity. The 1H NMR and C13 NMR indicated the formation of the desired product--- MOLNUPIRAVIR.

Example 10: Synthesis of Molnupiravir:
10 gms (0.02832M) of 5’-Isobutyl ester of Cytidine acetonide [Cytidine Acetonide 5’-Isobutyrate(Cytidine Acetonide 5’-(2-Methyl propanate)] is suspended in 50 ml water at 25-30°C.

In another flask 6.4 gms (0.0920M, 3.25 equivalents) of Hydroxylamine hydrochloride is dissolved in 60 ml isopropanol at 25-30°C. 5.1gms (0.09107M) of Potassium hydroxide in 25 ml isopropanol is added to the above Hydroxylamine hydrochloride solution, stirred for 30 minutes. The solids are filtered, washed with 20 ml Isopropanol and the entire filtrate is cooled to 10-15°C. Subsequently 5.48 gms (0.09133M) of Acetic acid is added dropwise in a duration of 4-10 minutes at 10-15°C. To this solution the previously prepared 10 gms of Cytidine-5’-Isobutyrate acetonide in 50 ml water is added dropwise at 10-15°C. After complete addition, 150 ml water is added & the reaction is stirred at 60-70°C for 12-16 hours till analysis indicates the completion of reaction. After 15 hours the solvent and water are evaporated under vacuum and the residue is stripped of the residual water by Toluene. The crude dry residue is treated with Formic acid and heated at 23-33°C for 3-5 hours till the TLC analysis indicated the completion of the reaction. After the completion of reaction, the solvent is evaporated under vacuum and stripped with Methanol, isopropanol & methyl tert-butyl ether repeatedly and the crude residue is purified by column chromatography using Ethyl Acetate: Methanol (0-15% gradient respectively). After evaporation 5.5 gms of the white solid is isolated. The HPLC analysis of the isolated compound MOLNUPIRAVIR indicates 98%+ purity.
,CLAIMS:
1. A chemo-enzymatic process for synthesis of compound of Formula I

the process comprises of:
(a) reacting compound of formula II with acetone in presence of sulphuric acid to obtain compound of formula III;

Formula III

(b) enzymatically converting the compound of Formula III to compound of Formula IV, and

Formula IV,

(c) Hydroxyaminating the Formula IV, followed by deprotection to obtain compound of Formula I,
wherein, the compound of Formula I has HPLC purity of more than 99% and wherein esterification is carried out in presence of a lipase.

2. The process as claimed in Claim 1, wherein, acyl donor in Step b is acetone isobutyryl oxime ester.

3. The process as claimed in Claim 2, wherein the acetone isobutyryl oxime ester (Formula IIIC) is obtained by reacting compound of formula IIIB with isobutanoyl chloride or isobutyric acid or isobutyric anhydride.

4. The process as claimed in Claim 1, wherein the lipase enzyme is Candida antarctica lipase B (CAL B) expressed in Pichia pastoris and immobilized to form Biocatalyst CALB .

5. The process as claimed in Claim 4, wherein range of immobilized enzyme CALB denoted Biocatalyst CAL B 1000 to Biocatalyst CALB 12000 is Candida antarctica lipase B (CAL B) expressed in Pichia pastoris immobilized on different polyacrylate porous hydrophobic polymer beads bearing none or either of functional groups being any of epoxy, aldehyde or ionic groups expressing enzyme activity ranging from 1000- 12000 u/g lipase units.

6. The process as claimed in Claim 1, wherein, the protection in step (a) is, optionally, done by using 2,2-dimethoxypropane.

7. The process as claimed in Claim 1, wherein the enzymatic reaction is carried out in a solvent containing mixture of tetrahydrofuran and triethylamine.

8. The process as claimed in Claim 1, wherein in step (c) hydroxyamination is carried out by using hydroxylamine sulphate or hydroxylamine hydrochloride.

9. The process as claimed in Claim 8, wherein the deprotection in step (c) is carried out by acids selected from formic acid or sulphuric acid.

10. The process as claimed in Claim 1, wherein the step (a) requires 20-120 volumes of Formula IIIA preferably 40-100 volumes more preferably 60-90 volumes of Formula IIIA.