Claims:
1. An improved, short process for preparation of Molnupiravir comprising;

i. Hydroxyamination of Cytidine (1) with 1.0-3.0 equivalents of hydroxylamine hydrochloride solution in presence of catalytic amount of acetic acid or 0.6-2.0 equivalents of hydroxylamine sulphate optionally in water to obtain 4N-hydroxy cytidine hydrate(2);

ii. Chemical acylation of 4N-hydroxy cytidine hydrate (2) of step (i) with isobutyric anhydride in solvent to obtain N-Hydroxy Cytidine isobutyryl ester (13);

and
iii. Enzymatic conversion of N-Hydroxy Cytidine isobutyryl ester (13) on immobilized Biocatalyst CAL B lipase enzyme TA 1000-10000 u/gm expressed in Pichia pastoristo obtain Molnupiravir,

wherein the desired product is obtained with HPLC purity of 99.9%.

2. The improved, short process as claimed in claim 1, wherein the process steps (ii) and (iii) may be carried out in-situ.

3. The improved, short process as claimed in claim 1, wherein the acylation of 4N-hydroxy cytidine hydrate (2) in step (ii) is carried out with 1.0 to 5.0 equivalents of isobutyric anhydride.

4. The improved, short process as claimed in claim 1, wherein the enzyme loading is in the range of 10-300% w/w with respect to N-Hydroxy Cytidine Isobutyryl ester (13).

5. The improved, short process as claimed in claim 1, wherein the solvent(s) for the process is selected from polar protic or aprotic solvents such as lower alcohols, ethers such as THF or 2-Methyl Tetrahydrofuran, ketones, esters or mixtures thereof.

6. The improved, short process as claimed in claim 2, comprising;

i. Reacting Cytidine (1) with 1.0-3.0 equivalents of hydroxylamine hydrochloride solution in presence of catalytic amount of acetic acid or 0.6-2.0 equivalents of hydroxylamine sulphate optionally in water to obtain 4N-hydroxy cytidine hydrate (2);
ii. Treating 4N-hydroxy cytidine hydrate (2) with isobutyric anhydride in solvent to formN-Hydroxy Cytidine Isobutyryl ester (13) followed by addition of isobutyric anhydride and immobilized Biocatalyst CAL B lipase enzyme TA 1000-10000 u/gm expressed in Pichia pastoris to obtain Molnupiravir;
wherein the desired product is obtained with HPLC purity of 99.9%.

7. The improved, short process as claimed in claim 6, wherein 4N-hydroxy cytidine hydrate (2) is reacted with 0.1-1.2 equivalents of isobutyric anhydride.

8. The improved short process as claimed in claim 6, wherein the enzyme loading is in the range of 10-100%w/w with respect to N-Hydroxy Cytidine hydrate (2).

9. The improved short process as claimed in claim 6, wherein the enzyme loading is in the range of 10-50%w/w with respect to N-Hydroxy Cytidine (2).

10. The improved short process as claimed in claim 6, wherein the solvent(s) for the process is selected from polar protic or aprotic solvents such as lower alcohols, ethers such as THF or 2-Methyl Tetrahydrofuran, ketones, esters or mixtures thereof.

11. Intermediate, N-Hydroxy Cytidine Isobutyryl ester (i.e. 4-{[(2-methylpropanoyl) oxy] amino} Cytidine) (13)of the formula;

, Description:FIELD OF THE INVENTION
The present invention relates to an economical and improved short process for synthesis of Molnupiravir from cytidine.The process of the present invention provides a novel intermediate N-Hydroxy Cytidine Isobutyryl ester (13)which can be enzymatically converted to Molnupiravir in good yield and purity.

BACKGROUND OF THE INVENTION
Molnupiravir (development codes MK-4482 and EIDD-2801), chemically is((2R,3S,4R,5R)-3,4-dihydroxy-5-(4-(hydroxyimino)-2-oxo-3,4-dihydro pyrimidin-1(2H)-yl)tetrahydrofuran-2-yl)methyl isobutyrate was developed originally by Emory University around 2000, and was further developed in partnership with Merck, for the treatment of influenza. Molnupiravir is SARS-CoV2 polymerase inhibitor. It is a prodrug of the synthetic nucleoside derivative N4-hydroxycytidine, and exerts its antiviral action through introduction of copying errors during viral RNA replication.

Recently, studies have revealed that Molnupiravir can effectively inhibit COVID -19 virus, which has caused the current pandemic and can prevent the viral replication in SARS CoV-2/COVID-19 patients within 24 hours of administration. Clinical trials havetherefore begun for the treatment of patients infected with the COVID -19 virus. The market prospects will jump up heavily if the trials are successful. Researchers around the world, therefore, have started developing processes to manufacture Molnupiravir by chemical or enzymatic routes to meet the demands of the market in future.
Molnupiravir has, conventionally, been produced from uridine or cytidine through a multi-step chemical or enzymatic processes.

To reduce the cost of raw material, use of cytidine was proposed by N. Vasudevan et.al in the article titled “Toward a Practical, Two-Step Process for Molnupiravir from Cytidine” in Medicines for All Institute, Virginia. The article reports the twostep synthesis of Molnupiravir comprising direct hydroxyamination of the cytidine ring and enzymatic esterification of the sugar’s primary alcohol without use of protecting or activating groups represented in the Scheme below.

In the article titled “A Concise Route to MK-4482 (EIDD-2801) from Cytidine: Part 2” by V. Gopalsamuthiram et al. published in Synlett2020, 31, A–C,DOI: 10.1055/a-1275-2848; Art ID: st-2020-v0498-l, the process for preparation of Molnupiravir is depicted in scheme below:

In said article, the chemical O-acylation is carried out with isobutyric anhydride of the compound (3) where the free hydroxy groups are protected as acetonide. The O-acylated compound is then converted to Molnupiravir over 4 steps with the yield of 44% and over 3 steps with the yield of 41%.
CN112608357 disclose a process for preparing Molnupiravir, which comprises condensing compound (1) with hydroxylamine hydrochloride to obtain (2);

esterification reaction between compound 2 and an esterification reagent under the catalysis of an esterase enzyme to obtain compound 4;

using a microchannel reaction technology to deprotect a solution of intermediate compound (4) and an acid solution in a microchannel reaction, and then neutralize it with an alkaline solution in a microchannel reactor to obtain Molnupiravir (5).

The alternate process disclosed in CN’357 relates to (a) esterifying compound (1) with an esterification reagent under the catalysis of an esterification enzyme to obtain compound 3;
(b) compound (3) is reacted and condensed with hydroxylamine hydrochloride under the combined action of organic base and condensing agent, compound 3 to obtain compound 4;

and converting compound (4) to Molnupiravir using a microchannel reaction technology as shown above.

The Applicant in their earlier filed application no. 201721034128 has disclosed the chemo-enzymatic process for synthesis of Molnupiravir from cytidine using a lipase for esterification reaction.

The inventors felt that there is a scope to provide an economical and improved short process for synthesis of Molnupiravir with good yield and purity. This remains objective of the invention.

SUMMARY OF THE INVENTION
In accordance with the above, the present invention provides an improved, short process for preparation of Molnupiravir via a key intermediate N-Hydroxy Cytidine Isobutyryl ester, i.e. 4-{[(2-methylpropanoyl) oxy] amino} Cytidine (13).
In an aspect, the improved process for synthesis of Molnupiravir from cytidine comprises:
(i) Hydroxyamination of Cytidine (1) with hydroxylamine hydrochloride solution in presence of catalytic amount of acetic acid or hydroxylamine sulphate optionally in water to obtain 4N-hydroxy cytidine hydrate (2);

(ii) Chemical acylation of compound (2) of step (i) with isobutyric anhydride in solvent to obtain N-Hydroxy Cytidine Isobutyryl ester (i.e., 4-{[(2-methylpropanoyl) oxy] amino} Cytidine) (13).

and
(iii) Enzymatic conversion of N-Hydroxy Cytidine Isobutyryl ester (13) on immobilized Biocatalyst CAL B TA 1000-10000 u/gm expressed in Pichia pastoristo obtain Molnupiravir.

In an aspect, the process steps (ii) and (iii) may be carried out in-situ without isolating the intermediate, N-Hydroxy Cytidine Isobutyryl ester (i.e., 4-{[(2-methylpropanoyl) oxy] amino} Cytidine) (13).

In another aspect, the enzyme is loaded in an amount of 10-300% with respect to the intermediate N-Hydroxy Cytidine Isobutyryl ester (13), preferably in an amount of 10-50%w/w of the intermediate N-Hydroxy Cytidine Isobutyryl ester (13).

In another aspect, the intermediates of step (i) and step (ii) are formed in high yield and purity which results in the formation of Molnupiravir with high yield and purity.

In an aspect, the present invention provide the intermediate, N-Hydroxy Cytidine Isobutyryl ester (i.e. 4-{[(2-methylpropanoyl) oxy] amino} Cytidine) (13) shown below;

DETAILED DESCRIPTION OF THE INVENTION
The present invention will now be described in detail with its preferred and optional embodiments so that the various aspects therein can be more clearly understood and appreciated.

In an embodiment, the present invention relates to an improved and short process for preparation of Molnupiravir comprising;
(i) Hydroxyamination of Cytidine (1) with 1.0-3.0 equivalents of hydroxylamine hydrochloride solution in presence of catalytic amount of acetic acid or 0.6-2.0 equivalents of hydroxylamine sulphate optionally in water to obtain 4N-hydroxy cytidine hydrate (2);

(ii) Chemical acylation of compound (2) of step (i) with isobutyric anhydride in solvent to obtain N-Hydroxy Cytidine Isobutyryl ester (i.e., 4-{[(2-methylpropanoyl) oxy] amino} Cytidine) (13).

and
(iii) Enzymatic conversion of N-Hydroxy Cytidine Isobutyryl ester (13) on immobilized Biocatalyst CAL B lipase enzyme TA 1000-10000 u/gm expressed in Pichia pastoristo obtain Molnupiravir.

In an embodiment, the process steps (ii) and (iii) may be carried out in-situ without isolating the intermediate, N-Hydroxy Cytidine Isobutyryl ester (i.e. 4-{[(2-methylpropanoyl) oxy] amino} Cytidine) (13).

In another embodiment, the enzyme is loaded in an amount of 10-300% with respect to the intermediate N-Hydroxy Cytidine Isobutyryl ester (13).

The process according to the embodiment of the present invention is depicted in the Scheme 1 below:
In another embodiment, the present invention discloses the intermediate compound N-Hydroxy Cytidine Isobutyryl ester(i.e.4-{[(2-methylpropanoyl) oxy] amino} Cytidine)(13) of the formula;

Accordingly, the process of the invention comprises the steps of:
Step (i): In the first step of the present process cytidine (1) was allowed to react with 1.0 to 3.0 equivalents of hydroxylamine hydrochloride solution in presence of catalytic amount of acetic acid or 0.6-2.0 equivalents of hydroxylamine sulphate optionally in water to obtain 4N-hydroxy cytidine hydrate (2).
The solvent for the process is selected from lower alcohols such as methanol or ethanol. The base in the reaction is selected from sodium hydroxide or potassium hydroxide. The process was carried out at a temperature ranging between 25-80oC until completion of the reaction. After work up and crystallization in water, the desired product in crystalline form was isolated in purified form for the next step.
Step (ii): According to step (ii), the compound 4N-hydroxy cytidine hydrate was acylated with1.0 to 5.0 equivalents of isobutyric anhydride in 2-200 volumes of the solvent selected from polar protic or aprotic solvent such as acetone, tetrahydrofuran (THF), 2-methyl tetrahydrofuran and the like alone or mixtures thereof. The mixture was heated at a temperature ranging from 35-75oC until completion of the reaction. After completion of the reaction, the solvent was evaporated under vacuum and the residue was treated with solvent selected from aliphatic or aromatic hydrocarbon such as n-hexane, n-heptane, toluene, xylene and the like alone or mixtures thereof at 20-40oC. The isolated compound N-Hydroxy Cytidine Isobutyryl ester(13) was filtered, washed and dried.

Step(iii): The N-Hydroxy Cytidine Isobutyryl ester (13) was dissolved in 2-100 volumes of the solvent selected from polar protic or aprotic solvent such as acetone, tetrahydrofuran (THF), 2-methyl tetrahydrofuran and the like alone or mixtures thereof. This was followed by addition of 10-300%w/w of Immobilized Biocatalyst CAL B TA 1000-10000u/gm was added along with 0.4-3.7 equivalents of Isobutyric anhydride(9). The reaction mass was gently stirred at 20-60oC until HPLC analysis indicated 65-95% formation of Molnupiravir. The reaction mass was filtered, washed and purified to obtain pure Molnupiravir.

In an embodiment, the process steps of the present invention comprising first the chemical acylation of 4N-hydroxy cytidine hydrate to obtain N-Hydroxy Cytidine Isobutyryl ester (13) followed by the enzymatic reaction of said intermediate provides preparation of Molnupiravir in two steps which makes the process economical and less cumbersome. The two step process reduces the use of solvents, reagents and the product can be obtained in high yield and purity which makes it industrially feasible.

In the alternate embodiment, the process relates to the in-situ preparation of Molnupiravir from cytidine comprising the following procedure.

Accordingly, 4N-hydroxy cytidine hydrate (2) obtained from cytidine (1) as in step (i) is heated with 1.0 to 3.7 equivalents, preferably 1.3 to 2.8equivalents of isobutyric anhydride in the solvent selected from polar protic or aprotic solvent such as acetone, tetrahydrofuran (THF), 2-methyl tetrahydrofuran and the like alone or mixtures thereof at a temperature ranging from 55-65oC to form N-Hydroxy Cytidine Isobutyryl ester (i.e. 4-{[(2-methylpropanoyl) oxy] amino} Cytidine) (13). After the reaction is complete, the reaction mass is cooled to 30-35oC. This is followed by adding 0.1-1.2 equivalents, preferably 0.3-1 equivalents of Isobutyric anhydride and finally 10-100% w/w of Immobilized Biocatalyst CAL B TA 10000 u/gm with reference to substrate (13), preferably 15-50 % w/w of the substrate (2)or more preferably 20-49 % w/w of substrate (2),is added and the reaction mass is gently stirred at a temperature ranging from 40-45oC until completion of reaction and formation of desired product, Molnupiravir. The reaction mass was filtered, and the solvent evaporated under vacuum, purified in the solvent and finally in water to yield Molnupiravir in high purity of about 99.9%.

The solvent for purification is selected from polar protic or aprotic solvents such as ethyl acetate, isopropyl acetate, methyl-tert-butyl ether, acetone, alcohols and the like alone or mixtures thereof.

In yet another embodiment, the intermediate compound N-Hydroxy Cytidine Isobutyryl ester (i.e 4-{[(2-methylpropanoyl) oxy] amino} Cytidine) (13) may be converted to Molnupiravir Isobutyryl ester (4) (i.e., Diacyl ester of 4N-Hydroxy Cytidine) by treating with Isobutyric anhydride (9) and Immobilized Biocatalyst CAL B TA 10000 u/gm in Tetrahydrofuran or 2-Methyl Tetrahydrofuran. The product Molnupiravir Isobutyryl ester (4) is further treated with Liquid CAL B BIOCATALYST 10L(Not less than 10000 TBU/ml) to synthesise Molnupiravir (5). The reaction scheme 2 depicting the conversion of (13) to (4) to (5) is given below.

In an embodiment, the CAL B enzyme 10000u/gm used in the process of the present invention expressed in Pichia pastorisis prepared in house The enzymes used for Biocatalysis are immobilized on polyacrylate supports.

Examples:
Example 1: General process for synthesis of 4N-Hydroxy Cytidine hydrate (2)
1 equivalent of Cytidine (1) was treated with 0.60 to 1.5 equivalents of Hydroxylamine sulphate in 3-20 volumes of water or 1-3 equivalents of Hydroxylamine hydrochloridewith catalytic amount of acetic acid in methanol and water or 0.6-2 equivalents of Hydroxylamine sulphate. The reaction mass was heated at 30-80°C for 2 to 50 hours. After work up and crystallization in water 80-95% yield of the desired product 4N-Hydroxy Cytidine hydrate (2) was isolated with an HPLC purity of 98%-99.7%.

Example 1A: Synthesis of 4N-Hydroxy Cytidine hydrate (2) using Hydroxylamine Sulphate
250 gms of Cytidine (1)(1.028 moles, 1 equivalent) was charged in 2-L three neck RB flask equipped with water condenser, followed by 0.6L RO water and hydroxylamine sulphate (0.823 moles,0.82 equivalents). The mixture was stirred and heated to 70-80ºC. The solid suspension was dissolved, and the reaction mixture became clear solution after 20 minutes. The reaction mixture was stirred for 12-15 hours at the same temperature 75ºC. The reaction was monitored with HPLC, 94% of cytidine was consumed. The heating was stopped, suspension allowed to slowly cool to ambient temperature (25ºC) over the course of approximately 1-2 hours, then cooled to 0-5°C using an ice-salt bath and stirred for additional 5 hours. The solid obtained was filtered and washed with ice-cold water (500 mL x 4) and dried under vacuum at (60 ºC) in rotavapor to afford a white crystalline solid with 85.9 % yield (245g).
Appearance: Colourless crystalline solid
HPLC Purity: ~98.5%
Melting Point: 167-170oC
Mass Spectra: (M+): 260; (M-): 258
1H NMR (400 MHz, CD3OD):d 7.17 (d, 1H), 5.86 (d, 1H), 5.61 (d, 1H), 4.17 (t, 1H), 4.12 (t, 1H), 3.94 (d, 1H), 3.80 (dd, 1H), 3.71 (dd, 1H) ppm.
13C NMR (MHz, CD3OD):d 151.8, 146.3, 132.2, 99.3, 89.7, 86.1, 74.6, 71.74, 62.8 ppm.

EXAMPLE 1B: Synthesis of 4N-Hydroxy Cytidine hydrate (2) using Hydroxylamine Hydrochloride.
500 gm (2.057mole, 1 Equivalent) of Cytidine (1) was dissolved in 4 litres of water at 25-30°C. In another flask 500 gms (7.19 moles, 3.5 equivalents) of Hydroxylamine hydrochloride was dissolved in 4 litres methanol at 25-30°C. 350 gms of Potassium hydroxide in 1.5litres methanol was added to the above Hydroxylamine hydrochloride solution, stirred for 30 minutes. The solids were filtered, washed with 300 ml Methanol and the entire filtrate was cooled to 10-15°C. Subsequently 355 gms of acetic acid was added dropwise in a duration of 10-30 minutes at 10-15°C. To this solution 500 gms of Cytidine in 4 litres of water was added dropwise at 10-15°C. After complete addition, the reaction was stirred at 40°C for 29-44 hours till analysis indicated completion of the reaction. After 40 hours the solvent and water were evaporated under vacuum and the residue was stripped of the residual water by Toluene. The dry residue was purified by adding 2.5 litres of water and cooling to 10-15°C for 2-3 hours. The solids were filtered and washed with 2.5 litres (500ml *5) of ice- cold water. The HPLC analysis indicated 95% purity. The solids were dried under vacuum at 50-60°C for 3-4 hours. The dried solids weigh 425 gms (78% yield).
Appearance: Colourless crystalline solid
HPLC Purity: ~95%
Melting Point: 167-170oC

Example 2: General Synthesis of N-Hydroxy Cytidine Isobutyryl ester(4-{[(2-methylpropanoyl) oxy]amino}Cytidine) (13)
1 equivalent of 4N-Hydroxy Cytidine hydrate (2) was treated with 1 equivalent to 5 equivalents of isobutyric anhydride (9) in 2-100 volumes of Tetrahydrofuran or 2-Methyl Tetrahydrofuran and heated at 40-70°C for 2 to 20 hours. After completion of reaction the solvent was evaporated under vacuum and the residue was treated with 3-100 volumes of toluene, stirred at 20-40°C for 2-10 hours. The isolated solids i.e., N-Hydroxy Cytidine Isobutyryl ester (13) were filtered and washed with 1-2 volumes of Toluene, dried under vacuum. The yields were 85-95% and the HPLC analysis indicated a purity of 96%-99%.
(400 MHz, D2O): d 7.49 (d, 1H), 5.88-5.894 (m, 2H), 4.175 (t,1H), 4.116 (t, 1H), 3.959 (d, 1H), 3.822-3.815 (m, 1H), 3.727-3.689 (m, 1H), 2.849-2.797 (m, 1H), 1.214 (d, 6H).
(100 MHz, D2O): 176.329, 151.171,150.845, 135.944, 97.357, 89.94, 86.296, 75.061, 71.630, 62.581, 33.544, 19.394.
LCMS (M+H+ ) calculated for C13H20N3O7: 330; found: 330

Example 2A:
100 gms (0.3610 moles, 1 equivalent) of 4N-Hydroxy Cytidine hydrate (2) was suspended in 500 ml Tetrahydrofuran. 114.1 gms (0.7220 moles, 2 equivalents) of Isobutyric anhydride (9) was added in one lot. The reaction mass was heated at 60-65°C for 2-4 hours. After 4 hours HPLC and TLC analysis indicated the completion of reaction. The reaction mass was cooled to 28-33°C. The solvent tetrahydrofuran was evaporated under vacuum and the residue was treated with 500 ml Toluene, stirred at 28-33°C for 2-4 hours. After 4 hours the solids were filtered, washed with 100 ml Toluene. The isolated solids were dried under vacuum at 65°C for 3-4 hours to obtain N-Hydroxy Cytidine Isobutyryl ester (i.e.4-{[(2-methylpropanoyl) oxy]amino}Cytidine) (13).
Yields: 114 gms
%Yields: 96%.
HPLC purity: >97%

EXAMPLE 2B:
100 gms (0.3610 moles, 1 equivalent) of 4N-Hydroxy Cytidine hydrate (2) was suspended in 500 ml Tetrahydrofuran. 68.45 gms (0.43321 moles, 1.2 equivalents) of Isobutyric anhydride was added in one lot. The reaction mass was heated at 60-65°C for 5-9 hours. After 7 hours HPLC and TLC analysis indicated the completion of reaction. The reaction mass was cooled to 28-33°C. The solvent Tetrahydrofuran was evaporated under vacuum and the residue was treated with 300 ml Toluene, stirred at 28-33°C for 2-4 hours. After 4 hours the solids were filtered, washed with 100 ml Toluene. The isolated solids were dried under vacuum at 65°C for 3-4 hours.
Yields: 110 gms
%Yields: 92.6%.
HPLC purity: >98%

EXAMPLE 2C:
100 gms (0.3610 moles, 1 equivalent) of 4N-Hydroxy Cytidine hydrate (2) was suspended in 300 ml 2-Methyl Tetrahydrofuran. 142.6gms (0.9025 moles, 2.5 equivalents) of Isobutyric anhydride was added in one lot. The reaction mass was heated at 60-65°C for 1-3 hours. After 3 hours HPLC and TLC analysis indicated the completion of reaction. The reaction mass was cooled to 28-33°C. The solvent 2-Methyl tetrahydrofuran was evaporated under vacuum and the residue was treated with 600 ml Toluene, stirred at 28-33°C for 2-4 hours. After 4 hours the solids were filtered, washed with 200 ml Toluene. The isolated solids were dried under vacuum at 65°C for 3-4 hours.
Yields: 114 gms
%Yields: 96%.
HPLC purity: >98%

EXAMPLE 2D:
100 gms (0.3610 moles, 1 equivalent) of 4N-Hydroxy Cytidine hydrate (2) was suspended in 500ml Tetrahydrofuran. 68.45 gms (0.43321 moles, 1.2 equivalents) of Isobutyric anhydride was added in one lot. The reaction mass was heated at 60-65°C for 5-9 hours. After 7 hours HPLCand TLC analysis indicated the completion of reaction. The reaction mass was cooled to 28-33°C. The solvent Tetrahydrofuran was evaporated under vacuum and the residue was treated with 200 ml of Isopropyl acetate, stirred at 28-33°C for 2-4 hours. After 4 hours the solids were filtered, washed with 100 ml Isopropyl acetate. The isolated solids were dried under vacuum at 65°C for 3-4 hours.
Yields: 105gms
%Yields: 88.37 %.
HPLC purity: >99%

Example 3: General procedure for Synthesis of Molnupiravir (5)
1equivalent of N-Hydroxy Cytidine Isobutyryl ester/4-{[(2-methylpropanoyl) oxy]amino}Cytidine (13) was dissolved in 2-100 volumes of Tetrahydrofuran, 10%- 300% w/w ofImmobilized Biocatalyst CAL B TA 1000-10000 u/gm {w.r.t the substrate N-Hydroxy Cytidine Isobutyryl ester (13)} was added, along with 0.4-3.0 equivalents of Isobutyric anhydride (9).The reaction mass was gently stirred at 20-60°C for 4-60 hours. HPLC analysis of the reaction mass after 4-60 hours indicated the formation of 65%-95% Molnupiravir. The reaction mass was filtered, washed with Tetrahydrofuran. The entire Tetrahydrofuran was evaporated, and the residue was purified in Toluene, Isopropyl acetate, Methyl tert-butyl ether and finally in 2-10 volumes of water to isolate pure Molnupiravir in 60%-90% of theoretical yields with a HPLC purity of 99-99.95%.

Example 3A:
100 gms (0.3039 moles,1 equivalent) of N-Hydroxy Cytidine Isobutyryl ester (4-{[(2-methylpropanoyl) oxy]amino}Cytidine) (13) was suspended in 400 ml of Tetrahydrofuran, 62.4gms (0.3951 moles, 1.3 equivalents) of Isobutyric anhydride (9) was added at 28-33°C. The reaction mass was stirred at 28-33°C for 10-15 minutes. After 15 minutes 50 gms (50% w/w of the substrate-13) of Immobilized Biocatalyst CAL B TA 10000 u/gm was added and the reaction mass was gently stirred at 40-45°C for 30-32 hours. After 32 hours the reaction mass indicated formation of Molnupiravir (5), absence of starting material (13) and 2-4% formation of 4N-Hydroxy Cytidine (2). The detailed HPLC analysis are reported in Table 1. The reaction mass was filtered, washed with Tetrahydrofuran. The solvent was evaporated under vacuum and the residue was suspended in 700 ml water, heated to 70-75°C till it dissolved. Subsequently the clear solution was cooled to 6-10°C, stirred at 6-10°C for 6 hours. The solids were filtered, washed with 200 ml water, dried under vacuum at 85°C for 6 hours.
Yield: 85 gms
% Yields: 85%
HPLC Purity: 99.9%.
Mass Spectra: (M+): 330 ; (M-): 328
1H NMR (400 MHz, CD3OD): d 6.92 (d, 1H), 5.82 (d, 1H), 5.62 (d, 1H), 4.29 (d, 2H), 4.14 (t, 1H), 4.10 (p,2H), 2.63 (p, 1H), 1.19 (d, 6H).
13C NMR (400 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.

EXAMPLE 3B:
100 gms (0.3039 moles,1 equivalent) of N-Hydroxy Cytidine Isobutyryl ester(4-{[(2-methylpropanoyl) oxy]amino}Cytidine) (13) was suspended in 400 ml of Tetrahydrofuran. 96.0324 gms (0.6078 moles, 2 equivalents) of Isobutyric anhydride (9) was added at 28-33°C. The reaction mass was stirred at 28-33°C for 10-15 minutes. After 15 minutes 10 gms (10% w/w of the substrate-13) of Immobilized Biocatalyst CAL B TA 10000 u/gm was added and the reaction mass was gently stirred at 40-45°C for 46-54 hours. After 54 hours the reaction mass indicated formation of Molnupiravir (5), 4% of starting material (13) and 7-9% formation of 4N-Hydroxy Cytidine (2). The detailed HPLC analysis are reported in Table 1. The reaction mass was filtered, washed with Tetrahydrofuran. The solvent was evaporated under vacuum and the residue was stripped with 300 ml (3*100ml) Toluene. The residue after stripping was suspended in 200 ml Toluene and stirred overnight at 28-33°C. After 12 hours the solids were filtered, washed with 100 ml Toluene, dried under vacuum. The resulting dry solid wassuspended in 700ml water, heated to 70-75°C till it dissolved. Subsequently the clear solution was cooled to 6-10°C, stirred at 6-10°C for 6 hours. The solids were filtered, washed with 300 ml water, dried under vacuum at 85°C for 6 hours.
Yield: 78 gms
% Yields: 78%
HPLC Purity: 99.9%.

EXAMPLE 3C
100 gms (0.3039 moles,1 equivalent) of N-Hydroxy Cytidine Isobutyryl ester/4-{[(2-methylpropanoyl) oxy]amino}Cytidine (13) was suspended in 400 ml of Tetrahydrofuran, 62.4 gms (0.3951 moles, 1.3 equivalents) of Isobutyric anhydride (9) was added at 28-33°C. The reaction mass was stirred at 28-33°C for 10-15 minutes. After 15 minutes 15gms (15% w/w of the substrate-13) of Immobilized Biocatalyst CAL B TA 10000 u/gm was added and the reaction mass was gently stirred at 40-45°C for 36-40 hours. After 40 hours the reaction mass indicated formation of Molnupiravir (5), 5% of starting material (13) and 4-6% of 4N-Hydroxy Cytidine (2). The detailed HPLC analysis are reported in Table 1. The reaction mass was filtered, washed with Tetrahydrofuran. The solvent was evaporated under vacuum and the residue was suspended in 1000 ml Ethyl acetate. The ethyl acetate suspension was washed with 600 ml (2*300 ml) water containing 2% EDTA. The Ethyl acetate layer was evaporated under vacuum and the residue was dissolved in 500 ml Isopropyl acetate, heated, and stirred at 60°C for 4 hours. After 4 hours the solution was cooled to 28-33°C, filtered, washed with chilled 100 ml Isopropyl acetate, dried under vacuum at 60°C.
Yield: 65 gms
% Yields: 65%
HPLC Purity: 99.9%.

EXAMPLE 3D
100 gms (0.3039 moles,1 equivalent) of N-Hydroxy Cytidine Isobutyryl ester/4-{[(2-methylpropanoyl) oxy] amino} Cytidine (13) was suspended in 600 ml of 2-MethylTetrahydrofuran, 62.4 gms (0.3951 moles, 1.3 equivalents) of Isobutyric anhydride (9) was added at 28-33°C. The reaction mass was stirred at 28-33°C for 10-15 minutes. After 15 minutes 25gms (25% w/w of the substrate-13) of Immobilized Biocatalyst CAL B TA 10000 u/gm was added and the reaction mass was gently stirred at 40-45°C for 26-32 hours. After 32 hours the reaction mass indicated formation of Molnupiravir (5), 3% of starting material (13) and 3-6% of 4N-Hydroxy Cytidine (2). The detailed HPLC analysis are reported in Table 1. The reaction mass was filtered, washed with 200 ml 2-Methyl Tetrahydrofuran. The organic layer was washed with 400ml (2*200ml) water, dried over anhydrous sodium sulphate and finally the solvent was evaporated under vacuum. The residue was stirred with 300 ml Toluene, filtered and the filtered solids were suspended in 400 ml water, heated to 60-70°C. The entire solid dissolvedand was stirred at 60-70°C for 30 minutes. The solution was gradually cooled to 5-10°C, stirred at 5-10°C for 2 hours. The solids were filtered washed with 200 ml ice cold water, dried under vacuum at 60°C.
Yield: 79 gms
% Yields: 79 %
HPLC Purity: 99.9%.

COMBINING STEP 2 & STEP 3 IN A SINGLE STEP
The example 3E combines the Step 2 & Step 3 in one single step without isolating the step 2 product i.e., N-Hydroxy Cytidine Isobutyryl ester (i.e.4-{[(2-methylpropanoyl) oxy] amino}Cytidine) (13) In this step 4N-Hydroxy Cytidine Hydrate (2) was treated with 1-3 equivalents preferably 1.3-2.5 equivalents of isobutyric anhydride in Tetrahydrofuran at 60-65°C to form N-Hydroxy Cytidine Isobutyryl ester(i.e. 4-{[(2-methylpropanoyl) oxy] amino} Cytidine) (13). After the reaction was complete the temperature of the reaction mass was cooled to 30-35°C. 0.1-1.5 equivalents preferably 0.3-1.3 equivalents of Isobutyric anhydride was added and finally 10-100 % w/w of Immobilized Biocatalyst CAL B TA 10000 u/gm with respect to the substrate (2), preferably 15-50 % w/w of substrate (2) or more preferably 20-40 % w/w of substrate (2), was added and the reaction mass was gently stirred at 40-45°C till completion of reaction and formation of desired product. The reaction mass was filtered, and the solvent evaporated under vacuum. The residue was then purified in either isopropyl acetate or Methyl-tert-butyl ether and finally in water to yield Molnupiravir in high purity.

Example 3E: In-situ process for synthesis of Molnupiravir
100 gms (0.3610 moles, 1 equivalent) of 4N-Hydroxy Cytidine hydrate (2) was suspended in 650 ml tetrahydrofuran. 114 gms (0.722 moles, 2 equivalents) of Isobutyric anhydride (9) was added and the reaction mass was heated at 60-65°C for 4-8 hours. After 6 hours the TLC and HPLC analysis indicated absence of starting material and formation of N-Hydroxy Cytidine Isobutyryl ester (i.e.4-{[(2-methylpropanoyl) oxy] amino} Cytidine) (13). The reaction mass was cooled to 28-33°C. 17.1 gms (0.1083 moles, 0.3 equivalents) of Isobutyric anhydride (9) was added, stirred at 28-33°C. 25 gms (25% w/w of the substrate-2) of Immobilized Biocatalyst CAL B TA 10000 u/gm was added and the reaction mass was gently stirred at 40-45°C for 26-32 hours. After 32 hours the HPLC analysis indicated formation of Molnupiravir (5), 0.5-2% of N-Hydroxy Cytidine Isobutyryl ester (i.e.4-{[(2-methylpropanoyl) oxy] amino} Cytidine (13) and 3-7% of 4N-Hydroxy Cytidine hydrate (2). The reaction mass was filtered, washed with 100-200 ml Tetrahydrofuran. The organic layer was evaporated under vacuum and the residue was treated with 300 ml of Methyl-tert-butyl ether, stirred at 40°C for 3-6 hours. After 6 hours the suspension was filtered, washed with 100 ml of Methyl-tert-butyl ether. The solids were then dissolved in 400 ml water at 60-70°C to get a clear solution. The clear solution was then cooled gradually to 5-10°C, stirred at 5-10°C for 3 hours. The solids formed were filtered, washed with 100 ml of water, dried under vacuum at 60-80°C for 3-6 hours.
Yield: 74 gms
% Yields: 74 %
HPLC Purity: 99.9%.
The 1HNMR, 13CNMR and Mass spectra matched with the spectra of Molnupiravir isolated in earlier examples.

General method for the conversion of N-Hydroxy Cytidine Isobutyryl ester (i.e 4-{[(2-methylpropanoyl) oxy] amino} Cytidine) (13) to Molnupiravir Isobutyryl ester (4) (i.e., Diacyl ester of 4N-Hydroxy Cytidine) and finally to Molnupiravir (5).
1 equivalent of N-Hydroxy Cytidine Isobutyryl ester (i.e 4-{[(2-methylpropanoyl) oxy] amino} Cytidine) (13) was suspended in 2-10 volumes of Tetrahydrofuran or 2-Methyl Tetrahydrofuran. 5%-100% of BIOCATALYST CAL B TA 10000 u/gm preferably 5-75%, more preferably 5-40% BIOCATALYST CAL B TA 10000 u/gm, heated to 25-65°C, 0.8 equivalents- 3 equivalents of Isobutyric anhydride was added and the reaction mixture was heated at 25-65°C till TLC or HPLC analysis indicates completion of the reaction and formation of Molnupiravir Isobutyryl ester (4) (i.e., Diacyl ester of 4N-Hydroxy Cytidine). The reaction is cooled to 5-30°C and treated with 50%-200% w/v preferably 80%-150% (with respect to substrate 13) Liquid CAL B BIOCATALYST 10L (Not less than 10000 TBU/ml), stirred at 5-30°C preferably 10-25°C till TLC or HPLC analysis indicates the absence of Molnupiravir Isobutyryl ester (4) and formation of Molnupiravir (5). The solvent was evaporated, and the residue was suspended in Ethyl acetate and washed with water. After evaporation of the organic solvent the residue is crystallized in water to isolate pure Molnupiravir (5).

EXAMPLE 3F:
100 gms (0.3039 moles,1 equivalent) of N-Hydroxy Cytidine Isobutyryl ester(4-{[(2-methylpropanoyl) oxy]amino}Cytidine)(13) was suspended in 400 ml of Tetrahydrofuran. 50.5 gms (0.3190 moles, 1.05 equivalents) of Isobutyric anhydride (9) was added at 28-33°C. The reaction mass was stirred at 40-50°C for 10-15 minutes. After 15 minutes 7 gms (7% w/w of the substrate-13) of Immobilized Biocatalyst CAL B TA 10000 u/gm was added and the reaction mass was gently stirred at 40-45°C for 2-9 hours. After 9 hours, HPLC of the reaction mass indicated formation of 97% Molnupiravir Isobutyryl ester (4), 0.5% of starting material (13) and 0.2 % formation of 4N-Hydroxy Cytidine (2). The reaction mass was filtered, washed with 100 ml Tetrahydrofuran, cooled to 15-25°C. 80 ml (80% w/v of substrate 13) of Liquid CAL B BIOCATALYST 10L (10000 TBU/ml) was added and the reaction mass was stirred at 15-25°C for 22 -30 hours or till HPLC analysis indicated the completion of reaction. HPLC analysis after 29 hours indicated 90-94% Molnupiravir and 3-7% of 4N-Hydroxy Cytidine (2).The solvent was evaporated under vacuum and the residue was stripped with 600 ml (3*200ml) Toluene. The residue was dissolved in 700 ml ethyl acetate, washed with 2*150 ml water. The organic layer was separated and evaporated under vacuum. The residue was treated with 300 ml water, heated to 70-75°C, maintained at 70-75°C for 2 hours, gradually cooled to 4-8°C, stirred at 4-8°C for 3 hours. The solids were filtered under vacuum, washed with 100 ml water, suck dried and finally dried under vacuum at 60°C for 1-3 hours till constant weight.
Yield: 76 gms
% Yields: 76%
HPLC Purity: 99.9%.

EXAMPLE 3G

100 gms (0.3039 moles,1 equivalent) of N-Hydroxy Cytidine Isobutyryl ester(4-{[(2-methylpropanoyl) oxy] amino} Cytidine) (13) was suspended in 400 ml of Tetrahydrofuran. 50.5 gms (0.3190 moles, 1.05 equivalents) of Isobutyric anhydride (9) was added at 28-33°C. The reaction mass was stirred at 40-50°C for 10-15 minutes. After 15 minutes 10 gms (10% w/w of the substrate-13) of Immobilized Biocatalyst CAL B TA 10000 u/gm was added and the reaction mass was gently stirred at 40-45°C for 2-6 hours. After 6 hours, HPLC of the reaction mass indicated formation of 97.4% Molnupiravir Isobutyryl ester (4), 0.5% of starting material (13) and 0.8 % formation of 4N-Hydroxy Cytidine (2). The reaction mass was filtered, washed with 100 ml Tetrahydrofuran, cooled to 15-25°C. 140 ml (140 % w/v of substrate 13) of Liquid CAL B BIOCATALYST 10L (10000 TBU/ml) was added and the reaction mass was stirred at 15-25°C for 18-20 hours or till HPLC analysis indicated completion of the reaction. HPLC analysis after 20 hours indicates 90-91% Molnupiravir and 6-9% of 4N-Hydroxy Cytidine (2).The solvent was evaporated under vacuum and the residue was stripped with 600 ml (3*200ml) Toluene. The residue was dissolved in 700 ml ethyl acetate, washed with 2*150 ml water. The organic layer was separated and evaporated under vacuum. The residue was treated with 500 ml water, heated to 70-75°C, maintained at 70-75°C for 2 hours, gradually cooled to 4-8°C, stirred at 4-8°C for 3 hours. The solids were filtered under vacuum, washed with 100 ml water, suck dried and finally dried under vacuum at 60°C for 1-3 hours till constant weight.
Yield: 80 gms
% Yields: 80%
HPLC Purity: 99.9%.

Table 1:
Example Enzyme loading w/w % with respect to substrate (13) Reaction Mass HPLC analysis % % Yields
post purification Remarks
(5) (2) (13)
3A 50% 94% 2-4% 0.5 85% Minor impurities present in crude mass
3B 10% 89% 7-9% 4% 78% Minor impurities present in crude mass
3C 15% (with respect to substrate 2) 85% 4-6% 5% 65% Minor impurities present in crude mass
3D 25% 86% 3-6% 3% 79% Minor impurities present in crude mass
3E 25% 86% 3-7% 0.5-2% 74% Minor impurities present in crude mass

Although the invention has been described in detail in the foregoing for the purpose of illustration, it is to be understood that such detail is solely for that purpose and that variations can be made therein by those skilled in the art without departing from the spirit and scope of the invention except as it may be limited by the claims.