Claims:
1. An enzymatic process for preparation of Molnupiravir intermediate, 5’-isobutylester of Cytidine Acetonide wherein the said process comprises;
a) reacting Cytidine Acetonide Sulphate or salts thereof with O-Isobutyl Acetone oxime in presence of an enzyme, a suitable solvent and a base at a temperature in range of 15°C – 100°C and
b) isolating pure 5’-isobutylester of Cytidine Acetonide.

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

3. The process as claimed in Claim 2, wherein the enzyme is immobilized on substrates selected from polyacrylate porous hydrophobic polymer beads bearing optionally, functional groups such as epoxy, aldehyde or ionic groups.

4. The process as claimed in Claim 3, wherein the enzyme activity is in range of 1000 - 12000 u/g lipase units.

5. The process as claimed in Claim 1, wherein the solvent is selected from methyl tert-butyl ether, diethyl ether, toluene, 1,4- dioxane, dichloromethane, ethylacetate, tetrahydrofuran, and combinations thereof.

6. The process as claimed in Claim 1, wherein the base is selected from triethylamine, diethyl amine, N, N-diethyl isopropyl amine, ammonium hydroxide, and combinations thereof.

7. The process as claimed in Claim 1, wherein the O-Isobutyl acetone Oxime is synthesised by treating acetone oxime with isobutryl chloride & triethyl amine.

8. The process as claimed in Claim 1, wherein the O-Isobutyl acetone Oxime is synthesised by treating acetone oxime with isobutyric acid & 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDCI).

9. The process as claimed in Claim 1, wherein the O-Isobutyl acetone Oxime is synthesised by treating acetone oxime with isobutyric anhydride & triethyl amine in dichloromethane or tetrahydrofuran.
, Description:Field of Invention:
The present invention relates to an enzymatic process for synthesis of Molnupiravir intermediate, 5’-isobutylester of cytidine acetonide (cytidine 5’-isobutyrate acetonide or cytidine -5’-(2-methylpropanoate) acetonide). The present invention further relates to process for preparation of O-Isobutyl acetone oxime, which is used for synthesis of 5’-isobutylester of cytidine acetonide.

Background of the Invention:
Molnupiravir, also known as MK-4482, EIDD-2801, was originally developed by Emory University and was further developed in partnership with Merck, for treatment of influenza.

Researchers have recently observed that treatment of SARS-CoV-2 infection (Covid-19) with Molnupiravir (MK-4482, EIDD-2801), helps in suppression of virus transmission. Therefore, Molnupiravir has been repurposed for the treatment of SARS-CoV-2 infection.

WO2002032920 broadly covers Molnupiravir along with pharmaceutical composition used for treatment or prophylaxis of host exhibiting a Flaviviridae, Orthomyxoviridae or Paramyxoviridae viral infection or abnormal cellular proliferation and the process for preparation of compounds thereof. The methods disclosed are chemical methods which employ N -protected-D-cytidine nucleoside as a starting material.

WO2019113462 relates to N4-hydroxycytidine derivatives, which are used to prepare pharmaceutical compositions used for the treatment of various viral diseases such as Ebola, Influenza, RSV, and Zika virus infection with the disclosed compounds. WO2019113462 further discloses Molnupiravir and its preparation method. The route employed in WO2019113462 uses uridine as a starting material and derives Molnupiravir through a chemical route. However, this process has some serious drawbacks like low yield (Around 17 % reported yield), poor regioselectivity and higher cost of raw material, uridine. This makes the process commercially unviable, as it increases the process cost. The key intermediate of the process which is 5’-isobutylester of uridine is prepared by esterification of uridine using DMAP and isobutyric anhydride in presence of Tri-ethyl amine (Et3N). The ester intermediate is then subjected to Hydroxyamination by using Hydroxylamine sulphate to get Molnupiravir.

As reported above, Molnupiravir has been repurposed for the treatment of SARS-CoV-2 infection. It has been widely reported that Researchers in the Institute for Biomedical Sciences at Georgia State University have discovered that treatment of SARS-CoV-2 infection with a new antiviral drug, MK-4482/EIDD-2801 or Molnupiravir, completely suppresses virus transmission within 24 hours. Hence, there is now a need to meet the demand and supply of Molnupiravir in larger quantities and at reasonably affordable price for the treatment of SARS-CoV-2 infection.

Therefore, there remains a need in the art to develop a process which is cost effective and at the same time is efficient in terms of regioselectivity. The present inventors have come up with an enzymatic process which results in higher yield with higher purity of the final product. Thus, helping in fulfilling the requirements of the availability of drug to the poorer countries and weaker sections of the society.

Summary of Invention:
In an aspect, the present invention discloses 5’-isobutylester of Cytidine Acetonide (cytidine 5’-isobutyrate acetonide or cytidine -5’-(2-methylpropanoate) acetonide) intermediate (353) of the below formula used in synthesis of Molnupiravir.

The present invention further relates to the processes of O-Isobutyl acetone oxime (143) preparation, which is used for synthesis of 5’-isobutylester of cytidine acetonide.

In an another aspect, the present invention provides a novel process for preparing 5’-isobutylester of cytidine acetonide, wherein the process consists of reacting Cytidine Acetonide Sulphate or salts thereof with O-Isobutyl Acetone oxime in presence of an enzyme, suitable solvent and a base; at a temperature in range of 15°C – 100°C, to get pure 5’-isobutylester of Cytidine Acetonide (cytidine 5’-isobutyrate acetonide or cytidine -5’-(2-methylpropanoate) acetonide.).

Brief description of figures:
To further establish and confirm the formation of the intermediate and the subsequent product various analytical tools and methods were employed. The results of these analyses are provided herewith in the form of Graphical representation derived from the respective tools. It is appreciated that these figures are only for the guidance purpose of the typical embodiments of the invention and are therefore not to be considered limiting of its scope.

Figure 1-A to 1-D is H1 NMR spectra of 5’-isobutylester of Cytidine Acetonide (cytidine 5’-isobutyrate acetonide or cytidine -5’-(2-methylpropanoate) acetonide).
Figure 2-A and 2-B is Mass spectra of 5’-isobutylester of cytidine acetonide.
Figure 3 HPLC spectra of 5’-isobutylester of cytidine acetonide.

Abbreviations:
DMAP - 4-dimethylaminopyridine
ET3N – Triethylamine
Biocatalyst CALB TA 10000 or Fermenta CAL B – Immobilized CALB enzyme, activity 10000 u/g expressed in Pichia pastoris.

Detailed Description of the Invention:
For the purpose of this invention, it is to be understood that various terminologies used herein is with the intentions of explaining the said embodiments only, and is not intended to be limiting.

The invention is now described in detail with respect to the preferred and optional embodiments. Accordingly, the present invention discloses enzymatic synthesis of 5’-isobutylester of Cytidine Acetonide. This compound is represented as Formula 353, which is subsequently used for the synthesis of Molnupiravir.

Formula 353

As employed herein, the terms TLC, HPLC, C13 NMR, H1 NMR, are the standard terms practiced in the industry which are apparent to one skilled in the art.

In an embodiment, the invention provides an enzymatic process for synthesis of 5’-isobutylester of cytidine acetonide (Formula 353), which uses Cytidine as a starting material.

The process of the invention can be performed in different solvents selected from Methyl tert-butyl ether, Diethyl ether, Toluene, THF, dichloromethane, dioxane using different bases such as Triethyl amine, Diethyl amine, N, N-Diethyl isopropyl amine, Ammonium Hydroxide and the like.

In accordance of the above embodiment, the esterification of cytidine acetonide is carried out using Biocatalyst CAL B TA10000. The CAL B enzyme used in the process of invention is expressed in Pichia pastoris. The enzyme used for biocatalysis is immobilized on solid polyacrylate support. The support is in form of porous hydrophobic polymer beads which may or may not contain active functional groups, such as, 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.

The Biocatalyst CALB TA 10000 and the variants thereof, represented by alphanumeric notation in bracket following it (C1, C2…) wherein difference in polymer support is 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.

In another embodiment, the invention further provides the process for preparation of O-Isobutyl acetone Oxime (Formula 143), which is used as esterification agent. Formula 143 is prepared by using acetone oxime wherein, acetone oxime is treated with Isobutryl chloride & triethyl amine.

In an alternative embodiment, O-Isobutyl acetone Oxime which is used as esterification agent is prepared using acetone oxime wherein, acetone oxime is treated with Isobutyric acid & 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDCI).

In yet another alternative embodiment, O-Isobutyl acetone Oxime, which is used as esterification agent, is prepared using acetone oxime wherein, acetone oxime is treated with Isobutyric anhydride & triethyl amine in Dichloromethane or Tetrahydrofuran.

In a preferred embodiment, Cytidine Acetonide Sulphate is treated with 1.1 equivalents- 5 equivalents of O-Isobutyl Acetone oxime (Isobutryl oxime ester) 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 Biocatalyst CALB 1000 -12000 selected from activity ranging from 1000-12000 u/g preferably with 50- 200% w/w of the substrate and more preferably with 100-200% w/w of the substrate. 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 5’-isobutylester of Cytidine Acetonide. HPLC analysis indicated the purified sample to be 95%-98.

REACTION SCHEME:

In a preferred embodiment, the process for preparing 5’-isobutylester of Cytidine Acetonide comprises the following steps.
i) reacting Cytidine Acetonide Sulphate with O-Isobutyl Acetone oxime (143) 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 in presence of an organic base and Enzyme CAL B at a temperature in range of 15°C – 100°C for 6-50 hours.
ii) TLC analysis after 6-50 hours indicates completion of reaction. HPLC analysis indicated 90% formation of the desired product.
iii) The reaction mass is filtered, washed with 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 and the filtrate is evaporated.
iv) The residue after evaporation is purified by column chromatography to give 5’-isobutylester of Cytidine Acetonide. HPLC analysis indicated the purified sample to be 95%-98% pure.

Examples/Experimental details:
Following examples are described below to illustrate the various embodiments of the present invention according to the disclosed subject matter. These examples are not intended to be limiting or inclusive of all aspects of the present invention, but rather for the illustrative purposes. Further, it is to be understood that by the persons skilled in the art that, these examples are not intended to exclude equivalents and variations of the present invention.

Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.) and the results obtained by various analytical methods to confirm the formation of intermediates/end product of the experiments performed by the inventors of the present invention There are possible variations and combinations of reaction conditions, e.g., solvents, reaction vessels, and other experimental conditions that are used for the optimum results obtained from the process as illustrated.

Example 1
100 gms (0.2624M) of Cytidine Acetonide Sulphate was treated with 3 equivalents of O-Isobutyl Acetone oxime (Isobutryl oxime ester) in 2000 ml of Tetrahydrofuran, 200 gms (200 % wt ) of BIOCATALYST CAL B TA 10000 26.5gms(0.2625M) of Triethylamine at 25-35°C for 18-24 hours. TLC analysis after 24 hours indicated completion of reaction. HPLC analysis indicated >90% formation of the desired product. The reaction mass was filtered, washed with tetrahydrofuran and the filtrate was evaporated. The residue was dissolved in Dichloromethane, washed with water, 1N Potassium hydrogen sulphate solution. The solvent was evaporated, and the residue was purified by column chromatography using Ethyl acetate: Methanol (0-10% gradient) to give 84 gms of 5’-isobutylester of Cytidine Acetonide. HPLC analysis indicated the purified sample to be 95%-99% pure.
(1H NMR: FIGURE 1, 13C NMR: FIGURE 2, MASS SPECTRA (M+): 354: FIGURE 3, HPLC SPECTRA
FiGURE 4, )
Mass Spectra: (M+): 354

Example 2
10 gms (0.02624M) of Cytidine Acetonide Sulphate was treated with 3 equivalents of O-Isobutyl Acetone oxime (Isobutryl oxime ester) in 200 ml of 1,4-Dioxane, 20 gms of BIOCATALYST CAL B 10000, 2.65 gms(0.02624M) of Triethylamine at 25-35°C for 42-45 hours. TLC analysis after 45 hours indicated completion of reaction. HPLC analysis indicated >88% formation of the desired product. The reaction mass was filtered, washed with 1,4-Dioxane and the filtrate was evaporated. The residue was dissolved in Dichloromethane, washed with water, 1N Potassium hydrogen sulphate solution. The solvent was evaporated, and the residue was purified by column chromatography using Ethyl acetate: Methanol (0-10%) to give 6.1 gms of 5’-isobutylester of Cytidine Acetonide. HPLC analysis indicated the purified sample to be 96%-98% pure.

Example 3
10 gms (0.02624M) of Cytidine Acetonide Sulphate was treated with 3 equivalents of O-Isobutyl Acetone oxime (Isobutryl oxime ester) in 200 ml of 1,4-Dioxane, 20 gms of BIOCATALYST CAL B 10000, 2.65gms (0.02624M) of Triethylamine at 50-55°C for 24-28 hours. TLC analysis after 28 hours indicated completion of reaction. HPLC analysis indicated >80% formation of the desired product. The reaction mass was filtered, washed with 1,4-Dioxane and the filtrate was evaporated. The residue was dissolved in Dichloromethane, washed with water, 1N Potassium hydrogen sulphate solution. The solvent was evaporated, and the residue was purified by column chromatography using Ethyl acetate: Methanol (0-10%) to give 7.4 gms of 5’-isobutylester of Cytidine Acetonide. HPLC analysis indicated the purified sample to be 95%-98% pure.

Example 4
10 gms (0.02624M) of Cytidine Acetonide Sulphate was treated with 3 equivalents of O-Isobutyl Acetone oxime (Isobutryl oxime ester) in 200 ml of Tetrahydrofuran, 10 gms of BIOCATALYST CAL B 10000, 2.65gms(0.02624M) of Triethylamine at 25-33°C for 34-40 hours. TLC analysis after 40 hours indicated completion of reaction. HPLC analysis indicated >70% formation of the desired product. The reaction mass was filtered, washed with tetrahydrofuran and the filtrate was evaporated. The residue was dissolved in dichloromethane, washed with water, 1N Potassium hydrogen sulphate solution. The solvent was evaporated, and the residue was purified by column chromatography using Ethyl acetate: Methanol (0-10%) to give 5.6 gms of 5’-isobutylester of cytidine acetonide. HPLC analysis indicated the purified sample to be 95%-98% pure.

Advantages of the invention:
Synthesis of Molnupiravir (MK-4482) proceeding through the cytidine acetonide followed by enzymatic esterification / hydroxy amination and final cleavage of acetonide has number of advantages over the disclosed route which utilises Uridine and progresses by chemical methods to prepare Molnupiravir
1) It eliminates potential of undesired O-acylation, thus simplifying the impurity profile.

2) Intermediates possessing the free diol are hydrophilic and obtaining high recovery from aqueous layers can be challenging thereby reducing the yield.

3) Even though chemical esterification is less expensive but regioselectivity is not as high as enzymatic one so enzymatic esterification is preferred one as it reduces side products such as O-diacylated product and conversion to the desired product (5’-isobutylester of Cytidine Acetonide) is more than 95% as compared to 78% by chemical esterification.

4) Enzymatic esterification is environmentally friendly step.