WO2022168107A1 - Synthèse enzymatique d'intermédiaire de molnupiravir - Google Patents

Synthèse enzymatique d'intermédiaire de molnupiravir Download PDF

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Publication number
WO2022168107A1
WO2022168107A1 PCT/IN2021/050490 IN2021050490W WO2022168107A1 WO 2022168107 A1 WO2022168107 A1 WO 2022168107A1 IN 2021050490 W IN2021050490 W IN 2021050490W WO 2022168107 A1 WO2022168107 A1 WO 2022168107A1
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WO
WIPO (PCT)
Prior art keywords
cytidine
acetonide
acetone oxime
isobutylester
molnupiravir
Prior art date
Application number
PCT/IN2021/050490
Other languages
English (en)
Inventor
Anupama Datla
Prashant NAGRE
Jagdish TAMORE
Manojkumar Sadanand PRABHU
Sreenath TRIVIKRAM
Trupti Krishnakant Ashar
Rina JAIN
Sachin Vasant KADAM
Original Assignee
Fermenta Biotech Limited
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Fermenta Biotech Limited filed Critical Fermenta Biotech Limited
Publication of WO2022168107A1 publication Critical patent/WO2022168107A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C12N9/20—Triglyceride splitting, e.g. by means of lipase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/38—Nucleosides
    • C12P19/385—Pyrimidine nucleosides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
    • C12Y301/01003—Triacylglycerol lipase (3.1.1.3)

Definitions

  • 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.
  • 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.
  • Molnupiravir has been repurposed for the treatment of SARS-CoV-2 infection.
  • W02002032920 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.
  • 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 Triethyl amine (Et3N). The ester intermediate is then subjected to Hydroxyamination by using Hydroxylamine sulphate to get Molnupiravir.
  • 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.
  • 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.
  • 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.).
  • Figure 1-A to 1-D is Hl 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.
  • 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.
  • 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.
  • the esterification of cytidine acetonide is carried out using Biocatalyst CAL B TAI 0000.
  • 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 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.
  • O-Isobutyl acetone Oxime which is used as esterification agent is prepared using acetone oxime wherein, acetone oxime is treated with Isobutyric acid & l-Ethyl-3 -(3 -dimethylaminopropyl) carbodiimide (EDCI).
  • 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.
  • 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.
  • Tri ethylamine 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.
  • 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.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • Virology (AREA)
  • Biomedical Technology (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

L'invention concerne le procédé enzymatique pour la synthèse d'un intermédiaire de Molnupiravir, du 5'-isobutylester d'acétonide de cytidine. Le procédé utilise de l'acétonide de cytidine en tant que matière première pour préparer du 5'-isobutylester d'acétonide de cytidine. L'estérification de l'acétonide de cytidine est effectuée à l'aide d'une enzyme CAL B exprimée chez Pichia pastoris. La présente invention concerne en outre des procédés de préparation d'oxime d'O-Isobutyl acétone.
PCT/IN2021/050490 2021-02-06 2021-05-20 Synthèse enzymatique d'intermédiaire de molnupiravir WO2022168107A1 (fr)

Applications Claiming Priority (2)

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IN202121005154 2021-02-06
IN202121005154 2021-02-06

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WO2022168107A1 true WO2022168107A1 (fr) 2022-08-11

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115785181A (zh) * 2022-11-30 2023-03-14 山东诚汇双达药业有限公司 一锅法制备莫匹拉韦中间体的方法

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019113462A1 (fr) * 2017-12-07 2019-06-13 Emory University N4-hydroxycytidine et dérivés et leurs utilisations anti-virales

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019113462A1 (fr) * 2017-12-07 2019-06-13 Emory University N4-hydroxycytidine et dérivés et leurs utilisations anti-virales

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
BENKOVICS TAMAS, MCINTOSH JOHN A, SILVERMAN STEVEN M, KONG JONGROCK, MALIGRES PETER, ITOH TETSUJI, YANG HAO, HUFFMAN MARK A, VERMA: "Evolving to an Ideal Synthesis of Molnupiravir, an Investigational Treatment for COVID19", CHEMRXIV. CAMBRIDGE : CAMBRIDGE OPEN ENGAGE, 23 December 2020 (2020-12-23), XP055960928, DOI: 10.26434/chemrxiv.13472373.v1 *
N. VASUDEVAN ET AL.: "A concise route to MK-4482 (EIDD-2801) from cytidine", CHEM. COMMUN., vol. 2020, no. 56, 2 October 2020 (2020-10-02), pages 13363 - 13364, XP055950766, DOI: https://doi.org/10.1039/DOCC05944G *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115785181A (zh) * 2022-11-30 2023-03-14 山东诚汇双达药业有限公司 一锅法制备莫匹拉韦中间体的方法
CN115785181B (zh) * 2022-11-30 2023-08-29 山东诚汇双达药业有限公司 一锅法制备莫匹拉韦中间体的方法

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