WO2022208508A1 - Scalable two step synthesis of molnupiravir - Google Patents
Scalable two step synthesis of molnupiravir Download PDFInfo
- Publication number
- WO2022208508A1 WO2022208508A1 PCT/IN2021/050458 IN2021050458W WO2022208508A1 WO 2022208508 A1 WO2022208508 A1 WO 2022208508A1 IN 2021050458 W IN2021050458 W IN 2021050458W WO 2022208508 A1 WO2022208508 A1 WO 2022208508A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- molnupiravir
- cytidine
- reaction
- hydroxy
- hydroxylamine
- Prior art date
Links
- 229940075124 molnupiravir Drugs 0.000 title claims abstract description 113
- HTNPEHXGEKVIHG-ZJTJHKMLSA-N molnupiravir Chemical compound CC(C)C(=O)OC[C@H]1O[C@H](C(O)C1O)N1C=C\C(NC1=O)=N\O HTNPEHXGEKVIHG-ZJTJHKMLSA-N 0.000 title claims abstract description 87
- 230000015572 biosynthetic process Effects 0.000 title claims abstract description 39
- 238000003786 synthesis reaction Methods 0.000 title claims abstract description 25
- 238000006243 chemical reaction Methods 0.000 claims abstract description 76
- UHDGCWIWMRVCDJ-UHFFFAOYSA-N 1-beta-D-Xylofuranosyl-NH-Cytosine Natural products O=C1N=C(N)C=CN1C1C(O)C(O)C(CO)O1 UHDGCWIWMRVCDJ-UHFFFAOYSA-N 0.000 claims abstract description 28
- UHDGCWIWMRVCDJ-PSQAKQOGSA-N Cytidine Natural products O=C1N=C(N)C=CN1[C@@H]1[C@@H](O)[C@@H](O)[C@H](CO)O1 UHDGCWIWMRVCDJ-PSQAKQOGSA-N 0.000 claims abstract description 28
- UHDGCWIWMRVCDJ-ZAKLUEHWSA-N cytidine Chemical compound O=C1N=C(N)C=CN1[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O1 UHDGCWIWMRVCDJ-ZAKLUEHWSA-N 0.000 claims abstract description 26
- 238000006184 hydroxyamination reaction Methods 0.000 claims abstract description 9
- XCUAIINAJCDIPM-XVFCMESISA-N N(4)-hydroxycytidine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=NO)C=C1 XCUAIINAJCDIPM-XVFCMESISA-N 0.000 claims description 67
- WYURNTSHIVDZCO-UHFFFAOYSA-N tetrahydrofuran Substances C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 57
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 51
- -1 Molnupiravir isobutyryl oxime ester Chemical class 0.000 claims description 50
- 108090000790 Enzymes Proteins 0.000 claims description 39
- 102000004190 Enzymes Human genes 0.000 claims description 39
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 35
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 26
- 238000000034 method Methods 0.000 claims description 26
- LSACYLWPPQLVSM-UHFFFAOYSA-N isobutyric acid anhydride Chemical compound CC(C)C(=O)OC(=O)C(C)C LSACYLWPPQLVSM-UHFFFAOYSA-N 0.000 claims description 20
- 239000007788 liquid Substances 0.000 claims description 20
- 102100021851 Calbindin Human genes 0.000 claims description 19
- 101000898082 Homo sapiens Calbindin Proteins 0.000 claims description 19
- 101001021643 Pseudozyma antarctica Lipase B Proteins 0.000 claims description 19
- 150000002148 esters Chemical class 0.000 claims description 15
- WTDHULULXKLSOZ-UHFFFAOYSA-N Hydroxylamine hydrochloride Chemical compound Cl.ON WTDHULULXKLSOZ-UHFFFAOYSA-N 0.000 claims description 13
- 241000235058 Komagataella pastoris Species 0.000 claims description 12
- RYZOWUQRHBLZMN-UHFFFAOYSA-N CC(C)COC(=O)C(C)C.CC(C)CC(C)(C)C(O)=O Chemical compound CC(C)COC(=O)C(C)C.CC(C)CC(C)(C)C(O)=O RYZOWUQRHBLZMN-UHFFFAOYSA-N 0.000 claims description 9
- 239000000203 mixture Substances 0.000 claims description 9
- 239000003795 chemical substances by application Substances 0.000 claims description 8
- NXPHCVPFHOVZBC-UHFFFAOYSA-N hydroxylamine;sulfuric acid Chemical group ON.OS(O)(=O)=O NXPHCVPFHOVZBC-UHFFFAOYSA-N 0.000 claims description 8
- 230000003197 catalytic effect Effects 0.000 claims description 7
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 6
- 150000002443 hydroxylamines Chemical class 0.000 claims description 6
- 241000222120 Candida <Saccharomycetales> Species 0.000 claims description 5
- 239000004367 Lipase Substances 0.000 claims description 5
- 238000003776 cleavage reaction Methods 0.000 claims description 5
- 239000003960 organic solvent Substances 0.000 claims description 5
- 230000007017 scission Effects 0.000 claims description 5
- 239000000758 substrate Substances 0.000 claims description 4
- 238000005917 acylation reaction Methods 0.000 claims description 3
- 101150076489 B gene Proteins 0.000 claims description 2
- 108090001060 Lipase Proteins 0.000 claims description 2
- 102000004882 Lipase Human genes 0.000 claims description 2
- 230000010933 acylation Effects 0.000 claims description 2
- 238000006911 enzymatic reaction Methods 0.000 claims description 2
- 235000019421 lipase Nutrition 0.000 claims description 2
- 238000011068 loading method Methods 0.000 claims description 2
- 229920005989 resin Polymers 0.000 claims description 2
- 239000011347 resin Substances 0.000 claims description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N ether Substances CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims 1
- 125000001033 ether group Chemical group 0.000 claims 1
- QWOJMRHUQHTCJG-UHFFFAOYSA-N CC([CH2-])=O Chemical compound CC([CH2-])=O QWOJMRHUQHTCJG-UHFFFAOYSA-N 0.000 abstract description 7
- 230000032050 esterification Effects 0.000 abstract description 4
- 238000005886 esterification reaction Methods 0.000 abstract description 4
- 150000003138 primary alcohols Chemical class 0.000 abstract description 3
- 150000002009 diols Chemical group 0.000 abstract description 2
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 45
- 238000004128 high performance liquid chromatography Methods 0.000 description 43
- 239000000047 product Substances 0.000 description 39
- 239000007787 solid Substances 0.000 description 38
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 24
- 238000004458 analytical method Methods 0.000 description 17
- 239000011942 biocatalyst Substances 0.000 description 17
- RXGUIWHIADMCFC-UHFFFAOYSA-N 2-Methylpropyl 2-methylpropionate Chemical compound CC(C)COC(=O)C(C)C RXGUIWHIADMCFC-UHFFFAOYSA-N 0.000 description 15
- BZLVMXJERCGZMT-UHFFFAOYSA-N Methyl tert-butyl ether Chemical compound COC(C)(C)C BZLVMXJERCGZMT-UHFFFAOYSA-N 0.000 description 12
- 239000011541 reaction mixture Substances 0.000 description 12
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 11
- 238000005160 1H NMR spectroscopy Methods 0.000 description 10
- DRTQHJPVMGBUCF-XVFCMESISA-N Uridine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-XVFCMESISA-N 0.000 description 10
- 238000004440 column chromatography Methods 0.000 description 10
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 9
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 8
- 239000002904 solvent Substances 0.000 description 8
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 6
- HTNPEHXGEKVIHG-QCNRFFRDSA-N molnupiravir Chemical compound C(OC(=O)C(C)C)[C@H]1O[C@H]([C@@H]([C@@H]1O)O)N1C(=O)N=C(NO)C=C1 HTNPEHXGEKVIHG-QCNRFFRDSA-N 0.000 description 6
- 239000012044 organic layer Substances 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- 239000007858 starting material Substances 0.000 description 6
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 5
- DRTQHJPVMGBUCF-PSQAKQOGSA-N beta-L-uridine Natural products O[C@H]1[C@@H](O)[C@H](CO)O[C@@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-PSQAKQOGSA-N 0.000 description 5
- 239000010410 layer Substances 0.000 description 5
- 238000001819 mass spectrum Methods 0.000 description 5
- 239000000741 silica gel Substances 0.000 description 5
- 229910002027 silica gel Inorganic materials 0.000 description 5
- 229960001866 silicon dioxide Drugs 0.000 description 5
- 238000001228 spectrum Methods 0.000 description 5
- DRTQHJPVMGBUCF-UHFFFAOYSA-N uracil arabinoside Natural products OC1C(O)C(CO)OC1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-UHFFFAOYSA-N 0.000 description 5
- 229940045145 uridine Drugs 0.000 description 5
- VHYFNPMBLIVWCW-UHFFFAOYSA-N 4-Dimethylaminopyridine Chemical compound CN(C)C1=CC=NC=C1 VHYFNPMBLIVWCW-UHFFFAOYSA-N 0.000 description 4
- UHDGCWIWMRVCDJ-XVFCMESISA-N cytidine Chemical compound O=C1N=C(N)C=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 UHDGCWIWMRVCDJ-XVFCMESISA-N 0.000 description 4
- 229910001873 dinitrogen Inorganic materials 0.000 description 4
- KQNPFQTWMSNSAP-UHFFFAOYSA-N isobutyric acid Chemical compound CC(C)C(O)=O KQNPFQTWMSNSAP-UHFFFAOYSA-N 0.000 description 4
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 3
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- HMFHBZSHGGEWLO-SOOFDHNKSA-N D-ribofuranose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H]1O HMFHBZSHGGEWLO-SOOFDHNKSA-N 0.000 description 3
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 3
- PYMYPHUHKUWMLA-LMVFSUKVSA-N Ribose Natural products OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 description 3
- 239000008186 active pharmaceutical agent Substances 0.000 description 3
- HMFHBZSHGGEWLO-UHFFFAOYSA-N alpha-D-Furanose-Ribose Natural products OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000007257 deesterification reaction Methods 0.000 description 3
- 238000009826 distribution Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 239000003480 eluent Substances 0.000 description 3
- 230000002255 enzymatic effect Effects 0.000 description 3
- 239000000706 filtrate Substances 0.000 description 3
- 238000002955 isolation Methods 0.000 description 3
- 238000012544 monitoring process Methods 0.000 description 3
- 150000002923 oximes Chemical class 0.000 description 3
- 238000010898 silica gel chromatography Methods 0.000 description 3
- 238000011282 treatment Methods 0.000 description 3
- GQHTUMJGOHRCHB-UHFFFAOYSA-N 2,3,4,6,7,8,9,10-octahydropyrimido[1,2-a]azepine Chemical compound C1CCCCN2CCCN=C21 GQHTUMJGOHRCHB-UHFFFAOYSA-N 0.000 description 2
- ZNBNBTIDJSKEAM-UHFFFAOYSA-N 4-[7-hydroxy-2-[5-[5-[6-hydroxy-6-(hydroxymethyl)-3,5-dimethyloxan-2-yl]-3-methyloxolan-2-yl]-5-methyloxolan-2-yl]-2,8-dimethyl-1,10-dioxaspiro[4.5]decan-9-yl]-2-methyl-3-propanoyloxypentanoic acid Chemical compound C1C(O)C(C)C(C(C)C(OC(=O)CC)C(C)C(O)=O)OC11OC(C)(C2OC(C)(CC2)C2C(CC(O2)C2C(CC(C)C(O)(CO)O2)C)C)CC1 ZNBNBTIDJSKEAM-UHFFFAOYSA-N 0.000 description 2
- 229960000549 4-dimethylaminophenol Drugs 0.000 description 2
- 101150041968 CDC13 gene Proteins 0.000 description 2
- 241001678559 COVID-19 virus Species 0.000 description 2
- OTMSDBZUPAUEDD-UHFFFAOYSA-N Ethane Chemical compound CC OTMSDBZUPAUEDD-UHFFFAOYSA-N 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- HEDRZPFGACZZDS-MICDWDOJSA-N Trichloro(2H)methane Chemical compound [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 2
- PXAJQJMDEXJWFB-UHFFFAOYSA-N acetone oxime Chemical compound CC(C)=NO PXAJQJMDEXJWFB-UHFFFAOYSA-N 0.000 description 2
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- 229910000378 hydroxylammonium sulfate Inorganic materials 0.000 description 2
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
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- HEWZVZIVELJPQZ-UHFFFAOYSA-N 2,2-dimethoxypropane Chemical compound COC(C)(C)OC HEWZVZIVELJPQZ-UHFFFAOYSA-N 0.000 description 1
- MFYSUUPKMDJYPF-UHFFFAOYSA-N 2-[(4-methyl-2-nitrophenyl)diazenyl]-3-oxo-n-phenylbutanamide Chemical compound C=1C=CC=CC=1NC(=O)C(C(=O)C)N=NC1=CC=C(C)C=C1[N+]([O-])=O MFYSUUPKMDJYPF-UHFFFAOYSA-N 0.000 description 1
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 1
- 108010031797 Candida antarctica lipase B Proteins 0.000 description 1
- 241000710781 Flaviviridae Species 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 1
- AVXURJPOCDRRFD-UHFFFAOYSA-N Hydroxylamine Chemical compound ON AVXURJPOCDRRFD-UHFFFAOYSA-N 0.000 description 1
- 108010084311 Novozyme 435 Proteins 0.000 description 1
- 241000712464 Orthomyxoviridae Species 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 241000711504 Paramyxoviridae Species 0.000 description 1
- 108020000999 Viral RNA Proteins 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 239000012345 acetylating agent Substances 0.000 description 1
- 125000002252 acyl group Chemical group 0.000 description 1
- 150000008064 anhydrides Chemical class 0.000 description 1
- 230000000840 anti-viral effect Effects 0.000 description 1
- 238000010504 bond cleavage reaction Methods 0.000 description 1
- 239000012267 brine Substances 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 235000019253 formic acid Nutrition 0.000 description 1
- 238000004817 gas chromatography Methods 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 206010022000 influenza Diseases 0.000 description 1
- 229910052816 inorganic phosphate Inorganic materials 0.000 description 1
- 238000007040 multi-step synthesis reaction Methods 0.000 description 1
- 150000003833 nucleoside derivatives Chemical class 0.000 description 1
- 239000008194 pharmaceutical composition Substances 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 150000005374 primary esters Chemical class 0.000 description 1
- 229940002612 prodrug Drugs 0.000 description 1
- 239000000651 prodrug Substances 0.000 description 1
- 238000011321 prophylaxis Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical compound O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 1
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- 238000005303 weighing Methods 0.000 description 1
Classifications
-
- 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
- A61P31/14—Antivirals for RNA viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
- A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
- A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
- A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
- A61K31/7068—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
- C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
- C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
- C07H19/06—Pyrimidine radicals
- C07H19/067—Pyrimidine radicals with ribosyl as the saccharide radical
Definitions
- the present invention relates to scalable two-step synthesis of molnupiravir.
- the reaction sequence involves the use of commercially easily available cytidine (1), and molnupiravir is formed through direct hydroxy amination of the cytidine followed by esterification of the primary alcohol without protecting dihydroxy groups (vicinal diols) i.e. without formation of acetonide.
- Molnupiravir also known as MK-4482, EIDD-2801
- MK-4482 EIDD-2801
- the Drug was first disclosed in W02002032920 for treatment or prophylaxis of host exhibiting a Flaviviridae, Orthomyxoviridae or Paramyxoviridae viral infection or abnormal cellular proliferation.
- Molnupiravir and its pharmaceutical compositions were claimed in Indian Patent Application N°s 201717025098 and 202017019418, corresponding to pending European Patent Application N° EP3236972 and EP3706762, respectively.
- Molnupiravir is effective in treatment of SARS CoV-2/COVID-19 virus, which is cause of current pandemic. It has been reported that Molnupiravir effectively inhibits viral replication in 24hrs of administration.
- 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 Ns 201717025098 and 202017019418.
- the processes as discussed therein is a chemical process which uses uridine as starting material.
- the uridine is a costly raw material which has a limited availability.
- the cytidine acetonide ester (4) is converted to N- hydroxy cytidine acetonide ester (5) by adding hydroxylamine sulfate to ester (4) followed by 70%IPA and heating to about 78°C. Finally, N-hydroxy cytidine acetonide ester (5), is reacted with formic acid to obtain Molnupiravir.
- the first step comprises Esterification of ribose catalyzed by Novozym 435 to give esterified ribose (1); Enzymatic cascade from (1) to 5-isobutyryl uridine (2) with a stoichiometric phosphate donor and inorganic phosphate removal system; and Conversion of 5’-isobutyryl uridine (2) to molnupiravir, as shown in scheme 2.
- the present invention provides a twostep process for synthesis of Molnupiravir, which process comprises; a) hydroxy amination of cytidine with Hydroxylamine salts in water or alcohol or mixture thereof optionally presence of catalytic quantity of Acetic acid to obtain hydrate of N-hydroxy cytidine (2); and b) acylation of the N-hydroxy cytidine (2) with acylating agents selected from Isobutyrie anhydride or Isobutyl isobutyrate (2-Methylpropyl-2-Methyl propanoate) or Isobutyryloxime ester in presence of immobilised CALB- Lipase (Candida antartica) enzyme and an organic solvent to obtain mixture of Molnupiravir and Molnupiravir isobutyryl oxime ester, followed by selective cleavage of Molnupiravir isobutyryl oxime ester using LIQUID CAL B to obtain Molnupir
- the present invention provides a twostep process for synthesis of Molnupiravir, which process comprises; a) hydroxy amination of cytidine with Hydroxylamine salts in water or alcohol or mixture thereof optionally presence of catalytic quantity of Acetic acid to obtain hydrate of N-hydroxy cytidine (2); and b) treating the N-hydroxy cytidine (2) with aeylating agents selected from Isobutyric anhydride or Isobutyl isobutyrate (2-Methylpropyl-2-Methyl propanoate) or Isobutyryloxime ester in presence of immobilised CALB-Lipase (Candida antartica) enzyme and an organic solvent to obtain mixture of Molnupiravir and Molnupiravir isobutyryl oxime ester, followed by selective cleavage of Molnupiravir isobutyryl oxime ester using LIQUID CAL B to obtain Molnupiravir.
- the Hydroxylamine salt is selected from Hydroxylamine sulphate or Hydroxylamine chloride.
- reaction of step a) can be carried out by treating cytidine with Hydroxylamine sulphate in water or isopropanol to obtain hydrate of N- hydroxy cytidine.
- reaction of step a) can be carried out by treating cytidine with Hydroxylamine chloride in methanol, water optionally presence of catalytic quantity of Acetic acid to obtain hydrate of N-hydroxy cytidine.
- a colourless crystalline hydrate of N-hydroxy cytidine (2) is obtained in high yield with purity (Y: ⁇ 85%, HPLC purity: 98.9%) through direct hydroxy amination of cytidine with Hydroxylamine salts selected from Hydroxylamine sulphate in water or Isopropanol or with Hydroxylamine chloride in Water , Methanol & in presence of catalytic quantity of Acetic acid.
- Hydroxylamine salts selected from Hydroxylamine sulphate in water or Isopropanol or with Hydroxylamine chloride in Water , Methanol & in presence of catalytic quantity of Acetic acid.
- the hydroxy amination of cytidine reaction can be carried out at a temperature range of 30 to 80°C.
- the N-Hydroxy Cytidine (2) thus obtained is treated with different aeylating agents selected from the group consisting of Isobutyric anhydride; (6) (CAS NO: [97-72-3]) or Isobutyl isobutyrate (2-Methylpropyl-2- Methyl propanoate) (5) (CAS no: [97-85-8] or Isobutyryloxime ester (3) in presence of immobilised CALB-Lipase (Candida antartica) enzyme and an ethereal organic solvent such as THF.
- the acylation reaction can be carried out at room temperature.
- the Enzyme loading was maintained at 50%-600% w/w of substrate.
- the desired product Molnupiravir is formed in 30-70% along with diacylated product (i.e. Molnupiravir isobutyryl oxime ester (4), (30-70%) was also formed.
- the yield of desired product was increased enzymatically (Biocatalyst CALB 10L, 1000-10000 TBU u/mL or LIQUID CALB 3000-20000 u/ml) promoted selective oxime ester cleavage which converted the by-product, Molnupiravir isobutyryl oxime ester (4) into desired product Molnupiravir.
- the desired product MOLNUPIRAVIR can be isolated by Column chromatography or by Crystallization of crude Molnupiravir in water to afford a purity above -99.5% with an overall yield of 60%.
- the IMMOBILIZED CAL B used in all the enzymatic reaction is: FERMENTA BIOTECH LTD manufactured IMMOBILIZED Biocatalyst CALB TA 10000 10000 u/g, Covalent resins, expressed in Pichia pastoris.
- the LIQUID CAL B gene was heterologous expressed in Pichia pastoris. Large quantities of the CAL B enzyme were produced by fermentation. The fermentation broth containing the enzyme was concentrated to an activity of 15000-20000 TBU/ml manufactured by Fermenta Biotech Ltd.
- the Liquid biocatalyst CALB 10L (NLT 10000 TBU/mL is also manufactured by Fermenta Biotech Ltd,
- Isobutyric anhydride and Isobutyl isobutyrate are readily available in the market.
- Isobutyric anhydride and Isobutyl isobutyrate are considerably cheaper than the synthesis cost of Isobutyryloxime ester.
- Isobutyryloxime ester is synthesised in a 2step procedure starting from Acetone- to Acetone Oxime to isobutyryloxime ester whereas Isobutyric anhydride is easily synthesised by reaction of isobutyric acid and acetic anhydride.
- the synthetic reaction for the preparation of Molnupiravir is shown in scheme 3 According to the invention, direct Hydroxyamination of cytidine with hydroxylamine sulphate in water yielded N-hydroxy cytidine in good yield and high purity.
- the biocatalyst CALB 10L (NLT 10000 TBU/mL) or Liquid CALB 3000-20000 u/mL, Fermenta Biotech Ltd), was quite selective for reaction with Molnupiravir oxime ester in preference to primary ester of Molnupiravir and consequently, Molnupiravir content was increased from 30% to a value above 80% with a marginal increase in NHC content.
- Table 2 The product distribution, post addition of the biocatalyst as cleavage reagent is shown in table 2 Table 2:
- Cytidine is treated with 0.6 equivalents- 4 equivalents of Hydroxylamine sulphate preferably 0 6-1 equivalents and more preferably 0.63 equivalents in 1- ⁇ 0 volumes of water preferably 2-6 volumes more preferably 2-3 volumes at 40-80°C preferably 50-70°C or 1- 3 equivalents of Hydroxylamine hydrochloride preferably 1 equivalent more preferably 1.3 equivalents in Methanol, water, and catalytic amount of Acetic acid at 30-60°C preferably at 45°C.
- the reaction is maintained at the desired temperatures till HPLC, or TLC analysis indicates completion of reaction. After work up the desired product is crystallised in water to yield 75-85% of pure product.
- EXAMPLE 1 EXAMPLE 1:
- Step 1
- Acetone isobutyryl oxime ester
- N-hydroxycytidine hydrate (NHC.1H20; 100.00 g, 0.361 mol, 1.0 equiv.), followed by tetrahydrofuran (500mL), isobutyryl oxime ester (155 g, 1.08 mol, 3.0 equiv.) and Biocatalyst CALB TA 10000,10000 u/g expressed in Pichia pastoris (Fermenta biotech Ltd.) (50.0 g, 50 wt%).
- the reaction mixture was stirred at room temperature. The reaction was continued for 24 hours monitoring the reaction by HPLC.
- reaction showed 1.05% for NHC, 33.6 % for product and 56.1% for diacylated product in HPLC at 215 nm.
- the reaction mass was filtered to separate enzyme from reaction mixture, and the enzyme was washed with tetrahydrofuran (2 X 250 mL) followed by water wash (500mL), enzyme recovered dried and found no loss of activity for reuse.
- N-hydroxycytidine hydrate (NHC.1H20; 10.0 g, 0.0361 mol, 1.0 equiv.), followed by tetrahydrofuran(50.0mL), isobutyryl oxime ester (15.5 g, 1.08 mol, 3.0 equiv.) and Biocatalyst CALB TA 10000,10000 u/g expressed in Pichia pastoris (Fermenta biotech Ltd.) (5.0 g, 50 wt%) .
- the reaction mixture was stirred at room temperature. The reaction was continued for ⁇ 24 hours monitoring the reaction by HPLC.
- reaction showed 0.49% for NHC, 32.9 % for product and 59.9% for over acylated product in HPLC at 215 nm.
- the reaction mass was filtered to separate enzyme from reaction mixture, and the enzyme was washed with tetrahydrofuran (2 X 50 mL) followed by water wash (2X100mL), enzyme recovered dried and found no loss of activity for reuse.
- N-hydroxycytidine hydrate (NHC.1H20; 10.0 g, 0.0361 mol, 1.0 equiv.), followed by tetrahydrofuran( 50.0mL), isobutyryl oxime ester (15.5 g, 1.08 mol, 3.0 equiv.) and Biocatalyst CALB TA 10000,10000 u/g expressed in Pichia pastoris(Fermenta biotech Ltd.) (5.0 g, 50 wt%) .
- the reaction mixture was stirred at room temperature. The reaction was continued for ⁇ 24 hours monitoring the reaction by HPLC.
- reaction showed 0.6% for NHC, 30.4 % for product and 60.2% for over acylated product in HPLC at 215 nm.
- the reaction mass was filtered to separate enzyme from reaction mixture, and the enzyme was washed with tetrahydrofuran (2 X 50 mL).
- the combined organic layer was concentrated in vacuo and the crude mass was purified by silicagel column chromatography using MDC: MeOH 98:2 as an eluent. Concentration of pure fractions collected gives yield of non-polar product (i.e: Molnupiravir oxime ester (7.0 g, %Y: 81%)
- N-Hydroxy cytidine hydrate (NHC.H20) is carried out as per the Examples 1A and 1 B.
- Step 2 Synthesis of MOLNUPIRAVIR (EIDD-2801 ):
- the HPLC analysis of the organic layer indicated 40-60% of Molnupiravir and 36-56% of Molnupiravir Isobutyryl Oxime ester (4).
- the tetrahydrofuran layer is then treated with LIQUID CAL B 3000-20000 u/ml preferably 7000-18000 u/ml, more preferably 12000-16000u/ml and stirred at 20-45 °C preferably 20-35 °C more preferably at 25-30°C for 6 hours.
- HPLC analysis indicated 78% - 85% Molnupiravir, 1-3% of Molnupiravir Isobutyryl Oxime ester (4) and 1-4% of 4-N-Hydroxy Cytidine (2).
- EXAMPLE 6 lOOgms (0.3610M, 1 equivalent) of N-4-Hydroxy cytidine (2) is suspended in 1200 ml of Tetrahydrofuran, 200 gms of Biocatalyst CALB TA 10000,10000 u/g (200%w/w) expressed in Pichia pastoris is added and the reaction mass is stirred at 86-102 RPM and 40-50°C. At 45°C 214 gms (1.35moles, 3.75 equivalents) of Isobutyric anhydride (6) is added in one lot and the reaction mass is stirred at 60- 67°C, 86-102 RPM for 3-5 hours.
- the yield of the isolated solids is 105 gms.
- the HPLC analysis of the isolated solid indicated 93% Molnupiravir and 4% of 4-N-Hydroxy Cytidine (2).
- the solids are purified over column chromatography using silica gel 60-200 mesh and Diehl orom ethane: methanol (99:1 and graded up to 90:10 respectively).
- the fractions containing pure solids are evaporated and dried under vacuum.
- EXAMPLE 7 lOOgms (0.3610M, 1 equivalent) of N-4-Hydroxy cytidine (2) is suspended in 1200 ml of Tetrahydrofuran, 100 gms of Biocatalyst CALB TA 10000,10000 u/g (100%w/w), expressed in Pichia pastoris is added and the reaction mass is stirred at 86-102 RPM and 40-50°C.
- Liquid CAL B is added 5100 u/gm of substrate (2) i.e 32 ml of LIQUID CAL B with strength 15500-16500 u/ml is added and the reaction is stirred at 22-28°C for 4-8 hours.
- the yield of the isolated solids is 104 gms.
- the HPLC analysis of the isolated solid indicated 92% Molnupiravir and 4% of 4-N-Hydroxy Cytidine (2).
- the solids are purified over column chromatography using silica gel 60-200 mesh and Diehl orom ethane: methanol (99:1 and graded up to 90:10 respectively).
- the fractions containing pure solids are evaporated and dried under vacuum.
- EXAMPLE 8 Repeat of example 2a but isolation and purification method changed. lOOgms (0.3610M, 1 equivalent) of N-4-Hydroxy cytidine (2) is suspended in 1200 ml of Tetrahydrofuran, 200 gms of Biocatalyst CALB TA 10000,10000 u/g (200%w/w) expressed in Pichia pastoris is added and the reaction mass is stirred at 86-102 RPM and 40-50°C. At 45°C 214 gms (1.35moles, 3.75 equivalents) of Isobutyric anhydride (6) is added in one lot and the reaction mass is stirred at 60- 67°C, 86-102 RPM for 3-5 hours.
- Tetrahydrofuran is evaporated under vacuum at 35-45 °C. 100 ml of Toluene is added to the residue and stirred at 30-35°C for 2 hours. Solids separates out, filtered, washed with 2* 100 ml of Toluene. The HPLC analysis of the filtered solids indicated 90 % Molnupiravir and 2% Molnupiravir Isobutyryl Oxime ester (4) plus some other impurities.
- the toluene layer is evaporated under vacuum and the residue weighing 70 gms is dissolved in 5 volumes of Tetrahydrofuran(350ml) and treated with 20 ml LIQUID CAL B with strength 15500-16500 u/ml. and stirred at 20-28°C for 5 hours. After 5 hours HPLC analysis indicated 90% of Molnupiravir, 2% of Molnupiravir Isobutyryl Oxime ester (4) and 5% of 4-N-Hydroxy Cytidine (2).
- N-Hydroxycytidine hydrate (NHC.H20) is carried out as per Examples 1 A and IB Step 2: Synthesis of MOLNUPIRAVIR(EIDD-2801):
- EXAMPLE 10 lOgms (0.03610M, 1 equivalent) of N-4-Hydroxy cytidine (2) is suspended in 1200 ml of Tetrahydrofuran, 20 gms of Biocatalyst CALB TA 10000,10000 u/g (200%w/w) expressed in Pichia pastoris is added and the reaction mass is stirred at 86-102 RPM and 40-50°C. At 45°C 78 gms (0.541M, 15 equivalents) of Isobutyl isobutyrate (5) is added in one lot and the reaction mass is stirred at 60-67°C, 86- 102 RPM for 3-5 hours.
- EXAMPLE 11 lOgms (0.03610M, 1 equivalent) of N-4-Hydroxy cytidine (2) is suspended in 1200 ml of Tetrahydrofuran, 60 gms of BIOCATALYST CALB 10000 10000 u/g(600%w/w) expressed in Pichia pastoris is added and the reaction mass is stirred at 86-102 RPM and 40-50°C. At 45°C 26 gms (0.1805m, 5 equivalents) of Isobutyl isobutyrate is added in one lot and the reaction mass is stirred at 60-67°C, 86-102 RPM for 70 hours.
Abstract
The present invention provides a scalable two-step synthesis of molnupiravir. The reaction sequence involves the use of commercially easily available cytidine (1), and molnupiravir is formed through direct hydroxy amination of the cytidine followed by esterification of the primary alcohol without protecting dihydroxy groups (vicinal diols) i.e. without formation of acetonide.
Description
“SCALABLE TWO STEP SYNTHESIS OF MOLNUPIRAVIR”
Technical filed:
The present invention relates to scalable two-step synthesis of molnupiravir. The reaction sequence involves the use of commercially easily available cytidine (1), and molnupiravir is formed through direct hydroxy amination of the cytidine followed by esterification of the primary alcohol without protecting dihydroxy groups (vicinal diols) i.e. without formation of acetonide.
Background and prior art:
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 W02002032920 for treatment or prophylaxis of host exhibiting a Flaviviridae, Orthomyxoviridae or Paramyxoviridae viral infection or abnormal cellular proliferation. Subsequently, Molnupiravir and its pharmaceutical compositions were claimed in Indian Patent Application N°s 201717025098 and 202017019418, corresponding to pending European Patent Application N° EP3236972 and EP3706762, respectively.
Recently, it has been reported that Molnupiravir is effective in treatment of SARS CoV-2/COVID-19 virus, which is cause of current pandemic. It has been reported that Molnupiravir effectively inhibits viral replication in 24hrs of administration.
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 Ns 201717025098 and 202017019418. The processes as discussed therein is a chemical process which uses uridine as starting material. The uridine is a costly raw material which has a limited availability.
A Short Synthesis of Molnupiravir was reported by D.R. Snead et.al (Commun. 2020, 56, 13363-13364). This article reports two step synthesis of Molnupiravir from cytidine. First the selective transesterification of cytidine was accomplished using immobilized Candida Antarctica lipase B (200 wt%, 1.5 mol%) and isobutyric oxime ester E as the acyl transfer agent. This is followed by a transamination of D using hydroxylamine sulfate to deliver the target molecule in 75% yield for the two steps. The process is depicted in scheme 1 below.
Another article by Gopalsamuthiram, Vijayagopal, Snead, David R. et al published in Thieme on 29.09.2020 with DOI 10.1055/s-1275-2848 which discloses synthesis of Cytidine Acetonide (3) from cytidine which comprises reacting cytidine with
2,2-dimethoxypropane in anhydrous acetone and adding neat sulfuric acid. The cytidine acetonide (3) is converted to its ester (4) by reacting with isobutyric anhydride in anhydrous acetonitrile, DMAP and DBU, thereby greatly reducing levels of diacylated product. The cytidine acetonide ester (4) is converted to N- hydroxy cytidine acetonide ester (5) by adding hydroxylamine sulfate to ester (4) followed by 70%IPA and heating to about 78°C. Finally, N-hydroxy cytidine acetonide ester (5), is reacted with formic acid to obtain Molnupiravir.
Another article by Tamas Benkovics et.al; Department of Process Research and Development, discloses three-step synthesis of Molnupiravir from ribose. The first step comprises Esterification of ribose catalyzed by Novozym 435 to give esterified ribose (1); Enzymatic cascade from (1) to 5-isobutyryl uridine (2) with a stoichiometric phosphate donor and inorganic phosphate removal system; and Conversion of 5’-isobutyryl uridine (2) to molnupiravir, as shown in scheme 2.
Scheme 2
N. Vasudevan et al published in October 2020 describes the preparation of Molnupiravir involves synthesis of the Isobutyryloxime ester, the synthesis of which again escalates the cost of the process.
As is evident from the above, the prior art documents proposes multistep synthesis of Molnupiravir, the synthesis of which results in low isolated yields of Molnupiravir and also involves tedious work up procedures. Moreover, the prior art processes involves Isobutyryloxime ester, which is expensive. Therefore, there remains a need in the art to provide a novel process for synthesis of Molnupiravir which is devoid of Isobutyryloxime ester, will comprise less number of steps and thus can be commercially scalable.
Summary of the invention:
In the light of the above, the present invention provides a twostep process for synthesis of Molnupiravir, which process comprises; a) hydroxy amination of cytidine with Hydroxylamine salts in water or alcohol or mixture thereof optionally presence of catalytic quantity of Acetic acid to obtain hydrate of N-hydroxy cytidine (2); and b) acylation of the N-hydroxy cytidine (2) with acylating agents selected from Isobutyrie anhydride or Isobutyl isobutyrate (2-Methylpropyl-2-Methyl propanoate) or Isobutyryloxime ester in presence of immobilised CALB- Lipase (Candida antartica) enzyme and an organic solvent to obtain mixture of Molnupiravir and Molnupiravir isobutyryl oxime ester, followed by selective cleavage of Molnupiravir isobutyryl oxime ester using LIQUID CAL B to obtain Molnupiravir.
Detailed description of the invention:
Accordingly, the present invention provides a twostep process for synthesis of Molnupiravir, which process comprises; a) hydroxy amination of cytidine with Hydroxylamine salts in water or alcohol or mixture thereof optionally presence of catalytic quantity of Acetic acid to obtain hydrate of N-hydroxy cytidine (2); and
b) treating the N-hydroxy cytidine (2) with aeylating agents selected from Isobutyric anhydride or Isobutyl isobutyrate (2-Methylpropyl-2-Methyl propanoate) or Isobutyryloxime ester in presence of immobilised CALB-Lipase (Candida antartica) enzyme and an organic solvent to obtain mixture of Molnupiravir and Molnupiravir isobutyryl oxime ester, followed by selective cleavage of Molnupiravir isobutyryl oxime ester using LIQUID CAL B to obtain Molnupiravir.
In an embodiment, the Hydroxylamine salt is selected from Hydroxylamine sulphate or Hydroxylamine chloride.
In an embodiment, the reaction of step a) can be carried out by treating cytidine with Hydroxylamine sulphate in water or isopropanol to obtain hydrate of N- hydroxy cytidine.
In another embodiment, the reaction of step a) can be carried out by treating cytidine with Hydroxylamine chloride in methanol, water optionally presence of catalytic quantity of Acetic acid to obtain hydrate of N-hydroxy cytidine.
In accordance with the process of the present invention, a colourless crystalline hydrate of N-hydroxy cytidine (2) is obtained in high yield with purity (Y:~85%, HPLC purity: 98.9%) through direct hydroxy amination of cytidine with Hydroxylamine salts selected from Hydroxylamine sulphate in water or Isopropanol or with Hydroxylamine chloride in Water , Methanol & in presence of catalytic quantity of Acetic acid. The hydroxy amination of cytidine reaction can be carried out at a temperature range of 30 to 80°C.
In another embodiment, the N-Hydroxy Cytidine (2) thus obtained is treated with different aeylating agents selected from the group consisting of Isobutyric anhydride; (6) (CAS NO: [97-72-3]) or Isobutyl isobutyrate (2-Methylpropyl-2- Methyl propanoate) (5) (CAS no: [97-85-8] or Isobutyryloxime ester (3) in presence of immobilised CALB-Lipase (Candida antartica) enzyme and an ethereal organic solvent such as THF. The acylation reaction can be carried out at room temperature.
The Enzyme loading was maintained at 50%-600% w/w of substrate. The desired product Molnupiravir is formed in 30-70% along with diacylated product (i.e. Molnupiravir isobutyryl oxime ester (4), (30-70%) was also formed. The yield of desired product was increased enzymatically (Biocatalyst CALB 10L, 1000-10000 TBU u/mL or LIQUID CALB 3000-20000 u/ml) promoted selective oxime ester cleavage which converted the by-product, Molnupiravir isobutyryl oxime ester (4) into desired product Molnupiravir. The desired product MOLNUPIRAVIR can be isolated by Column chromatography or by Crystallization of crude Molnupiravir in water to afford a purity above -99.5% with an overall yield of 60%.
In an embodiment, the IMMOBILIZED CAL B used in all the enzymatic reaction is: FERMENTA BIOTECH LTD manufactured IMMOBILIZED Biocatalyst CALB TA 10000 10000 u/g, Covalent resins, expressed in Pichia pastoris. The LIQUID CAL B gene was heterologous expressed in Pichia pastoris. Large quantities of the CAL B enzyme were produced by fermentation. The fermentation broth containing the enzyme was concentrated to an activity of 15000-20000 TBU/ml manufactured by Fermenta Biotech Ltd. The Liquid biocatalyst CALB 10L (NLT 10000 TBU/mL is also manufactured by Fermenta Biotech Ltd,
The Advantages of using Isobutyric anhydride and Isobutyl isobutyrate in the present invention over reported /prior art Isobutyryloxime ester, are provided herein below.
1. Isobutyric anhydride and Isobutyl isobutyrate are readily available in the market.
2. Isobutyric anhydride and Isobutyl isobutyrate are considerably cheaper than the synthesis cost of Isobutyryloxime ester.
3. Isobutyryloxime ester is synthesised in a 2step procedure starting from Acetone- to Acetone Oxime to isobutyryloxime ester whereas Isobutyric anhydride is easily synthesised by reaction of isobutyric acid and acetic anhydride.
The synthetic reaction for the preparation of Molnupiravir is shown in scheme 3
According to the invention, direct Hydroxyamination of cytidine with hydroxylamine sulphate in water yielded N-hydroxy cytidine in good yield and high purity. Further the esterification of cytidine’ s primary alcohol with isobutyryloxime ester in THF solvent using immobilised CALB-Lipase (Candida antartica) enzyme gave not only desired product (Molnupiravir) but also good amount of over acetylated (diacylated) product i.e. Molnupiravir isobutyryl oxime ester, as shown in scheme 4.
The results (Product distribution prior to addition of oxime ester cleavage reagent) obtained from the use of various acetylating agents are discussed in table 1.
Though the yield of the desired product under the above conditions appears low, we wondered whether the over acylated product could be directly converted to Molnupiravir. The oxime ester bond seems to be easily cleaved enzymatically. Therefore, to prove this concept of oxime ester bond cleavage, Liquid- CAL B enzyme has been added to acylated Molnupiravir reaction mixture. To the inventor’s surprise, it has been observed that selective cleavage of Molnupiravir oxime ester bond and increased content of desired product Molnupiravir in the reaction mass. The biocatalyst CALB 10L (NLT 10000 TBU/mL) or Liquid CALB 3000-20000 u/mL, Fermenta Biotech Ltd), was quite selective for reaction with Molnupiravir oxime ester in preference to primary ester of Molnupiravir and consequently, Molnupiravir content was increased from 30% to a value above 80% with a marginal increase in NHC content. However, there remains a room for further improvements, which the present inventors are working upon. The product distribution, post addition of the biocatalyst as cleavage reagent is shown in table 2 Table 2:
Having this excellent reaction outcome, the attention was turned into scale-up reaction and isolation of the API Molnupiravir from reaction solution. Accordingly following scale up batches have been carried out and its experimental details are given below.
Experimental:
Synthesis of 4-N-Hydroxy Cytidine
One equivalent of Cytidine is treated with 0.6 equivalents- 4 equivalents of Hydroxylamine sulphate preferably 0 6-1 equivalents and more preferably 0.63 equivalents in 1-Ί0 volumes of water preferably 2-6 volumes more preferably 2-3 volumes at 40-80°C preferably 50-70°C or 1- 3 equivalents of Hydroxylamine hydrochloride preferably 1 equivalent more preferably 1.3 equivalents in Methanol, water, and catalytic amount of Acetic acid at 30-60°C preferably at 45°C. The reaction is maintained at the desired temperatures till HPLC, or TLC analysis indicates completion of reaction. After work up the desired product is crystallised in water to yield 75-85% of pure product.
EXAMPLE 1:
EXAMPLE 1A
N-Hydroxycytidine hydrate (NHC.H20):
(243) (277)
Hydroxylamine
1 Hydrochloride/ 2
MeOH/AcOH/Water
Cytidine(500g,2.05mol) was charged in 3-L three neck RB flask equipped with water condenser, followed, by 1.2L RO water and hydroxylamine sulphate (215g, 0.8 equiv. 1.64mol). The mixture was stirred and heated to 70-80 °C. The solid suspension was dissolved, and the reaction mixture became clear solution after 20 minutes. The reaction mixture was stirred total for 12-15 hours at the same temperature 75 °C. The reaction was monitored with HPLC, 94% of cytidine was consumed. The heating was stopped, suspension allowed to slowly cool to ambient temperature (25 °C) 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 °C) in rotavapor to afford a white crystalline solid with 83.7% yield (475g). Appearance: Colourless crystalline solid HPLC Purity: -98%
M.Pt: 165-170°C Mass Spectra: (M+): 260
(M-): 258
1H NMR (400 MHz, CD30D): 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, CD30D): d 151.8, 146.3, 132.2, 99.3, 89.7, 86.1, 74.6, 71.74, 62 8 ppm. Data matched with those reported earlier.
EXAMPLE 1 B:
Hydroxyamination using Hydroxylamine Hydrochloride
100 GMS (0.411 M, 1 Equivalent) OF CYTIDINE (1) is dissolved in 2 litres of water at 25-30°C. In another flask 100 gms of Hydroxylamine hydrochloride is dissolved in 875 ml methanol at 25-30°C. 70 gms of Potassium hydroxide in 315 ml Methanol is added to the above Hydroxylamine hydrochloride solution, stirred for 30 minutes. The solids are filtered, washed with 60 ml Methanol and the entire filtrate is cooled to 10-15°C. Subsequently 71 gms of Acetic acid is added dropwise in a duration of 4-10 minutes at 10-15°C. To this solution 100 gms of Cytidine in 2 litres of water is added dropwise at 10-15°C. After complete addition, the reaction is stirred at 40°C for 29-44 hours till analysis indicates the completion of reaction. After 40 hours the solvent and water are evaporated under vacuum and the residue is stripped of the residual water by Toluene. The dry residue is purified by adding 450 ml water and cooling to 10-15°C for 2-3 hours. The solids are filtered and washed with 500ml of Ice- cold water. The HPLC analysis indicates 90% purity. The solids is dried under vacuum at 50-60°C for 3-4 hours. The dried solids weigh 85 gms (78% yield).
The 1H NMR, C13 NMR, and the Mass analysis of (2) indicates the formation of desired product.
(88) (73) 3 (143)
To a 5-L three-neck round bottom flask equipped with an overhead stirrer, nitrogen gas inlet was charged acetone oxime (300.0 g, 4.11 mol, 1.0 equiv.), dichloromethane (3600 mL) followed by Isobutyric acid (414mL, 4.52mol) and EDC. HC1 (942g, 4.92 mol, 1.2 equiv.) was added followed by DMAP (42g, 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 H20 (750 mLX4) and brine solution (750 mLX2). The organic layer was dried over anhydrous Na2S04, evaporated under vacuum in rotavapor to give desired oxime ester as light-yellow oil (585 g, quantitative yield, >98% purity).
1H NMR (400 MHz, CDC13): d 1.17 (d, Hz, 6H), 1.93 (s, 3 H), 1.98 (s, 3 H), 2.60 (7.0 Hz, 1H);
13C NMR (400 MHz, CDC13): d 16.8, 19.0 (2C), 22.0, 33.0, 163.9, 174.2 ppm. Mass Spectra: (M+): 144
(M-): 142
The 1HNMR, C13 NMR & the mass spectra of the isolated matched with the structure of the compound isolated by the above reaction.
Example 2:
Synthesis of Molnupiravir (MK-4482; EIDD-2801):
Example 2a:
In a 2-L three-neck round bottom flask equipped with an overhead stirrer and nitrogen gas inlet was charged N-hydroxycytidine hydrate (NHC.1H20; 100.00 g, 0.361 mol, 1.0 equiv.), followed by tetrahydrofuran (500mL), isobutyryl oxime ester (155 g, 1.08 mol, 3.0 equiv.) and Biocatalyst CALB TA 10000,10000 u/g
expressed in Pichia pastoris (Fermenta biotech Ltd.) (50.0 g, 50 wt%). The reaction mixture was stirred at room temperature. The reaction was continued for 24 hours monitoring the reaction by HPLC. The reaction showed 1.05% for NHC, 33.6 % for product and 56.1% for diacylated product in HPLC at 215 nm. The reaction mass was filtered to separate enzyme from reaction mixture, and the enzyme was washed with tetrahydrofuran (2 X 250 mL) followed by water wash (500mL), enzyme recovered dried and found no loss of activity for reuse.
The combined organic layer was concentrated in vacuo and the residual mass, dissolved in 1 L RO water was charged in a 2-L round bottom flask with nitrogen inlet and Liquid biocatalyst CALB 10L (NLT 10000 TBU/mL Fermenta Biotech Ltd), 30 mL approx. 50% wt of diacylated product) was added. The mixture was stirred at room temperature for 24 hours. The HPLC analysis showed 1.48% for NHC, 83.4 % for product and 1.66 % for diacylated product in the reaction mixture. The water was removed under vacuo and residual mass was striped once toluene(500mL) and again charged with toluene(500mL), solid precipitated, stirred and decanted the solvent and then again charged with methyl tert-butyl ether (MTBE; 1000 mL), stirred at room temperature for 1-2 hours. The solid obtained was filtered, washed with MTBE (2 X 250 mL). The obtained solid was purified by silicagel column chromatography using MDC: MeOH (95:5) as an eluent and isolated a desired product as colourless solid (72g, %Y:61%)
Appearance: Colourless solid M.Pt: 155-158°C Mass Spectra: (M+): 330 (M-): 328
Purity: 99.8% (assessed by HPLC).
1H MR (400 MHz, CD30D): 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. Data matched with those previously reported.
Example 2b:
In a 250mL three-neck round bottom flask equipped with an overhead stirrer and nitrogen gas inlet was charged N-hydroxycytidine hydrate (NHC.1H20; 10.0 g, 0.0361 mol, 1.0 equiv.), followed by tetrahydrofuran(50.0mL), isobutyryl oxime ester (15.5 g, 1.08 mol, 3.0 equiv.) and Biocatalyst CALB TA 10000,10000 u/g expressed in Pichia pastoris (Fermenta biotech Ltd.) (5.0 g, 50 wt%) . The reaction mixture was stirred at room temperature. The reaction was continued for ~24 hours monitoring the reaction by HPLC. The reaction showed 0.49% for NHC, 32.9 % for product and 59.9% for over acylated product in HPLC at 215 nm. The reaction mass was filtered to separate enzyme from reaction mixture, and the enzyme was washed with tetrahydrofuran (2 X 50 mL) followed by water wash (2X100mL), enzyme recovered dried and found no loss of activity for reuse.
The combined organic layer was concentrated in vacuo and the residual mass, dissolved in 100 mL RO water was charged in a 250mL round bottom flask with nitrogen inlet and liquid CALB (3000-20000 u/mL Fermenta Biotech Ltd), 3.0 mL approx 50% wt of diacylated product) was added. The mixture was stirred at room temperature for 24 hours. The HPLC analysis showed 1.0% for NHC, 85.9 % for product, Molnupiravir and 1.2 % for diacylated product in the reaction mixture. The water was removed under vacuo and residual mass was striped once toluene(lOOmL) and again charged with toluene(lOOmL), solid precipitated, stirred and decanted the solvent and then again charged with methyl tert-butyl ether (MTBE; 100 mL), stirred at room temperature for 1-2 hours. The solid obtained was filtered, washed with MTBE (2 X 25 mL). The obtained solid was purified by silicagel column chromatography using MDC: MeOH (95:5) as an eluent and isolated a desired product as colourless solid (7.5g, %Y:65% with purity >98%) Data matched with those reported earlier.
Example 3:
In a 250mL three-neck round bottom flask equipped with an overhead stirrer and nitrogen gas inlet was charged N-hydroxycytidine hydrate (NHC.1H20; 10.0 g, 0.0361 mol, 1.0 equiv.), followed by tetrahydrofuran( 50.0mL), isobutyryl oxime ester (15.5 g, 1.08 mol, 3.0 equiv.) and Biocatalyst CALB TA 10000,10000 u/g expressed in Pichia pastoris(Fermenta biotech Ltd.) (5.0 g, 50 wt%) . The reaction mixture was stirred at room temperature. The reaction was continued for ~24 hours monitoring the reaction by HPLC. The reaction showed 0.6% for NHC, 30.4 % for product and 60.2% for over acylated product in HPLC at 215 nm. The reaction mass was filtered to separate enzyme from reaction mixture, and the enzyme was washed with tetrahydrofuran (2 X 50 mL). The combined organic layer was concentrated in vacuo and the crude mass was purified by silicagel column chromatography using MDC: MeOH 98:2 as an eluent.
Concentration of pure fractions collected gives yield of non-polar product (i.e: Molnupiravir oxime ester (7.0 g, %Y: 81%)
Appearance: white solid,
HPLC purity: 99.7%
Mass Spectra: (M+): 400
(M-): 398
1H NMR (400 MHz, DMSO): d 7.2 (d, 1H), 5.83 (d, 1H), 5.75 (d, 1H), 4.29 (d, 2H), 4.17(t, 1H), 4.11 (p,2H), 2.85(p,lH) ,2.64 (p, 1H), 1.25 (d, 6H), 1.17(d,6H)
Based on the above spectral data, the structure of molecule is confirmed as predicted i.e. Molnupiravir oxime ester.
Further elution of column with solvent MDC: MeOH 95:5 and 90:10 gives the pure fractions of desired product Molnupiravir. Concentration of pure fractions in vacuo gave pure API Molnupiravir as white solid (3.0g). HPLC purity: > 98%
Analytical data matched with those previously reported.
Example 4:
To a solution of Molnupiravir oxime ester (7g) in RO water (70mL) in a 250 mLround bottom flask, Liquid biocatalyst CALB 10L (NLT 10000 TBU/mL Fermenta Biotech Ltd), 7 mL approx. 100% wt of diacylated product was added. The mixture was stirred at room temperature for 12-15 hours. The HPLC analysis showed complete conversion (>90%) of Molnupiravir oxime ester to desired API Molnupiravir with marginal formation of NHC 1-2 %. Water was removed under vacuo and charged with toluene (lOOmL), solid precipitated removed the solvent and again charged with methyl tert-butyl ether (MTBE; 100 mL), stirred at room temperature for 1-2 hours. The solid obtained was filtered, washed with MTBE (2 X 25 mL). The obtained solid was dried in vacuo to get Molnupiravir (4.9 g, %Y :85%).
HPLC purity >98%. Analytical data matched with those previously reported.
Reaction Scheme 6: USING ISOBUTYRIC ANHYDRIDE as a ACEYLATING AGENT
Step 1: Synthesis of 4-N-Hydroxy Cytidine (2)
The synthesis of N-Hydroxy cytidine hydrate (NHC.H20) is carried out as per the Examples 1A and 1 B.
Step 2: Synthesis of MOLNUPIRAVIR (EIDD-2801 ):
1 equivalent of 4-N-Hydroxy Cytidine (2) is suspended in 5-15 volumes of Tetrahydrofuran preferably 12 volumes. 50-200% w/w preferably 60-200% w/w of BIOCATALYST CAL B is added, and the reaction is stirred at 45-50°C. 2-10 equivalents preferably 2.5-7 equivalents more preferably 2.5-6 equivalents of Isobutyric anhydride (6) is added and the reaction is stirred at 80-110 RPM & 40- 65°C for 3- 20 hours. After HPLC and TLC analysis indicates the completion of reaction the reaction mass is filtered, washed with Tetrahydrofuran. The filtered enzyme is washed with 1 litre of water, dried and reused for the next similar reaction. The HPLC analysis of the organic layer indicated 40-60% of Molnupiravir and 36-56% of Molnupiravir Isobutyryl Oxime ester (4). The tetrahydrofuran layer is then treated with LIQUID CAL B 3000-20000 u/ml preferably 7000-18000 u/ml, more preferably 12000-16000u/ml and stirred at 20-45 °C preferably 20-35 °C more preferably at 25-30°C for 6 hours. After 6 hours HPLC analysis indicated 78% - 85% Molnupiravir, 1-3% of Molnupiravir Isobutyryl Oxime ester (4) and 1-4% of 4-N-Hydroxy Cytidine (2). The solvent is evaporated under vacuum and treated with Toluene, stirred at 30-35°C for 1-2 hours, the solid separated is filtered, washed with Toluene and dried. The HPLC analysis indicated 90-93% of Molnupiravir & 3-5% of 4-N-Hydroxy Cytidine (2). This crude mass is purified over column chromatography to yield the desired product Molnupiravir.
EXAMPLE 6: lOOgms (0.3610M, 1 equivalent) of N-4-Hydroxy cytidine (2) is suspended in 1200 ml of Tetrahydrofuran, 200 gms of Biocatalyst CALB TA 10000,10000 u/g (200%w/w) expressed in Pichia pastoris is added and the reaction mass is stirred at
86-102 RPM and 40-50°C. At 45°C 214 gms (1.35moles, 3.75 equivalents) of Isobutyric anhydride (6) is added in one lot and the reaction mass is stirred at 60- 67°C, 86-102 RPM for 3-5 hours. HPLC analysis indicated completion of reaction & formation of Molnupiravir (60%) and 38% of Molnupiravir Isobutyryl Oxime ester (4). The reaction mass is cooled, filtered, washed with 300 ml Tetrahydrofuran. To the combined THF layer is added LIQUID CAL B with strength 15500-16500 u/ml. The mentioned Liquid CAL B is added 5100 u/gm of substrate (2) i.e 25 ml of LIQUID CAL B with strength 15500-16500 u/ml is added and the reaction is stirred at 22-28°C for 4-8 hours. HPLC analysis indicated presence of 10% of Molnupiravir Isobutyryl Oxime ester (4). An additional 6.5 ml of LIQUID CAL B is added, and the reaction is stirred at 22-28°C for additional 2 hours. HPLC analysis indicated 87% Molnupiravir, 3% of Molnupiravir Isobutyryl Oxime ester (4) and 4% of 4-N-Hydroxy Cytidine (2). The reaction mass is evaporated under vacuum and the residue is treated with 200 ml Toluene & stirred overnight. The solid formed are filtered and washed with 100 ml Toluene, dried under vacuum at 45-50°C.
The yield of the isolated solids is 105 gms. The HPLC analysis of the isolated solid indicated 93% Molnupiravir and 4% of 4-N-Hydroxy Cytidine (2). The solids are purified over column chromatography using silica gel 60-200 mesh and Diehl orom ethane: methanol (99:1 and graded up to 90:10 respectively). The fractions containing pure solids are evaporated and dried under vacuum.
The HPLC analysis of the isolated pure MOLNUPIRAVIR indicated 99.4-99.6% pure.
Yield: 85 gms % Yield: 71%.
The 1H NMR, C13 NMR analysis and Mass analysis indicated the formation of MOLNUPIRAVIR and matches with the spectra of Molnupiravir isolated in earlier examples.
EXAMPLE 7: lOOgms (0.3610M, 1 equivalent) of N-4-Hydroxy cytidine (2) is suspended in 1200 ml of Tetrahydrofuran, 100 gms of Biocatalyst CALB TA 10000,10000 u/g (100%w/w), expressed in Pichia pastoris is added and the reaction mass is stirred at 86-102 RPM and 40-50°C. At 45°C 214 gms (1.35moles, 3.75 equivalents) of Isobutyric anhydride (6) is added in one lot and the reaction mass is stirred at 60- 67°C, 86-102 RPM for 3-5 hours. HPLC analysis indicated completion of reaction & formation of 40% Molnupiravir and 56% of Molnupiravir Isobutyryl Oxime ester (4). The reaction mass is cooled, filtered, washed with 300 ml Tetrahydrofuran. To the THF layer is added LIQUID CAL B with strength 15500- 16500 u/ml. The mentioned Liquid CAL B is added 5100 u/gm of substrate (2) i.e 32 ml of LIQUID CAL B with strength 15500-16500 u/ml is added and the reaction is stirred at 22-28°C for 4-8 hours.
HPLC analysis indicated 84% Molnupiravir, 2% of Molnupiravir Isobutyryl Oxime ester (4) and 5% of 4-N-Hydroxy Cytidine (2). The reaction mass is evaporated under vacuum and the residue is treated with 200 ml Toluene & stirred overnight. The solid formed are filtered and washed with 100 ml Toluene, dried under vacuum at 45-50°C.
The yield of the isolated solids is 104 gms. The HPLC analysis of the isolated solid indicated 92% Molnupiravir and 4% of 4-N-Hydroxy Cytidine (2). The solids are purified over column chromatography using silica gel 60-200 mesh and Diehl orom ethane: methanol (99:1 and graded up to 90:10 respectively). The fractions containing pure solids are evaporated and dried under vacuum.
The HPLC analysis of the isolated pure MOLNUPIRAVIR indicated 99.4-99.6% pure.
Yield: 82 gms % Yield: 69%.
The 1H NMR, C13 NMR analysis and Mass analysis indicated the formation of MOLNUPIRAVIR and matches with the spectra of Molnupiravir isolated in earlier examples.
EXAMPLE 8: Repeat of example 2a but isolation and purification method changed. lOOgms (0.3610M, 1 equivalent) of N-4-Hydroxy cytidine (2) is suspended in 1200 ml of Tetrahydrofuran, 200 gms of Biocatalyst CALB TA 10000,10000 u/g (200%w/w) expressed in Pichia pastoris is added and the reaction mass is stirred at 86-102 RPM and 40-50°C. At 45°C 214 gms (1.35moles, 3.75 equivalents) of Isobutyric anhydride (6) is added in one lot and the reaction mass is stirred at 60- 67°C, 86-102 RPM for 3-5 hours. HPLC analysis indicated completion of reaction & formation of Molnupiravir (58%) and 34% of Molnupiravir Isobutyryl Oxime ester (4). The reaction mass is cooled, filtered, washed with 300 ml Tetrahydrofuran.
The entire Tetrahydrofuran is evaporated under vacuum at 35-45 °C. 100 ml of Toluene is added to the residue and stirred at 30-35°C for 2 hours. Solids separates out, filtered, washed with 2* 100 ml of Toluene. The HPLC analysis of the filtered solids indicated 90 % Molnupiravir and 2% Molnupiravir Isobutyryl Oxime ester (4) plus some other impurities.
The yield of the isolated solid after drying is 50 gms. (Part A)
The toluene layer is evaporated under vacuum and the residue weighing 70 gms is dissolved in 5 volumes of Tetrahydrofuran(350ml) and treated with 20 ml LIQUID CAL B with strength 15500-16500 u/ml. and stirred at 20-28°C for 5 hours. After 5 hours HPLC analysis indicated 90% of Molnupiravir, 2% of Molnupiravir Isobutyryl Oxime ester (4) and 5% of 4-N-Hydroxy Cytidine (2). The reaction is stopped, and the solvent evaporated under vacuum and the residue along with the
Part A 50 gms solid is purified over column chromatography using silica gel 60- 200 mesh and Dichloromethane: methanol (99:1 and graded up to 90:10 respectively). The fractions containing pure solids are evaporated and dried under vacuum.
The HPLC analysis of the isolated pure MOLNUPIRAVIR indicated 99.4-99.6% pure.
Yield: 86 gms % Yield: 72%.
The 1H NMR, C13 NMR analysis and Mass analysis indicated the formation of MOLNUPIRAVIR and matches with the spectra of Molnupiravir isolated in earlier examples.
Reaction Scheme 7: USING ISOBUTYL ISOBUTYRATE ((2-METHYL-2- METHYL PROPANOATE) as ACEYLATING AGENT
Molnupiravir
(329)
Example 9:
Synthesis of 4-N-Hydroxy Cytidine (2)
The synthesis of N-Hydroxycytidine hydrate (NHC.H20) is carried out as per Examples 1 A and IB
Step 2: Synthesis of MOLNUPIRAVIR(EIDD-2801):
1 equivalent of 4-N-Hydroxy Cytidine (2) is suspended in 5-15 volumes of Tetrahydrofuran preferably 12 volumes. 100-800% w/w preferably 200-600% w/w of BIOCATALYST CAL B is added, and the reaction is stirred at 45-50°C.2-30 equivalents preferably 2-25 equivalents more preferably 2-155 equivalents of Isobutyl Isobutyrate is added, and the reaction is stirred at 80-110 RPM & 40-65°C for 2- 70 hours. After HPLC and TLC analysis indicates the completion of reaction the reaction mass is filtered, washed with Tetrahydrofuran. The filtered enzyme is washed with 1 litre of water, dried and reused for the next similar reaction. The HPLC analysis of the organic layer indicated 40-60% of Molnupiravir and 36- 56% of 4-N-Hydroxy Cytidine (2). The Tetrahydrofuran layer is then evaporated under vacuum and purified by column chromatography. The product and the starting material are separated by column chromatography to yield the desired product in pure form.
Examples of Step 2;
Crystallization or 329 column chromatography Molnupiravir 329
Molnupiravir
EXAMPLE 10 lOgms (0.03610M, 1 equivalent) of N-4-Hydroxy cytidine (2) is suspended in 1200 ml of Tetrahydrofuran, 20 gms of Biocatalyst CALB TA 10000,10000 u/g
(200%w/w) expressed in Pichia pastoris is added and the reaction mass is stirred at 86-102 RPM and 40-50°C. At 45°C 78 gms (0.541M, 15 equivalents) of Isobutyl isobutyrate (5) is added in one lot and the reaction mass is stirred at 60-67°C, 86- 102 RPM for 3-5 hours. HPLC analysis indicated completion formation of Molnupiravir (52%) and 43% of 4 N- Hydroxy Cytidine (2). The reaction mass is cooled, filtered, washed with 300 ml Tetrahydrofuran. The enzyme is separately washed with 1 litre water, dried and reused again. The Filtrate is evaporated under vacuum and the residue is purified by column chromatography using silica gel 60- 200 mesh and using Diehl or omethane: Methanol 99:1 to 90: 10 gradient system respectively. The desired product Molnupiravir and the unreacted starting material is recovered to be re-used again.
Yields & Purity:
1. Molnupiravir a) Yield: 5.34 gms b) HPLC ANALYSIS: 99%+ c) % Yields based on recovery of starting material: 75%
2. 4-N-Hydroxy Cytidine a) Yield: 4 gms b) HPLC ANALYSIS: 90%+
ANALYSIS:
The 1H NMR, C13 NMR analysis and Mass analysis indicated the formation of MOLNUPIRAVIR and matches with the spectra of Molnupiravir isolated in earlier examples.
EXAMPLE 11: lOgms (0.03610M, 1 equivalent) of N-4-Hydroxy cytidine (2) is suspended in 1200 ml of Tetrahydrofuran, 60 gms of BIOCATALYST CALB 10000 10000 u/g(600%w/w) expressed in Pichia pastoris is added and the reaction mass is stirred at 86-102 RPM and 40-50°C. At 45°C 26 gms (0.1805m, 5 equivalents) of Isobutyl isobutyrate is added in one lot and the reaction mass is stirred at 60-67°C, 86-102 RPM for 70 hours. HPLC analysis indicated completion formation of Molnupiravir
(42%) and 54% of 4 N- Hydroxy Cytidine (2). The reaction mass is cooled, filtered, washed with 300 ml Tetrahydrofuran. The enzyme is separately washed with 1 litre water, dried and reused again. The Filtrate is evaporated under vacuum and the residue is purified by column chromatography using silica gel 60-200 mesh and using Dichloromethane: Methanol 99:1 to 90: 10 gradient system, respectively. The desired product Molnupiravir and the unreacted starting material is recovered to be re-used again.
Yield & Purity:
3. Molnupiravir d) Yield: 4.0 gms e) HPLC ANALYSIS: 99%+ f) % Yields based on recovery of starting material: 74%
4. 4-N-Hydroxy Cytidine c) Yield: 5.5 gms
HPLC ANALYSIS: 90%+
ANALYSIS:
The 1H NMR, C13 NMR analysis and Mass analysis indicated the formation of MOLNUPIRAVIR and matches with the spectra of Molnupiravir isolated in earlier examples.
Claims
1. A twostep process for synthesis of Molnupiravir, which process comprises; a) hydroxy amination of cytidine by reacting with a Hydroxylamine salt optionally presence of catalytic quantity of Acetic acid to obtain hydrate of N-hydroxy cytidine; and b) acylation of the N-hydroxy cytidine by reacting with acylating agents selected from the group consisting of Isobutyric anhydride or Isobutyl isobutyrate (2-Methylpropyl-2-Methyl propanoate) or Isobutyryloxime ester in presence of immobilised CALB Lipase (Candida antartica) enzyme, in an organic solvent to obtain mixture of Molnupiravir and Molnupiravir isobutyryl oxime ester, followed by selective cleavage of Molnupiravir isobutyryl oxime ester using LIQUID CAL B to obtain Molnupiravir.
2. The process as claimed in claim 1, wherein, the Hydroxylamine salt is selected from Hydroxylamine sulphate or Hydroxylamine chloride.
3. The process as claimed in claim 1, wherein, the reaction of step a) is carried out by treating cytidine with Hydroxylamine sulphate in water or isopropanol to obtain hydrate of N-hydroxy cytidine.
4. The process as claimed in claim 1, wherein, the reaction of step a) is carried out by treating cytidine with Hydroxylamine chloride in methanol, water optionally presence of catalytic quantity of Acetic acid to obtain hydrate of N-hydroxy cytidine.
5. The process as claimed in claim 1, wherein, the acylating agents are selected from the group consisting of Isobutyric anhydride; Isobutyl isobutyrate (2- Methylpropyl-2- Methyl propanoate) or Isobutyryloxime ester.
6. The process as claimed in claim 1, wherein, the organic solvent is an ether, preferably THF.
7. The process as claimed in claim 1, wherein the enzyme loading is maintained at 50%-600% w/w of the substrate.
8. The process as claimed in claim 1, wherein, the IMMOBILIZED CAL B used in the enzymatic reaction is CALB TA 10000 10000 u/g, Covalent resins, expressed in Pichia pastoris.
9. The process as claimed in claim 1, wherein, the LIQUID CAL B gene is heterologous, expressed in Pichia pastoris.
10. The process as claimed in claim 1, wherein, the reaction of step a) is carried out at a temperature range of 30 to 80°C.
11. The process as claimed in claim 1, wherein, the reaction of step b) is carried out at room temperature.
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