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EP4590685A1 - Nouveaux nucléosides phosphates stabilisés et leurs analogues - Google Patents

Nouveaux nucléosides phosphates stabilisés et leurs analogues

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Publication number
EP4590685A1
EP4590685A1 EP23782602.9A EP23782602A EP4590685A1 EP 4590685 A1 EP4590685 A1 EP 4590685A1 EP 23782602 A EP23782602 A EP 23782602A EP 4590685 A1 EP4590685 A1 EP 4590685A1
Authority
EP
European Patent Office
Prior art keywords
mmol
compound
equiv
solution
stirred
Prior art date
Legal status (The legal status 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 status listed.)
Pending
Application number
EP23782602.9A
Other languages
German (de)
English (en)
Inventor
Marija Prhavc
Yi Jin
Mahesh Ramaseshan
Renee Desjarlais
Ferdinand Hermann LUTTER
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Janssen Biotech Inc
Original Assignee
Janssen Biotech Inc
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Filing date
Publication date
Application filed by Janssen Biotech Inc filed Critical Janssen Biotech Inc
Publication of EP4590685A1 publication Critical patent/EP4590685A1/fr
Pending legal-status Critical Current

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Classifications

    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/645—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having two nitrogen atoms as the only ring hetero atoms
    • C07F9/6509—Six-membered rings
    • C07F9/6512—Six-membered rings having the nitrogen atoms in positions 1 and 3
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6558—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system
    • C07F9/65586—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system at least one of the hetero rings does not contain nitrogen as ring hetero atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6561—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing systems of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring or ring system, with or without other non-condensed hetero rings
    • 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
    • C07H19/16—Purine radicals

Definitions

  • the present disclosure relates generally to the field of chemistry, molecular biology and genetic engineering, including oligonucleotides and nucleic acid moieties and molecules useful for stabilizing oligonucleotides, and the synthesis and use of the oligonucleotides and nucleic acid moieties and molecules.
  • RNAi Efficient gene silencing by RNAi in vivo requires the recognition and binding of the 5’-phosphate of the guide strand of an siRNA by the Argonaute protein. It has been demonstrated that incorporation of a 5’-E-vinyl-phosphonate 5 ’-(E)- VP increases siRNA accumulation, extends duration of silencing, and protects siRNA from 5 ’-3 ’-exonuclease.
  • X is selected from a 3- to 5-membered cycloalkyl, -CHCH-, a 3- to 5-membered heterocycle, and -CHR 3 CHR 3 -;
  • Y is selected from O or NR’;
  • R’ is a counterion, H or a protecting group;
  • Z is selected from H, a counterion, an activating group, and an oligonucleotide;
  • a and the dashed lines connected are optional, and when present, A is selected from O, S, and CR 4 R 4 ;
  • B is a nucleobase; each R is independently selected from an oligonucleotide, a counterion, H and a protecting group, such as C 1 -C 5 alkyl and POM or C 1 - C 5 alkyl;
  • each R 1 and R 2 is independently selected from H, F, OH, and an optionally substituted O-alkyl, or R 1 and R 2 form an optionally substituted ox
  • R 1 and R 2 are not each H.
  • the compounds represented by the compound of formula (Ia) and (Ib) are represented by the following: (Ia’) and (Ib’).
  • the compound of formula (Ia) and (Ib) are represented by the following: (Ia’’) and (Ib’’).
  • the compound of formula (Ia) is represented by the following: or .
  • X is a cyclobutyl.
  • the cyclobutyl is represented by the following: or wherein the dashed lines represent connection points to adjacent atoms.
  • X is -CHR 3 CHR 3 -, and R 3 is a methylene.
  • X is selected from the following: and wherein the dashed lines represent connection points to adjacent atoms.
  • X is a 3- to 5-membered heterocycle selected from: wherein the dashed lines represent connection points to adjacent atoms.
  • R 2 is an optionally substituted oxetine.
  • X is -CHCH-.
  • Y is O.
  • Z is an activating group.
  • the activating group is represented by: , wherein the dashed line represents a connection point to the adjacent atom.
  • Z is an oligonucleotide.
  • the oligonucleotide is an antisense strand of RNA, preferably an antisense strand of siRNA.
  • the 5’-end of the oligonucleotide is connected to Y.
  • A is O.
  • A is CH 2 or CHF.
  • X is - CHCH-.
  • B is a uracil or thymine.
  • R 1 is H and R 2 is OMe, OEt, MOE, or F.
  • R is a protecting group, such as POM, Et, and Z is an activating group.
  • R is H and Z is an oligonucleotide.
  • R and Z are each oligonucleotide, e.g., the compound of the present disclosure is not at terminal end of an oligonucleotide.
  • R 5 is XP(O)(OR) 2 .
  • R 6 is XP(O)(OR) 2 .
  • the compound is selected from:
  • any numerical value such as a % sequence identity or a % sequence identity range described herein, are to be understood as being modified in all instances by the term “about.”
  • a numerical value typically includes ⁇ 10% of the recited value.
  • a dosage of 10 mg includes 9 mg to 11 mg.
  • the use of a numerical range expressly includes all possible subranges, all individual numerical values within that range, including integers within such ranges and fractions of the values unless the context clearly indicates otherwise.
  • the conjunctive term “and/or” between multiple recited elements is understood as encompassing both individual and combined options.
  • a first option refers to the applicability of the first element without the second.
  • a second option refers to the applicability of the second element without the first.
  • a third option refers to the applicability of the first and second elements together. Any one of these options is understood to fall within the meaning, and therefore satisfy the requirement of the term “and/or” as used herein. Concurrent applicability of more than one of the options is also understood to fall within the meaning, and therefore satisfy the requirement of the term “and/or.” [0015] Unless otherwise indicated, the term “at least” preceding a series of elements is to be understood to refer to every element in the series.
  • nucleobases can include unmodified, natural or modified nucleobases. “Unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).
  • Modified nucleobases include other synthetic and natural nucleobases such as 5- methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2- aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2- thiocytosine, 5-halouracil and cytosine, 5-propynyl (-C ⁇ C-CH 3 ) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8- substituted adenines and guanines
  • nucleobases include tricyclic pyrimidines such as phenoxazine cytidine(1H-pyrimido[5,4-b][l,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][l,4]benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g.9-(2-am-oe1hoxy)-H-pyrimido[5,4-b][l,4]benzoxazin- 2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H- pyrido[3,2 ,5]pyrrolo[2,3-d]pyrimidin-2-one).
  • Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7- deaza-adenine, 7-deazaguanosine, 2-aminopyridine, and 2-pyridone.
  • Alkyl refers to an optionally substituted saturated straight or branched chain hydrocarbon radical. Examples include without limitation methyl, ethyl, propyl, iso-propyl, butyl, iso-butyl, tert-butyl, n-pentyl and n-hexyl.
  • Cycloalkyl refers to cyclic alkyl groups of from 3 to 10 carbon atoms having single or multiple cyclic rings including, by way of example, adamantyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclooctyl and the like. In some embodiments, an optionally substituted alkyl is substituted with hone or more halogen, such as F. [0020] As used herein "heterocycle” refers to a saturated, unsaturated, or aromatic ring system of 3 to 18 atoms that includes at least one N, O, S, or P. In some embodiments, the heterocycle includes one N, O, or S.
  • the heterocycle includes two N, O, or S.
  • “protecting group” refers to a chemical modification of a functional group to obtain chemoselectivity in a subsequent chemical reaction. Examples of protecting groups include those disclosed, e.g., in Greene Wuts, Protective Groups in Organic Synthesis.
  • the term “activating group” includes a moiety that increases the capability of the group to form a covalent bond with another molecule. Activating groups include phosphite- triester, phosphotriester, H-phosphonate, or preferably phosphoramidite group on at least one of the oxygen atoms of the sugar moiety.
  • the activating group is on the C-3′ oxygen or C-5′ oxygen of the nucleic acid monomer.
  • the activating group is on the C-3′ oxygen of the nucleic acid monomer, for synthesizing probes in the 3′ ⁇ 5′ direction, with the oligonucleotide attached to the support via the 3′-end.
  • the activating group is on the C-5′ oxygen of the nucleic acid monomer, for synthesizing probes in the 5′ ⁇ 3′ (“reverse”) direction, with the oligonucleotide attached to the support via the 5′-end.
  • the present disclosure relates compound represented by formula (Ia) or (Ib): O (Ia) and (Ib), wherein: X is selected from a 3- to 5-membered cycloalkyl, -CHCH-, a 3- to 5-membered heterocycle, and -CHR 3 CHR 3 -; Y is selected from O or NR’; R’ is a counterion, H or a protecting group; Z is selected from H, a counterion, an activating group, and an oligonucleotide; A and the dashed lines connected are optional, and when present, A is selected from O, S, and CR 4 R 4 ; B is a nucleobase; each R is independently selected from an oligonucleotide, a counterion, H and a protecting group, such as C 1 -C 5 alkyl and POM or C 1 -C 5 alkyl; each R 1 and R 2 is independently
  • R 1 and R 2 are not each H.
  • the compound of formula (Ia) and (Ib) are represented by the following: [0026] (Ia’) and (Ib’). [0027] In some embodiments, the compound of formula (Ia) and (Ib) are represented by the following: [0028] (Ia’’) and (Ib’’). [0029] In some embodiments, the compound of formula (Ia) is represented by the following: or .
  • X is a cyclobutyl.
  • the cyclobutyl is represented by the following: or , wherein the dashed lines represent connection points to adjacent atoms.
  • X is -CHR 3 CHR 3 -, and R 3 is a methylene.
  • X is selected from the following: , , , and , wherein the dashed lines represent connection points to adjacent atoms.
  • X is a 3- to 5-membered heterocycle has a structure: or , where the curved line includes one or more heteroatom (e.g., N, O, or S).
  • X is a 3- to 5-membered heterocycle selected from: , , , , , , , , wherein the dashed lines represent connection points to adjacent atoms.
  • Y is O.
  • Y is O and X is not cyclopropyl.
  • Z is an activating group.
  • Z is a phosphoramidite or functionally similar moiety.
  • the activating group is represented by: , wherein the dashed line represents A connection point to the adjacent atom.
  • Z is an oligonucleotide.
  • the oligonucleotide is an antisense strand of RNA, preferably an antisense strand of siRNA. In some embodiments, the 5’-end of the oligonucleotide is connected to Y. [0033] In some embodiments, A is O. In some embodiments, A is O and X is not cyclopropyl. In some embodiments, A is CH 2 or CHF.
  • R 1 and R 2 are independently selected from H, F, OH, and an optionally substituted O-alkyl, provided that when X is selected from a 3- to 5-membered cycloalkyl, -CHCH-, a 3- to 5-membered heterocycle, and -CHR 3 CHR 3 - then R 1 and R 2 are not each H, or R 1 and R 2 form an optionally substituted oxetine.
  • the substituted O-alkyl includes —OMe, –OEt, – CH 2 CH 2 OCH 3 (or MOE), –CF 2 CH 2 OCH 3 , – CH 2 CF 2 OCH 3 , –CH 2 CH 2 OCF 3 , –CF 2 CF 2 OCH 3 , –CH 2 CF 2 OCF 3 , –CF 2 CH 2 OCF 3 , –CF 2 CF 2 OCF 3 , —CHFCH 2 OCH 3 , –CHFCHFOCH 3 , –CHFCH 2 OCFH 2 , –CHFCH 2 OCHF 2 and – CH 2 CHFOCH 3 , and the like.
  • B is a uracil.
  • R 1 is H and R 2 is OMe, OEt, MOE, or F.
  • R is a protecting group, such as POM, C 1 -C 5 alkyl (e.g., Et), and Z is an activating group.
  • R is H and Z is an oligonucleotide.
  • the compound is selected from:
  • R 2 is selected from H, F, OH, and an optionally substituted O-alkyl, such as OMe, OEt, MOE, etc. In some embodiments, R 2 is -OMe. In some embodiments, each R is independently selected from an oligonucleotide, a counterion, H and a protecting group, such as POM or C 1 -C 5 alkyl.
  • R is a protecting group, such as POM or C 1 -C 5 alkyl (e.g., Et) and Z is an activating group, such as a phosphoramidite or functionally similar moiety.
  • the activating group is represented by: .
  • R is H and Z is an oligonucleotide.
  • Example 2 Synthesis of Phosphonates
  • Example 2-1 To a solution of potassium tert-butoxide (65.5 g, 583.8mmol, 3.0 equiv) in tert- Butyl methyl ether (1300 mL) with an inert atmosphere of argon was added sec-butyllithium (450 mL, 583.8 mmol, 3.0 equiv) dropwise was stirring at -78°C for 2.5 hours. The resulting solution was added Lithium bromide (101.5 g, 1167.5 mmol, 6.0 equiv) in tetrahydrofuran (1000 mL) dropwise with stirring at -78 °C.
  • the resulting solution was stirring for 30min at - 15 °C.
  • the resulting solution was cooled to -78 °C.
  • the resulting solution was added copper(I) bromide-dimethyl sulfide (60 g, 291.9 mmol, 1.5 equiv) in 6-O-(triisopropylsilyl)- d-galactal cyclic carbonate (420 mL) at -78°C.
  • the resulting solution was stirred at -78°C for 1 hour.
  • the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under vacuum.
  • the crude product was purified by Flash-Prep-HPLC with the following conditions (IntelFlash-1): Column, C18 silica gel; mobile phase, water and acetonitrile (30% acetonitrile up to 100% in 10 min and hold 100% for 5 min); Detector, UV 254 nm. The fraction was dilute with equal volume of dichloromethane. The organic phase layer was separated. The aqueous phase was extracted with 3 x 50mL of dichloromethane. The organic phase was combined and dried over anhydrous sodium sulfate. The solid was filtered out.
  • Example 2-2 To a solution of (3aR,5S,6R,6aR)-5-((R)-2,2-dimethyl-1,3-dioxolan-4-yl)-2,2- dimethyltetrahydrofuro [2,3-d][1,3]dioxol-6-ol (400 g, 1.54 mol, 1.0 equiv) in 4 L of tetrahydrofuran with an inert atmosphere of nitrogen was added sodium hydride (55.2 g, 2.31 mol, 1.5 equiv) at 0°C. The resulting solution was stirred for 20 min at 0°C.
  • the reaction mixture was stirred at room temperature for 12 hours and diluted with 3000 mL of dichloromethane, washed with 2 x 1000 mL of saturated aqueous sodium bicarbonate and 2 x 1000 mL of saturated aqueous sodium chloride respectively.
  • the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure.
  • the residue was purified by Flash-Prep- HPLC with the following conditions: Column, C18 silica gel; mobile phase, water (containing 0.1% FA) and acetonitrile (10% acetonitrile up to 100% in 12 min), UV 254 nm.
  • the solid was filtered out and concentrated under reduced pressure.
  • the crude product was purified by Flash-Prep-HPLC with the following conditions (IntelFlash-1): Column, C18 silica gel; mobile phase, water (containing 0.04% NH 4 HCO 3 ) and acetonitrile (10% acetonitrile up to 100% in 15 min and hold 100% for 5 min); Detector, UV 254 nm.90 g (70%) of 48 was obtained as a light yellow solid.
  • the resulting solution was stirred at room temperature for 12 hours.
  • the resulting solution was extracted with ethyl acetate and the organic layers combined.
  • the organic layer was washed with 1 x 500 mL water and 2 x 500 mL saturated sodium chloride respectively.
  • the mixture was dried over anhydrous sodium sulfate and concentrated under reduced pressure.
  • the crude product was purified by Flash-Prep-HPLC with the following conditions (IntelFlash-1): Column, C18 silica gel; mobile phase, water (containing 0.04% NH 4 HCO 3 ) and acetonitrile (30% acetonitrile up to 100% in 10 min and hold 100% for 5 min); Detector, UV 254 nm.12.5 g (50%) of 201 was obtained as a light yellow solid.
  • the crude product was purified by Flash-Prep-HPLC with the following conditions: Column, C18 silica gel; mobile phase, water (containing 0.04% NH 4 HCO 3 ) and acetonitrile (30% acetonitrile up to 100% in 10 min and hold 100% for 5 min); Detector, UV 254 nm.8.5 g (65%) of 91 was obtained as a light yellow solid.
  • the resulting solution was allowed to react, with stirring, for an additional 1 h at 60°C.
  • the reaction mixture was cooled to 0°C, quenched by the addition of 100 mL saturned aqueous sodium bicarbonate.
  • the resulting solution was extracted with 2 x 300 mL ethyl acetate and the organic layers combined and dried over anhydrous sodium sulfate and concentrated under reduced pressure.
  • the crude product was purified by Flash-Prep-HPLC with the following conditions: Column, C18 silica gel; mobile phase, water (containing 0.04% NH 4 HCO 3 ) and acetonitrile (45% acetonitrile up to 100% in 10 min and hold 100% for 5 min); Detector, UV 254 nm.9 g (60%) of 83 was obtained as a white solid.
  • the crude product was purified by Flash-Prep- HPLC with the following conditions: Column, C18 silica gel; mobile phase, water (containing 0.04% NH 4 HCO 3 ) and acetonitrile (5% acetonitrile up to 50% in 15 min and hold 100% for 5 min); Detector, UV 254 nm.3 g (60%) of the mixture was obtained as a white solid and separated by SFC.1.5 g of 417 was obtained as white solid. MS m/z [M+H] + (ESI):419.00. 31 P NMR (162 MHz, DMSO) ⁇ 30.27.
  • the reacting solution was diluted with 100 mL of dichloromethane, washed with 1 x 20 mL saturated aqueous sodium bicarbonate and 1 x 20 mL saturated aqueous sodium chloride respectively.
  • the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated till no residual solvent left under reduced pressure.
  • the residue was purified by Flash-Prep-HPLC with the following conditions: Column, C18 silica gel; mobile phase, water (containing 0.04% NH 4 HCO 3 ) and acetonitrile (20% acetonitrile up to 100% in 10 min and 100% acetonitrile hold 5 min).
  • the organic phase layer was separated.
  • the aqueous phase was extracted with 3 x 50mL of dichloromethane.
  • the reacting solution was diluted with 20 mL of dichloromethane, washed with saturated aqueous sodium bicarbonate and saturated aqueous sodium chloride respectively.
  • the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated till no residual solvent left under reduced pressure.
  • the residue was purified by Flash-Prep-HPLC with the following conditions: Column, C18 silica gel; mobile phase, water (containing 0.04% NH 4 HCO 3 ) and acetonitrile (20% acetonitrile up to 100% in 10 min and 100% acetonitrile hold 5 min).
  • the organic phase layer was separated.
  • the aqueous phase was extracted with 3 x 50mL of dichloromethane.
  • Example 2-3 To a solution of (3R,4S,5R)-5-(hydroxymethyl) tetrahydrofuran-2,3,4-triol (600 g, 3.997 mol, 1.0 equiv) in 6 L of methanol with an inert atmosphere of argon was slowly added Sulfuric acid (39.19 g, 0.4 mol, 0.1 equiv) at 0°C. The reaction mixture was allowed to warm to room temperature and stirred overnight. The mixture was adjusted to pH 7 with sodium bicarbonate, then filtered and concentrated under reduced pressure to afford 21 (520 g, crude) as yellow oil. MS m/z [M+H] + (ESI): 165.07.
  • the crude product was purified by Flash-Prep-HPLC with the following conditions (IntelFlash-1): Column, C18 silica gel; mobile phase, water (containing 0.04% NH 4 HCO 3 ) and acetonitrile (15% acetonitrile up to 100% in 25 min and hold 100% for 5 min); Detector, UV 254 nm.27 g (62%) of 4-53 was obtained as a yellow solid. MS m/z [M+H] + (ESI): 620.30.
  • the crude product was purified by Flash-Prep-HPLC with the following conditions (IntelFlash-1): Column, C18 silica gel; mobile phase, water (containing 0.04% NH 4 HCO 3 ) and acetonitrile (15% acetonitrile up to 100% in 25 min and hold 100% for 5 min); Detector, UV 254 nm.12 g (68%) of 4-24 was obtained as a yellow oil. MS m/z [M+H] + (ESI): 618.24.
  • reaction mixture was stirred overnight at room temperature and diluted with 200 mL of dichloromethane, washed with 2 x 200 mL of saturated aqueous sodium bicarbonate and 2 x 200 mL of saturated aqueous sodium chloride respectively.
  • the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure.
  • the reacting solution was diluted with 300 ml of dichloromethane, washed with 2 x100 mL of saturated aqueous sodium bicarbonate and 1 x 100 mL of saturated aqueous sodium chloride respectively.
  • the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated till no residual solvent left under reduced pressure.
  • the crude product was purified by Flash-Prep-HPLC with the following conditions (IntelFlash-1): Column, C18 silica gel; mobile phase, water (containing 0.04% NH 4 HCO 3 ) and acetonitrile (30% acetonitrile up to 100% in 15 min and hold 100% for 5 min); Detector, UV 254 nm.
  • Example 2-4 To a solution of L-ascorbic acid (125 g, 0.71 mol, 1.0 equiv) in 1 L of water with an inert atmosphere of argon was slowly added calcium carbonate (125 g, 1.25 mol, 1.76 equiv) over 30 min. The resulting solution was added hydrogen peroxide(250 mL, 30% aq.) dropwise over 1 h with stirring at 0°C. The reaction mixture was allowed to warm to room temperature and stirred overnight. After then, the reaction mixture was filtered and the filter cake was washed with 2 x 100 mL of water. The filtrate was treated with activated carbon (25 g), then heated to 70°C.
  • activated carbon 25 g
  • the organic layer was washed with 3 x 100 mL of water, 3 x 100 mL of saturated aqueous sodium bicarbonate aqueous and 3 x 100 mL of saturated aqueous sodium chloride respectively.
  • the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure.
  • the crude was purified by Flash-Prep-HPLC with the following conditions: Column, C18 silica gel; mobile phase, water (containing 0.1%FA) and acetonitrile (20% acetonitrile up to 100% in 10 min and 100% acetonitrile hold 8 min), UV 254 nm.
  • the fractions were diluted with dichloromethane and dried over anhydrous sodium sulfate.
  • the reaction mixture was stirred for overnight at room temperature and diluted with 300 mL of dichloromethane, washed with 2 x 100 mL of saturated aqueous sodium bicarbonate and 2 x 100 mL of saturated aqueous sodium chloride respectively.
  • the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure.
  • the residue was purified by Flash-Prep- HPLC with the following conditions: Column, C18 silica gel; mobile phase, water (containing 0.1% FA) and acetonitrile (10% acetonitrile up to 100% in 12 min), UV 254 nm.
  • the reaction mixture was quenched by 10 mL of methanol, diluted with 500 mL of dichloromethane, washed with 2 x 200 mL of saturated aqueous sodium bicarbonate and 2 x 200 mL of saturated aqueous sodium chloride respectively.
  • the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure.
  • the residue was dissolved in 400 mL of ammonia (7M NH 3 in methanol) and stirred for 24 h at room temperature with an inert atmosphere of argon.
  • the reaction mixture was concentrated under reduced pressure.
  • the reacting solution was diluted with 200 mL of dichloromethane, washed with 2 x100 mL of saturated aqueous sodium bicarbonate and 1 x 100 mL of saturated aqueous sodium chloride respectively.
  • the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated till no residual solvent left under reduced pressure.
  • the residue was purified by Flash-Prep-HPLC with the following conditions: Column, C18 silica gel; mobile phase, water (containing 0.04% NH 4 HCO 3 ) and acetonitrile (20% acetonitrile up to 100% in 10 min and 100% acetonitrile hold 5 min).
  • the fraction was diluted with 500 mL of dichloromethane.
  • Example 3 Exemplary synthesis of oxetane VP [00126] Under a N 2 atmosphere 1 (Prepared according to: T. Jonckers et al. J. Med. Chem. 2016, 59, 5790 ⁇ 5798) (10.0 g, 25.6 mmol), imidazole (34.9 g, 512 mmol) and DMAP (0.94 g, 7.7 mmol) were dissolved in DMF (100 ml). TBSCl (30.9 g, 205 mmol) was added portion wise at 0 °C. After complete addition the mixture was stirred at 80 °C for 16 h. To the reaction was added sat. aq.
  • reaction mixture was quenched by addition H 2 O 350 mL at 0 °C, then extracted with Ethyl acetate 600 mL (200 mL ⁇ 3). The combined organic layers were washed with brine 200 mL, dried over Na 2 SO 4 , filtered, and concentrated under reduced pressure. The crude was purified by flash silica gel chromatography (PE/EtOAc, gradient from 100:0 to 0:100) to afford compound 25 (21.0 g, 85% yield).
  • the crude was purified by flash silica gel chromatography (heptane with 0.15% TEA/EtOAc with 0.15% TEA, gradient from 100:0 to 0:100) affording 30 (163 mg, 30%) and another fraction of 30 (279 mg, 36%, 70% purity).
  • the syringe was filled with 200 uL of 0.1 M solution of phosphonate amidite in dry acetonitrile and 200 uL of 0.25 M 5-(thylthio)-1H-tetrazole solution in acetonitrile.
  • the syringe was tightly closed and left gently shaken for 40 min at the ambient temperature. Solvents were removed and the operation repeated second time. Solvents were removed and CPG was washed with 0.6 ml of acetonitrile 6 times.
  • 0.05 M Solution of iodine in pyridine/water 9:1 (v:v) was added and kept for 15 min, repeated twice.
  • Step 3 Dry CPG in the syringe was treated with 250 uL of 3.5% solution of trimethylsilyl iodide in acetonitrile/pyridine 50:1 (v/v) for 20 min at ambient temperature. This treatment was repeated 3 times.
  • Step 4 Solvent was removed and CPG was washed 6 times with 0.6 ml of acetonitrile, then treated with solution of 4% mercaptoethanol in pyridine/triethylamine 1:1 (v:v) for 20 min at ambient temperature. Solvent was removed and CPG was washed 6 times with 0.6 ml of acetonitrile, 3 times with dry DCM, then dried in vacuum for 30 min. An aliquot of CPG (2 mg) was taken, treated with AMA for 10 min at 60 °C, and MW of the obtained oligo was checked by LCMS. If the deprotection was not running up to completion the step 3 was repeated. [00158] Step 4.
  • the compound of embodiment 11, wherein the oligonucleotide is an antisense strand of RNA, preferably an antisense strand of siRNA. 13.
  • the compound of embodiment 15, wherein X is -CHCH-.
  • B is a uracil.
  • each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc.
  • all language such as “up to,” “at least,” “greater than,” “less than,” and the like, include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above.
  • a range includes each individual member.

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Abstract

La présente invention comprend des oligonucléotides et des compositions d'acide nucléique ayant des fractions de phosphate stabilisé. Formule (Ia) et Formule (Ib), dans lesquelles : X est choisi parmi un cycloalkyle ayant 3 à 5 chaînons, -CHCH-, un hétérocycle ayant 3 à 5 chaînons, et -CH3CH3-.
EP23782602.9A 2022-09-21 2023-09-21 Nouveaux nucléosides phosphates stabilisés et leurs analogues Pending EP4590685A1 (fr)

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WO2011139699A2 (fr) * 2010-04-28 2011-11-10 Isis Pharmaceuticals, Inc. Nucléosides modifiés en 5' et composés oligomères préparés à partir de ceux-ci
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