Classifications

• • 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
  • C07F9/6527—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having nitrogen and oxygen atoms as the only ring hetero atoms
  • C07F9/6533—Six-membered rings
• • 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
  • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
  • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical

Definitions

• the present invention relates to a method for producing a novel oligonucleic acid compound.
• a solid-phase method and a liquid-phase method are known as methods for producing an oligonucleic acid compound.
• the solid-phase method is a heterogeneous reaction method in which a nucleic acid is extended while a substrate supported on a solid-phase carrier and a solution containing a reaction reagent are brought into contact with each other.
• a so-called batch method is used in which a reaction vessel with a filter is used and the reaction is carried out in the vessel (see, for example, Non-Patent Document 1 and Patent Document 1).
• the liquid phase method is a homogeneous reaction method in which nucleic acids are elongated by reaction in a solution containing both substrates and reaction reagents.
• the liquid phase method also uses a batch method in which the reaction is carried out in a container (see, for example, Patent Documents 2 and 3).
• oligonucleic acid compounds In any of the solid-phase, liquid-phase, batch, and pseudo-flow synthesis methods, the chemical synthesis of oligonucleic acid compounds involves the removal of protective groups for oxygen atoms or amino groups on nucleic acid compounds. Nucleic acid is elongated by repeating the "deprotection” reaction and the “condensation” reaction that forms a bond between the deprotected oxygen or nitrogen atom and the phosphorus atom.
• oligonucleic acid compounds When oligonucleic acid compounds are used as raw materials for pharmaceuticals, oligonucleic acid compounds must be of high purity. Therefore, it is necessary to improve the reaction efficiency and reaction rate while suppressing the formation of by-products in each step of the extension reaction. However, it was difficult because the solvents and reagents that can be used are limited.
• An object of the present invention is to identify impurities that affect the purity of oligonucleic acid compounds and to provide a novel production method for controlling the generation of such impurities.
• the present inventors In the elongation reaction of an oligonucleic acid compound, the present inventors have found that the deprotection reaction and the condensation reaction proceed efficiently and that the pretreatment of the condensation reaction proceeds efficiently by extensively studying the treatment of reaction raw materials and intermediates before condensation. The present inventors have completed the present invention by discovering that impurities produced by the method affect the purity of oligonucleic acid compounds.
• the present invention relates to the following.
• BP is independently an optionally protected nucleobase
• R 1 is trityl, monomethoxytrityl, or dimethoxytrityl
• X is O or S
• Y is dialkylamino or alkoxy
• n is any integer within the range of 1 to 99, preferably any integer within the range of 15 to 30, more preferably any integer within the range of 18 to 28 can be
• L is hydrogen, acyl, or formula (IV): is a group represented by]
• Formula (III) [Wherein, L, B P , X, Y, and n are as defined above]
• halogen e.g., chloro, bromo, iodo
• BP is independently an optionally protected nucleobase
• R 1 is trityl, monomethoxytrityl, or dimethoxytrityl
• X is O or S
• Y is dialkylamino or alkoxy
• n is any integer within the range of 1 to 99, preferably any integer within the range of 15 to 30, more preferably any integer within the range of 18 to 28.
• L is hydrogen, acyl, or formula (IV): is a group represented by], Formula (II): [Wherein, L, B P , X, Y, R 1 and n are as defined above] a compound of removing R 1 from a compound of formula (II) by treatment with a solution containing an acid and a scavenger; Formula (III): [Wherein, L, B P , X, Y, and n are as defined above] a step of forming a compound of Formula (III): [Wherein, L, B P , X, Y, and n are as defined above] a compound of treating with a solution comprising an organic amine and an aprotic polar solvent, and formula (VIII): [Wherein, B P , X, Y, Z, and R 1 are as defined above] a compound of A method for producing a morpholino nucleic acid oligomer, comprising the step of treating with a solution containing an organic amine
• BP is independently an optionally protected nucleobase
• R 1 is trityl, monomethoxytrityl, or dimethoxytrityl
• X is O or S
• Y is dialkylamino or alkoxy
• n is any integer within the range of 1 to 99, preferably any integer within the range of 15 to 30, more preferably any integer within the range of 18 to 28 can be
• L is hydrogen, acyl, or formula (IV): is a group represented by]
• ⁇ 9> The method for producing a morpholino nucleic acid oligomer according to any one of ⁇ 1> to ⁇ 8>, wherein the solution containing an acid and a scavenger is a solution containing trifluoroacetic acid and triisopropylsilane.
• ⁇ 10> The solution according to any one of ⁇ 1> to ⁇ 9>, wherein the solution containing the acid and scavenger is a solution containing trifluoroacetic acid, triethylamine, triisopropylsilane, 2,2,2-trifluoroethanol, and dichloromethane.
• the solution containing the acid and scavenger is a solution containing trifluoroacetic acid, triethylamine, triisopropylsilane, 2,2,2-trifluoroethanol, and dichloromethane.
• ⁇ 11> The morpholino nucleic acid oligomer according to any one of ⁇ 1> to ⁇ 9>, wherein the solution containing an organic amine and an aprotic polar solvent is N-ethylmorpholine and 1,3-dimethyl-2-imidazolidinone. Production method.
• the solid support is swelling polystyrene, non-swelling polystyrene, PEG chain-linked polystyrene, fixed pore glass, oxalylated - fixed pore glass, TentaGel support - aminopolyethylene glycol derivatized support, or Poros - polystyrene/divinyl
• the linker is short-chain alkylene, long-chain alkylene, amino-short-chain alkylene, amino-long-chain alkylene, diacyl-short-chain alkylene (e.g., succinyl), diacyl-long-chain alkylene, or dialkylenesulfonyl, ⁇ 1>-
• the present invention enables economical, high-yield, high-purity production of morpholino nucleic acid oligomers.
• the morpholino nucleic acid oligomer has the formula: [Wherein, Base represents a nucleic acid base, and X and Y are as described above] It is an oligomer having a group represented by as a constituent unit.
• nucleobase can include, for example, adenine, guanine, hypoxanthine, cytosine, thymine, uracil, or modified bases thereof.
• Such modified bases include, for example, pseudouracil, 3-methyluracil, dihydrouracil, 5-alkylcytosine (eg, 5-methylcytosine), 5-alkyluracil (eg, 5-ethyluracil), 5-halouracil (eg, , 5-bromouracil, 5-fluorouracil), 6-azapyrimidine, 6-alkylpyrimidine (e.g., 6-methyluracil), 2-thiouracil, 4-thiouracil, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil , 5′-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, 1-methyladenine, 1-methylhypoxanthine, 2,2-dimethylguanine, 3-methylcytosine, 2-methyladenine, 2 -methylguanine, N 6 -methyladenine, 7-methylguanine, 5-methoxyaminomethyl-2-thiouracil, 5-methyl
• One aspect of the invention is a compound of formula (I): [In the formula, Base, X, Y are as defined above; n is any integer within the range of 1-99, preferably any integer within the range of 15-30, more preferably any integer within the range of 18-28 ] is a method for producing a morpholino nucleic acid oligomer (hereinafter referred to as a morpholino nucleic acid oligomer (I)).
• One aspect of the invention is a morpholino nucleic acid oligomer extension reaction wherein a compound of formula (II) is reacted with a compound of formula (VIII) to give a compound of formula (II'):
• BP is independently an optionally protected nucleobase
• R 1 is trityl, monomethoxytrityl, or dimethoxytrityl
• X is O or S
• Y is OH, lower alkoxy, mono-lower alkylamino, or di-lower alkylamino
• Z is halogen (e.g.
• n is any integer within the range of 1 to 99, preferably any integer within the range of 15 to 30, more preferably any integer within the range of 18 to 28 can be;
• L is hydrogen, acyl, or formula (IV): is a group represented by], a method for producing a morpholino nucleic acid oligomer.
• optionally protected nucleobases include, for example, both unprotected “nucleobases” and protected “nucleobases”, such as amino and/or hydroxyl are unprotected or protected, adenine, guanine, hypoxanthine, cytosine, thymine, uracil, and the like.
• the amino group-protecting group is not particularly limited as long as it is used as a nucleic acid-protecting group, and specific examples include benzoyl, 4-methoxybenzoyl, acetyl, propionyl, and butyryl. , isobutyryl, phenylacetyl, phenoxyacetyl, 4-tert-butylphenoxyacetyl, 4-isopropylphenoxyacetyl, (dimethylamino)methylene. Benzoyl, acetyl, phenoxyacetyl, and 4-tert-butylphenoxyacetyl are preferred as amino-protecting groups.
• hydroxyl-protecting groups include 2-cyanoethyl, 4-nitrophenethyl, phenylsulfonylethyl, methylsulfonylethyl, trimethylsilylethyl, and substituents substituted with 1 to 5 electron-withdrawing groups at any substitutable position.
• phenyl diphenylcarbamoyl, dimethylcarbamoyl, diethylcarbamoyl, methylphenylcarbamoyl, 1-pyrrolidinylcarbamoyl, morpholinocarbamoyl, 4-(tert-butylcarboxy)benzyl, 4-[(dimethylamino)carboxy]benzyl, 4-( phenylcarboxy)benzyl (see, eg, WO 2009/064471 A1).
• Preferred hydroxyl-protecting groups are 2-cyanoethyl, 4-nitrophenethyl and 4-(tert-butylcarboxy)benzyl. 2-cyanoethyl is preferable as the protective group for the hydroxyl group at the 6-position of guanine.
• the protected nucleobase has, for example, the formula: [Wherein, Pg represents a protecting group] group.
• the protected nucleobases are, for example, adenine with a benzoyl-protected amino group (A Bz ), cytosine with a benzoyl-protected amino group (C Bz ), 2-cyanoethyl and the amino group is protected with phenoxyacetyl (G CE ), and the like, but are not limited to these.
• the solid-phase carrier is not particularly limited as long as it can be used for solid-phase reaction of nucleic acids.
• the solid phase carrier is, for example, swelling polystyrene (for example, aminomethyl polystyrene resin 1% divinylbenzene crosslinked (200-400 mesh) (2.4-3.0 mmol/g) (Tokyo Chemical Industry Co., Ltd.
• swelling polystyrene for example, aminomethyl polystyrene resin 1% divinylbenzene crosslinked (200-400 mesh) (2.4-3.0 mmol/g) (Tokyo Chemical Industry Co., Ltd.
• AMINOMETHYLATED Polystyrene Resin / HCL [1 % of dibinyl venzene, 100 to 200 mesh], AMINOMETHYL RESIN [Polystyrene 1 % DIVINYLB ENZENE COPOLYMER, 200-400 MESH] (manufactured by Watanabe Chemical Co., Ltd.)
• Non-swelling polystyrene e.g., Primer Support (manufactured by GE Healthcare)
• PEG chain-bonded polystyrene e.g., NH2-PEG resin (manufactured by Watanabe Chemical Industry Co., Ltd.)
• TentaGel resin controlled pore glass (CPG)
• CPG controlled pore glass
• oxalylated-constant pore glass see, for example, Alul et al., Nucleic Acids Research, Vol. 19, 1527 (1991)
• the linker can be a known linker used for linking nucleic acids or morpholino nucleic acid derivatives, such as short-chain alkylene, long-chain alkylene, amino-short-chain alkylene, amino - long chain alkylene, diacyl short chain alkylene (e.g. succinyl), diacyl long chain alkylene, dialkylenesulfonyl, preferably 3-aminopropyl, succinyl, 2,2'-diethanolsulfonyl, long chain alkylamino (LCAA) can be mentioned.
• LCAA long chain alkylamino
• L is represented by formula (IV): A group represented by the formula (IV-1): or a group represented by formula (IV-2): It may be a group represented by
• Formula (II): [Wherein, L, B P , X, Y, R 1 and n are as defined above] can be prepared by methods known in the art (see, eg, WO2012/043730).
• a step of producing a compound of formula (VI) (hereinafter referred to as compound (VI)) by reacting a compound of formula (V) with an acylating agent:
• an acylating agent e.g., succinyl
• diacyl long-chain alkylene e.g., succinyl
• diacyl long-chain alkylene e.g., short-chain alkylene, long-chain alkylene, or dialkylenesulfonyl
• R 6 is hydroxyl, halogen, or amino
• This step can be carried out by introducing a known linker using compound (V) as a starting material.
• formula (VIa): [Wherein, B P and R 1 are as defined above] can be prepared by carrying out a method known as an esterification reaction using compound (V) and succinic anhydride.
• a condensing agent or the like can include
• compound (VI) and a solid phase carrier can be used for production by a method known as condensation reaction.
• the compound of formula (VIII) includes, for example, compounds listed in Table 1 below.
• washing step before deprotection step> ⁇ (2) deprotection step>, ⁇ (3) Neutralization step>, ⁇ (4) Washing step after neutralization step>, ⁇ (5) Washing step before condensation>, ⁇ (6) condensation step>, ⁇ (7) Washing step 1 after condensation step> and ⁇ (8) Washing step 2 after condensation step>
• a method for producing a morpholino nucleic acid oligomer comprising at least one step selected from the group consisting of
• This step comprises formula (II): [wherein L, B P , X, Y, R 1 and n are as defined above], (1) Including treatment with a solution containing alcohols and/or halogen solvents.
• the solution in step (1) is not particularly limited as long as it contains alcohols and/or halogen solvents, such as 2,2,2-trifluoroethanol, difluoroethanol, chloroform, and dichloromethane. At least one selected from the group consisting of can be used, preferably 2,2,2-trifluoroethanol and dichloromethane can be used.
• alcohols and/or halogen solvents such as 2,2,2-trifluoroethanol, difluoroethanol, chloroform, and dichloromethane.
• At least one selected from the group consisting of can be used, preferably 2,2,2-trifluoroethanol and dichloromethane can be used.
• the solution of step (1) contains an alcohol relative to the halogen solvent, for example, 0.01 to 100 times by volume, preferably 0.1 to 2 times (v/v ) can be used in
• the amount of the solution used in step (1) is not particularly limited. and preferably within the range of 2 to 100 times the amount.
• the time of step (1) is not particularly limited, and is, for example, 0.1 minute to 24 hours, preferably 1 minute to 5 hours, more preferably 3 minutes to 1 hour.
• the temperature in step (1) is not particularly limited, and is, for example, 0°C to 40°C, preferably 10°C to 35°C, more preferably 15°C to 30°C.
• the number of times of step (1) is not particularly limited, and is, for example, 1 to 10 times, preferably 1 to 5 times.
• This step comprises formula (II): [wherein L, B P , X, Y, R 1 and n are as defined above], (2) removing R 1 from the compound of formula (II) by treatment with a solution containing an acid and a scavenger; Formula (III): wherein L, B P , X, Y, and n are as defined above.
• the acid can be, for example, hydrochloric acid, trifluoroacetic acid, dichloroacetic acid, or trichloroacetic acid, preferably trifluoroacetic acid.
• the amount of the acid used in (2) is, for example, in the range of 1 to 500-fold, preferably 2 to 100-fold, in terms of molar ratio relative to 1 mol of compound (II). is within.
• the acid can be diluted with a suitable solvent to a concentration within the range of 0.1% to 30%.
• the solvent to be used is not particularly limited as long as it does not participate in the reaction. , or mixtures thereof.
• organic amine can be used with the acid.
• organic amine include, but are not limited to, triethylamine, N,N-diisopropylethylamine, N-ethylmorpholine, pyridine and the like, preferably triethylamine.
• the amount of the organic amine used in (2) is, for example, 0.01 to 10 times, preferably 0.1 to 2 times, the molar ratio of 1 mol of the acid. within the range.
• ⁇ Ratio of acid to organic amine in (2)> when a salt or mixture of an acid and an organic amine is used, it can include, for example, a salt or mixture of trifluoroacetic acid and triethylamine, for example, to 1.2 equivalents of trifluoroacetic acid A mixture of 1 equivalent of triethylamine can be mentioned.
• scavengers are, for example, alcohols (including, for example, methanol, ethanol, 2,2,2-trifluoroethanol), thiols (mercaptoethanol, dithiothreitol, mercaptosuccinic acid), organosilicon selected from the group consisting of compounds (including, for example, triisopropylsilane, triethylsilane, trimethylsilane), and aromatic compounds (including, for example, 1-hydroxybenzotriazole, pyrrole, indole, anisole, and thioanisole) is at least one, preferably an organosilicon compound.
• alcohols including, for example, methanol, ethanol, 2,2,2-trifluoroethanol
• thiols mercaptoethanol, dithiothreitol, mercaptosuccinic acid
• organosilicon selected from the group consisting of compounds (including, for example, triisopropylsilane, triethylsilane, tri
• scavengers are, for example, methanol, ethanol, mercaptoethanol, 2,2,2-trifluoroethanol, triisopropylsilane, triethylsilane, trimethylsilane, 1-hydroxybenzotriazole, pyrrole, indole, anisole, and It is at least one selected from the group consisting of thioanisole, preferably one selected from the group consisting of triisopropylsilane and ethanol, more preferably triisopropylsilane.
• the amount of the scavenger to be used is, for example, in a molar ratio of 0.1 to 1000 times, preferably 0.5 times, relative to 1 mol of compound (II). It is in the range of 1 to 100 times the amount, more preferably in the range of 1 to 20 times the amount.
• reaction time is, for example, within the range of 0.1 minutes to 24 hours, preferably within the range of 1 minute to 3 hours.
• reaction temperature is, for example, preferably within the range of 0°C to 40°C, more preferably within the range of 10°C to 35°C, and even more preferably within the range of 15°C to 30°C. be.
• the number of times of step (2) is not particularly limited, and is, for example, 1 to 10 times, preferably 1 to 8 times.
• Formula (II) [wherein L, B P , X, Y, R 1 and n are as defined above], (1) treatment with a solution containing alcohols and/or halogen solvents, followed by (2) treatment with a solution containing an acid and a scavenger to remove R1
• Formula (III) [wherein L, B P , X, Y, and n are as defined above]. Steps (1) and (2) are as described above and can be combined as appropriate.
• This process is Formula (III): [wherein L, B P , X, Y, and n are as defined above], (3) Including treatment with a solution containing a base and a solvent (alcohols and halogen solvents).
• the base is not particularly limited as long as it does not affect the protecting group, and examples thereof include triethylamine, N,N-diisopropylethylamine, pyridine and the like, preferably N,N-diisopropylethylamine.
• the amount of base used in (3) can be diluted with an appropriate solvent so as to have a concentration within the range of 0.1% (v/v) to 30% (v/v).
• the amount of the solution used in step (3) is not particularly limited. and preferably within the range of 2 to 100 times the amount.
• the solvent is at least one selected from the group consisting of alcohols, halogen solvents, polar solvents, ether solvents, and mixtures thereof, preferably mixtures of alcohols and halogen solvents.
• alcohols are ethanol, isopropyl alcohol, 2,2,2-trifluoroethanol, etc., preferably isopropyl alcohol.
• the halogen solvent is chloroform, dichloromethane, tetrachloroethane, tetrachloroethylene, and the like, preferably dichloromethane.
• the polar solvent is acetonitrile, dimethylsulfoxide, and the like.
• ether solvents are tetrahydrofuran, cyclopentyl methyl ether, and the like.
• reaction time varies depending on the type of base used and the reaction temperature, but is, for example, within the range of 0.1 minute to 24 hours, preferably within the range of 1 minute to 5 hours.
• reaction temperature is, for example, preferably in the range of 0° C. to 40° C., more preferably in the range of 1° C. to 35° C., still more preferably in the range of 3° C. to 20° C. be.
• the number of times of step (3) is not particularly limited, and is, for example, 1 to 10 times, preferably 1 to 5 times.
• It can be treated with a solution containing a base and a solvent (alcohols and halogen solvents). Steps (2) and (3) are as described above and can be combined as appropriate.
• This step comprises formula (III): [wherein L, B P , X, Y, and n are as defined above], (4) including treatment with a solution containing a halogen solvent;
• the solution in step (4) is not particularly limited as long as it contains a halogen solvent, for example, at least one selected from the group consisting of chloroform, dichloromethane, tetrachloroethane, and tetrachloroethylene. and is preferably dichloromethane.
• a halogen solvent for example, at least one selected from the group consisting of chloroform, dichloromethane, tetrachloroethane, and tetrachloroethylene. and is preferably dichloromethane.
• the amount of the solution used in step (4) is not particularly limited, and is, for example, in the range of 1 to 1000 times the weight of 1 g of compound (III). , preferably in the range of 2 to 100 times the amount.
• the time of step (4) is not particularly limited, and is, for example, 0.1 minute to 2 hours, preferably 1 minute to 60 minutes, more preferably 3 minutes to 30 minutes.
• the temperature in step (4) is not particularly limited, and is, for example, 0°C to 35°C, preferably 10°C to 30°C, more preferably 15°C to 25°C.
• the number of times of step (4) is not particularly limited, and is, for example, 1 to 10 times, preferably 1 to 5 times.
• Formula (II) [wherein L, B P , X, Y, R 1 and n are as defined above], (2) removing R 1 from the compound of formula (II) by treatment with a solution containing an acid and a scavenger;
• Steps (1), (2), and (3) are as described above, and can be combined as appropriate.
• This step comprises formula (III): [wherein L, B P , X, Y, and n are as defined above], (5) treatment with a solution containing an organic amine and an aprotic polar solvent;
• the organic amine is, for example, at least one selected from the group consisting of N,N-diisopropylethylamine, triethylamine, and N-ethylmorpholine, preferably N-ethylmorpholine.
• the amount of the organic amine used in (5) is, for example, in the range of 1 to 1000 times, preferably 1 to 100 times the molar ratio of the compound of the formula (III) to 1 mol. It is within the double dose range.
• the aprotic polar solvent is selected from the group consisting of, for example, 1,3-dimethyl-2-imidazolidinone, N-methylpyrrolidone, N,N-dimethylformamide, tetrahydrofuran, or mixtures thereof. and preferably 1,3-dimethyl-2-imidazolidinone.
• the amount of the aprotic polar solvent used in (5) is, for example, in the range of 1 to 1000 times (v/v), preferably 3 times the volume of the organic amine. It is in the range of ⁇ 100 times the amount.
• the amount of the solution used in step (5) is not particularly limited. within the range, preferably within the range of 1 to 50 times the amount.
• the reaction time varies depending on the type of organic amine used and the reaction temperature, but is usually within the range of 0.1 minute to 2 hours, preferably within the range of 1 minute to 60 minutes.
• reaction temperature is, for example, preferably in the range of 0°C to 100°C, more preferably in the range of 10°C to 50°C.
• the number of times of step (5) is not particularly limited, and is, for example, 1 to 10 times, preferably 1 to 5 times.
• This step is represented by formula (VIII): [wherein B P , X, Y, Z, and R 1 are as defined above], (6) treatment with a solution containing an organic amine and an aprotic polar solvent, for example dissolving in a mixed solution containing an organic amine and an aprotic polar solvent;
• the organic amine is, for example, at least one selected from the group consisting of N,N-diisopropylethylamine, triethylamine, and N-ethylmorpholine, preferably N-ethylmorpholine.
• the amount of the organic amine to be used is, for example, in the range of 0.1 to 1000 times the molar ratio, preferably 1 time, relative to 1 mol of the compound of the formula (VIII). amount to 100 times the amount.
• the aprotic polar solvent is selected from the group consisting of, for example, 1,3-dimethyl-2-imidazolidinone, N-methylpyrrolidone, N,N-dimethylformamide, tetrahydrofuran, or mixtures thereof. and preferably 1,3-dimethyl-2-imidazolidinone.
• the amount of the aprotic polar solvent used in (6) is, for example, in the range of 1 to 1000 times (v/v), preferably 3 times the volume of the organic amine. It is in the range of ⁇ 100 times the amount.
• the treatment time varies depending on the type of organic amine used and the reaction temperature, but it is usually in the range of 0.1 minute to 48 hours, preferably in the range of 1 minute to 24 hours.
• the treatment temperature is, for example, preferably in the range of 0°C to 50°C, more preferably in the range of 10°C to 40°C. be.
• This step further comprises formula (III): [Wherein, L, B P , X, Y, and n are as defined above] and in the presence of an organic amine, Formula (VIII): reacting a compound of [wherein X, Y, Z, B P and R 1 are as defined above], Formula (II''): wherein L, B P , X, Y, R 1 , and n are as defined above.
• the compound of formula (VIII) is, for example, in a molar ratio of 1 to 10 times, preferably 1 time, relative to 1 mol of the compound of formula (III). It is within the range of the amount to 5 times the amount.
• the organic amine is, for example, at least one selected from the group consisting of N,N-diisopropylethylamine, triethylamine, and N-ethylmorpholine, preferably N-ethylmorpholine.
• the amount of the organic amine to be used is, for example, in the range of 0.1 to 1000-fold, preferably 1-fold, relative to 1 mol of the compound of the formula (VIII). It is in the range of ⁇ 100 times the amount.
• the reaction time varies depending on the type of organic amine used and the reaction temperature, but it is usually within the range of 10 minutes to 48 hours, preferably within the range of 30 minutes to 24 hours.
• the reaction temperature is, for example, preferably within the range of 0°C to 100°C, more preferably within the range of 10°C to 50°C.
• the number of times of step (6) is not particularly limited, and is, for example, 1 to 3 times, preferably 1 time.
• Formula (III) [wherein L, B P , X, Y, and n are as defined above], (5) treated with a solution containing an organic amine and an aprotic polar solvent, and formula (VIII): [wherein B P , X, Y, Z, and R 1 are as defined above], (6) after treatment with a solution containing an organic amine and an aprotic polar solvent, Formula (III): In the presence of a compound of [wherein L, B P , X, Y, and n are as defined above] and an organic amine, Formula (VIII): [wherein X, Y, Z, B P and R 1 are as defined above], Formula (II'): [wherein L, B P , X, Y, R 1 and n are as defined above]. Steps (5) and (6) are as described above and can be combined as appropriate.
• This step is represented by the formula (II'): (7) treating the compound of wherein L, B P , X, Y, R 1 , and n are as defined above with a solution comprising a halogen solvent;
• the cleaning agent in step (7) is not particularly limited as long as it contains a halogen solvent, for example, at least one selected from the group consisting of chloroform, dichloromethane, tetrachloroethane, and tetrachloroethylene. can be used, preferably dichloromethane.
• the amount of the detergent to be used in the step (7) is not particularly limited. and preferably within the range of 2 to 100 times the amount.
• the time of step (7) is not particularly limited, and is, for example, 0.1 minute to 24 hours, preferably 1 minute to 5 hours, more preferably 3 minutes to 1 hour.
• the temperature in step (7) is not particularly limited, and is, for example, 0°C to 40°C, preferably 10°C to 35°C, more preferably 15°C to 30°C.
• This step is represented by the formula (II'): (8) treating the compound of [wherein L, B P , X, Y, R 1 and n are as defined above] with a solution comprising an alcohol and/or a halogen solvent;
• the solution in step (8) is not particularly limited as long as it contains alcohols and/or halogen solvents, such as 2,2,2-trifluoroethanol, difluoroethanol, chloroform, and dichloromethane. At least one selected from the group consisting of can be used, preferably 2,2,2-trifluoroethanol and dichloromethane can be used.
• alcohols and/or halogen solvents such as 2,2,2-trifluoroethanol, difluoroethanol, chloroform, and dichloromethane.
• At least one selected from the group consisting of can be used, preferably 2,2,2-trifluoroethanol and dichloromethane can be used.
• the solution of the step (8) uses an alcohol at a ratio of, for example, 0.01-fold to 100-fold, preferably 0.1-fold to 2-fold (v/v) with respect to the halogen solvent. can do.
• the amount of the solution used in step (8) is not particularly limited. within the range of 2 to 100 times the amount.
• the time of step (8) is not particularly limited, and is, for example, 0.1 minute to 24 hours, preferably 1 minute to 5 hours, more preferably 3 minutes to 1 hour.
• the temperature in step (8) is not particularly limited, and is, for example, 0°C to 40°C, preferably 10°C to 35°C, more preferably 15°C to 30°C.
• the number of times of step (8) is not particularly limited, and is, for example, 1 to 10 times, preferably 1 to 5 times.
• One aspect of the present invention is the extension reaction: Followinged by, ⁇ (A) protecting group removal step>, ⁇ (B) Step of removing protecting group of morpholino nitrogen> and ⁇ (C) Purification step>
• a method for producing a morpholino nucleic acid oligomer comprising at least one step selected from
• this step is a step of obtaining a compound of formula (I'') from a compound of formula (II''): [Wherein, Base, B P , R 1 , L, n, X and Y are as defined above] including.
• this step is a step of obtaining a compound of formula (I) from a compound of formula (II''): [Wherein, Base, B P , R 1 , L, n, X and Y are as defined above] including.
• this step is a step of obtaining a compound of formula (I'''') from a compound of formula (II''): [Wherein, B P , R 1 , L, n, X, and Y are as defined above] including.
• this step is a step of obtaining a compound of formula (I'''') from a compound of formula (II''): [Wherein, B P , R 1 , L, n, X, and Y are as defined above] including.
• L in the compound of formula (II'') and the optionally protected nucleobase protecting group in the compound of formula (II''); and L in the compound of formula (II''') and the formula ( The protecting group of the nucleobase which may be protected in the compound of II'') is deprotected according to the type or property of the solid phase carrier, linker, and protecting group to obtain the desired compound. can be manufactured. For example, according to the deprotection method described in "Green's PROTECTIVE GROUPS in ORGANIC SYNTHESIS, 4th Edition, 2006", all protecting groups possessed by the compound can be removed. If desired, the compound of interest can be subjected to further chemical structural transformations.
• L in the compound of formula (II''); and L in the compound of formula (II''') can produce the desired compound by carrying out deprotection treatment depending on the type or property of the solid phase support and linker.
• the target compound can be produced according to the method described herein or the deprotection method described in "Green's PROTECTIVE GROUPS in ORGANIC SYNTHESIS, 4th Edition, 2006", all protecting groups possessed by the compound can be removed. .
• the compound of interest can be subjected to further chemical structural transformations.
• the amount of (1) ammonia water, (2) ammonia water/ethanol or (3) methylamine-methanol solution/water mixed solution is, for example, 1 g of the compound of formula (II'') or
• the weight ratio is in the range of 1 to 1000 times, preferably in the range of 3 to 100 times the weight of 1 g of the compound of formula (II''').
• the reaction time varies depending on the reaction temperature and the like, but is in the range of 10 minutes to 120 hours, preferably in the range of 30 minutes to 72 hours, more preferably in the range of 5 hours to 48 hours. is within.
• the reaction temperature is, for example, within the range of 5°C to 100°C, preferably within the range of 10°C to 70°C, and more preferably within the range of 15°C to 50°C.
• this step comprises removing R 1 in the compound of formula (II') from the compound of formula (II') to obtain a compound of formula (II'''): [wherein, B P , L, n, R 1 , X and Y are as defined above. ] including.
• this step comprises removing R 1 in the compound of formula (I') from the compound of formula (I') to obtain the compound of formula (I): [In the formula, Base, X, Y, n, and R 1 are as defined above. ] including.
• the step of removing R 1 in the compound of I′) to obtain the compound of formula (I); and the conditions used in steps (1) to (4) above can be applied.
• this step separates the morpholino nucleic acid oligomer (I) from the reaction mixture by conventional separation and purification means such as extraction, concentration, neutralization, filtration, centrifugation, precipitation, recrystallization, C1 to C18 . Isolated by using means such as reversed phase column chromatography, cation exchange column chromatography, anion exchange column chromatography, gel filtration column chromatography, high performance liquid chromatography, dialysis, ultrafiltration, etc. alone or in combination (see, eg, WO 1991/09033 A1).
• a mixed solution of, for example, 20 mM triethylamine/acetic acid buffer and acetonitrile can be used as an elution solvent.
• a mixed solution of 1 M saline and 10 mM sodium hydroxide aqueous solution or 0.3 M of 50 mM phosphate buffer Saline can be used.
• the production method of the present invention includes ⁇ (2) deprotection step>.
• the production method of the present invention includes ⁇ (2) deprotection step> and ⁇ (3) neutralization step>. including.
• the production method of the present invention includes ⁇ (2) deprotection step>, ⁇ (3) neutralization step>, and ⁇ (4) washing step after neutralization step>. including.
• the production method of the present invention includes ⁇ (5) washing step before condensation> and ⁇ (6) condensation step>.
• the production method of the present invention includes ⁇ (5) washing step before condensation>, ⁇ (6) condensation step>, and ⁇ (7) washing step 1 after condensation step> and/or ⁇ (8) Washing step 2> after the condensation step.
• the production method of the present invention includes ⁇ (2) Deprotection step>, ⁇ (5) Washing step before condensation>, and ⁇ (6) Condensation step>.
• the production method of the present invention includes ⁇ (2) deprotection step>, ⁇ (3) neutralization step>, ⁇ (5) washing step before condensation>, and ⁇ (6) condensation step >.
• the production method of the present invention comprises ⁇ (1) Washing step before deprotection step>, ⁇ (2) deprotection step>, ⁇ (3) Neutralization step>, ⁇ (4) Washing step after neutralization step>, ⁇ (5) Washing step before condensation>, ⁇ (6) condensation step>, ⁇ (7) Washing step 1 after condensation step> and ⁇ (8) Washing step 2 after condensation step> including.
• the production method of the present invention does not include ⁇ (5) washing step before condensation>.
• the production method of the present invention comprises ⁇ (1) Washing step before deprotection step>, ⁇ (2) deprotection step>, ⁇ (3) Neutralization step>, ⁇ (4) Washing step after neutralization step>, ⁇ (6) condensation step>, ⁇ (7) Washing step 1 after condensation step> and ⁇ (8) Washing step 2 after condensation step> including.
• the production method of the present invention optionally does not include a ⁇ capping step>.
• formula (I) The morpholino nucleic acid oligomer of [wherein Base, X, Y, and n are as defined above] is disclosed in, for example, , WO2021/132591, WO2021/172498, etc.) or a phosphorodiamidate mopholino oligomer (PMO) that can be recognized by a person skilled in the art based on the description in the international publication, preferably human dystrophin gene 51, 53 , 45th, 55th, 44th, and 50th exons. No.: 2055732-84-6).
• formula (I-1) Morpholino nucleic acid oligomers [wherein Base, X, Y, and n are as defined above] are disclosed, for example, in International 057350, WO2014/144978, WO2014/153240, etc.) or a phosphorodiamidate mopholino oligomer (PMO) that can be recognized by a person skilled in the art based on the description in the international publication, preferably 51 of the human dystrophin gene, A phosphorodiamidate mopholino oligomer (PMO) capable of skipping at least one selected from the group consisting of 53rd, 45th, 55th, 44th and 50th exons, specifically Eteplirsen (WO2006/000057, WO2010/050801 , see WO2014/144978; CAS Registry Number: 1173755-55-9), Golodirsen (see WO2001/083740, WO2006/000057; CAS Registry Number:
• the morpholino nucleic acid oligomer obtained by the production method of the present invention has the formula (I-2): [wherein Base, X, Y and n are as defined above].
• Reference Example 1 Morpholino Monomer Compounds Table 2 shows structural formulas and abbreviations of morpholino monomer compounds.
• a p , C p , and T p were either commercially available or prepared by methods commonly used in the art (see WO2008/008113).
• G CE and G POB were prepared by methods commonly used in the art (see WO2012/043730, WO2009/064471).
• UV measurement conditions Equipment: U-2910 (Hitachi, Ltd.) Solvent: methanesulfonic acid Wavelength: 409 nm ⁇ value: 45000
• the number of extension reaction synthesis cycles was defined as the number corresponding to the chain length to be synthesized.
• the amount of the morpholino monomer compound is 1.3 to 2.2 relative to the oligomer obtained in the immediately preceding extension reaction synthesis cycle so that the base sequence of the morpholino nucleic acid oligomer is 5′-CCTCCGGTTCTGAAGGTGTTC-3′. Used in molar equivalents.
• wash solution 1 30% 2,2,2-trifluoroethanol (TFE)/dichloromethane (DCM) (30% TFE in DCM) was used.
• wash solution 2 DCM was used.
• washing solution 3 the pretreatment solution 1 described later was used.
• Deprotection solution 1 was a mixture of trifluoroacetic acid (1.2 eq) and triethylamine (1 eq) to 5% (w/v) with 1% (v/v) triisopropylsilane (TIPS). and dissolved in a DCM solution containing 20% (v/v) TFE.
• IPA isopropyl alcohol
• DIPEA N,N-diisopropylethylamine
• DCM 353:75:1065 (v/v) ratio
• pretreatment liquid 1 a mixture of 1,3-dimethyl-2-imidazolidinone (DMI) containing N-ethylmorpholine (NEM) was used (5-6% NEM solution (v/v) in DMI). .
• DMI 1,3-dimethyl-2-imidazolidinone
• NEM N-ethylmorpholine
• a p , C p , T p , and G CE listed in Table 1 were used as morpholino monomer compounds.
• a morpholino monomer compound was dissolved in pretreatment liquid 1 to prepare a 0.10 to 0.14 M morpholino monomer compound solution (condensation solution 1) in pretreatment liquid 1, which was used as condensation solution 1.
• step 5 After step 4, wash solution 3 (5.3% NEM solution in DMI (v/ v)) was added, stirred for 5 minutes and the solution in the reaction vessel was drained. 25 mL of Wash Solution 3 was then added to the reaction vessel, stirred for 5 minutes, and the solution was drained.
• step 6 After step 5, 25 mL of condensation solution 1 (0.12 M G CE solution in pretreatment solution 1) was added to the reaction vessel and stirred for 18 hours.
• step 7 After step 6, the solution in the reaction vessel was drained. Subsequently, 50 mL of Wash Solution 2 was added to the reaction vessel, stirred for 5 minutes, and the solution in the reaction vessel was drained.
• Example 2 1. Extension reaction 4- ⁇ [(2S,6R)-6-(4-benzamido-2-oxopyrimidin-1-yl)-4-tritylmorpholin-2-yl]methoxy ⁇ -4 supported on aminomethyl polystyrene resin - 3.0 g (1.5 mmol) of oxobutanoic acid (synthesized in the same manner as in Reference Example 3) was transferred to the reactor, and the synthesis cycle of Table 4 was started. The number of extension reaction synthesis cycles was defined as the number corresponding to the chain length to be synthesized.
• the amount of the morpholino monomer compound is 1.3 to 2.2 relative to the oligomer obtained in the immediately preceding extension reaction synthesis cycle so that the base sequence of the morpholino nucleic acid oligomer is 5′-CCTCCGGTTCTGAAGGTGTTC-3′.
• the reactor was purged with nitrogen gas. Specifically, after step 4, after the liquid was drained, a nitrogen balloon was attached to the lower part of the filter of the filter reactor, the cock was opened, and nitrogen gas was introduced twice.
• wash solution 1 30% 2,2,2-trifluoroethanol (TFE)/dichloromethane (DCM) (30% TFE in DCM) was used.
• DCM diichloromethane
• Deprotection solution 1 was a mixture of trifluoroacetic acid (1.2 eq) and triethylamine (1 eq) to 5% (w/v) with 1% (v/v) triisopropylsilane (TIPS). and dissolved in a DCM solution containing 20% (v/v) TFE.
• IPA isopropyl alcohol
• DIPEA N,N-diisopropylethylamine
• DCM 353:75:1065 (v/v) ratio
• a p , C p , T p , and G CE listed in Table 1 were used as morpholino monomer compounds.
• the morpholino monomer compound was dissolved in a 5-6% DIPEA solution (v/v) in DMI to prepare a 0.10-0.13 M morpholino monomer compound solution (condensation solution 2) and used as condensation solution 2.
• capping solution 1 a mixture of acetic anhydride/DIPEA/DCM (ratio 0.5:1.25:98.25 (v/v)) was used.
• step 2 After step 1, 45 mL of deprotection solution 1 was added to an aminomethyl polystyrene resin supporting a 17-mer oligomer whose base sequence was 5′-CCTCCGGTTCTGAAGGT-3′ and whose ends were protected with a trityl group. Add, stir for 5 minutes, and drain the solution in the reaction vessel. This process was repeated 5 more times.
• step 3 After Step 2, 50 mL of Neutralization Solution 1 was added to the reaction vessel, stirred for 5 minutes, and the solution in the reaction vessel was drained. This process was repeated two more times.
• step 4 After step 3, 50 mL of Wash Solution 2 was added to the reaction vessel, stirred for 5 minutes, and the solution in the reaction vessel was drained. This process was repeated one more time.
• step 5 After step 4, after purging the reaction vessel with nitrogen, 25 mL of condensation solution 2 (0.12 M G CE solution in 5.3% DIPEA solution (v/v) in DMI) was added, and 6 Stirred for an hour.
• step 6 After step 5, the solution in the reaction vessel was drained. 50 mL of Wash Solution 2 was then added to the reaction vessel, stirred for 5 minutes, and the solution was drained.
• step 7 After step 6, the solution in the reaction vessel was drained. 50 mL of Capping Solution 1 was then added to the reaction vessel, stirred for 10 minutes, and the solution was drained.
• each condensation step 2.0 to 3.0 of the morpholino monomer compound is added to the oligomer obtained in the immediately preceding extension reaction synthesis cycle so that the base sequence of the morpholino nucleic acid oligomer is 5′-CCTCCGGTTCTGAAGGTGTTC-3′. Used in molar equivalents.
• wash solution 2 DCM was used.
• cleaning solution 4 a pretreatment solution 2, which will be described later, was used.
• Deprotection solution 2 was a mixture of trifluoroacetic acid (2 eq.) and triethylamine (1 eq.) to 3% (w/v), 1% (v/v) ethanol and 10% (v/v ) dissolved in a DCM solution containing TFE.
• IPA/DIPEA/DCM 25:5:70 (v/v) ratio
• a DIPEA/DMI mixture (10:90 (v/v) ratio) was used as the pretreatment liquid 2.
• a p , C p , T p , and G CE listed in Table 1 were used as morpholino monomer compounds.
• a morpholino monomer compound was dissolved in pretreatment liquid 2 to prepare a 0.16 M morpholino monomer compound solution (condensation solution 2) in pretreatment liquid 2 and used as condensation solution 2.
• Test Example 1 Comparison of the Morpholino Nucleic Acid Oligomers Prepared in Examples 1 and 2 and the Morpholino Nucleic Acid Oligomer Prepared in Comparative Example 1
• a morpholino nucleic acid oligomer having a nucleotide sequence of 5'-CCTCCGGTTCTGAAGGTGTTC-3' was synthesized by extension reaction.
• Approximately 30 mg of each of the aminomethyl polystyrene resins supporting morpholino nucleic acid oligomers of Example 1 and Comparative Example 1 obtained by drying was placed in a reaction vessel, 0.75 mL of deprotection solution 1 was added, and the mixture was heated at room temperature. React for 40 minutes.
• Deprotection Solution 1 was removed and the resin was washed with Neutralization Solution 1 and dichloromethane and dried under vacuum at room temperature. 28% aqueous ammonia-ethanol (4/1) was added to the dried resin and stirred at room temperature for 17 hours. The supernatant was diluted and subjected to HPLC (C18 ion pair analysis) to determine the purity of morpholino nucleic acid oligomers in the crude mixture. By comparing the morpholino nucleic acid oligomer prepared in Examples 1 and 2 with the morpholino nucleic acid oligomer prepared in Comparative Example 1, it was confirmed that the purity of the morpholino nucleic acid oligomer was improved by the production methods of Examples 1 and 2. bottom.
• Test Example 2 Comparison of condensation reaction yields of the morpholino nucleic acid oligomer prepared in Example 1 and the morpholino nucleic acid oligomer prepared in Comparative Example 1 Example 1-1. After each elongation cycle of the elongation reaction, a small amount of the morpholino nucleic acid oligomer-supported aminomethylpolystyrene resin was collected, a 40% methylamine-methanol solution/water mixture was added, and the resin was reacted at 65°C for 10 minutes. cut out from Similarly, 1. of Comparative Example 1.
• Condensation reaction yield (%) 100 - peak area value of raw material * / (peak area value of raw material + main peak area value after condensation reaction)
• the raw material refers to a morpholino nucleic acid oligomer having no trityl group at the end and having a chain length shorter by one residue than the chain length obtained by the condensation reaction.
• TFE 2,2,2-trifluoroethanol
• DCM dichloromethane
• TFA trifluoroacetic acid
• TEA triethylamine
• TIPS triisopropylsilane
• IPA isopropyl alcohol
• DIPEA N,N-diisopropylethylamine
• NEM N-ethylmorpholine
• DMI 1,3 -dimethyl-2-imidazolidinone

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