Method for producing oligonucleic acid compound

Abstract

The present invention relates to a method for producing a morpholino nucleic acid oligomer (each symbol is as defined in the description), the method including a step for treating a compound of formula (II) using a solution that contains an acid and a scavenger in an elongation reaction of a morpholino nucleic acid oligomer represented by the formula and removing R1 from the compound of formula (II) to produce a compound of formula (III).

Description

Method for producing oligonucleic acid compound

[0001] The present invention relates to a method for producing a novel oligonucleic acid compound.

[0002] Known methods for producing oligonucleic acid compounds include solid-phase and liquid-phase methods. The solid-phase method is a heterogeneous reaction method in which nucleic acids are extended by contacting a substrate supported on a solid support with a solution containing a reaction reagent. The solid-phase method uses a so-called batch method in which a reaction is carried out in a reaction vessel equipped with a filter (see, for example, Non-Patent Document 1 and Patent Document 1). A pseudo-flow synthesis method, similar to that used in automated nucleic acid synthesizers (e.g., DNA and RNA synthesizers), is also known in which a solid support is placed in a column and a solution containing a reaction reagent is passed through the column to carry out the reaction. Meanwhile, the liquid-phase method is a homogeneous reaction method in which nucleic acids are extended by reaction in a solution containing both a substrate and a reaction reagent. The liquid-phase method also uses a batch method in which the reaction is carried out in a vessel (see, for example, Patent Documents 2 and 3).

[0003] Whether solid-phase, liquid-phase, batch, or pseudo-flow synthesis is used, chemical synthesis of oligonucleic acid compounds involves repeatedly repeating a "deprotection" reaction, which removes the protecting groups on the oxygen atoms or amino groups on the nucleic acid compound, and a "condensation" reaction, which forms a bond between the oxygen atom or nitrogen atom that has been deprotected and is now reactive, and the phosphorus atom, thereby extending the nucleic acid.

[0004] When using oligonucleic acid compounds as pharmaceutical raw materials, high purity is required for the oligonucleic acid compounds. Therefore, it is necessary to improve the reaction efficiency and reaction rate while suppressing the production of by-products in each step of the extension reaction. However, this is difficult because the solvents and reagents that can be used are limited.

[0005] International Publication No. 1991 / 09033A1 International Publication No. 2014 / 077292A1 International Publication No. 2013 / 122236A1

[0006] Acc. Chem. Res. , Vol. 24, 278-284, 1991

[0007] The elongation reaction of an oligonucleic acid compound involves repeated deprotection and condensation reactions, resulting in the generation of complex impurities. An object of the present invention is to provide a novel production method that identifies impurities that affect the purity of an oligonucleic acid compound and controls the generation of such impurities.

[0008] The present inventors have thoroughly investigated the treatment of reaction raw materials and intermediates before condensation in the elongation reaction of an oligonucleic acid compound, and have found that the deprotection reaction and condensation reaction proceed efficiently, and further that impurities generated by pretreatment for the condensation reaction affect the purity of the oligonucleic acid compound, thereby completing the present invention.

[0009] That is, the present invention relates to the following:

[0010] <1> Morpholino nucleic acid oligomer extension reaction: [In the formula, B Ｐ are 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 in the range of 1 to 99, preferably any integer in the range of 15 to 30, and more preferably any integer in the range of 18 to 28; L is hydrogen, acyl, or a group represented by formula (IV): In the formula (II): [In the formula, L, B Ｐ , X, Y, R １ and n is as defined above] is treated with a solution containing an acid and a scavenger to convert R １ to obtain a compound of formula (III): [In the formula, L, B Ｐ , X, Y, and n are as defined above.

[0011] <2> Further, the formula (III): [In the formula, L, B Ｐ, X, Y, and n are as defined above] with a solution containing a base, an alcohol, and a halogenated solvent.

[0012] <3> Further, the formula (III): [In the formula, L, B Ｐ , X, Y, and n are as defined above] with a solution containing an organic amine and an aprotic polar solvent.

[0013] <4> Further, the formula (VIII): [In the formula, B Ｐ , X, Y, and R １ is as defined above, and Z is a halogen (e.g., chloro, bromo, or iodo), with a solution containing an organic amine and an aprotic polar solvent.

[0014] <5> Further, the formula (III): [In the formula, L, B Ｐ , X, Y, and n are as defined above] in the presence of an organic amine, [In the formula, X, Y, Z, B Ｐ and R １ is as defined above] to obtain a compound of formula (II'): [In the formula, L, B Ｐ , X, Y, R １ and n is as defined above.

[0015] <6> Further, the formula (II′): [In the formula, L, B Ｐ , X, Y, R １ and n is as defined above] with a solution containing an alcohol and / or a halogenated solvent.

[0016] <7> Morpholino nucleic acid oligomer extension reaction: [In the formula, B Ｐare 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 in the range of 1 to 99, preferably any integer in the range of 15 to 30, and more preferably any integer in the range of 18 to 28; L is hydrogen, acyl, or a group represented by formula (IV): In the formula (II): [In the formula, L, B Ｐ , X, Y, R １ and n is as defined above] is treated with a solution containing an acid and a scavenger to convert R １ to obtain a compound of formula (III): [In the formula, L, B Ｐ , X, Y, and n are as defined above, [In the formula, L, B Ｐ , X, Y, and n are as defined above] with a solution containing an organic amine and an aprotic polar solvent, and [In the formula, B Ｐ , X, Y, Z, and R １ is as defined above] with a solution containing an organic amine and an aprotic polar solvent.

[0017] <8> Morpholino nucleic acid oligomer extension reaction: [In the formula, B Ｐ are 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 in the range of 1 to 99, preferably any integer in the range of 15 to 30, and more preferably any integer in the range of 18 to 28; L is hydrogen, acyl, or a group represented by formula (IV): In the formula (II): [In the formula, L, B Ｐ , X, Y, R １ and n is as defined above] is treated with a solution containing an acid and a scavenger to convert R １ to obtain a compound of formula (III): [In the formula, L, B Ｐ , X, Y, and n are as defined above, [In the formula, L, B Ｐ , X, Y, and n are as defined above] with a compound of formula (VIII): [In the formula, B Ｐ , X, Y, Z, and R １ is as defined above] with a solution containing an organic amine and an aprotic polar solvent.

[0018] <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.

[0019] <10> The method for producing a morpholino nucleic acid oligomer according to any one of <1> to <9>, wherein the solution containing an acid and a scavenger is a solution containing trifluoroacetic acid, triethylamine, triisopropylsilane, 2,2,2-trifluoroethanol, and dichloromethane.

[0020] <11> The method for producing a 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.

[0021] <12> The method according to any one of <1> to <11>, wherein the solid phase support is swellable polystyrene, non-swellable polystyrene, PEG-chain-bonded polystyrene, pore-controlled glass, oxalated pore-controlled glass, TentaGel support-aminopolyethylene glycol-derivatized support, or Poros-polystyrene / divinylbenzene copolymer.

[0022] <13> The production method according to any one of <1> to <12>, wherein the linker is a short-chain alkylene, a long-chain alkylene, an amino-short-chain alkylene, an amino-long-chain alkylene, a diacyl short-chain alkylene (for example, succinyl), a diacyl long-chain alkylene, or a dialkylenesulfonyl.

[0023] The present invention makes it possible to produce morpholino nucleic acid oligomers economically, in good yield, and with high purity.

[0024] The present invention will be described in detail below.

[0025] <Morpholino Nucleic Acid Oligomer> In one embodiment of the present invention, a morpholino nucleic acid oligomer has the formula: [wherein Base represents a nucleic acid base, and X and Y are as defined above] as a constituent unit.

[0026] <Nucleobase> In one embodiment of the present invention, examples of the "nucleobase" include adenine, guanine, hypoxanthine, cytosine, thymine, uracil, and modified bases thereof. Examples of such modified bases include pseudouracil, 3-methyluracil, dihydrouracil, 5-alkylcytosine (e.g., 5-methylcytosine), 5-alkyluracil (e.g., 5-ethyluracil), 5-halouracil (e.g., 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-methylaminomethyluracil, 5-methylcarbonylmethyluracil, 5-methyloxyuracil, 5-methyl-2-thiouracil, 2-methylthio-N ６ Examples of bases include, but are not limited to, 2-isopentenyladenine, uracil-5-oxyacetic acid, 2-thiocytosine, purine, 2,6-diaminopurine, 2-aminopurine, isoguanine, indole, imidazole, and xanthine, provided that the amino group or hydroxyl group of the nucleic acid base of Base may be protected.

[0027] <Method of Producing a Morpholino Nucleic Acid Oligomer> One aspect of the present invention is a method for producing a morpholino nucleic acid oligomer comprising the steps of: [wherein Base, X, and Y are as defined above; and n is any integer within the range of 1 to 99, preferably any integer within the range of 15 to 30, and more preferably any integer within the range of 18 to 28] (hereinafter referred to as morpholino nucleic acid oligomer (I)).

[0028] One aspect of the present invention is a method for the extension reaction of a morpholino nucleic acid oligomer, in which a compound of formula (II) is reacted with a compound of formula (VIII) to obtain a compound of formula (II′): [In the formula, B Ｐ are 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., chloro, bromo, iodo); n is any integer in the range of 1 to 99, preferably any integer in the range of 15 to 30, and more preferably any integer in the range of 18 to 28; L is hydrogen, acyl, or a group represented by formula (IV): The present invention is a method for producing a morpholino nucleic acid oligomer comprising the above-mentioned compound, wherein the compound is a group represented by the formula:

[0029] In one aspect of the present invention, the optionally protected nucleobases include, for example, both unprotected and protected "nucleobases," such as adenine, guanine, hypoxanthine, cytosine, thymine, uracil, etc., in which the amino group and / or hydroxyl group is unprotected or protected.

[0030] In one embodiment of the present invention, the amino-protecting group is not particularly limited as long as it is a group that can be used as a protecting group for nucleic acids, and specific examples thereof include benzoyl, 4-methoxybenzoyl, acetyl, propionyl, butyryl, isobutyryl, phenylacetyl, phenoxyacetyl, 4-tert-butylphenoxyacetyl, 4-isopropylphenoxyacetyl, and (dimethylamino)methylene. Preferred amino-protecting groups are benzoyl, acetyl, phenoxyacetyl, and 4-tert-butylphenoxyacetyl. Examples of hydroxyl-protecting groups include 2-cyanoethyl, 4-nitrophenethyl, phenylsulfonylethyl, methylsulfonylethyl, trimethylsilylethyl, phenyl optionally substituted with 1 to 5 electron-withdrawing groups at any substitutable position, diphenylcarbamoyl, dimethylcarbamoyl, diethylcarbamoyl, methylphenylcarbamoyl, 1-pyrrolidinylcarbamoyl, morpholinocarbamoyl, 4-(tert-butylcarboxy)benzyl, 4-[(dimethylamino)carboxy]benzyl, and 4-(phenylcarboxy)benzyl (see, for example, WO 2009 / 064471 A1). Preferred hydroxyl-protecting groups include 2-cyanoethyl, 4-nitrophenethyl, and 4-(tert-butylcarboxy)benzyl. The preferred protecting group for the 6-hydroxyl group of guanine is 2-cyanoethyl.

[0031] In one embodiment of the invention, the protected nucleobase may be, for example, a nucleobase of the formula: wherein Pg represents a protecting group.

[0032] In one embodiment of the present invention, the protected nucleobase is, for example, adenine (A) whose amino group is protected with benzoyl. Ｂｚ ), cytosine whose amino group is protected with benzoyl (C Ｂｚ ), guanine (G) in which the hydroxyl group is protected with 2-cyanoethyl and the amino group is protected with phenoxyacetyl; ＣＥ ) and the like, but are not limited to these.

[0033] In one embodiment of the present invention, the solid phase carrier is not particularly limited as long as it is a carrier that can be used in solid phase reactions of nucleic acids, but it is desirable that the solid phase carrier be, for example, (i) practically insoluble in reagents that can be used in the synthesis of morpholino nucleic acid oligomers (e.g., dichloromethane, acetonitrile, N-ethylmorpholine, N,N-diisopropylethylamine, N-methylimidazole, pyridine, acetic anhydride, 2,6-lutidine, 2,4-lutidine, trifluoroacetic acid), (ii) chemically stable against reagents that can be used in the synthesis of morpholino nucleic acid derivatives, (iii) capable of chemical modification, (iv) capable of being loaded with a desired morpholino nucleic acid derivative, (v) strong enough to withstand high pressures applied during processing, and (vi) have a certain particle size range and distribution.

[0034] In one embodiment of the present invention, the solid phase carrier is, for example, a swellable polystyrene (e.g., Aminomethyl Polystyrene Resin 1% Divinylbenzene Crosslinked (200-400 mesh) (2.4-3.0 mmol / g) (Tokyo Chemical Industry Co., Ltd.), Aminomethylated Polystyrene Resin.HCl [Divinylbenzene 1%, 100-200 mesh] (Peptide Institute), Aminomethyl Resin [Polystyrene 1% Divinylbenzene Copolymer, 200-400 mesh] (Watanabe Chemical Industry Co., Ltd.)), a non-swellable polystyrene (e.g., Primer Support (GE Healthcare)), a PEG-chain-linked polystyrene (e.g., NH2-PEG Examples of such supports include TentaGel resin (manufactured by Watanabe Chemical Industry Co., Ltd.), TentaGel resin), controlled pore glass (CPG) (e.g., manufactured by CPG Corporation), oxalated controlled pore glass (see, for example, Allul et al., Nucleic Acids Research, Vol. 19, 1527 (1991)), TentaGel support-aminopolyethylene glycol derivatized support (see, for example, Wright et al., Tetrahedron Letters, Vol. 34, 3373 (1993)), and Poros-polystyrene / divinylbenzene copolymer.

[0035] In one embodiment of the present invention, the linker can be a known linker used for linking nucleic acids or morpholino nucleic acid derivatives, and examples thereof include 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, and dialkylenesulfonyl, preferably 3-aminopropyl, succinyl, 2,2′-diethanolsulfonyl, and long-chain alkylamino (LCAA).

[0036] In one embodiment of the present invention, L is a group represented by formula (IV): A group represented by formula (IV-1): or a group represented by formula (IV-2): It may be a group represented by the following formula:

[0037] In one embodiment of the present invention, a compound of formula (II): [In the formula, L, B Ｐ , X, Y, R １ and n is as defined above] can be prepared by methods known in the art (see, for example, WO2012 / 043730).

[0038] <Binding to solid phase and linker>

[0039] In one embodiment of the present invention, there is provided a compound (II) represented by formula (IIa), in which n is 1 and L is a group represented by formula (IV): [In the formula, B Ｐ , R １ , the linker, and the solid phase carrier are as described above] (hereinafter referred to as compound (IIa)).

[0040] In one embodiment of the present invention, a compound of formula (VI) (hereinafter referred to as compound (VI)) is produced by reacting a compound of formula (V) with an acylating agent: [In the formula, B Ｐ and R １, is as defined above, and the linker is diacyl short chain alkylene (e.g., succinyl), diacyl long chain alkylene, short chain alkylene, long chain alkylene, or dialkylenesulfonyl; R ６ is a hydroxyl group, a halogen, or an amino.

[0041] This step can be carried out by a known linker introduction reaction using compound (V) as a starting material.

[0042] In one embodiment of the present invention, a compound of formula (VIa): [In the formula, B Ｐ , R １ is as defined above] can include a step of producing the compound of formula (V) by carrying out a method known as an esterification reaction using compound (V) and succinic anhydride.

[0043] In one embodiment of the present invention, a step of producing compound (IIa) by reacting compound (VI) with a solid support by treating the compound with a condensing agent or the like: [In the formula, B Ｐ , R ６ , R １ , the linker, and the solid phase support are as described above.

[0044] This step can be carried out by a method known as a condensation reaction using compound (VI) and a solid support.

[0045] <Raw materials> In one embodiment of the present invention, a compound of formula (VIII): [In the formula, B Ｐ , X, Y, Z, and R １ are as defined above] are commercially available or can be prepared and used by methods known in the art.

[0046] In one embodiment of the present invention, examples of the compound of formula (VIII) include the compounds listed in Table 1 below.

[0047]

[0048] One aspect of the present invention is a method for producing a morpholino nucleic acid oligomer, wherein the extension reaction includes at least one step selected from the group consisting of <(1) a washing step before the deprotection step>, <(2) a deprotection step>, <(3) a neutralization step>, <(4) a washing step after the neutralization step>, <(5) a washing step before the condensation>, <(6) a condensation step>, <(7) a first washing step after the condensation step>, and <(8) a second washing step after the condensation step>.

[0049] The steps (1) to (8) are explained below.

[0050] <(1) Washing step before deprotection step> This step is carried out by washing a compound of formula (II): [In the formula, L, B Ｐ , X, Y, R １ and n is as defined above] with a solution containing an alcohol and / or a halogenated solvent.

[0051] <Solution of (1)> In one embodiment, the solution of the step (1) is not particularly limited as long as it contains an alcohol and / or a halogenated solvent, and for example, at least one selected from the group consisting of 2,2,2-trifluoroethanol, difluoroethanol, chloroform, and dichloromethane can be used, and preferably 2,2,2-trifluoroethanol and dichloromethane can be used.

[0052] <Solution of (1)> In one embodiment, the solution of step (1) can be prepared by using an alcohol in a volume ratio of, for example, 0.01 to 100 times, preferably 0.1 to 2 times (v / v) the volume ratio of the alcohol to the halogenated solvent.

[0053] <Amount of Solution in (1) Used> In one embodiment, the amount of the solution in step (1) used is not particularly limited, and is, for example, within a range of 1 to 1000 times, and preferably within a range of 2 to 100 times, the weight ratio of which is based on 1 g of compound (II).

[0054] <Treatment Time of (1)> In one embodiment, the time of the step (1) is not particularly limited and is, for example, 0.1 minute to 24 hours, preferably 1 minute to 5 hours, and more preferably 3 minutes to 1 hour.

[0055] <Treatment Temperature in (1)> In one embodiment, the temperature in the step (1) is not particularly limited, and is, for example, 0°C to 40°C, preferably 10°C to 35°C, and more preferably 15°C to 30°C.

[0056] <Number of Times of Step (1)> In one embodiment, the number of times of step (1) is not particularly limited, and is, for example, 1 to 10 times, and preferably 1 to 5 times.

[0057] <(2) Deprotection Step> This step is carried out by deprotecting a compound of formula (II): [In the formula, L, B Ｐ , X, Y, R １ and n is as defined above], (2) treating a compound of formula (II) with a solution containing an acid and a scavenger to convert R １ to obtain a compound of formula (III): [In the formula, L, B Ｐ , X, Y, and n are as defined above.

[0058] <Acid (2)> In one embodiment, the acid may be, for example, hydrochloric acid, trifluoroacetic acid, dichloroacetic acid, or trichloroacetic acid, and is preferably trifluoroacetic acid.

[0059] <Amount of Acid (2) Used> In one embodiment, the amount of the acid used is, for example, in a molar ratio within a range of 1 to 500 times, and preferably within a range of 2 to 100 times, per 1 mole of compound (II).

[0060] <Concentration of Acid (2)> In one embodiment, the acid can be used after being diluted with a suitable solvent to a concentration within the range of 0.1% to 30%.

[0061] <Solvent for diluting the acid (2)> In one embodiment, the solvent to be used is not particularly limited as long as it is not involved in the reaction, and examples thereof include dichloromethane, toluene, acetonitrile, alcohols (ethanol, isopropyl alcohol, 2,2,2-trifluoroethanol, etc.), water, and mixtures thereof.

[0062] <Organic Amine (2)> In one embodiment, an “organic amine” can be used together with the acid. The organic amine is not particularly limited, but examples thereof include triethylamine, N,N-diisopropylethylamine, N-ethylmorpholine, pyridine, etc., and preferably triethylamine.

[0063] <Amount of Organic Amine (2) Used> In one embodiment, the amount of organic amine used is, for example, in a molar ratio range of 0.01 to 10 times, and preferably 0.1 to 2 times, the amount of 1 mole of the acid.

[0064] <Ratio of Acid to Organic Amine in (2)> In one embodiment, when a salt or mixture of an acid and an organic amine is used, for example, a salt or mixture of trifluoroacetic acid and triethylamine can be used, such as a mixture of 1.2 equivalents of trifluoroacetic acid and 1 equivalent of triethylamine.

[0065] <Scavenger (2)> In one embodiment, the scavenger is at least one selected from the group consisting of alcohols (for example, methanol, ethanol, and 2,2,2-trifluoroethanol), thiols (mercaptoethanol, dithiothreitol, and mercaptosuccinic acid), organosilicon compounds (for example, triisopropylsilane, triethylsilane, and trimethylsilane), and aromatic compounds (for example, 1-hydroxybenzotriazole, pyrrole, indole, anisole, and thioanisole), and is preferably an organosilicon compound.

[0066] <Scavenger (2)> In one embodiment, the scavenger is, for example, at least one selected from the group consisting of methanol, ethanol, mercaptoethanol, 2,2,2-trifluoroethanol, triisopropylsilane, triethylsilane, trimethylsilane, 1-hydroxybenzotriazole, pyrrole, indole, anisole, and thioanisole, preferably one selected from the group consisting of triisopropylsilane and ethanol, and more preferably triisopropylsilane.

[0067] <Amount of Scavenger (2) Used> In one embodiment, the amount of the scavenger used is, for example, in a molar ratio of 0.1 to 1000 times, preferably 0.5 to 100 times, and more preferably 1 to 20 times, relative to 1 mole of compound (II).

[0068] <Reaction Time of (2)> In one embodiment, the reaction time is, for example, in the range of 0.1 minute to 24 hours, and preferably in the range of 1 minute to 3 hours.

[0069] <Reaction Temperature of (2)> In one embodiment, the reaction temperature is, for example, preferably in the range of 0°C to 40°C, more preferably in the range of 10°C to 35°C, and even more preferably in the range of 15°C to 30°C.

[0070] <Number of Times of Step (2)> In one embodiment, the number of times of step (2) is not particularly limited, and is, for example, 1 to 10 times, and preferably 1 to 8 times.

[0071] <(1) Washing before deprotection and (2) Sequential reaction of deprotection> In one embodiment of the present invention, a compound of formula (II): [In the formula, L, B Ｐ , X, Y, R １ and n is as defined above] is treated with a solution containing an alcohol and / or a halogenated solvent, and then with a solution containing an acid and a scavenger to obtain R １ to obtain a compound of formula (III): [In the formula, 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.

[0072] <(3) Neutralization Step> This step is carried out by neutralizing a compound of formula (III): [In the formula, L, B Ｐ , X, Y, and n are as defined above] with a solution containing a base and a solvent (an alcohol and a halogenated solvent).

[0073] <Base (3)> In one embodiment, the base is not particularly limited as long as it does not affect the protecting group. Examples of the base include triethylamine, N,N-diisopropylethylamine, and pyridine, and N,N-diisopropylethylamine is preferred.

[0074] <Amount of Base Used in (3)> In one embodiment, the base can be used by diluting it with an appropriate solvent to a concentration within the range of 0.1% (v / v) to 30% (v / v).

[0075] <Amount of Solution in (3) Used> In one embodiment, the amount of the solution in step (3) used is not particularly limited, and is, for example, within a range of 1 to 1000 times, and preferably within a range of 2 to 100 times, the weight ratio of 1 g of compound (III).

[0076] <Solvent (3)> In one embodiment, the solvent is at least one selected from the group consisting of alcohols, halogenated solvents, polar solvents, ether-based solvents, and mixtures thereof, and is preferably a mixture of alcohols and halogenated solvents.

[0077] In one embodiment, the alcohols are ethanol, isopropyl alcohol, 2,2,2-trifluoroethanol, etc., and preferably isopropyl alcohol.

[0078] In one embodiment, the halogen solvent is chloroform, dichloromethane, tetrachloroethane, tetrachloroethylene, or the like, preferably dichloromethane.

[0079] In one embodiment, the polar solvent is acetonitrile, dimethyl sulfoxide, or the like.

[0080] In one embodiment, the ethereal solvent is tetrahydrofuran, cyclopentyl methyl ether, or the like.

[0081] <Reaction Time of (3)> In one embodiment, the reaction time varies depending on the type of base used and the reaction temperature, but is, for example, in the range of 0.1 minute to 24 hours, and preferably in the range of 1 minute to 5 hours.

[0082] <Reaction Temperature of (3)> In one embodiment, the 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, and even more preferably in the range of 3°C to 20°C.

[0083] <Number of Times of Step (3)> In one embodiment, the number of times of step (3) is not particularly limited, and is, for example, 1 to 10 times, and preferably 1 to 5 times.

[0084] <(2) Deprotection and (3) Neutralization Sequential Reactions> In one embodiment of the present invention, a compound of formula (II): [In the formula, L, B Ｐ , X, Y, R １ and n is as defined above], (2) treating a compound of formula R with a solution containing an acid and a scavenger to form a compound of formula R １ to obtain a compound of formula (III): [In the formula, L, B Ｐ , X, Y, and n are as defined above], and then reacting the compound of formula (III): [In the formula, L, B Ｐ , X, Y, and n are as defined above] can be treated with a solution containing a base and a solvent (an alcohol and a halogenated solvent). Steps (2) and (3) are as described above and can be combined as appropriate.

[0085] <(4) Cleaning step after neutralization step>

[0086] This step is carried out by reacting a compound represented by formula (III): [In the formula, L, B Ｐ, X, Y, and n are as defined above] with a solution containing a halogenated solvent.

[0087] <Solution of (4)> In one embodiment, the solution of the step (4) is not particularly limited as long as it contains a halogen solvent, and for example, at least one selected from the group consisting of chloroform, dichloromethane, tetrachloroethane, and tetrachloroethylene can be used, and preferably dichloromethane.

[0088] <Amount of Solution in (4) Used> In one embodiment, the amount of the solution in step (4) used is not particularly limited, and is, for example, within a range of 1 to 1000 times, and preferably within a range of 2 to 100 times, the amount by weight of 1 g of compound (III).

[0089] <Treatment Time in (4)> In one embodiment, the time for the step (4) is not particularly limited and is, for example, 0.1 minutes to 2 hours, preferably 1 minute to 60 minutes, and more preferably 3 minutes to 30 minutes.

[0090] <Treatment Temperature in Step (4)> In one embodiment, the temperature in step (4) is not particularly limited and is, for example, 0°C to 35°C, preferably 10°C to 30°C, and more preferably 15°C to 25°C.

[0091] <Number of Times of Step (4)> In one embodiment, the number of times of step (4) is not particularly limited, and is, for example, 1 to 10 times, and preferably 1 to 5 times.

[0092] <(2) Deprotection, (3) Neutralization, and (4) Sequential Reactions of Washing After Neutralization> In one embodiment of the present invention, a compound represented by formula (II): [In the formula, L, B Ｐ , X, Y, R １ and n is as defined above], (2) treating a compound of formula (II) with a solution containing an acid and a scavenger to convert R １ to obtain a compound of formula (III): [In the formula, L, B Ｐ , X, Y, R １ and n is as defined above], followed by reacting a compound of formula (III): [In the formula, L, B Ｐ , X, Y, R １ and n is as defined above] can be treated with (3) a solution containing a base and a solvent (an alcohol and a halogenated solvent), and then (4) a solution containing a halogenated solvent. Steps (1), (2), and (3) are as described above and can be combined as appropriate.

[0093] <(5) Washing step before condensation> This step is carried out by washing a compound of formula (III): [In the formula, L, B Ｐ , X, Y, and n are as defined above] with a solution containing an organic amine and an aprotic polar solvent.

[0094] <Organic Amine (5)> In one embodiment, the organic amine is, for example, at least one selected from the group consisting of N,N-diisopropylethylamine, triethylamine, and N-ethylmorpholine, and is preferably N-ethylmorpholine.

[0095] <Amount of Organic Amine (5) Used> In one embodiment, the amount of the organic amine used is, for example, in the range of 1 to 1000 times, and preferably in the range of 1 to 100 times, in terms of molar ratio, relative to 1 mole of the compound of formula (III).

[0096] <Aprotic Polar Solvent (5)> In one embodiment, the aprotic polar solvent is, for example, at least one selected from the group consisting of 1,3-dimethyl-2-imidazolidinone, N-methylpyrrolidone, N,N-dimethylformamide, tetrahydrofuran, or a mixture thereof, and preferably 1,3-dimethyl-2-imidazolidinone.

[0097] <Amount of Aprotic Polar Solvent (5) Used> In one embodiment, the amount of the aprotic polar solvent used is, for example, in a volume ratio of 1 to 1000 times (v / v) the amount of the organic amine, and preferably in a volume ratio of 3 to 100 times.

[0098] <Amount of Solution in (5) Used> In one embodiment, the amount of the solution in step (5) used is not particularly limited, and is, for example, within a range of 0.5 to 100 times, and preferably within a range of 1 to 50 times, the weight ratio of which is based on 1 g of compound (II).

[0099] <Treatment Time of (5)> In one embodiment, the reaction time varies depending on the type of organic amine used and the reaction temperature, but is usually within a range of 0.1 minutes to 2 hours, and preferably within a range of 1 minute to 60 minutes.

[0100] <Treatment Temperature of (5)> In one embodiment, the reaction temperature is, for example, preferably in the range of 0°C to 100°C, and more preferably in the range of 10°C to 50°C.

[0101] <Number of Times of Step (5)> In one embodiment, the number of times of step (5) is not particularly limited, and is, for example, 1 to 10 times, and preferably 1 to 5 times.

[0102] <(6) Condensation step>

[0103] This step is carried out by reacting a compound represented by formula (VIII): [In the formula, B Ｐ , X, Y, Z, and R １ (6) treating a compound of formula (I) above with a solution containing an organic amine and an aprotic polar solvent, for example, dissolving the compound in a mixed solution containing an organic amine and an aprotic polar solvent.

[0104] <Organic Amine (6)> In one embodiment, the organic amine is, for example, at least one selected from the group consisting of N,N-diisopropylethylamine, triethylamine, and N-ethylmorpholine, and is preferably N-ethylmorpholine.

[0105] <Amount of Organic Amine (6) Used> In one embodiment, the amount of the organic amine used is, for example, in a molar ratio within a range of 0.1 to 1000 times, and preferably within a range of 1 to 100 times, with respect to 1 mole of the compound of formula (VIII).

[0106] <Aprotic Polar Solvent (6)> In one embodiment, the aprotic polar solvent is, for example, at least one selected from the group consisting of 1,3-dimethyl-2-imidazolidinone, N-methylpyrrolidone, N,N-dimethylformamide, tetrahydrofuran, or a mixture thereof, and preferably 1,3-dimethyl-2-imidazolidinone.

[0107] <Amount of Aprotic Polar Solvent (6) Used> In one embodiment, the amount of the aprotic polar solvent used is, for example, in a volume ratio of 1 to 1000 times (v / v) the amount of the organic amine, and preferably in a volume ratio of 3 to 100 times.

[0108] <Treatment Time of (6)> In one embodiment, the treatment time varies depending on the type of organic amine used and the reaction temperature, but is usually in the range of 0.1 minute to 48 hours, and preferably in the range of 1 minute to 24 hours.

[0109] <(6) Treatment Temperature> In one embodiment, the treatment temperature is, for example, preferably in the range of 0°C to 50°C, and more preferably in the range of 10°C to 40°C.

[0110] This step further comprises reacting a compound of formula (III): [In the formula, L, B Ｐ , X, Y, and n are as defined above] in the presence of an organic amine, [In the formula, X, Y, Z, B Ｐ and R １ is as defined above] to obtain a compound of formula (II″): [In the formula, L, B Ｐ , X, Y, R １ and n is as defined above.

[0111] In one embodiment, the amount of the compound of formula (VIII) is, for example, in a molar ratio within a range of 1 to 10 times, and preferably within a range of 1 to 5 times, with respect to 1 mole of the compound of formula (III).

[0112] In one embodiment, the organic amine is, for example, at least one selected from the group consisting of N,N-diisopropylethylamine, triethylamine, and N-ethylmorpholine, and is preferably N-ethylmorpholine.

[0113] In one embodiment, the amount of the organic amine used is, for example, in a molar ratio within a range of 0.1 to 1000 times, and preferably within a range of 1 to 100 times, 1 mole of the compound of formula (VIII).

[0114] In one embodiment, the reaction time varies depending on the type of organic amine used and the reaction temperature, but is usually within the range of 10 minutes to 48 hours, and preferably within the range of 30 minutes to 24 hours.

[0115] In one embodiment, the 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.

[0116] <Number of Times of Step (6)> In one embodiment, the number of times of step (6) is not particularly limited, and is, for example, 1 to 3 times, and preferably 1 time.

[0117] <Continuous reaction of washing, pretreatment, and condensation> In one embodiment of the present invention, a hydroxybenzoate of formula (III): [In the formula, L, B Ｐ , X, Y, and n are as defined above] is treated with a solution containing an organic amine and an aprotic polar solvent, and a compound of formula (VIII): [In the formula, B Ｐ , X, Y, Z, and R １ (6) treating a compound of formula (III): with a solution containing an organic amine and an aprotic polar solvent; [In the formula, L, B Ｐ , X, Y, and n are as defined above], in the presence of an organic amine, [In the formula, X, Y, Z, B Ｐ and R １ is as defined above] to form a compound of formula (II'): [In the formula, L, B Ｐ, X, Y, R １ and n is as defined above. Steps (5) and (6) are as defined above and can be combined as appropriate.

[0118] <(7) Washing step 1 after condensation step>

[0119] This step is carried out by reacting a compound represented by formula (II'): [In the formula, L, B Ｐ , X, Y, R １ and n is as defined above] with a solution containing a halogenated solvent.

[0120] <Solution of (7)> ​​In one embodiment, the cleaning agent in the step (7) is not particularly limited as long as it contains a halogen solvent, and for example, at least one selected from the group consisting of chloroform, dichloromethane, tetrachloroethane, and tetrachloroethylene can be used, and preferably dichloromethane.

[0121] <Amount of Solution (7) Used> In one embodiment, the amount of the detergent used in the step (7) is not particularly limited, and is, for example, in a range of 1 to 1000 times, and preferably 2 to 100 times, the weight ratio of 1 g of compound (II').

[0122] <Treatment Time in (7)> ​​In one embodiment, the time for step (7) is not particularly limited and is, for example, 0.1 minute to 24 hours, preferably 1 minute to 5 hours, and more preferably 3 minutes to 1 hour.

[0123] <Treatment Temperature in Step (7)> ​​In one embodiment, the temperature in step (7) is not particularly limited, and is, for example, 0°C to 40°C, preferably 10°C to 35°C, and more preferably 15°C to 30°C.

[0124] <Continuous reaction of condensation and washing after condensation> In one embodiment of the present invention, (6) a compound represented by formula (III): [In the formula, L, B Ｐ , X, Y, and n are as defined above] in the presence of an organic amine, [In the formula, X, Y, Z, B Ｐand R １ is as defined above] to produce a compound of formula (II'): [In the formula, L, B Ｐ , X, Y, R １ and n are as defined above], followed by (7) treating with a solution containing a halogenated solvent. Steps (6) and (7) are as defined above and can be combined as appropriate.

[0125] <(8) Washing step 2 after condensation step>

[0126] This step is carried out by reacting a compound represented by formula (II'): [In the formula, L, B Ｐ , X, Y, R １ and n is as defined above] with a solution containing an alcohol and / or a halogenated solvent.

[0127] <Solution of (8)> In one embodiment, the solution of the step (8) is not particularly limited as long as it contains an alcohol and / or a halogenated solvent, and for example, at least one selected from the group consisting of 2,2,2-trifluoroethanol, difluoroethanol, chloroform, and dichloromethane can be used, and preferably 2,2,2-trifluoroethanol and dichloromethane can be used.

[0128] <Solution of (1)> In one embodiment, the solution of the step (8) can be prepared by using an alcohol in a ratio of, for example, 0.01 to 100 times, preferably 0.1 to 2 times (v / v) the alcohol relative to the halogenated solvent.

[0129] <Amount of Solution in (1) Used> In one embodiment, the amount of the solution in step (8) used is not particularly limited, and is, for example, in the range of 1 to 1000 times, and preferably 2 to 100 times, the weight ratio of 1 g of compound (II').

[0130] <Treatment Time in (8)> In one embodiment, the time for step (8) is not particularly limited and is, for example, 0.1 minute to 24 hours, preferably 1 minute to 5 hours, and more preferably 3 minutes to 1 hour.

[0131] <Treatment Temperature in (8)> In one embodiment, the temperature in the step (8) is not particularly limited, and is, for example, 0°C to 40°C, preferably 10°C to 35°C, and more preferably 15°C to 30°C.

[0132] <Number of Times of Step (8)> In one embodiment, the number of times of step (8) is not particularly limited, and is, for example, 1 to 10 times, and preferably 1 to 5 times.

[0133] <Condensation, Post-Condensation Washing, and Continuous Reaction> In one embodiment of the present invention, (6) a compound of formula (III): [In the formula, L, B Ｐ , X, Y, and n are as defined above] and a compound of formula (VIII): [In the formula, X, Y, Z, B Ｐ and R １ is as defined above] to produce a compound of formula (II'): [In the formula, L, B Ｐ , X, Y, R １ and n are as defined above], followed by (7) treating with a solution containing a halogen solvent, and then (8) treating with a solution containing an alcohol and / or a halogen solvent. Steps (6), (7), and (8) are as defined above and can be combined as appropriate.

[0134] One aspect of the present invention is a method for producing a nucleotide sequence comprising the steps of: and (C) a purification step.

[0135] Steps (A) to (C) will be described below.

[0136] <(A-1) Step of Removing Protecting Group> In one embodiment, this step is a step of obtaining a compound of formula (I″) from a compound of formula (II″): [In the formula, Base, B Ｐ , R １ , L, n, X, and Y are as defined above.

[0137] <(A-2) Step of Removing Protecting Group> In one embodiment, this step is a step of obtaining a compound of formula (I) from a compound of formula (II'''): [In the formula, Base, B Ｐ , R １ , L, n, X, and Y are as defined above.

[0138] <(A-3) Step of Removing Protecting Group> In one embodiment, this step is a step of obtaining a compound of formula (I'''') from a compound of formula (II''): [In the formula, B Ｐ , R １ , L, n, X, and Y are as defined above.

[0139] <(A-4) Step of Removing Protecting Group> In one embodiment, this step is a step of obtaining a compound of formula (I'''") from a compound of formula (II'''): [In the formula, B Ｐ , R １ , L, n, X, and Y are as defined above.

[0140] In one embodiment, the target compound can be produced by carrying out a deprotection treatment depending on the type or nature of the solid phase support, linker, and protecting group for L in the compound of formula (II") and the protecting group of the optionally protected nucleobase in the compound of formula (II"); and L in the compound of formula (II'") and the protecting group of the optionally protected nucleobase in the compound of formula (II'"). For example, all protecting groups in the compound can be removed according to the deprotection method described in "Green's PROTECTIVE GROUPS in ORGANIC SYNTHESIS, 4th Edition, 2006". If necessary, the target compound can be subjected to further chemical structure transformation.

[0141] In one embodiment, the target compound can be produced by carrying out a deprotection treatment of L in the compound of formula (II") and L in the compound of formula (II"') according to the type or nature of the solid phase support and linker. Furthermore, B in the compound of formula (I"') Ｐ and R １and B in the compound of formula (I'''''). Ｐ The target compound can be produced by carrying out a deprotection treatment according to the type or nature of the protecting group. For example, all protecting groups in the compound can be removed according to the method described herein or the deprotection method described in "Green's PROTECTIVE GROUPS in ORGANIC SYNTHESIS, 4th Edition, 2006." If necessary, the target compound can be subjected to further chemical structural transformations.

[0142] In one embodiment, for example, by treating with (1) aqueous ammonia, (2) aqueous ammonia / ethanol, or (3) a mixed solution of methylamine-methanol solution / water, preferably by treating with (2) a mixed solution of aqueous ammonia / ethanol, L in the compound of formula (II") and the protecting group of the nucleobase which may be protected in the compound of formula (II"); and L in the compound of formula (II"') and the protecting group of the nucleobase which may be protected in the compound of formula (II"') can be removed.

[0143] In one embodiment, the amount of (1) aqueous ammonia, (2) aqueous ammonia / ethanol, or (3) mixed solution of methylamine-methanol solution / water used is, for example, within a range of 1 to 1000 times, and preferably within a range of 3 to 100 times by weight, relative to 1 g of the compound of formula (II") or 1 g of the compound of formula (II'")

[0144] In one embodiment, 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, and more preferably in the range of 5 hours to 48 hours.

[0145] In one embodiment, the reaction temperature is, for example, in the range of 5°C to 100°C, preferably in the range of 10°C to 70°C, and more preferably in the range of 15°C to 50°C.

[0146] <(B-1) Step of Removing the Protecting Group of the Morpholino Nitrogen> In one embodiment, this step is carried out by removing the R １to obtain a compound of formula (II'"): [In the formula, B Ｐ , L, n, R １ , X and Y are as defined above.]

[0147] <(B-2) Step of Removing the Protecting Group of the Morpholino Nitrogen> In one embodiment, this step is carried out by removing the R １ to obtain a compound of formula (I): [In the formula, Base, X, Y, n, R １ is as defined above.]

[0148] In one embodiment, from the compound of formula (II'), R in the compound of formula (II') １ to obtain a compound of formula (II'''); and １ to obtain a compound of formula (I); and the conditions used in the above steps (1) to (4) can be applied to the steps.

[0149] <(11) Purification Step> In one embodiment, this step is a step in which the morpholino nucleic acid oligomer (I) is separated from the reaction mixture by a conventional separation and purification method, such as extraction, concentration, neutralization, filtration, centrifugation, precipitation, recrystallization, C １ From C １８ and isolating the compound by using, alone or in combination, means such as reverse phase column chromatography, cation exchange column chromatography, anion exchange column chromatography, gel filtration column chromatography, high performance liquid chromatography, dialysis, and ultrafiltration (see, for example, WO 1991 / 09033 A1).

[0150] In one embodiment, when the desired compound is purified using reverse phase chromatography, a mixed solution of 20 mM triethylamine / acetic acid buffer and acetonitrile can be used as the elution solvent.

[0151] In one embodiment, when ion exchange chromatography is used to purify the desired compound, for example, a mixture of 1 M saline and 10 mM aqueous sodium hydroxide or 0.3 M saline in 50 mM phosphate buffer can be used.

[0152] In one embodiment of the present invention, the production method of the present invention includes <(2) a deprotection step>.

[0153] In one embodiment of the present invention, the production method of the present invention includes <(2) a deprotection step> and <(3) a neutralization step>.

[0154] In one embodiment of the present invention, the production method of the present invention includes <(2) a deprotection step>, <(3) a neutralization step>, and <(4) a washing step after the neutralization step>.

[0155] In one embodiment of the present invention, the production method of the present invention includes <(5) a washing step before condensation> and <(6) a condensation step>.

[0156] In one embodiment of the present invention, the production method of the present invention includes <(5) a washing step before condensation>, <(6) a condensation step>, and <(7) a washing step 1 after the condensation step> and / or <(8) a washing step 2 after the condensation step>.

[0157] In one embodiment of the present invention, the production method of the present invention includes <(2) a deprotection step>, <(5) a washing step before condensation>, and <(6) a condensation step>.

[0158] In one embodiment of the present invention, the production method of the present invention includes <(2) a deprotection step>, <(3) a neutralization step>, <(5) a washing step before condensation>, and <(6) a condensation step>.

[0159] In one aspect of the present invention, the production method of the present invention includes: <(1) a washing step before the deprotection step>, <(2) a deprotection step>, <(3) a neutralization step>, <(4) a washing step after the neutralization step>, <(5) a washing step before the condensation>, <(6) a condensation step>, <(7) a washing step 1 after the condensation step>, and <(8) a washing step 2 after the condensation step>.

[0160] In one embodiment of the present invention, the production method of the present invention does not include <(5) the washing step before condensation>.

[0161] In one aspect of the present invention, the production method of the present invention includes: <(1) a washing step before the deprotection step>, <(2) a deprotection step>, <(3) a neutralization step>, <(4) a washing step after the neutralization step>, <(6) a condensation step>, <(7) a washing step 1 after the condensation step>, and <(8) a washing step 2 after the condensation step>.

[0162] In one embodiment of the present invention, the production method of the present invention does not include a capping step, as the case may be.

[0163] According to the method for producing a morpholino nucleic acid oligomer of the present invention, a compound represented by the formula (I): [wherein Base, X, Y, and n are as defined above], a morpholino nucleic acid oligomer of formula (I-1): [wherein Base, X, Y, and n are as defined above], for example, a morpholino nucleic acid oligomer of formula (I-2): A morpholino nucleic acid oligomer of the formula: [wherein Base, X, Y, and n are as defined above] can be obtained.

[0164] In one embodiment of the present invention, a compound of formula (I): [In the formula, Base, X, Y, and n are as defined above] Morpholino nucleic acid oligomers are described, for example, in International Publications (WO2012 / 029986, WO2013 / 100190, WO2015 / 137409, WO2015 / 194520, WO2017 / 047707, WO2021 / 132591, WO2021 / 172498, etc.), or can be synthesized by a person skilled in the art based on the description in the International Publications. (PMO), preferably a phosphorodiamidate mopholino oligomer (PMO) capable of skipping at least one exon selected from the group consisting of exons 51, 53, 45, 55, 44, and 50 of the human dystrophin gene, and a specific example thereof is Viltepso (see WO2012 / 029986; CAS registration number: 2055732-84-6).

[0165] In one embodiment of the present invention, a compound of formula (I-1): [wherein Base, X, Y, and n are as defined above] morpholino nucleic acid oligomers are described, for example, in international publications (WO2001 / 083740, WO2006 / 000057, WO2010 / 048586, WO2010 / 050801, WO2011 / 057350, WO2014 / 144978, WO2014 / 153240, etc.), or can be synthesized by a phosphorodiamidate morpholino oligomer that a person skilled in the art can recognize based on the description in the international publications. (PMO), preferably a phosphorodiamidate mopholino oligomer capable of skipping at least one exon selected from the group consisting of exons 51, 53, 45, 55, 44, and 50 of the human dystrophin gene. (PMO), and specific examples thereof include Eteplirsen (see WO2006 / 000057, WO2010 / 050801, WO2014 / 144978; CAS registration number: 1173755-55-9), Golodirsen (see WO2001 / 083740, WO2006 / 000057; CAS registration number: 1422959-91-8), and Casimersen (see WO2001 / 083740, WO2006 / 000057, WO2011 / 057350; CAS registration number: 1422958-19-7).

[0166] In one embodiment of the present invention, the morpholino nucleic acid oligomer obtained by the production method of the present invention is represented by formula (I-2): [wherein Base, X, Y, and n are as defined above].

[0167] Reference Example 1: Morpholino Monomer Compounds The structural formulas and abbreviations of the morpholino monomer compounds are shown in Table 2. ｐ , C ｐ , T ｐ was either commercially available or prepared by methods commonly used in the art (see WO2008 / 008113). ＣＥ and G ＰＯＢ was prepared by a method commonly used in the art (see WO2012 / 043730, WO2009 / 064471).

[0168]

[0169] Reference Example 2: Preparation of 4-{[(2S,6R)-6-(4-benzamido-2-oxopyrimidin-1-yl)-4-tritylmorpholin-2-yl]methoxy}-4-oxobutanoic acid 4-{[(2S,6R)-6-(4-benzamido-2-oxopyrimidin-1-yl)-4-tritylmorpholin-2-yl]methoxy}-4-oxobutanoic acid was prepared by a method commonly used in the art (see WO2012 / 043730).

[0170] Reference Example 3: Preparation of 4-{[(2S,6R)-6-(4-benzamido-2-oxopyrimidin-1-yl)-4-tritylmorpholin-2-yl]methoxy}-4-oxobutanoic acid supported on aminomethylpolystyrene resin. 10.0 g of aminomethylpolystyrene resin (manufactured by Watanabe Chemical Co., Ltd.) was placed in 130 mL of pyridine, and 12.1 g of 4-{[(2S,6R)-6-(4-benzamido-2-oxopyrimidin-1-yl)-4-tritylmorpholin-2-yl]methoxy}-4-oxobutanoic acid prepared in Reference Example 2, 17.3 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 2.7 g of 4-DMAP were added, followed by washing in with 10 mL of pyridine. Next, 15.3 mL of triethylamine and 20 mL of pyridine were added, and the mixture was stirred at room temperature for 1 day. After the reaction, the resin was filtered off. The resulting resin was washed with pyridine, dichloromethane, methanol, and tetrahydrofuran, in that order. 125 mL of tetrahydrofuran, 15.6 mL of 2,6-lutidine, and 12.7 g of benzoyl chloride were added to the resulting resin, and the mixture was stirred at room temperature for 1 hour. The resin was filtered off, washed with tetrahydrofuran, methanol, and dichloromethane, in that order, and dried under reduced pressure to obtain 13.6 g of the title compound. The loading amount of the target compound was determined by measuring the molar amount of trityl per 1 g of resin by UV absorbance at 409 nm using a known method. The loading amount of the resin was 550 μmol / g.

[0171] UV measurement conditions: Instrument: U-2910 (Hitachi) Solvent: methanesulfonic acid Wavelength: 409 nm ε value: 45,000

[0172] Example 1: Preparation of oligomer Synthesis of morpholino nucleic acid oligomer having the base sequence 5'-CCTCCGGTTCTGAAGGTGTTC-3'

[0173] 1. Elongation Reaction 3.0 g (1.7 mmol) of 4-{[(2S,6R)-6-(4-benzamido-2-oxopyrimidin-1-yl)-4-tritylmorpholin-2-yl]methoxy}-4-oxobutanoic acid (Reference Example 3) supported on aminomethyl polystyrene resin was transferred to a reaction vessel, and the synthesis cycle shown in Table 3 was initiated. The number of extension reaction synthesis cycles was defined as the number corresponding to the chain length to be synthesized. In each condensation step, 1.3 to 2.2 molar equivalents of the morpholino monomer compound were used relative to the oligomer obtained in the immediately preceding extension reaction synthesis cycle, so that the base sequence of the morpholino nucleic acid oligomer would be 5'-CCTCCGGTTCTGAAGGTGTTC-3'.

[0174]

[0175] 30% 2,2,2-trifluoroethanol (TFE) / dichloromethane (DCM) (containing 30% TFE in DCM) was used as cleaning solution 1. DCM was used as cleaning solution 2. Pretreatment solution 1, which will be described later, was used as cleaning solution 3.

[0176] Deprotection solution 1 was prepared by dissolving a mixture of trifluoroacetic acid (1.2 equivalents) and triethylamine (1 equivalent) in a DCM solution containing 1% (v / v) triisopropylsilane (TIPS) and 20% (v / v) TFE to a concentration of 5% (w / v).

[0177] As the neutralization solution 1, isopropyl alcohol (IPA) / N,N-diisopropylethylamine (DIPEA) / DCM (ratio of 353:75:1065 (v / v)) was used.

[0178] As pretreatment solution 1, a mixture of 1,3-dimethyl-2-imidazolidinone (DMI) containing N-ethylmorpholine (NEM) was used (5-6% NEM solution in DMI (v / v)).

[0179] As the morpholino monomer compound, A shown in Table 1 ｐ , C ｐ , T ｐ , and G ＣＥ A morpholino monomer compound was dissolved in pretreatment solution 1 to prepare a 0.10 to 0.14 M morpholino monomer compound solution in pretreatment solution 1 (condensation solution 1), which was used as condensation solution 1.

[0180] <Steps 5 to 7 in preparing an 18-mer having the base sequence 5'-CCTCCGGTTCTGAAGGTG-3'>

[0181] Step 5: After Step 4, 25 mL of Washing Solution 3 (5.3% NEM solution in DMI (v / v)) was added to the aminomethylpolystyrene resin carrying the 17-mer oligomer having the base sequence 5'-CCTCCGGTTCTGAAGGT-3' in the reaction vessel, followed by stirring for 5 minutes, and then the solution in the reaction vessel was drained. Subsequently, 25 mL of Washing Solution 3 was added to the reaction vessel, followed by stirring for 5 minutes, and then the solution was drained.

[0182] Step 6 After Step 5, add Condensation Solution 1 (0.12 M G in Pretreatment Solution 1) to the reaction vessel. ＣＥ 25 mL of the solution was added and stirred for 18 hours.

[0183] Step 7: After Step 6, the solution in the reaction vessel was drained. Subsequently, 50 mL of washing solution 2 was added to the reaction vessel, stirred for 5 minutes, and the solution in the reaction vessel was drained.

[0184] The aminomethylpolystyrene resin carrying the morpholino nucleic acid oligomer synthesized above was washed twice with DCM, recovered from the reaction vessel, and dried under reduced pressure at 60° C. for 6 hours, yielding 14.6 g of aminomethylpolystyrene resin carrying the morpholino nucleic acid oligomer.

[0185] Example 2 1. Elongation Reaction 3.0 g (1.5 mmol) of 4-{[(2S,6R)-6-(4-benzamido-2-oxopyrimidin-1-yl)-4-tritylmorpholin-2-yl]methoxy}-4-oxobutanoic acid supported on aminomethyl polystyrene resin (synthesized in the same manner as in Reference Example 3) was transferred to a reaction vessel, and the synthesis cycle shown in Table 4 was initiated. The number of extension reaction synthesis cycles was defined as the number corresponding to the chain length to be synthesized. In each condensation step, 1.3 to 2.2 molar equivalents of morpholino monomer compound were used relative to the oligomer obtained in the immediately preceding extension reaction synthesis cycle, so that the base sequence of the morpholino nucleic acid oligomer would be 5'-CCTCCGGTTCTGAAGGTGTTC-3'. In Example 2, the reactor was purged with nitrogen gas instead of Step 5 of Example 1. Specifically, after step 4, the liquid was drained, and then a nitrogen balloon was attached to the lower part of the filter of the filter reactor, the cock was opened, and nitrogen was purged twice.

[0186]

[0187] 30% 2,2,2-trifluoroethanol (TFE) / dichloromethane (DCM) (30% TFE in DCM) was used as cleaning solution 1. DCM was used as cleaning solution 2.

[0188] Deprotection solution 1 was prepared by dissolving a mixture of trifluoroacetic acid (1.2 equivalents) and triethylamine (1 equivalent) in a DCM solution containing 1% (v / v) triisopropylsilane (TIPS) and 20% (v / v) TFE to a concentration of 5% (w / v).

[0189] As the neutralization solution 1, isopropyl alcohol (IPA) / N,N-diisopropylethylamine (DIPEA) / DCM (ratio of 353:75:1065 (v / v)) was used.

[0190] As the morpholino monomer compound, A shown in Table 1 ｐ , C ｐ , T ｐ , and G ＣＥThe morpholino monomer compound was dissolved in a 5 to 6% DIPEA solution (v / v) in DMI to prepare a 0.10 to 0.13 M morpholino monomer compound solution (condensation solution 2), which was used as condensation solution 2.

[0191] As capping solution 1, a mixture of acetic anhydride / DIPEA / DCM (ratio of 0.5:1.25:98.25 (v / v)) was used.

[0192] <Steps 2 to 7 in preparing an 18-mer having the base sequence 5'-CCTCCGGTTCTGAAGGTG-3'>

[0193] Step 2: After Step 1, 45 mL of deprotection solution 1 was added to the aminomethylpolystyrene resin in the reaction vessel carrying the 17-mer oligomer having the base sequence 5'-CCTCCGGTTCTGAAGGT-3' and protected at the ends with trityl groups, followed by stirring for 5 minutes and draining the solution from the reaction vessel. This process was repeated five more times.

[0194] 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.

[0195] 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 once more.

[0196] Step 5: After step 4, the reaction vessel was purged with nitrogen, and then condensation solution 2 (0.12 M G in 5.3% DIPEA solution in DMI (v / v)) was added. ＣＥ 25 mL of the solution was added and stirred for 6 hours.

[0197] Step 6: After Step 5, the solution in the reaction vessel was drained. Then, 50 mL of Wash Solution 2 was added to the reaction vessel, stirred for 5 minutes, and the solution was drained.

[0198] Step 7: After Step 6, the solution in the reaction vessel was drained. Then, 50 mL of capping solution 1 was added to the reaction vessel, stirred for 10 minutes, and the solution was drained.

[0199] The aminomethylpolystyrene resin carrying the synthesized morpholino nucleic acid oligomer was washed three times with DCM, recovered from the reaction vessel, and dried under reduced pressure at 60° C. for 6 hours, yielding 11.7 g of aminomethylpolystyrene resin carrying the morpholino nucleic acid oligomer.

[0200] Comparative Example 1: Preparation of oligomer Synthesis of morpholino nucleic acid oligomer having the base sequence 5'-CCTCCGGTTCTGAAGGTGTTC-3'

[0201] 1. Elongation Reaction 50 g (23 mmol) of 4-{[(2S,6R)-6-(4-benzamido-2-oxopyrimidin-1-yl)-4-tritylmorpholin-2-yl]methoxy}-4-oxobutanoic acid supported on aminomethyl polystyrene resin (resin loading: 458 μmol / g), prepared by the same method as in Reference Example 2, was transferred to a reaction vessel, and the synthesis cycle shown in Table 5 was initiated. In each condensation step, 2.0 to 3.0 molar equivalents of the morpholino monomer compound were used relative to the oligomer obtained in the immediately preceding elongation reaction synthesis cycle, so that the base sequence of the morpholino nucleic acid oligomer would be 5'-CCTCCGGTTCTGAAGGTGTTC-3'.

[0202]

[0203] DCM was used as cleaning solution 2. Pretreatment solution 2, which will be described later, was used as cleaning solution 4.

[0204] Deprotection solution 2 was prepared by dissolving a mixture of trifluoroacetic acid (2 equivalents) and triethylamine (1 equivalent) in a DCM solution containing 1% (v / v) ethanol and 10% (v / v) TFE to a concentration of 3% (w / v).

[0205] As neutralization solution 2, IPA / DIPEA / DCM (ratio of 25:5:70 (v / v)) was used.

[0206] As pretreatment solution 2, a mixture of DIPEA / DMI (ratio of 10:90 (v / v)) was used.

[0207] As the morpholino monomer compound, A shown in Table 1 ｐ, C ｐ , T ｐ , and G ＣＥ The morpholino monomer compound was dissolved in the pretreatment solution 2 to prepare a 0.16 M solution of the morpholino monomer compound in the pretreatment solution 2 (condensation solution 2), which was used as the condensation solution 2.

[0208] As capping solution, a mixture of acetic anhydride / 2,6-lutidine / DCM (ratio 20:30:50 (v / v)) was used.

[0209] The aminomethylpolystyrene resin carrying the synthesized morpholino nucleic acid oligomer was recovered from the reaction vessel and dried under reduced pressure at room temperature for two days, yielding 258.0 g of aminomethylpolystyrene resin carrying the morpholino nucleic acid oligomer.

[0210] Test Example 1: Comparison of the morpholino nucleic acid oligomers prepared in Examples 1 and 2 with the morpholino nucleic acid oligomer prepared in Comparative Example 1. Morpholino nucleic acid oligomers having the base sequence 5'-CCTCCGGTTCTGAAGGTGTTC-3' were synthesized by an extension reaction and dried to obtain aminomethyl polystyrene resins carrying the morpholino nucleic acid oligomers of Example 1 and Comparative Example 1. Approximately 30 mg of each of these resins was placed in a reaction vessel, and 0.75 mL of deprotection solution 1 was added and reacted at room temperature for 40 minutes. Deprotection solution 1 was then removed, and the resin was washed with neutralization solution 1 and dichloromethane and dried under reduced pressure at room temperature. 28% aqueous ammonia-ethanol (4 / 1) was added to the dried resin, and the mixture was stirred at room temperature for 17 hours. The supernatant was diluted and subjected to HPLC (C18 ion pair analysis) to measure the purity of the morpholino nucleic acid oligomer in the unpurified mixture. By comparing the morpholino nucleic acid oligomers 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.

[0211]

[0212] Test Example 2 Comparison of Condensation Reaction Yields Between the Morpholino Nucleic Acid Oligomer Prepared in Example 1 and the Morpholino Nucleic Acid Oligomer Prepared in Comparative Example 1 After each extension cycle of the extension reaction in Example 1-1, a small amount of the aminomethyl polystyrene resin carrying the morpholino nucleic acid oligomer was sampled, and a 40% methylamine-methanol solution / water mixture was added. The mixture was reacted at 65°C for 10 minutes to separate the morpholino nucleic acid oligomer from the resin. Similarly, after each extension cycle of the extension reaction in Comparative Example 1-1, a small amount of the aminomethyl polystyrene resin carrying the morpholino nucleic acid oligomer was sampled, and a 28% aqueous ammonia-ethanol mixture was added. The mixture was reacted overnight at 55°C to separate the morpholino nucleic acid oligomer from the resin. Each supernatant was diluted and subjected to HPLC (C18 ion pair analysis), and the condensation reaction yield was calculated according to the following formula.

[0213] Condensation reaction yield (%) = 100 - peak area value of raw material* / (peak area value of raw material + main peak area value after condensation reaction)

[0214] *The raw material refers to a morpholino nucleic acid oligomer that does not have a trityl group at its end and has a chain length that is one residue shorter than the chain length obtained by the condensation reaction.

[0215] The condensation reaction yield in Example 1 was improved compared to Comparative Example 1.

[0216] Abbreviations: 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

Claims

1. Elongation reaction of morpholino nucleic acid oligomers: 【Chemical 1】 [wherein, B Ｐ is, independently, a nucleobase that may be protected; 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; L is hydrogen, acyl, or a group represented by formula (IV): [Chemical 2] In, Formula (II): 【Chemical Formula 3】 [wherein, L, B Ｐ , X, Y, R １ , and n are as defined above] Of the compound, Treat with a solution containing an acid and a scavenger to remove R from the compound of formula (II) １ and obtain Formula (III): 【Chemical 4】 [wherein, L, B Ｐ , X, Y, and n are as defined above] The step of making the compound of; The step of treating the compound of formula (III) with a solution containing an organic amine and an aprotic polar solvent; Formula (VIII): 【Chemical Formula 7】 [wherein, BP, X, Y, and R1 are as described above, and Z is halogen] The step of treating the compound of with a solution containing an organic amine and an aprotic polar solvent; and In the presence of an organic amine, reacting the compound of formula (III) with the compound of formula (VIII) to obtain a compound of formula (II’): 【Chemical Formula 10】 [wherein, L, BP, X, Y, R1, and n are as described above] A method for producing a morpholino nucleic acid oligomer, comprising the step of.

2. Furthermore, formula (II’): 【Chemical Formula 11】 [wherein, L, B Ｐ , X, Y, R １ , and n are as defined above] Of the compound, The step of treating with a solution containing alcohols and / or halogen solvents, The production method according to claim 1.

3. The method for producing a morpholino nucleic acid oligomer according to claim 1 or 2, wherein the solution containing the acid and the scavenger is a solution containing trifluoroacetic acid and triisopropylsilane.

4. The method for producing a morpholino nucleic acid oligomer according to claim 1 or 2, wherein the solution containing the acid and the scavenger is a solution containing trifluoroacetic acid, triethylamine, triisopropylsilane, 2,2,2-trifluoroethanol, and dichloromethane.

5. The method for producing a morpholino nucleic acid oligomer according to claim 1 or 2, wherein the solution containing the organic amine and the aprotic polar solvent is a solution containing N-ethylmorpholine and 1,3-dimethyl-2-imidazolidinone.

6. The method for producing a morpholino nucleic acid oligomer according to claim 1 or 2, wherein the solid phase carrier is swellable polystyrene, non-swellable polystyrene, PEG-linked polystyrene, controlled pore glass, oxalylated-controlled pore glass, Tentagel support - aminopolyethylene glycol derivatized support, or a copolymer of Poros - polystyrene / divinylbenzene.

7. The method for producing a morpholino nucleic acid oligomer according to claim 1 or 2, wherein the linker is a 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 dialkylene sulfonyl.

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