Nucleoside derivatives and uses thereof

Abstract

To provide nucleoside derivatives, and to provide RNA drugs using the nucleoside derivatives.SOLUTION: Provided is a derivative represented by the formula (1), (R1 represents a hydroxyl group, a protected hydroxyl group, and the like; R2 represents a hydrogen atom, a protecting group for a hydroxyl group, and the like; R3 represents a hydrogen atom, a protecting group for a hydroxyl group, and the like; R4 represents a hydroxyl group, a protected hydroxyl group, and the like; and B represents a purine base or a pyrimidine base.)SELECTED DRAWING: None

Description

Technical Field

[0001] This specification relates to nucleoside derivatives and their uses.

Background Art

[0002] As one of the RNA drugs designed to hybridize with mRNA and suppress gene expression, siRNA, which utilizes RNA interference, and antisense nucleic acids (ASO) that utilize RNaseH interaction have been developed.

[0003] In order to effectively suppress gene expression using these drugs, in addition to the selection of the target site, the ability to hybridize with the target RNA of the drug, nuclease resistance, cell membrane permeability, and the ability to suppress gene expression based on the gene expression suppressing action inherent to the drug are required. Various modifications to the backbone portion have been attempted to enhance the effectiveness of such drugs (Patent Document 1).

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] Although various modified forms have been created, specific knowledge regarding the stereoisomerism of such modified forms and the above-mentioned various functions is extremely scarce. Furthermore, the relationship between the introduction sites into the modified forms in siRNA and ASO and the above-mentioned various functions is not necessarily clear, and it has become an extremely difficult situation to predict the introduction sites suitable for specific modified forms.

[0006] The inventors have hitherto studied the modification of the aminoalkyl group at the 4'-position carbon atom of ribose and various modifications at the 5'-position carbon atom thereof. In the process, it has been found that in the 4'-aminoalkyl modification, although it has excellent cell membrane permeability performance and nuclease resistance performance, its ability to form a double strand with the target RNA is reduced due to its N-type-S-type conformational isomerism.

[0007] In addition, in the 5'-aminoalkyl modification, both the R-form and the S-form exist. Regarding the R-form, it has been reported that it has excellent gene expression inhibitory ability, but regarding the S-form, nothing has been reported.

[0008] This specification provides a nucleoside derivative and an RNA drug using the nucleoside derivative.

Means for Solving the Problems

[0009] When the inventors attempted to introduce a certain type of aminoalkyl group to the 5'-position carbon atom of ribose, as a result of various studies, the S-form for the 5'-position was obtained. When such an S-form was used as the introduced species of siRNA, it was surprisingly found that it exhibits high affinity for the target RNA and also excellent nuclease resistance. It was difficult to predict such an action of the S-form.

[0010] Furthermore, when the S-form was introduced into the 5'-terminal region of the passenger strand, the siRNA exhibited excellent nuclease resistance performance and gene expression inhibitory ability. Based on these findings, this specification provides the following means.

[0011] [1] A nucleoside derivative, A derivative represented by the following formula (1).

Chemical formula

[10] A method for a nucleoside derivative represented by formula (1), comprising a step of introducing a purine base or a pyrimidine base into the 1'-position carbon atom of an intermediate represented by the following formula (4).

Chemical formula

Brief Description of Drawings

[0012]

Figure 1

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Figure 8

Figure 9

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Figure 17

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Figure 20

Figure 21

Mode for Carrying Out the Invention

[0013] The disclosure of this specification relates to nucleoside derivatives useful for RNA drugs. Specifically, it relates to a nucleoside derivative having an aminopropyl group at the 5'-position of ribose in a specific enantiomer (S-form) (hereinafter also referred to as the S-form), and an RNA drug having this S-form at a specific site.

[0014] According to the disclosure in this specification, the S-form, which is a specific enantiomer, is selected as the introduced species of the RNA drug and introduced into the RNA drug site-specifically. Thereby, the double-stranded hybridizing ability, nuclease resistance ability, and gene expression inhibitory ability of the RNA drug can be maximized.

[0015] Hereinafter, representative and non-limiting specific examples of the present invention will be described in detail with appropriate reference to the drawings. This detailed description is simply intended to show those skilled in the art the details for implementing preferred examples of the present invention and is not intended to limit the scope of the present invention. Also, the additional features and inventions disclosed below can be used separately or together with other features and inventions in order to provide further improved nucleoside derivatives and their uses.

[0016] Also, the combinations of features and steps disclosed in the following detailed description are not essential for carrying out the present invention in the broadest sense and are described only for explaining representative specific examples of the present invention in particular. Furthermore, the various features of the above and below representative specific examples, as well as the various features described in the independent and dependent claims, do not have to be combined as shown in the specific examples described here or in the order listed when providing additional and useful embodiments of the present invention.

[0017] All features described in this specification and / or claims are intended to be disclosed individually and independently of one another, as distinct from the configuration of features described in the examples and / or claims, as limitations on the original disclosure and the specific matters claimed. Further, all descriptions of numerical ranges and groups or populations are made with the intention of disclosing intermediate configurations as limitations on the original disclosure and the specific matters claimed.

[0018] Hereinafter, the nucleoside derivatives or salts thereof disclosed in this specification and their uses will be described. For convenience of explanation, the nucleoside derivatives will be described first, and then the RNA drug design containing such nucleoside derivatives will be described.

[0019] In the following, the meaning of "lower" in the substituents of the compounds described in this specification means that the number of carbon atoms constituting the substituent is at most 10. For example, usually 1 to 6 carbon atoms, or 1 to 5 carbon atoms are exemplified. Also, for example, it is 1 or more and 4 or less carbon atoms, and also for example, it is 1 or more and 3 or less carbon atoms.

[0020] <Nucleoside derivative> The nucleoside derivative (S-form) disclosed in this specification is represented by the following formula (1). The S-form is an enantiomer in which the substituent adopts an S-configuration with respect to the 5'-carbon atom which is an asymmetric carbon atom represented by formula (1). Further, as the substituent, a nitrogen atom (N)-containing propyl group is bonded to the 5'-carbon atom to a carbon atom.

[0021] [Chemical formula]

[0022] [R 1 [Regarding] In formula (1), R 1 represents a hydroxyl group, a hydroxyl group in which a hydrogen atom is substituted with an alkyl group or an alkenyl group, or a hydroxyl group protected by a protecting group.

[0023] <Alkyl group> Examples of the alkyl group include saturated hydrocarbon groups that are linear, branched, cyclic, or combinations thereof. Usually, lower alkyl groups are preferred. For example, lower alkyl groups having 1 to 6 carbon atoms, or more preferably lower alkyl groups having 1 to 5 carbon atoms, and even more preferably lower alkyl groups having 1 to 4 carbon atoms or 1 to 3 carbon atoms can be mentioned. Examples of linear alkyl groups having 1 to 4 carbon atoms include methyl group, ethyl group, n-propyl group, and n-butyl group. Among them, methyl group, ethyl group, and n-propyl group are preferred, and for example, methyl group and ethyl group are preferred, and for example, methyl group is preferred. Examples of branched alkyl groups having 1 to 4 carbon atoms include isopropyl group, isobutyl group, s-butyl group, and t-butyl group. Among them, isopropyl group is a particularly preferred example. Examples of cyclic alkyl groups having 1 to 4 carbon atoms include cyclopropyl group, cyclobutyl group, or cyclopropylmethyl group.

[0024] <Alkenyl group> Examples of the alkenyl group include saturated hydrocarbon groups that are linear, branched, cyclic, or combinations thereof. Usually, lower alkenyl groups are preferred. Examples of lower alkenyl groups include ethenyl group, 1-propenyl group, 2-propenyl group, 1-methyl-2-propenyl group, 1-methyl-1-propenyl group, 2-methyl-1-propenyl group, 1-butenyl group, 2-butenyl group, and the like.

[0025] <Protecting group for hydroxyl group> As protecting groups for hydroxyl groups, those well-known to those skilled in the art can be referred to, for example, Protective Groups in Organic Synthesis (John Wiley and Sons, 2007 edition). Representative examples of protecting groups for hydroxyl groups include, for example, aliphatic acyl groups, aromatic acyl groups, lower alkoxymethyl groups, oxycarbonyl groups which may have appropriate substituents, tetrahydropyranyl groups which may have appropriate substituents, tetrathiopyranyl groups which may have appropriate substituents, methyl groups substituted with one to three substituted or unsubstituted aryl groups in combination (wherein the substituents in the aforementioned substituted aryl mean lower alkyl, lower alkoxy, halogen atoms, or cyano groups), or silyl groups, etc.

[0026] Examples of alkoxy groups include saturated alkyl ether groups that are linear, branched, cyclic, or combinations thereof. Lower alkoxy groups are preferred. Examples of lower alkoxy groups include lower alkoxy groups having 1 to 6 carbon atoms, or lower alkoxy groups having 1 to 5 carbon atoms. Further, alkoxy groups having 1 to 4 carbon atoms, or alkoxy groups having 1 to 3 carbon atoms are preferred, and alkoxy groups having 1 to 4 carbon atoms are particularly preferred. Preferred examples of alkoxy groups having 1 to 4 carbon atoms include methoxy group, ethoxy group, n-propoxy group, or n-butoxy group, etc. Also, isopropoxy group, isobutoxy group, s-butoxy group, or t-butoxy group, etc. are also preferred examples. Also, cyclopropoxy group, cyclobutoxy group are preferred, and cyclopropylmethoxy group is also a preferred example.

[0027] Among these, aliphatic acyl groups, aromatic acyl groups, and silyl groups are particularly preferred examples. Also, methyl groups substituted with one to three substituted or unsubstituted aryl groups in combination (wherein the substituents in the substituted aryl are as described above) are also preferred examples.

[0028] Examples of the aliphatic acyl group include an alkylcarbonyl group, a carboxyalkylcarbonyl group, a halogeno-lower alkylcarbonyl group, or a lower alkoxy-lower alkylcarbonyl group.

[0029] The alkyl in the alkylcarbonyl group is as described above. That is, examples of the alkylcarbonyl group include a formyl group, an acetyl group, a propionyl group, a butyryl group, an isobutyryl group, a pentanoyl group, a pivaloyl group, a valeryl group, an isovaleryl group, an octanoyl group, a nonanoyl group, a decanoyl group, a 3-methylnonanoyl group, an 8-methylnonanoyl group, a 3-ethyloctanoyl group, a 3,7-dimethyloctanoyl group, an undecanoyl group, a dodecanoyl group, a tridecanoyl group, a tetradecanoyl group, a pentadecanoyl group, a hexadecanoyl group, a 1-methylpentadecanoyl group, a 14-methylpentadecanoyl group, a 13,13-dimethyltetradecanoyl group, a heptadecanoyl group, a 15-methylhexadecanoyl group, an octadecanoyl group, a 1-methylheptadecanoyl group, a nonadecanoyl group, an icosanoyl group, or a henicosanoyl group. Among these, an acetyl group, a propionyl group, a butyryl group, an isobutyryl group, a pentanoyl group, and a pivaloyl group are preferable examples, and an acetyl group is a particularly preferable example. Further, the alkyl in the carboxylated alkylcarbonyl group is as described above. The substitution position of carboxylation and the like can also be appropriately selected. That is, examples of the carboxylated alkylcarbonyl group include a succinoyl group, a glutaryl group, and an adipoyl group.

[0030] Regarding the halogen, lower, and alkyl in the halogeno-lower alkylcarbonyl group, they are as described above. The substitution position of the halogen and the like can also be appropriately selected. That is, examples of the halogeno-lower alkylcarbonyl group include a chloroacetyl group, a dichloroacetyl group, a trichloroacetyl group, and a trifluoroacetyl group.

[0031] In the lower alkoxy lower alkylcarbonyl group, alkoxy and alkyl, and further lower are as described above. The position where the lower alkoxy substitutes can also be appropriately selected. That is, examples of the lower alkoxy lower alkylcarbonyl group include a methoxyacetyl group.

[0032] Examples of the aromatic acyl group include an arylcarbonyl group, a halogenoarylcarbonyl group, a lower alkylated arylcarbonyl group, a lower alkoxylated arylcarbonyl group, a carboxylated arylcarbonyl group, a nitrated arylcarbonyl group, or an arylated arylcarbonyl group.

[0033] Examples of the arylcarbonyl group include a benzoyl group, an α-naphthoyl group, a β-naphthoyl group, and more preferably a benzoyl group. Examples of the halogenoarylcarbonyl group include a 2-bromobenzoyl group and a 4-chlorobenzoyl group. Examples of the lower alkylated arylcarbonyl group include a 2,4,6-trimethylbenzoyl group, a 4-toluoyl group, a 3-toluoyl group, and a 2-toluoyl group. Examples of the lower alkoxylated arylcarbonyl group include a 4-anisoyl group, a 3-anisoyl group, and a 2-anisoyl group.

[0034] Examples of the carboxylated arylcarbonyl group include a 2-carboxybenzoyl group, a 3-carboxybenzoyl group, and a 4-carboxybenzoyl group. Examples of the nitrated arylcarbonyl group include a 4-nitrobenzoyl group, a 3-nitrobenzoyl group, and a 2-nitrobenzoyl group. Examples of the arylated arylcarbonyl group include a 4-phenylbenzoyl group.

[0035] Examples of the lower alkoxymethyl group include a methoxymethyl group, a 1,1-dimethyl-1-methoxymethyl group, an ethoxymethyl group, a propoxymethyl group, an isopropoxymethyl group, a butoxymethyl group, and a t-butoxymethyl group. Particularly preferred is methoxymethyl Examples of the base include.

[0036] Examples of the optionally substituted oxycarbonyl group include a lower alkoxycarbonyl group, a lower alkoxycarbonyl group substituted with a halogen or a silyl group, or an alkenyloxycarbonyl group.

[0037] Examples of the lower alkoxycarbonyl group include a methoxycarbonyl group, an ethoxycarbonyl group, a t-butoxycarbonyl isobutoxycarbonyl group. Examples of the lower alkoxycarbonyl group substituted with the halogen or the silyl group include a 2,2-trichloroethoxycarbonyl group and a 2-(trimethylsilyl)ethoxycarbonyl group.

[0038] Examples of the alkenyloxycarbonyl group include a vinyloxycarbonyl group. Examples of the optionally substituted tetrahydropyranyl group include, for example, a tetrahydropyran-2-yl group or a 3-bromotetrahydropyran-2-yl group, and particularly preferably a tetrahydropyran-2-yl group.

[0039] Examples of the optionally substituted tetrathiopyranyl group include a tetrahydrothiopyran-2-yl group and a 4-methoxytetrahydrothiopyran-4-yl group, and more preferably a tetrahydrothiopyran-2-yl group. In the methyl group substituted with one to three substituted or unsubstituted aryl groups, the substituents in the aforementioned substituted aryl mean a lower alkyl, a lower alkoxy, a halogen, or a cyano group.

[0040] Examples of the methyl group substituted with one to three substituted or unsubstituted aryl groups include a benzyl group, an α-naphthylmethyl group, a β-naphthylmethyl group, a diphenylmethyl group, a triphenylmethyl group, and an α-naphthyldiphenylmethyl group. Preferred examples include a benzyl group and a triphenylmethyl group. Other examples include a 9-anthrylmethyl 4-methylbenzyl group, a 2,4,6-trimethylbenzyl group, and a 3,4,5-trimethylbenzyl group. Preferred examples include a 2,4,6-trimethylbenzyl group and a 3,4,5-trimethylbenzyl group. Other types include, for example, a 4-methoxybenzyl group, a 4-methoxyphenyldiphenylmethyl group, and a 4,4'-dimethoxytriphenylmethyl group. Preferred examples include a 4-methoxybenzyl group, a 4-methoxyphenyldiphenylmethyl group, and a 4,4'-dimethoxytriphenylmethyl group. Further examples include a 4-chlorobenzyl group and a 4-bromobenzyl group. Additionally, a 4-cyanobenzyl group is also mentioned as a preferred example.

[0041] Examples of the silyl group include a trimethylsilyl group, a triethylsilyl group, an isopropyldimethylsilyl group, a t-butyldimethylsilyl group, a methyldiisopropylsilyl group, a methyldi-t-butylsilyl group, a triisopropylsilyl group, a diphenylmethylsilyl group, a diphenylbutylsilyl group, a diphenylisopropylsilylphenyldiisopropylsilyl group. Among these, more preferred examples include a trimethylsilyl group, a t-butyldimethylsilyl group, a triisopropylsilyl group, and a diphenylmethylsilyl group. Particularly preferred examples include a trimethylsilyl group, a t-butyldimethylsilyl group, and a diphenylmethylsilyl group.

[0042] As a protecting group for a hydroxyl group, it may mean a substituent that is cleaved and removed by either a chemical method (e.g., hydrogenolysis, hydrolysis, electrolysis, or photolysis, etc.) or a biological method (e.g., hydrolysis in the human body. Imagine induction by microorganisms, etc.). As a protecting group for a hydroxyl group, in particular, a substituent that is removed by hydrogenolysis or hydrolysis is preferably exemplified. Note that the protected hydroxyl group can be referred to as a hydroxyl group in which the hydrogen atom is substituted by such a protecting group.

[0043] [R 2 with respect to] R 2 represents a hydrogen atom, a protecting group for a hydroxyl group, a phosphate group, a protected phosphate group, a phosphorothioate group, a protected phosphorothioate group, or -P(=O) n R 5 R 6 as described above. The protecting group for a hydroxyl group is as described above. The phosphorothioate group is a group in which one oxygen atom of the phosphate group is substituted by a sulfur atom.

[0044] <protected phosphate group> The protecting groups in the protected phosphate group are known to those skilled in the art. Examples include a lower alkyl group, a lower alkyl group substituted with a cyano group, an ethyl group substituted with a silyl group, a lower alkyl group substituted with a halogen, a lower alkenyl group, a lower alkenyl group substituted with a cyano group, a cycloalkyl group, a lower alkenyl group substituted with a cyano group, an aralkyl group, an aralkyl group in which the aryl ring is substituted with a nitro group, an aralkyl group in which the aryl ring is substituted with a halogen, an aryl group substituted with a lower alkyl group, an aryl group substituted with a halogen, or an aryl group substituted with a nitro group.

[0045] The lower alkyl group is as described above. Examples of the lower alkyl group substituted with a cyano group include a 2-cyanoethyl group and a 2-cyano-1,1-dimethylethyl group, and particularly preferably, a 2-cyanoethyl group. Examples of the ethyl group substituted with a silyl group include a 2-methyldiphenylsilylethyl group, a 2-trimethylsilylethyl group, and a 2-triphenylsilylethyl group.

[0046] Examples of the lower alkyl group substituted with a halogen include a 2,2,2-trichloroethyl group, a 2,2,2-tribromoethyl group, a 2,2,2-trifluoroethyl group, and a 2,2,2-trichloroethyl group, and particularly preferably, a 2,2,2-trichloroethyl group. Examples of the lower alkenyl group include an ethenyl group, a 1-propenyl group, a 2-propenyl group, a 1-methyl-2-propenyl group, a 1-methyl-1-propenyl group, a 2-methyl-1-propenyl group, a 1-butenyl group, and a 2-butenyl group.

[0047] Examples of the lower alkenyl group substituted with a cyano group include a 2-cyanoethyl group, a 2-cyanopropyl group, and a 2-cyanobutenyl group. Examples of the aralkyl group include a benzyl group, an α-naphthylmethyl group, a β-naphthylmethyl group, an indenylmethyl group, a phenanthrenylmethyl group, an anthracenylmethyl group, a diphenylmethyl group, a triphenylmethyl group, a 1-phenethyl group, a 2-phenethyl group, a 1-naphthylethyl group, a 2-naphthylethyl group, a 1-phenylpropyl group, a 2-phenylpropyl group, a 3-phenylpropyl group, a 1-naphthylpropyl, a 2-naphthylpropyl, a 3-naphthylpropyl, a 1-phenylbutyl group, a 2-phenylbutyl group, a 3-phenylbutyl group, and a 4-phenylbutyl group. More preferably, a benzyl group, a diphenylmethyl group, a triphenylmethyl group, a 1-phenethyl group, and a 2-phenethyl group are included, and particularly preferably, a benzyl group is included.

[0048] Examples of the aralkyl group in which the aryl ring is substituted with a nitro group include a 2-(4-nitrophenyl)ethyl group, an o-nitrobenzyl group, a 4-nitrobenzyl group, a 2,4-dinitrobenzyl group, a 4-chloro-2-nitrobenzyl group, and the like.

[0049] The protecting group of phosphoric acid may mean a substituent that is cleaved and eliminated by either a chemical method (e.g., hydrogenolysis, hydrolysis, electrolysis, or photolysis, etc.) or a biological method (e.g., hydrolysis in the human body, etc. Imagine induction by microorganisms, etc.). As the protecting group of phosphoric acid, in particular, substituents that are eliminated by hydrogenolysis or hydrolysis are preferably exemplified.

[0050] <-P(=O)n(R 5 )R 6 > R of the S form 2 may be -P(=O)n(R 5 )R 6 n represents 0 or 1, and R 5 and R 6 are the same as or different from each other and each represents a hydrogen atom, a hydroxyl group, a protected hydroxyl group, a mercapto group, a protected mercapto group, a lower alkoxy group, a cyano lower alkoxy group, an amino group, or a substituted amino group. However, when n is 1, R 5 and R 6 do not both become hydrogen atoms at the same time. The protected hydroxyl group and the lower alkoxy group are as described above.

[0051] <protected mercapto group> The protected mercapto group is well-known to those skilled in the art. Examples of the protected mercapto group include, in addition to those exemplified as the protecting group of the above hydroxyl group, for example, an alkylthio group, an arylthio group, an aliphatic acyl group, and an aromatic acyl group. Preferably, an aliphatic acyl group and an aromatic acyl group are exemplified, and particularly preferably, an aromatic acyl group is exemplified. Examples of the aromatic acyl group include a benzoyl group.

[0052] Examples of the alkylthio group include saturated alkylthio groups that are linear, branched, cyclic, or a combination thereof. A lower alkylthio group is preferred. Examples of the lower alkylthio group include, for example, a lower alkylthio group having 1 to 6 carbon atoms, or a lower alkylthio group having 1 to 5 carbon atoms, and more preferably, a lower alkylthio group having 1 to 4 carbon atoms, or an alkylthio group having 1 to 3 carbon atoms. Examples of the saturated alkylthio group having 1 to 4 carbon atoms include, for example, a methylthio group, an ethylthio group, an n-propylthio group, an n-butylthio group, etc. As the alkylthio group, a lower alkylthio group is preferred, and examples thereof include, for example, methylthio, ethylthio, and t-butylthio groups. Also, an isopropylthio group, an isobutylthio group, an s-butylthio group, a t-butylthio group, etc. are also exemplified as preferred examples. Further, a cyclopropylthio group or a cyclobutylthio group is exemplified as a preferred example, and a cyclopropylmethylthio group is further exemplified as a more preferred example.

[0053] Examples of the arylthio group include, for example, benzylthio.

[0054] Examples of the cyano lower alkoxy group include, for example, an alkoxy group having 1 to 5 carbon atoms which is linear, branched, cyclic, or a combination thereof substituted with a cyano group (counting without including the number of carbon atoms in the cyano group). Specifically, for example, cyanomethoxy, 2-cyanoethoxy, 3-cyanopropoxy, 4-cyanobutoxy, 3-cyano-2-methylpropoxy, or 1-cyanomethyl-1,1-dimethylmethoxy, etc. are included, and particularly preferably, a 2-cyanoethoxy group is included.

[0055] R 5 and R 6 can be selected as a substituted amino group. The substituent of the amino group represents any one of a lower alkoxy group, a lower alkylthio group, a cyano lower alkoxy group, or a lower alkyl group. Note that the above R 5 and R 6When both are substituted amino groups, they may be different substituted amino groups from each other as the substituted amino groups. The lower alkoxy group, lower alkylthio group, cyano lower alkoxy group, and lower alkyl group are as described above.

[0056] -P(=O)n(R 5 )R 6 More specifically, the phosphoramidite group, H-phosphonate group, or phosphonyl group is preferably exemplified, and the phosphoramidite group is particularly preferably exemplified.

[0057] -P(=O)n(R 5 )R 6 In, when n is 0 and at least one of R 5 and R 6 is a substituted amino group and the other may be anything, it becomes a phosphoramidite group. As the phosphoramidite group, a phosphoramidite group in which one of R 5 and R 6 is a substituted amino group and the other is a lower alkoxy group or a cyano lower alkoxy group has good reaction efficiency in the condensation reaction and is particularly preferred. Examples of the substituted amino group include, for example, diethylamino group, diisopropylamino group, dimethylamino group, etc., and particularly preferably diisopropylamino group is exemplified. Also, as the lower alkoxy group in the other substituent of R 5 and R 6 , a methoxy group is preferably exemplified. Also, as the cyano lower alkoxy group, a 2-cyanoethyl group is preferably exemplified. Specifically, as the phosphoramidite group, -P(OC2H4CN)(N(CH(CH3)2), or -P(OCH3)(N(CH(CH3)2) is preferably exemplified.

[0058] -P(=O)n(R 5 )R 6 In, when n is 1 and R 5 and R 6When at least one of them is a hydrogen atom and the other can be anything other than a hydrogen atom, it becomes an H-phosphonate group. Examples of the substituent other than hydrogen include a hydroxyl group, a methyl group, a methoxy group, a thiol group, etc., and a hydroxyl group is particularly preferably exemplified.

[0059] Also, in -P(=O)n(R 5 )R 6 when n is 1 and both R 5 and R 6 are lower alkoxy groups, it becomes a phosphonyl group. Note that the lower alkoxy groups in R 5 and R 6 may be the same as or different from each other. Preferred examples of the lower alkoxy group include a methoxy group, an ethoxy group, etc. Specifically, examples of the phosphonyl group include -P(=O)(OCH3)2.

[0060] As R 2 in the S form, for example, it is particularly preferably -P(=O)n(R 5 )R 6 . Preferred examples of -P(=O)n(R 5 )R 6 include a phosphoramidite group, an H-phosphonate group, or a phosphonyl group. As R2, it is also preferably a phosphate group or a protected phosphate group. Further, as R 2 , it is also preferably a hydrogen atom or a protecting group for a hydroxyl group.

[0061] R 2 Specific other examples preferably include a hydrogen atom, an acetyl group, a benzoyl group, a benzyl group, a p-methoxybenzyl group, a trimethylsilyl group, a tert-butyldiphenylsilyl group, -P(OC2H4CN)(N(CH(CH3)2), -P(OCH3)(N(CH(CH3)2), or a phosphonyl group.

[0062] [Regarding R 3 R 3 ​represents a hydrogen atom, a protecting group for a hydroxyl group, a phosphate group, a protected phosphate group, a phosphorothioate group or a protected phosphorothioate group. These are as described above. R 3 Specific examples thereof include a hydrogen atom, a methyl group, a benzyl group, a p-methoxybenzyl group, a dimethoxytrityl group, a monomethoxytrityl group, a tert-butyldiphenylsilyl group, or a trimethylsilyl group as preferred examples.

[0063] [R 4 with respect to] R 4 represents an azide group (N3-), a hydroxyl group, a protected hydroxyl group, NHR 7 . R 7 includes a hydrogen atom or a protecting group for an amino group. The protected hydroxyl group is as already described.

[0064] Protecting groups for amino groups are well-known to those skilled in the art, and reference may be made to the aforementioned references. Specifically, in addition to those mentioned above as protecting groups for hydroxyl groups, for example, benzyl group, methylbenzyl group, chlorobenzyl group, dichlorobenzyl group, fluorobenzyl group, trifluoromethylbenzyl group, nitrobenzyl group, methoxyphenyl group, methoxymethyl (MOM) group, N-methylaminobenzyl group, N,N-dimethylaminobenzyl group, phenacyl group, acetyl group, trifluoroacetyl group, pivaloyl group, benzoyl group, phthalimide group, allyloxycarbonyl group, 2,2,2-trichloroethoxycarbonyl group, benzyloxycarbonyl group, t-butoxycarbonyl (Boc) group, 1-methyl-1-(4-biphenyl)ethoxycarbonyl (Bpoc) group, 9-fluorenylmethoxycarbonyl group, benzyloxymethyl (BOM) group, or 2-(trimethylsilyl)ethoxymethyl (SEM) group, etc. can be mentioned. More preferably, benzyl group, methoxyphenyl group, acetyl group, trifluoroacetyl (TFA) group, pivaloyl group, benzoyl group, t-butoxycarbonyl (Boc) group, 1-methyl-1-(4-biphenyl)ethoxycarbonyl (Bpoc) group, 9-fluorenylmethoxycarbonyl group, benzyloxymethyl (BOM) group, or 2-(trimethylsilyl)ethoxymethyl (SEM) group can be mentioned, and particularly preferably, benzyl group, methoxyphenyl group, acetyl group, benzoyl group, benzyloxymethyl group can be mentioned.

[0065] In the present invention, the protecting group for an amino group may mean a substituent that can be cleaved and removed by either a chemical method (e.g., hydrogenolysis, hydrolysis, electrolysis, or photolysis, etc.) or a biological method (e.g., hydrolysis in the human body, etc. imaginatively, induction by microorganisms, etc.). In particular, a substituent that can be removed by hydrogenolysis or hydrolysis is preferred as the protecting group for an amino group.

[0066] [Regarding B] Examples of B in the S body include purine bases or pyrimidine bases. For example, as B, a purin-9-yl group, a 2-oxo-pyrimidin-1-yl group, a substituted purin-9-yl group, or a substituted 2-oxo-pyrimidin-1-yl group can be selected.

[0067] Also, for example, as B, 2,6-dichloropurin-9-yl or 2-oxo-pyrimidin-1-yl can be mentioned. Further, 2-oxo-4-methoxy-pyrimidin-1-yl, 4-(1H-1,2,4-triazol-1-yl)-pyrimidin-1-yl, or 2,6-dimethoxypurin-9-yl can be mentioned.

[0068] Also, for example, 2-oxo-4-amino-pyrimidin-1-yl with the amino group protected, 2-amino-6-bromopurin-9-yl with the amino group protected, 2-amino-6-hydroxypurin-9-yl with the amino group protected, 2-amino-6-hydroxypurin-9-yl with the amino group and / or hydroxyl group protected, 2-amino-6-chloropurin-9-yl with the amino group protected, 6-aminopurin-9-yl with the amino group protected, or 4-amino-5-methyl-2-oxo-pyrimidin-1-yl group with the amino group protected can be mentioned. The protecting groups for the hydroxyl group and the amino group will be described later.

[0069] Also, for example, 6-aminopurin-9-yl (adenine), 2-amino-6-hydroxypurin-9-yl (guanidine), 2-oxo-4-amino-pyrimidin-1-yl (cytosine), 2-oxo-4-hydroxy-pyrimidin-1-yl (uracil), or 2-oxo-4-hydroxy-5-methylpyrimidin-1-yl (thymine) can be mentioned.

[0070] Also, for example, 4-amino-5-methyl-2-oxo-pyrimidin-1-yl (methylcytosine), 2,6-diaminopurin-9-yl, 6-amino-2-fluoropurin-9-yl, 6-mercaptopurin-9-yl, 4-amino-2-oxo-5-chloro-pyrimidin-1-yl, or 2-oxo-4-mercapto-pyrimidin-1-yl can be mentioned.

[0071] Also, for example, 6-amino-2-methoxypurin-9-yl, 6-amino-2-chloropurin-9-yl, 2-amino-6-chloropurin-9-yl, or 2-amino-6-bromopurin-9-yl can be mentioned.

[0072] The substituent in each of the substituted purin-9-yl group or the substituted 2-oxo-pyrimidin-1-yl group is any one of a hydroxyl group, a protected hydroxyl group, a lower alkoxy group, a mercapto group, a protected mercapto group, a lower alkylthio group, an amino group, a protected amino group, an amino group substituted with a lower alkyl group, a lower alkyl group, a lower alkoxymethyl group, or a halogen atom, or any combination of a plurality thereof. These substituents will be described later.

[0073] As B in the S form, the substituents in the substituted purin-9-yl group or the substituted 2-oxo-pyrimidin-1-yl group will be described later, but in addition to this, it is also preferable that a triazole group and a lower alkoxymethyl group are added.

[0074] Preferable examples of the substituted purin-9-yl group include, for example, 6-aminopurin-9-yl, 2,6-diaminopurin-9-yl, 2-amino-6-chloropurin-9-yl, 2-amino-6-bromopurin-9-yl, 2-amino-6-hydroxypurin-9-yl, 6-amino-2-methoxypurin-9-yl, 6-amino-2-chloropurin-9-yl, 6-amino-2-fluoropurin-9-yl, 2,6-dimethoxypurin-9-yl, 2,6-dichloropurin-9-yl, or 6-mercaptopurin-9-yl, etc. If there are amino groups or hydroxyl groups among the above-mentioned substituents, examples of preferable substituents in which those amino groups and / or hydroxyl groups are protected can be mentioned.

[0075] Examples of the substituted 2-oxo-pyrimidin-1-yl include 2-oxo-4-amino-pyrimidin-1-yl, 1H-(1,2,4-triazol-1-yl)-pyrimidin-1-yl, 4-1H-1,4-amino-2-oxo-5-chloro-pyrimidin-1-yl, 2-oxo-4-methoxy-pyrimidin-1-yl, 2-oxo-4-mercapto-pyrimidin-1-yl, 2-oxo-4-hydroxy-pyrimidin-1-yl, 2-oxo-4-hydroxy-5-methylpyrimidin-1-yl, or 4-amino-5-methyl-2-oxo-pyrimidin-1-yl, etc. Also, 2-oxo-4-methoxy-pyrimidin-1-yl, or 4-(1H-1,2,4-triazol-1-yl)-pyrimidin-1-yl are mentioned as preferred examples.

[0076] Among these Bs, if the substituent contains an amino group or a hydroxyl group, the substituents in which those amino groups or hydroxyl groups are protected are mentioned as preferred examples.

[0077] The S form may be a salt. The form of the salt is not particularly limited. In addition to the salt with a base such as phosphoric acid, acid addition salts are exemplified and may take the form of an internal counter ion. Also, for example, depending on the type of substituent, a base addition salt may be formed. As the salt, a pharmaceutically acceptable salt is preferred. The types of acids and bases that form pharmaceutically acceptable salts are well known to those skilled in the art, and for example, those described in J.Pharm.Sci., 1-19 (1977) etc. can be referred to. For example, as the acid addition salts, mineral acid salts and organic acid salts are included. Also, when one or more substituents contain an acidic moiety, base addition salts are also mentioned as preferred examples.

[0078] Examples of the mineral acid salts include hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, and hydrogen phosphate. Usually, hydrochloride and phosphate are preferred examples. Examples of the organic acid salts include acetate, trifluoroacetate, gluconate, lactate, salicylate, citrate, tartrate, ascorbate, succinate, maleate, fumarate, formate, benzoate, methanesulfonate, ethanesulfonate, or p-toluenesulfonate. Usually, acetate and the like are preferred examples. Examples of the base addition salts include salts of alkali metals, salts of alkaline earth metals, organic amine salts, and addition salts of amino acids.

[0079] Examples of the salts of the alkali metals include sodium salts and potassium salts. Examples of the salts of the alkaline earth metals include magnesium salts and calcium salts. Examples of the organic amine salts include triethylamine salts, pyridine salts, procaine salts, picoline salts, dicyclohexylamine salts, diethanolamine salts, triethanolamine salts, and tris(hydroxymethyl)aminomethane salts. Examples of the addition salts of amino acids include arginine salts, lysine salts, ornithine salts, serine salts, glycine salts, aspartate salts, and glutamate salts.

[0080] The S-form or its salt may exist as a hydrate or a solvate, and these substances are also included in the scope of the disclosure of this specification. The S-form or its salt can be synthesized based on the synthesis methods described below.

[0081] <Synthesis Method of S-Form> The S-form can be synthesized, for example, by the following method. The following shows synthesis examples divided into an S-form having uracil, which is a base having a secondary amino group -NH as a base, and an S-form having adenine and guanine, which are bases having a primary amino group as a base.

[0082] (Uridine Derivative, Cytidine Derivative) As shown below, starting from uridine, an S-form as a derivative of uridine can be obtained according to the following scheme (Ref: Bioorg. Med. Chem., 2013, 21, 6171Bioorg. Med. Chem., 2013, 21, 6171 ). In the following scheme, compounds 35 and later are included in the S-form. Details of the reactions in the scheme are disclosed in the examples.

[0083]

Chemical formula

Chemical formula

[0084] Also, the S-form as a cytidine derivative can be obtained according to the following scheme. Details of the reactions in the scheme are disclosed in the examples. In the following scheme, compounds 36 and later are included in the S-form.

Chemical formula

[0085] (Adenosine derivative, guanosine derivative) For nucleosides having these bases, a common intermediate of the S-form is prepared in advance according to the following scheme. By introducing a base into this intermediate, deprotection of the protecting group of the amino group of these bases can be avoided, and the S-form can be obtained (Ref: Org. Lett., 2001, 22, 3583.) . This intermediate can also be used for uridine nucleoside and thymidine nucleoside. For example, in the following scheme, the intermediates disclosed in this specification are compounds 49 to 52, but compounds 47 to 48 are also useful as their precursors.

[0086]

Chemical formula

[0087] Adenosine derivatives can be obtained according to the following scheme. For example, in the following scheme, compounds 53 and later are included in the S form. Details of the reactions in the scheme are disclosed in the examples.

Chemical formula

[0088] Guanosine derivatives can be obtained according to the following scheme. For example, in the following scheme, compounds 63 and later are included in the S form. Details of the reactions in the scheme are disclosed in the examples.

[0089]

Chemical formula

[0090] The S form described above is useful as a ribonucleoside or ribonucleotide for forming a structural unit of an oligonucleotide.

[0091] The S form is introduced into a basic skeleton formed by linking known ribonucleotides and / or deoxyribonucleotides, etc. as phosphoramidite forms and / or in a form bound to a solid phase according to, for example, known DNA or RNA synthesis methods in siRNA. Thereby, a base-containing unit which is a structural unit constituting the skeleton of the oligonucleotide can be constituted.

[0092] Note that the ribonucleotides and / or deoxyribonucleotides constituting the skeleton of the RNA drug may contain natural ribonucleotides and / or deoxyribonucleotides, or may contain various known chemically modified ribonucleotides and / or deoxyribonucleotides.

[0093] Examples of the chemically modified ribonucleotides and / or deoxyribonucleotides include LNA, BNA, PNA, modified bases, etc., and also, for example, nucleotide derivatives represented by the following formula (2).

[0094]

Chem.

[0095] In the above formula (2), R 1 , R 2 , R 3 and B are synonymous with these groups in formula (1), except that R 1 represents a fluorine atom. Typically, ribonucleotides in which R 1 is a fluorine atom, ribonucleotides in which it is OCH3, ribonucleotides in which R 2 is thiolated, etc. can be mentioned. Any of these known chemical modifications can be synthesized appropriately by those skilled in the art, and can also be commercially available.

[0096] <Intermediate for Obtaining S-Isomer and Method for Synthesizing S-Isomer> According to the present specification, an intermediate for obtaining an S-isomer represented by the following formula (4) is provided. Also, an intermediate precursor represented by the following formula (5) is provided. Furthermore, according to the present specification, a method for synthesizing an S-isomer using this intermediate is provided.

[0097]

Chem.

[0098] In formula (4), R 7 represents an acetyl group or a cyclic structure in which oxygen atoms bonded to each other are linked by -C(CH3)2- to R 7 , R 8 represents a hydrogen atom or a protecting group for a hydroxyl group, R 9 represents a hydrogen atom or a protecting group for a hydroxyl group, and R 10 represents a hydroxyl group, a protected hydroxyl group, or an azide group. Regarding the protecting group for the hydroxyl group in formula (4), the same embodiments as the protecting group for the hydroxyl group already described in the nucleoside derivative represented by formula (1) are applicable.

Chem.

[0099] In formula (5), R 7 represents a cyclic structure in which oxygen atoms that are bonded to each other and bonded to R 7 are linked by -C(CH3)2-, R 8 represents a protecting group for a hydroxyl group, and R 9 represents a hydrogen atom or a protecting group for a hydroxyl group. Regarding the protecting group for the hydroxyl group in formula (5), the same embodiments as those of the protecting group for the hydroxyl group already described in the nucleoside derivative represented by formula (1) are applicable.

[0100] As the intermediate represented by formula (4), compounds 49 to 52 in the synthesis scheme of the common intermediate of the aforementioned adenosine nucleoside and guanosine nucleoside are included. Such a common intermediate is, for example, in formula (4), R 7 represents an acetyl group or a cyclic structure in which oxygen atoms that are bonded to each other and bonded to R 7 are linked by -C(CH3)2-, R 8 represents a protecting group for a hydroxyl group, R 9 represents a protecting group for a hydroxyl group, R 10 represents a compound that represents a hydroxyl group, a protected hydroxyl group, or an azide group. The protecting group in such a common intermediate is appropriately selected from the aforementioned protecting groups and the like.

[0101] As the direct starting material for the adenosine derivative and the guanosine derivative, compound 52 can be used. By introducing a compound constituting the base portion into compound 52, adenosine derivatives and guanosine derivatives useful for efficiently synthesizing oligoribonucleotides can be synthesized. For example, a common intermediate such as compound 52 is, in formula (4), R 7 represents an acetyl group, R 8 represents a protecting group for a hydroxyl group, R 9 represents a protecting group for a hydroxyl group, R 10 represents a compound that represents an azide group. The protecting group in such a common intermediate is appropriately selected from the aforementioned protecting groups and the like.

[0102] As the intermediate precursor represented by formula (5), compounds 47 to 48 in the synthesis scheme of the aforementioned common intermediate are included. Compounds 47 and 48 have a 2-propenyl group (allyl group) with an S configuration at the 5'-position carbon atom of ribose, and various substituents can be introduced by subsequent addition reactions. The protecting group in such an intermediate precursor is appropriately selected from the aforementioned protecting groups and the like.

[0103] According to this specification, a method for synthesizing a nucleoside represented by formula (1) and having a purine base such as adenine or guanine as a base using the common intermediate represented by formula (4) is also provided. That is, a step of introducing a purine base, a pyrimidine base, or a purine base or a pyrimidine base in which an amino group is protected into the 1'-position carbon atom of the common intermediate represented by formula (4), especially a step of introducing a purine base in which an amino group is protected, into the common intermediate represented by formula (4), in which R 7 represents an acetyl group, R 8 represents a protecting group for a hydroxyl group, R 9 represents a protecting group for a hydroxyl group, R 10 represents an azide group. By doing so, elimination of the protecting group of the amino group in the purine base can be avoided or suppressed to obtain the S form.

[0104] Introduction of a purine base or the like into the 1'-position carbon atom of the common intermediate represented by formula (4) is possible by a glycosylation reaction using tin(IV) chloride or the like, for example. By introducing a base, one embodiment of the S form represented by formula (1) can be obtained. Furthermore, by performing introduction and elimination of various protecting groups and the like by known methods, a desired S form can be obtained.

[0105] <Oligonucleotide containing ribonucleotide> The oligonucleotides disclosed in this specification contain at least one or more base-containing units represented by at least the following formula (3). In addition to the base-containing units represented by the following formula (3), the oligonucleotides can contain ribonucleotides and / or deoxyribonucleotides. As the ribonucleotides, one or more base-containing units (hereinafter, also simply referred to as S-form-containing units) in which the ribonucleotides represented by the aforementioned formula (2) are linked are included.

[0106] [Chemical formula]

[0107] In formula (3), R 1 represents a hydroxyl group, or a hydroxyl group in which a hydrogen atom is substituted with an alkyl group or an alkenyl group. These are synonymous with the definitions already described with respect to formula (1). Also, B is synonymous with the definition already described with respect to formula (1).

[0108] In formula (3), X 1 represents an oxygen atom or a sulfur atom, and X 2 represents OH (or O - ) or SH (S - ).

[0109] The oligonucleotide is single-stranded, but can be an oligoribonucleotide or a hybrid of ribonucleotides and deoxyribonucleotides. The base length of the oligonucleotide is not particularly limited, and can have a base length corresponding to the RNA drug described later.

[0110] As described above, the oligonucleotide can be chemically synthesized by known DNA / RNA synthesis methods.

[0111] (siRNA) This specification discloses the passenger strand and guide strand of siRNA as well as siRNA. Figure 1 exemplifies siRNA having a 21-base-long passenger strand and guide strand respectively, and having 2-base-long dangling ends on their respective 3'-terminal sides.

[0112] (Passenger strand) From the perspective of the siRNA activation ability, the passenger strand can include, for example, one or more S-form-containing units within the first base to the ninth base from its 3'-terminal. Also, the passenger strand can include one or more S-form-containing units within the first base to the sixth base from its 5'-terminal.

[0113] By the passenger strand including at least one S-form-containing unit in either or both of these regions, the gene expression inhibitory ability of siRNA, that is, the siRNA activation ability, can be maintained or improved.

[0114] For example, it may include one, two, or three S-form-containing units in the first base to the third base from the 3'-terminal, and for example, it may include one, two, three, or four S-form-containing units in the first base to the fourth base from the 3'-terminal. Also for example, it may include one, two, or three S-form-containing units in the first base to the third base from the 5'-terminal, and for example, it may include one, two, three, or four S-form-containing units in the first base to the fourth base from the 5'-terminal. Even in such introduction forms, the siRNA activation ability can be maintained or improved. In the case of such sequence design of the S-form-containing unit, it can also include S-form-containing units on both the 3'-terminal side and the 5'-terminal side.

[0115] In contrast, in the region other than these 3'-terminal side region and 5'-terminal side region, that is, in the intermediate region sandwiched between the sixth base counted from the 5'-terminal side and the ninth base counted from the 3'-terminal side, by including one or more S-form-containing units, the siRNA activity decreases.

[0116] From the perspective of nuclease resistance performance, the passenger strand can include, for example, one or more S-form-containing units within the first to fifth bases from its 3'-end. By including the S-form-containing unit in this region, the nuclease resistance performance can be improved. Nucleotide

[0117] For example, within the first to third bases from the 3'-end of the passenger strand, for example, one to three, for example, two or three S-form-containing units can be included starting from the side closer to the 3'-end. Also, for example, within the first to fourth bases from the 3'-end of the passenger strand, for example, one to four, for example, three or four S-form-containing units can be included starting from the side closer to the 3'-end. Also, for example, within the first to fifth bases from the 3'-end of the passenger strand, for example, one to five, for example, four or five S-form-containing units can be included starting from the side closer to the 3'-end. By doing so, higher nuclease resistance performance can be ensured.

[0118] From the above, from the perspectives of siRNA activation ability and nuclease resistance performance, the passenger strand can continuously include, for example, three, four, or five S-form-containing units within the first to fifth bases from the 3'-end. Also, from the perspective of siRNA activation ability, one or more S-form-containing units can be included within the first to sixth bases from the 5'-end. Also, for example, three or four S-form-containing units can be included.

[0119] (Guide strand) From the perspective of siRNA activation ability, the guide strand can include an S-form-containing unit at the position of the seventh base from the 5'-end of the guide strand. By including the S-form-containing unit at this position, the siRNA activation ability can be maintained or improved.

[0120] On the other hand, by providing one or more S-form-containing units at sites other than the above-described seventh base, the siRNA activation ability will be reduced. For example, by providing S-form-containing units at the positions of the first to sixth bases and the eighth base from the 5'-end, the siRNA activation ability will also be reduced.

[0121] In the present specification, the base lengths of the passenger strand and the guide strand of siRNA are not particularly limited. For example, they are each an oligoribonucleotide strand of 21 bases or more and 25 bases or less. Further, as siRNA, typically, the passenger strand and the guide strand each have a 2-base-long dangling end on the 3'-end side.

[0122] The guide strand of siRNA can have 2'-OMe-modified ribonucleotides at the first and third bases from the 5'-end. Further, it can have a 2'-F-modified ribonucleotide at the second base from the 5'-end.

Example

[0123] Hereinafter, examples will be described as specific examples in order to more specifically explain the disclosure of the present specification. The following examples are for explaining the disclosure of the present specification and do not limit the scope thereof.

Example

[0124] (Synthesis of 5'-C-Aminoalkyl-Modified Nucleoside Analogue: (S)-5'-C-Aminopropyl-2'-O-methyluridine) The synthesis scheme is shown below. First, the 5'-hydroxyl group of the nucleoside sugar moiety is oxidized to an aldehyde, and an allyl group is introduced to the 5'-position of the sugar moiety using a Mukaiyama-Sakurai allylation reaction with allyltrimethylsilane in the presence of a Lewis acid. Then, the allyl group is converted to an azidopropyl group and led to the corresponding phosphoramidite form. That is, based on a known synthesis method using commercially available uridine as a starting material 73,74 Compound 27 in which the 3'-hydroxyl group of the sugar moiety was protected with a TBDMS group was synthesized.

[0125] The 5'-hydroxyl group of the sugar moiety was converted to an aldehyde by Pfitzner-Moffatt oxidation, and compounds 29 and 30 having an allyl group at the 5'-position of the sugar moiety were obtained by reacting allyltrimethylsilane in the presence of a Lewis acid. 1 By 1H-NMR analysis, it was confirmed that compounds 29 and 30 are diastereomers with different configurations at the 5'-position.

[0126] The 5'-hydroxyl group of compound 29 with the desired stereochemistry was protected with a DMTr group, and compound 32 having a hydroxypropyl group at the 5'-position was obtained by an oxidation reaction with hydrogen peroxide following Brown hydroboration. The resulting hydroxyl group was tosylated, and compound 34 azidated with NaN3 was synthesized. Subsequently, the azide group was reduced to an amino group by the Staudinger reaction, and the resulting amino group was protected with a trifluoroacetyl group. Finally, the 3'-TBDPS group was deprotected, and the resulting hydroxyl group was phosphitylated to obtain the target amidite 37.

[0127]

Chemical Structure

[0128] The reagents and conditions were as follows: (a) EDC HCl, CHCl2CO2H, DMSO / CH2Cl2 (1:1 v / v), -5 °C, 1.5 h; (b) allyltrimethylsilane, BF3OEt2, CH2Cl2, 0 °C, 1 h, 29: 43% (in 2 steps); 30: 2% (in 2 steps); (c) DMTrCl, 2,6-lutidine, pyridine, 40 °C, 67 h, 83%; (d) 9-BBN, THF then 30% H2O2aq., 3N NaOH aq., 30 °C, 15 min, 49%; (e) p-TsCl, pyridine, CH2Cl2, r.t., 7 h, 56%; (f) NaN3, DMF, 60 °C, 11 h, 90%; (g) (i) Ph3P, H2O, THF, 40 °C, 16 h, (ii) CF3CO2Et, Et3N, CH2Cl2, r.t., 5 h, 94% (in 2 steps); (h) TBAF / THF, THF, r.t., 14 h, 84%; (i) 2-cyanoethyl-N, N, N´, N´-tetraisopropylphosphoroamidite, 1H-tetrazole, 1-methylimidazole, DMF, r.t., 1 h, 89%.

[0129] The amidite form of the R enantiomer, which is the enantiomer of this S form, was synthesized according to the pamphlet of International Publication No. WO2018 / 110678. Furthermore, using these amidite forms, solid-phase carriers 39 and 41 linked with the S form and the R form were synthesized according to a conventional method.

[0130] [Chemical formula] [Examples]

[0131] (Chemical synthesis of RNA and evaluation of the synthesized RNA) Using the amidite compound synthesized in Example 1, the solid-phase carrier, etc., single-stranded RNAs having the base sequences (SEQ ID NOS: 1 and 2) and configurations shown in FIG. 2 were synthesized according to the known solid-phase phosphoramidite method to prepare double-stranded RNAs. For these double-stranded RNAs, the double-strand forming ability and thermodynamic parameters were calculated.

[0132] (Evaluation of double-strand forming ability) When the synthesized and prepared double-stranded RNA was heated from 15 °C to 70 °C, the increase in absorbance at 260 nm was measured, and the double-strand 50% melting temperature (T m ) was calculated from the obtained sigmoid curve.

[0133] As shown in FIG. 2, as a result of measuring the T m of each oligonucleotide, it was revealed that T m decreased compared to the natural oligonucleotide regardless of the position where the aminopropyl group was introduced. However, when comparing between nucleoside analogs, in the RNA containing the S-form and (S)-5'-AP-U, the decrease was 1.0 °C per modification, which was larger than that of the R-form and 4'AP-form.

[0134] (Calculation of thermodynamic parameters) Next, the thermodynamic parameters at 310 K were calculated using a van't Hoff plot. Since the double-strand forming ability of an oligonucleotide is determined by the balance between the enthalpy change and the entropy change, the thermodynamic evaluation of the double-strand is an important factor. This time, a method was used that utilized the fact that the equilibrium between the formation and dissociation of the double-strand changes depending on the total concentration of the oligonucleotide. The concentration of the oligonucleotide was adjusted to 8 levels (1, 3, 6, 12, 15, 21, 30, 60 M) to measure T m , and according to the conventional method, ΔH° and ΔS° were calculated from the slope and intercept. Then, the thermodynamic parameter (ΔD°) was calculated according to the conventional method.

[0135] As shown in Fig. 2, the absolute value of the ΔG° value increases in the order of natural (dsRNA 1), (S)-5'-AP-U (dsRNA 4), 4'-AP-U (dsRNA 2), and (R)-5'-AP-U (dsRNA 3). Therefore, the above-mentioned T m showed a correlation with the results.

Example

[0136] (Evaluation of nuclease resistance) The effect of the S-form introduced oligonucleotide on nuclease resistance was verified using bovine serum (BS). The oligonucleotide duplexes (SEQ ID NOs: 1-2) shown in Fig. 3 and BS were incubated at 37°C, and samples were taken over time. Thereafter, RNA fragments were separated by polyacrylamide electrophoresis (PAGE), and full-length RNA was evaluated by observing the fluorescence of fluorescein in the gel. The results are also shown in Fig. 3.

[0137] As shown in Fig. 3, in the case of natural RNA 1, the full-length RNA disappeared after 30 minutes, whereas in the case of RNAs 2-4 (4'-AP-U (dsRNA 2), (R)-5'-AP-U (dsRNA 3), (S)-5'-AP-U (dsRNA 4)) into which each analog was introduced, a clear band of full-length could still be confirmed even at the 360-minute time point. Furthermore, when the residual rate of the full-length at 360 minutes of RNAs 2-4 was calculated, it was found to be RNA 2: 56%, RNA 3: 42%, and RNA 4: 67%. Therefore, it was revealed that the introduction of the analog can significantly improve the nuclease resistance of RNA, and in particular, it was confirmed that the introduction of (S)-5'-AP-U can confer higher nuclease resistance to RNA than (R)-5'-AP-U and 4'-AP-U.

Example

[0138] (Evaluation of siRNA containing the S-form) It has been described that the newly synthesized S-form (here, (S)-5'-AP-U) forms a duplex with RNA having a complementary base sequence similar to 4'-AP-U, and can further enhance nuclease resistance. Subsequently, it was verified how the introduction of (S)-5'-AP-U into siRNA, a functional RNA, affects its properties.

[0139] First, thermally stable siRNA duplexes (siRNA5-8, SEQ ID NOs: 3-4) were formed using (S)-5'-AP-U, (R)-5'-AP-U, and 4'-AP-U (see Figure 4). Using these siRNA5-8, nuclease resistance was evaluated in the same manner as in Example 3. The results are shown together with Figure 5.

[0140] As shown in Figure 4, natural siRNA5 was completely degraded at the 30-minute time point, whereas no degradation of siRNA was confirmed even after 360 minutes for siRNA6-8 into which each analog was introduced. Therefore, it was revealed that (R)-5'-AP-U and (S)-5'-AP-U can significantly improve the nuclease resistance of siRNA, similar to 4'-AP-U.

Example

[0141] (Evaluation of the gene expression inhibitory ability of siRNA containing the S-form) Using (S)-5'-AP-U, (R)-5'-AP-U, and 4'-AP-U, siRNAs (SEQ ID NOs: 3-4) into which these analogs were introduced at the base sequences, introduction sites, and number of introductions shown in Figures 5-7, respectively, were synthesized. Using these siRNAs, their gene expression inhibitory ability was evaluated by Dual Luciferase Reporter Assay. In this experiment, siRNAs targeting Renilla Luciferase were designed, and the RNAi activity (siRNA concentrations of 1 nM and 10 nM) was evaluated by calculating the ratio of the luminescence intensities of Renilla Luciferase and Firefly Luciferase when introduced into cells. The control used in this study was one into which no siRNA was introduced. The results are shown together with Figures 5-7.

[0142] Figure 5 shows the evaluation results of the gene expression inhibitory ability of siRNA in which only one S body or the like synthesized on the passenger strand decomposed during RISC formation was introduced into the illustrated locations (1 to 3). As shown in Figure 5, it was suggested that siRNA containing any of the modified nucleic acids showed RNAi activity equivalent to that of the natural type, and the introduction of (R)-5'-AP-U and (S)-5'-AP-U into the passenger strand did not affect the RNAi activity.

[0143] Figure 6 shows the evaluation results of the gene expression inhibitory ability of siRNA in which only one S body or the like was introduced into the illustrated locations (4 to 6) of the guide strand. As shown in Figure 6, generally, the RNAi activity tended to decrease compared with the introduction into the passenger strand. The 2nd to 8th bases from the 5′ end of the guide strand are called the seed region and are strictly recognized by Argonaute-2 during RISC formation. Therefore, chemical modification of the seed region generally often reduces the RNAi activity. First, in the siRNA with an analog introduced at the 8th base from the 5′ end, the S body showed a higher gene expression inhibitory ability than the R body and was comparable to that of 4'-AP-U. Next, in the siRNA with an analog introduced at the 11th position from the 5′ end of the guide strand, the S body had the highest gene expression inhibitory ability. Finally, in the siRNA with an analog introduced at the 20th position from the 5′ end of the guide strand, all the analogs showed gene expression inhibitory ability equivalent to that of the natural type.

[0144] In addition, Figure 7 shows the evaluation results of the gene expression ability of siRNA in which S bodies or the like were introduced into three illustrated locations of the passenger strand and the 3′ end of the illustrated guide strand. As shown in Figure 7, it was confirmed that any of the siRNAs could suppress the expression of the target gene at a level equivalent to that of natural siRNA. From this result, it was suggested that simultaneous modification of both strands of siRNA is possible if the modification positions on both the passenger strand and the guide strand allow the influence of chemical modification.

Example

[0145] (Synthesis of various S bodies) (1) Synthesis of (S)-5′-C-Aminopropyl-2′-O-methylcytidine (S)-5′-AP-C-phosphoramidite 50 was synthesized. During the synthesis of (S)-5′-AP-U-phoshoramidite 35, the carbonyl group at the 4-position of uracil in the synthetic intermediate 35 was activated using 2,4,6-triisopropylbenzenesulfonyl chloride (TPSCl), and then treated with aqueous ammonia to convert the 4-position carbonyl group to an amino group. Subsequently, it was reacted with acetic anhydride in pyridine to acetyl-protect the amino group, the TBDMS group at the 3′-position was deprotected, and the resulting hydroxyl group was phosphitylated to achieve the synthesis of the target (S)-5′-AP-C-phosphoramidite 50.

[0146]

Chemical Structure

[0147] The reagents and conditions were as follows: (a) TPSCl, DMAP, Et3N, CH3CN, r.t., 2 h; (b) 28% NH3aq., r.t., 2 h; (c) Ac2O, pyridine, r.t., 12 h, 86% (3 steps); (d) TBAF (1 M in THF), THF, r.t., 6 h, 89%; (e) 2-cyanoethyl-N, N-diisopropylchlorophosphoroamidite, DIPEA, THF, r.t., 1.5 h, 88%.

[0148] (2) Synthesis of (S)-5′-C-Aminopropyl-2′-O-methyl-adenosine and -guanosine For the synthesis of these S-forms, a common intermediate was synthesized. This is because the protecting groups applied to the bases of these analogs are likely to be deprotected under basic conditions.

[0149] (Common intermediate) The synthesis of the common intermediate was carried out as follows. The scheme is shown below. First, using diacetone-D-glucose as the starting material, aldehyde 51 was synthesized based on a known method. 80 Similar to the synthesis of the (S)-5′-AP-U-amidite, in the presence of a Lewis acid, allyltrimethylsilane was reacted to synthesize ribofuranose derivatives 52 and 53 having an allyl group at the 5-position of the sugar moiety. Regarding the stereochemistry of the 5-position carbon atom of the sugar moiety, it was determined that compound 52 has the S configuration and 53 has the R configuration.

[0150] Subsequently, in order to protect the 5-position hydroxyl group of ribofuranose derivative 52 with a Bn group, the hydroxyl group was activated with NaH and then reacted with BnBr. Contrary to expectations, compound 54 in which the 3-position hydroxyl group was protected with Bn and the 5-position hydroxyl group was protected with TBDPS was obtained. The allyl group of compound 54 was converted to a hydroxypropyl group by an oxidation reaction with hydrogen peroxide following hydroboration with Brown's reagent, the hydroxyl group was tosylated, and then reacted with NaN3 to convert it to compound 57 having an azidopropyl group at the (S)-5-position. After deprotecting the 1,2-isopropylidene group in an aqueous solution of 50% trifluoroacetic acid, compound 58 in which the 1,2-position hydroxyl groups were acetylated with acetic anhydride was synthesized.

[0151]

Chemical formula

[0152] The reagents and conditions in the above scheme were as follows. (a) allyltrimethylsilane, BF3OEt2, CH2Cl2, 40 °C, 30 min, 82% (compound 52:compound 53 = 4:1); (b) BnBr, NaH, DMF, r.t., 16 h, 78%; (c) 9-BBN, THF then 30% H2O2 aq., 3N NaOH aq., 40 °C, 1 h, 91%; (d) p-TsCl, pyridine, CH2Cl2, r.t., 16 h, 95%; (e) NaN3, DMF, 60 °C, 8 h, 90%; (f)(i) 50% CF3CO2H aq., r.t., 4.5 h, (ii) Ac2O, pyridine, r.t., 24 h, 86% (2 steps).

[0153] Subsequently, the synthesized compound 58 was subjected to a glycosylation reaction with N 6 -benzoyladenine in the presence of tin(IV) chloride to synthesize the adenosine derivative 59. Subsequently, the 2'-acetyl group was removed by treatment with K2CO3 in CH3OH, and the compound 61 in which the 2'-hydroxyl group was methylated using CH3I was obtained in a yield of 72%. Next, the 5'-TBDPS group and the 3'-Bn group were deprotected, and the compound 64 in which the 3'-hydroxyl group was selectively protected with the TBDPS group was synthesized. After tritylating the 5'-hydroxyl group by reacting with DMTrCl in the presence of silver(I) nitrate, the azide group was converted to an amino group by the Staudinger reaction and the resulting amino group was protected with a trifluoroacetyl group. Finally, after deprotecting the 3'-TBDPS group of the compound 66, phosphitylation was carried out to synthesize the target (S)-5'-AP-A-Phosphoramidite 68 in a yield of 80%.

[0154] [Chemical formula]

[0155] The reagents and conditions in the above scheme were as follows. (a) N 6 -benzoyl adenine, SnCl4 / CH2Cl2, CH3CN, r.t., 2 h, 70%; (b) K2CO3, CH3OH, 0 °C, 30 min, 92%; (c) CH3I, NaH, THF, 0 °C, 2.5 h, 72%; (d) TBAF / THF, THF, r.t., 16 h, 92%; (e) BCl3 / CH2Cl2, CH2Cl2, -78 °C, 2 h, 89%; (f) TBDPSCl, imidazole, DMF, r.t., 24 h, 76%; (g) DMTrCl, AgNO3, pyridine, THF, 40 °C, 12 h, 72%; (h) (i) Ph3P, H2O, THF, 45 °C, 12 h, (ii) CF3CO2Et, Et3N, CH2Cl2, r.t., 24 h, 93%; (i) TBAF / THF, THF, r.t., 24 h, 99%; (j) 2-cyanoethyl-N,N-diisopropylchlorophosphoroamidite, DIPEA, THF, r.t., 1 h, 80%.

[0156] Subsequently, the synthesis of (S)-5′-AP-G-Phosphoramidite 79 was carried out. First, compound 58 was subjected to a glycosylation reaction with 2-amino-6-chloropurine in the presence of TMSOTf to synthesize compound 69 in a yield of 75%. Subsequently, after deprotecting the 2′-acetyl group, compound 71 in which the 2′-hydroxyl group was methylated with CH3I was synthesized. By treating compound 71 with 3-hydroxypropionitrile and NaH, a guanosine derivative 72 in which the chlorine atom at the 6-position was replaced with an oxygen atom was obtained in a yield of 85%. A compound 73 in which the exocyclic amino group was protected with an isobutyryl (iBu) group was synthesized, and then the guanosine derivative 73 was converted to (S)-5′-AP-G phosphoramidite 79 through the same synthetic route as (S)-5′-AP-A phosphoramidite 68.

[0157]

Chemical Structure

[0158] The reagents and conditions in the above scheme were as follows. (a) 2-Amino-6-chloropurine, N,O-bis(trimethylsilyl)acetamide, TMSOTf, toluene, 80 °C, 15 h, 75%; (b) K2CO3, CH3OH, 0 °C, 30 min, 93%; (c) CH3I, NaH, DMF, 0 °C, 7 h, 69%; (d) 3-Hydroxypropionitrile, NaH, THF, 0 °C, 6 h, 85%; (e) Isobutyric anhydride, DMAP, DMF, 60 °C, 13 h, 77%; (f) TBAF / THF, THF, r.t., 46 h, 83% (g) (i) BCl3 / CH2Cl2, CH2Cl2, -78 °C, 4 h; (ii) TBDPSCl, imidazole, DMF, 0 °C, 29 h, 50% (2 steps); (h) DMTrCl, AgNO3, pyridine, THF, 40 °C, 12 h, 92%; (i) (i) Ph3P, H2O, THF, 45 °C, 22 h, (ii) CF3CO2Et, Et3N, CH2Cl2, r.t., 19 h, 77% (2 steps); (j) TBAF / THF, THF, r.t., 24 h, 70%; (k) 2-Cyanoethyl-N,N-diisopropylchlorophosphoroamidite, DIPEA, THF, r.t., 1 h, 73%. [Example]

[0159] (Evaluation of gene expression inhibitory ability of siRNA containing various S-forms; passenger strand) Using the various S-forms of (S)-5′-AP-U, (S)-5′-AP-C, (S)-5′-AP-A, and (S)-5′-AP-G synthesized in the previous example, the relationship between the introduction position of the S-form and the gene expression inhibitory ability of siRNA was evaluated. As a control, 2'-OMe ribonucleotides were used. siRNAs were synthesized according to the base sequences (SEQ ID NOs: 3 to 4) shown in each of FIGS. 8 to 11 and the introduction position of the S-form, and the gene expression inhibitory ability was evaluated according to the method of Example 5. As a control, 2'-OMe ribonucleotides were used instead of the S-form.

[0160] FIG. 8 shows the evaluation results of the gene expression inhibitory ability of siRNAs in which three consecutive sites (sites 31 to 33) from the 3′-end of the passenger strand were modified with the S-form or 2'-OMe modification. As shown in FIG. 8, it was revealed that the siRNA containing three consecutive S-form modifications in the 3'-terminal region of the passenger strand had a gene expression inhibitory ability equivalent to that of the siRNA containing 2'-OMe modification at the same position.

[0161] FIG. 9 shows the evaluation results of the gene expression inhibitory ability of siRNAs in which three consecutive sites (sites 34 to 36) starting from the 10th base from the 3′-end of the passenger strand were modified with the S-form or 2'-OMe modification. As shown in FIG. 9, in the siRNA in which the S-form was introduced into the central region of the passenger strand, the gene expression inhibitory ability was significantly reduced compared to the siRNA in which 2′-OMe was introduced at the same position.

[0162] FIG. 10 shows the evaluation results of the gene expression inhibitory ability of siRNAs in which three consecutive sites (site 37) from the 5′-end of the passenger strand were modified with the S-form or 2'-OMe modification. As shown in FIG. 10, it was revealed that the siRNA containing three consecutive S-form modifications in the 5'-terminal region of the passenger strand had a gene expression inhibitory ability equivalent to that of the siRNA containing 2'-OMe modification at the same position.

[0163] Figure 11 shows the evaluation results of the gene expression inhibitory ability of siRNAs containing 3, 4, and 5 consecutive S-form modifications at the 3'-end and 5'-end of the passenger strand. As shown in Figure 11, siRNAs containing 3 or 4 consecutive S-form modifications at the 3'-end and 5'-end of the passenger strand, respectively, showed almost the same gene expression inhibitory ability as siRNAs containing 2'-OMe modification at the same position. On the other hand, the siRNA containing 5 consecutive S-form modifications showed a significant decrease in gene expression inhibitory ability compared to the siRNA containing 2'-OMe modification at the same position.

[0164] From the above, as shown in Figure 12, it was confirmed that siRNAs with S-form introduced at the 3'-end and 5'-end of the passenger strand showed the same gene expression inhibitory ability as natural siRNAs and siRNAs containing 2'-OMe modification at the same position. Also, it was found that when S-form was introduced into the central part of the passenger strand, the decrease in gene expression inhibitory ability was greater than that in the case of 2'-OMe modification.

Example

[0165] (Evaluation of gene expression inhibitory ability of siRNAs containing various S-forms; guide strand) Similar to Example 7, using S-form, the relationship between the introduction position of S-form and the gene expression inhibitory ability was evaluated for siRNAs with S-form introduced into the guide strand. siRNAs were synthesized according to the base sequences (SEQ ID NO: 3 - 4) and the introduction positions of S-form shown in each of Figures 13 - 14, and the gene expression inhibitory ability was evaluated according to the method of Example 5.

[0166] Figure 13 shows the evaluation results of the gene expression inhibitory ability of siRNAs with 1 S-form modification or 2'-OMe modification at each site from the 1st to 4th bases from the 5'-end of the guide strand. As shown in Figure 13, it was found that introducing S-form into the 5'-end region of the guide strand tended to significantly decrease the gene expression inhibitory ability compared to 2-OMe.

[0167] Figure 14 shows the evaluation results of the gene expression inhibitory ability of siRNAs with one S-form modification or 2'-OMe modification at each site from the 5th to 8th bases from the 5'-end of the guide strand. As shown in Figure 14, it was revealed that when the S-form was introduced at the 7th base in the 5'-terminal region of the guide strand, it could exhibit a gene expression inhibitory ability equivalent to that of natural RNA and 2'-OMe form.

[0168] From the above, the introduction of the S-form from the 5'-end of the guide strand to the 1st and 2nd bases from the 5'-end of the guide strand completely abolishes the gene expression inhibitory ability, and the introduction of the S-form to the 3rd and 4th bases also significantly reduces the gene expression ability to a large extent. Furthermore, it was revealed that the introduction of the S-form to the 5th, 6th, and 8th bases reduces the gene expression inhibitory ability to a certain extent, and it was clear that these positions should avoid the introduction of the S-form.

[0169] On the other hand, when the S-form was introduced at the 7th base from the 5'-end of the guide strand, since it showed the same activity as natural siRNA, it was considered effective to introduce the S-form at this site. The 2nd to 8th positions from the 5'-end of the guide strand are called the Seed region, and it has been reported that in this region, it interacts with many amino acid residues of Argonaute-2. Therefore, modified nucleic acids that can be introduced into the Seed region were limited to those with sterically small substituents such as 2'-OMe modification, 2'-F modification, and Phosphorothioate modification. However, if chemical modification of the Seed region is possible, the nuclease resistance of siRNA can be efficiently enhanced. In this regard, the S-form was considered to have advantages.

Example

[0170] (Evaluation of nuclease resistance of siRNAs containing various S-forms) The relationship between the introduction position of the S-form and the nuclease resistance of siRNA was evaluated. siRNAs were synthesized according to the base sequences (SEQ ID NOs: 3 to 4) and the introduction positions of the S-form shown in each of Figures 15 to 17, and the nuclease resistance performance was evaluated according to the method of Example 3.

[0171] Figures 15 and 16 show a combination of the introduction positions of the S-form and 2'-OMe form into the passenger strand and the guide strand, and the evaluation results of the nuclease resistance performance of siRNAs with the S-form and the like introduced at various positions. Note that although chemical modification of the guide strand is also important for imparting nuclease resistance, introduction of the S-form into the guide strand reduces the gene expression inhibitory ability. Therefore, phosphorothioate modification, introduction of the 2'-OMe form, and introduction of the 2'-F form were performed on the guide strand.

[0172] As shown in Figure 15, by introducing the S-form from the 1st to the 3rd bases from the 5'-end of the passenger strand and from the 1st to the 3rd bases from the 3'-end, siRNAs with the full-length remained even after 24 hours. In contrast, for siRNAs with the 2'-OMe form introduced at the same positions, no band of full-length siRNA was confirmed at the 24-hour time point, and the siRNAs were completely degraded. Also, as shown in Figure 16, by introducing the S-form from the 1st to the 4th bases and the 5th base from the 5'-end of the passenger strand and from the 1st to the 4th bases and the 5th base from the 3'-end, siRNAs with the full-length remained even after 24 hours. In contrast, for siRNAs with the 2'-OMe form introduced at the same positions, as in Figure 15, the siRNAs were completely degraded. Therefore, it was revealed that the S-form improves the nuclease resistance of siRNAs more than the 2'-OMe form analog.

[0173] Next, in order to examine in detail the effect of the S-form on the nuclease resistance performance of siRNAs, the nuclease resistance performance of siRNAs containing three consecutive S-forms at the 3'-end or 5'-end of the passenger strand was evaluated. The results are shown in Figure 17.

[0174] As shown in Figure 17, the band corresponding to the full-length siRNA of the siRNA with three consecutive S-forms introduced into the 3'-terminal region could be confirmed even after 12 hours. On the other hand, for the siRNA with three consecutive S-forms introduced into the 5'-terminal region, the band corresponding to the full-length siRNA was gradually degraded, and the same result was obtained for the same region of the passenger strand as that of the native siRNA.

[0175] From the above results, it was clarified that the introduction of the S form into the 3′-terminal region of siRNA can effectively improve the resistance to nucleases in serum compared to the case where (S)-5′-AP modification is introduced into the 5′-terminal region. In addition, the siRNA used in this example contains a UA sequence in the 3′-terminal region of the passenger strand, and the siRNA containing the S form near this UA sequence showed improved stability in serum. Therefore, it was clarified that the nuclease resistance can be efficiently improved by introducing the S form into the position where siRNA is easily degraded, particularly the sequence where U and A are consecutive near the terminal.

[0176] In addition, from the introduction status of the 2′-OMe form and 2′-F form into the guide strand of the siRNA used in this example, regarding the nuclease resistance performance, the modified form shown in Fig. 18 was considered as one of the preferred examples.

[0177] In addition, from the previous experimental results and known findings (such as the Ui-Tei rule), regarding the gene expression inhibitory ability and nuclease resistance performance of siRNA, the modified form shown in Fig. 19 was considered as one of the preferred examples.

Example

[0178] (Measurement of KNTC2 gene expression level by quantitative RT-PCR) In this example, siRNAs 47 - 49 against the KNTC2 gene were prepared using the sequences (SEQ ID NOs: 5 - 6) and chemical modifications shown in Fig. 20, and transfected into human colon cancer HCT116 cells using Lipofectamine RNAiMAX (Invitrogen), and their gene expression inhibitory ability was evaluated. In addition, siRNA against GL3 as an internal standard gene (SEQ ID NOs: 7 - 8) was also prepared and evaluated in the same manner. The KNTC2 gene is an important factor in chromosome segregation during the mitotic phase (M phase) of the cell cycle, and its expression inhibition can induce cell death in cells with active proliferation such as cancer cells.

[0179] On the day before transfection, HCT116 cells were seeded at 2.0×10 5 cells / well in a 12-well plate (1 mL / well of medium). siRNA against the KNTC2 gene (final concentration 10 nM) was transfected into HCT116 cells at a final concentration of 10 nM using Lipofectamine RNAiMAX (Invitrogen). After incubating with KNTC2 siRNA for 24 hours, the cells were re-seeded into a new plate and cultured for another 24 hours. Next, total RNA of the cells was extracted using the NucleoSpin RNA Plus Kit (Takara Bio), and cDNA was synthesized using PrimeScript RT Master Mix (Takara Bio) with the extracted RNA as a template. For the synthesized cDNA, quantitative RT-PCR was performed using the Thermal Cycler Dice Real Time System (Takara Bio) and TB Green Premix ExTaqII (Takara Bio) with the following primers. Human ACTB gene: 5'-GGAGCAATGATCTTGATCTT-3 and 5'-CCTTCCTGGGCATGGAGTCCT-3' (SEQ ID NOs: 9-10) Human KNTC2 gene: 5'-CCTCTCCATGCAGGAGTTAAGA-3 and 5'-GGTCTCGGGTCCTTGATTTTCT-3' (SEQ ID NOs: 11-12) The PCR reaction was performed in duplicate for each sample, and the relative gene expression levels were determined by the ΔCT method. The results are shown in Figure 21.

[0180] As shown in Figure 21, it was found that introducing an S-form modification to the 3'-terminal side of the passenger strand did not interfere with the RNAi activity (gene expression inhibitory ability) of siRNA.

Sequence Listing Free-Text

[0181] SEQ ID NOs: 1, 2: Single-stranded RNA SEQ ID NOs: 3, 5, 7: Passenger strand of siRNA SEQ ID NOs: 4, 6, 8: Guide strand of siRNA SEQ ID NOs: 9-12: Primers

Claims

1. A nucleoside derivative, A derivative represented by the following formula (1). 【Chemical 24】 (In the above formula (1), R １ represents a hydroxyl group, a hydroxyl group in which a hydrogen atom is substituted with an alkyl group or an alkenyl group, or a protected hydroxyl group; R 2 is a hydrogen atom, a protecting group for a hydroxyl group, a phosphate group, a protected phosphate group, a protected phosphorothioate group, or -P(=O) n R 5 R 6 (n represents 0 or 1, R 5 and R 6 are the same or different and each represents a hydrogen atom, a hydroxyl group, a protected hydroxyl group, a mercapto group, a protected mercapto group, a lower alkoxy group, a cyano lower alkoxy group, an amino group, or a substituted amino group. However, when n is 1, R 5 and R 6 and R are not hydrogen atoms. 3 represents a hydrogen atom, a protecting group for a hydroxyl group, a phosphate group, a protected phosphate group, or a phosphorothioate group; R 4 is a hydroxyl group, a protected hydroxyl group, an azide group, or NHR ７ represents R ７ represents a hydrogen atom or a protecting group for an amino group, and B represents a purine base or a pyrimidine base.

2. The aforementioned R １ is a methoxy group, and the derivative according to claim 1.

3. The derivative according to Claim 1, wherein B is adenine, cytosine, guanine, thymine or uracil.

4. An oligonucleotide comprising at least a base-containing unit represented by the following formula (3). 【Chemical Formula 25】 (In the above formula (3), R １ represents a hydroxyl group, a hydroxyl group in which a hydrogen atom is substituted with an alkyl group or an alkenyl group, X 1 represents an oxygen atom or a sulfur atom, X ２ is OH (or O － ), SH (or S － ), and B represents a purine base or a pyrimidine base.)

5. In the range from the 1st base to the 9th base inclusive counting from the 3'-end of the passenger strand and / or in the range from the 1st base to the 6th base inclusive counting from the 5'-end of the passenger strand, the following formula (3); 【Chemical 26】 (In the above formula (3), R １ represents a hydroxyl group, a hydroxyl group in which a hydrogen atom is substituted with an alkyl group or an alkenyl group, X 1 represents an oxygen atom or a sulfur atom, X ２ is OH (or O － ), SH (or S － ), and B represents a purine base or a pyrimidine base.) A passenger strand of siRNA having one or more base-containing units represented by

6. At the position of the 7th base counting from the 5'-end of the guide strand, the following formula (3); 【Chemical 27】 (In the above formula (3), R １ represents a hydroxyl group, a hydroxyl group in which a hydrogen atom is substituted with an alkyl group or an alkenyl group, X 1 represents an oxygen atom or a sulfur atom, X ２ is OH (or O － ), SH (or S － ), and B represents a purine base or a pyrimidine base.) A guide strand of siRNA having a base-containing unit represented by

7. An siRNA comprising the passenger strand according to Claim 5 and / or the guide strand according to Claim 6.

8. A compound represented by the following formula (4). 【Chemical 28】 (In the above formula (4), R 7 represents an acetyl group or a cyclic structure in which oxygen atoms bonded to each other are linked by -C(CH ７ ) 3 ) 2 to R 8 represents a hydrogen atom or a protecting group for a hydroxyl group, R ９ represents a hydrogen atom or a protecting group for a hydroxyl group, R １０ represents a hydroxyl group, a protected hydroxyl group, or an azide group.)

9. A method for synthesizing a nucleoside derivative represented by formula (1), The synthesis method includes a step of introducing a purine base or a pyrimidine base to the 1'-position carbon atom of an intermediate represented by the following formula (4). 【Chemical 29】 (In the above formula (4), R 7 represents an acetyl group, R 8 represents a protecting group for a hydroxyl group, R ９ represents a protecting group for a hydroxyl group, R １０ represents an azide group)

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