JP2018184351A - Nucleoside or derivative of the nucleotide, rna derivative containing the same as constitutional unit, nucleic acid medicine, and method for producing rna derivative or rna - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a nucleoside in which 2'-position hydroxy group is protected or a nucleotide derivative thereof in which RNA synthesis efficiency is satisfactory and can be deprotect under a mild condition, and purification after deprotection is easy.SOLUTION: There is provide a nucleoside expressed by formula (1) or a derivative of the nucleotide. (Xand Xseverally independently denote H, substituted/unsubstituted silyl group, 4-methoxytrityl group, 4,4'-dimethoxytrityl group or the like; Bdenotes modified/unmodified nucleic acid base; a formula (P1) is exemplified as R; R denotes substituent having electron-donating property such as methoxy group; Rand Rseverally independently denote H or alkyl group).SELECTED DRAWING: Figure 2

Description

  The present invention relates to a nucleoside or a derivative of a nucleotide thereof, an RNA derivative containing the same as a constituent unit, a nucleic acid pharmaceutical, and a method for producing an RNA derivative or RNA.

  A nucleic acid drug is a drug based on a synthesized oligonucleotide. Nucleic acid drugs are expected to provide new therapeutic methods for diseases that have been difficult to treat so far, such as having the action of suppressing the expression of specific proteins. Antisense methods, RNA interference methods, decoy methods, and the like have been proposed as technologies that can be applied as such nucleic acid drugs. Among these, the RNA interference method is a technique using a relatively short RNA called 21-23 mer called siRNA, and is attracting attention because it can be realized even with chemically synthesized RNA.

  However, chemical synthesis of RNA cannot be said to have a sufficient synthesis efficiency compared to that of DNA, and it is also difficult. Unlike the DNA, the nucleoside that constitutes RNA has a hydroxyl group at the 2 ′ position, so a protective group must be introduced into this, but the efficiency of introducing the protective group is poor, and the protection introduced at the 2 ′ position is also necessary. The reason is that the reaction efficiency of chain extension is poor due to steric hindrance of the group.

  From such a background, for example, Patent Document 1 proposes a nucleoside in which the 2'-position hydroxyl group is protected with a cyanoethyl group or a nucleotide thereof for RNA synthesis. According to Patent Document 1, it is said that a cyanoethyl group as a protecting group can be efficiently introduced into a nucleoside or a 2'-position hydroxyl group in a nucleotide thereof under mild conditions. According to Patent Document 2, the cyanoethyl group introduced as a protecting group at the 2'-position can be easily deprotected by using tetrabutylammonium fluoride (TBAF).

  Non-Patent Document 1 proposes protecting the 2'-position hydroxyl group with a 4-nitrobenzyloxymethylene group. By using such a ribonucleoside phosphoramidite into which a protecting group has been introduced, it is said that a chain elongation rate and a chain elongation efficiency equivalent to those of the phosphoramidite of deoxyribonucleoside used in DNA synthesis can be realized.

International Publication No. 2005/085271 JP 2006-248929 A

Jacek Cieslak et al. Org. Lett. , 2007, 9, 671-674

  According to the methods described in Patent Documents 1 and 2 and Non-Patent Document 1, it is possible to efficiently synthesize RNA in which the 2'-position hydroxyl group is protected. However, when this is deprotected, when a cyanoethyl group is used as the protecting group for the 2′-position hydroxyl group, TBAF is used in an aqueous solution, so that purification of the deprotected RNA is difficult. When a nitrobenzyloxymethylene group was used, severe conditions of heating at 90 ° C. in a 0.1 M acetic acid aqueous solution had to be used following the nitro group reduction operation. In addition, in order to deliver a drug to a site of action while avoiding degradation in the body, it is necessary to modify the RNA serving as the drug with an appropriate protecting group in the nucleic acid drug, but in order to deprotect as described above The necessary 90 ° C heating cannot be performed in the human body.

  The present invention has been made in view of the above situation, and the 2′-position hydroxyl group is easily protected after deprotection and can be deprotected under mild conditions with good RNA synthesis efficiency. It is an object to provide a modified nucleoside or a derivative of its nucleotide.

  As a result of intensive studies in order to solve the above problems, the present inventors have made nitroarylmethoxymethylene, such as 4-nitrobenzyloxymethylene group described in Non-Patent Document 1, a hydroxyl group at the 2 ′ position. Deprotection is completed at a temperature of about 37 ° C. after reducing the nitro group by introducing an electron donating group such as a methoxy group into the aromatic ring. In addition, it was found that RNA obtained after deprotection can be purified very easily. If deprotection is possible under such temperature conditions, it is useful for RNA synthesis as well as deprotection at body temperature, and is also useful as a nucleic acid drug. The present invention has been made based on the above findings, and provides the following.

(1) The present invention is a nucleoside represented by the following general formula (1) or a nucleotide derivative thereof.
(In the above general formula (1), X ¹ and X ² are each independently a hydrogen atom, an optionally substituted silyl group, a 4-methoxytrityl group, a 4,4′-dimethoxytrityl group, or represents the general formula (2), ^{B a} represents a nucleic acid base which may have been modified, ^{R p} is represented by the following general formula (P1), representing the (P2) or (P3).)
(In the general formula (2), R ³ and R ⁴ each independently represents an alkyl group having 1 to 7 carbon atoms, R ⁵ represents a protecting group for phosphoric acid, and R ³ and R ⁴ are linked to each other. To form a ring structure.)
(In the general formula (P1), each R independently represents an alkyl group having 1 to 5 carbon atoms, an alkoxy group having 1 to 5 carbon atoms, an alkylthio group having 1 to 5 carbon atoms, or —Si (OR ⁶ ) _{3. ^{, -SiR 6 (oR 6) 2}} , -SiR 6 2 (oR 6), - SiR 6 3, or an ^-NR _{6 2,} ^{R 1} and ^{R 2} each independently represent a hydrogen atom, or a carbon number 1 to 5 represents an alkyl group, each R ⁶ is independently an alkyl group having 1 to 5 carbon atoms, p, q, and r are each independently 0 or 1, and (p + q + r) is 1 or more, and n is (It may be an integer of 1 to (5-pqr), but n may be 0, provided that at least one of R ¹ and R ² is an alkyl group having 1 to 5 carbon atoms.)
(In the general formula (P2), each R independently represents an alkyl group having 1 to 5 carbon atoms, an alkoxy group having 1 to 5 carbon atoms, an alkylthio group having 1 to 5 carbon atoms, or —Si (OR ⁶ ) _{3. ^{, -SiR 6 (oR 6) 2}} , -SiR 6 2 (oR 6), - SiR 6 3, or an ^-NR _{6 2,} ^{R 1} and ^{R 2} each independently represent a hydrogen atom, or a carbon number 1 to 5 represents an alkyl group, each R ⁶ is independently an alkyl group having 1 to 5 carbon atoms, X is O or S, p and q are each independently 0 or 1, and (p + q) is 1 N is an integer of 1 to (3-pq), but n may be 0 on condition that at least one of R ¹ and R ² is an alkyl group having 1 to 5 carbon atoms. )
(In the above general formula (P3), R is an alkyl group having 1 to 5 carbon atoms, an alkoxy group having 1 to 5 carbon atoms, an alkylthio group having 1 to 5 carbon atoms, -Si (OR ⁶ ) ₃ , -SiR ⁶ ( _{^{oR 6) 2, -SiR 6 2}} (oR 6), - SiR 6 3, or an ^-NR _{6 2,} ^{R 1} and ^{R 2} each independently represent a hydrogen atom, or an alkyl group having 1 to 5 carbon atoms Each R ⁶ is independently an alkyl group having 1 to 5 carbon atoms, R ⁷ is an alkyl group having 1 to 5 carbon atoms, p and q are each independently 0 or 1, and (p + q) is 1 and n is 1, provided that at least one of R ¹ and R ² is an alkyl group having 1 to 5 carbon atoms, n may be 0, and when n is 0 (p + q ) May be 2.)

(2) Moreover, this invention is the derivative | guide_body of the nucleoside of the (1) description represented by following General formula (1a), or its nucleotide.
(In the general formula (1a), X ¹ and X ² are each independently a hydrogen atom, an optionally substituted silyl group, a 4-methoxytrityl group, a 4,4′-dimethoxytrityl group, or the following general formula Represents formula (2), Ba represents an ^optionally modified nucleobase.)
(In the general formula (2), R ³ and R ⁴ each independently represents an alkyl group having 1 to 7 carbon atoms, R ⁵ represents a protecting group for phosphoric acid, and R ³ and R ⁴ are linked to each other. To form a ring structure.)

(3) The present invention is also an RNA derivative containing a nucleoside derivative represented by the following general formula (3) as a structural unit.
(In the above general formula (3), each single bond with a wavy line binds to a phosphorus atom that forms a phosphodiester bond of an RNA derivative (or a hydrogen atom at the terminal when it is the terminal of an RNA derivative), and ^Ba is A nucleobase which may be modified is represented, and R ^p represents the following general formula (P1), (P2) or (P3).
(In the general formula (P1), each R independently represents an alkyl group having 1 to 5 carbon atoms, an alkoxy group having 1 to 5 carbon atoms, an alkylthio group having 1 to 5 carbon atoms, or —Si (OR ⁶ ) _{3. ^{, -SiR 6 (oR 6) 2}} , -SiR 6 2 (oR 6), - SiR 6 3, or an ^-NR _{6 2,} ^{R 1} and ^{R 2} each independently represent a hydrogen atom, or a carbon number 1 to 5 represents an alkyl group, each R ⁶ is independently an alkyl group having 1 to 5 carbon atoms, p, q, and r are each independently 0 or 1, and (p + q + r) is 1 or more, and n is (It may be an integer of 1 to (5-pqr), but n may be 0, provided that at least one of R ¹ and R ² is an alkyl group having 1 to 5 carbon atoms.)
(In the general formula (P2), each R independently represents an alkyl group having 1 to 5 carbon atoms, an alkoxy group having 1 to 5 carbon atoms, an alkylthio group having 1 to 5 carbon atoms, or —Si (OR ⁶ ) _{3. ^{, -SiR 6 (oR 6) 2}} , -SiR 6 2 (oR 6), - SiR 6 3, or an ^-NR _{6 2,} ^{R 1} and ^{R 2} each independently represent a hydrogen atom, or a carbon number 1 to 5 represents an alkyl group, each R ⁶ is independently an alkyl group having 1 to 5 carbon atoms, X is O or S, p and q are each independently 0 or 1, and (p + q) is 1 N is an integer of 1 to (3-pq), but n may be 0 on condition that at least one of R ¹ and R ² is an alkyl group having 1 to 5 carbon atoms. )
(In the above general formula (P3), R is an alkyl group having 1 to 5 carbon atoms, an alkoxy group having 1 to 5 carbon atoms, an alkylthio group having 1 to 5 carbon atoms, -Si (OR ⁶ ) ₃ , -SiR ⁶ ( _{^{oR 6) 2, -SiR 6 2}} (oR 6), - SiR 6 3, or an ^-NR _{6 2,} ^{R 1} and ^{R 2} each independently represent a hydrogen atom, or an alkyl group having 1 to 5 carbon atoms Each R ⁶ is independently an alkyl group having 1 to 5 carbon atoms, R ⁷ is an alkyl group having 1 to 5 carbon atoms, p and q are each independently 0 or 1, and (p + q) is 1 and n is 1, provided that at least one of R ¹ and R ² is an alkyl group having 1 to 5 carbon atoms, n may be 0, and when n is 0 (p + q ) May be 2.)

(4) Moreover, this invention is an RNA derivative as described in the (3) term which contains the ribonucleoside derivative represented by the following general formula (3a) as a structural unit.
(In the above general formula (3a), each single bond with a wavy line is bonded to a phosphorus atom that forms a phosphodiester bond of RNA (or a hydrogen atom at the terminal if it is the terminal of RNA), and ^Ba is modified. Represents a nucleobase that may be present.)

  (5) The present invention is also a nucleic acid pharmaceutical comprising the RNA derivative according to the item (3) or (4).

  (6) The present invention is also a method for producing an RNA derivative, comprising a step of extending an RNA chain using the nucleotide derivative described in the item (1) or (2).

(7) The present invention (6) against RNA derivative obtained in the RNA derivative production method according to claim, deprotecting the protecting group R ^p subjected to 2 'position hydroxyl group of RNA by performing a reduction operation process It is also a manufacturing method of RNA characterized by comprising.

  According to the present invention, there is provided a nucleoside or a nucleotide derivative thereof in which the 2'-position hydroxyl group is protected, which can be deprotected under mild conditions with good RNA synthesis efficiency and easy to purify. Is done.

FIG. 1 is an HPLC chart showing the change over time when ON2 deprotection reaction is carried out with titanium trichloride. FIG. 2 is an HPLC chart showing a change with time when ON3 deprotection reaction is performed with titanium trichloride. FIG. 3 is an HPLC chart showing the change with time when the deprotection reaction of ON1 by nitroreductase is performed. FIG. 4 is an HPLC chart showing the change over time when the deprotection reaction of ON2 by nitroreductase is performed. FIG. 5 is an HPLC chart showing the change over time when ON3 deprotection reaction with nitroreductase was performed. FIG. 6 is an HPLC chart showing a change with time when ON4 deprotection reaction is carried out with nitroreductase.

  Hereinafter, an embodiment of a nucleoside or nucleotide derivative of the present invention, an embodiment of an RNA derivative, an embodiment of a nucleic acid pharmaceutical, an embodiment of a method for producing an RNA derivative, and an embodiment of a method for producing RNA will be described. However, the present invention is not limited to the following embodiments and embodiments, and can be implemented with appropriate modifications within the scope of the present invention.

[Nucleoside or nucleotide derivative]
The nucleoside or nucleotide derivative of the present invention (hereinafter also simply referred to as a nucleoside derivative) is a compound represented by the following general formula (1). This compound is preferably used for RNA synthesis. In particular, when it is a phosphoramidite, a high reaction rate and reaction efficiency can be realized in a chain extension reaction by a solid phase synthesis method.

The present invention is characterized in that a protective group (R ^p ) unique to the 2′-position of the ribonucleoside represented by the general formula (1) is provided. First, R ^{p serving as} a protecting group will be described. The general formula R ^p is a monovalent group represented by the following general formula (P1), (P2), or (P3).

In the general formula (P1), each R is independently an alkyl group having 1 to 5 carbon atoms, an alkoxy group having 1 to 5 carbon atoms, an alkylthio group having 1 to 5 carbon atoms, -Si (OR ⁶ ) ₃ , _{^{-SiR 6 (oR 6) 2,}} -SiR 6 2 (oR 6), - SiR 6 3, or an ^-NR _{6 2.} Examples of the alkyl group having 1 to 5 carbon atoms include methyl group, ethyl group, propyl group, isopropyl group, butyl group, isobutyl group, tert-butyl group, and pentyl group. Examples of the alkoxy group having 1 to 5 carbon atoms include a methoxy group, an ethoxy group, a propoxy group, an isopropoxy group, a butoxy group, a tert-butoxy group, and a pentoxy group. Examples of the alkylthio group having 1 to 5 carbon atoms include methylthio group, ethylthio group, propylthio group, isopropylthio group, butylthio group, isobutylthio group, tert-butylthio group, and pentylthio group. Each R ⁶ is independently an alkyl group having 1 to 5 carbon atoms. Examples of the alkyl group having 1 to 5 carbon atoms are the same as those exemplified above. Among these, as R, a methoxy group is preferable. p, q, and r are each independently 0 or 1, and (p + q + r) is 1 or more. That is, in the above aromatic ring (ie, benzene ring), at least one nitro group is present, and is 1 atom away (ie adjacent) or 3 atoms away from the carbon atom on the aromatic ring bonded to the methylene carbon. Will be bonded to the carbon atom. n is an integer of 1 or more and (5-pqr) or less, and n is 0 on condition that at least one of R ¹ and R ² described later is an alkyl group having 1 to 5 carbon atoms. But you can.

  As shown above, each R is an electron donating group. The point of the present invention is that the nitro group and such an electron donating group are bonded to the aromatic ring. Next, this will be described. Schemes 1 and 2 below embody the above general formula (P1) as an example for explanation. As shown in the following schemes 1 and 2, in the nucleoside derivative of the present invention, when a nitro group is converted to an amino group by a reduction operation, a lone pair on a nitrogen atom contained in the amino group moves to an aromatic ring. The bond between the oxygen atom and the methylene carbon is broken. At this time, the nitrogen atom contained in the amino group is positively charged. However, the positively charged structure is stabilized by the electron-donating group bonded to the aromatic ring. As a result, this reaction is considered to proceed easily. As can be seen with reference to Scheme 1 below, this reaction involves a nitro group (reduced to an amino group) attached to the methylene carbon contained in the leaving group and to a carbon atom three atoms away from the carbon atom on the aromatic ring. )) Will proceed if combined. As can be seen by referring to Scheme 2 below, a nitro group is bonded to a carbon atom that is one atom away from the carbon atom on the aromatic ring (that is, adjacent) bonded to the methylene carbon contained in the leaving group. Similarly, the reaction proceeds. In Schemes 1 and 2 below, the aromatic ring is a benzene ring, but any structure of the aromatic ring can write a similar resonance structure by satisfying such a condition. Even if it is a furan ring, a thiophene ring or an imidazole ring, the deprotection reaction is promoted in the same manner because the electron donating group is bonded to the aromatic ring. The general formula (P2) and general formula (P3) to be described later are such examples.

Note that, as shown in Scheme 1 and 2, after the aromatic ring is eliminated, -CH ₂ O which is part of the R ^{p -} but will remain bound to the hydroxyl group at the 2 'position, the -CH ₂ O ⁻ quickly desorbs as formaldehyde.

In the general formula (P1), R ¹ and R ² are each independently a hydrogen atom or an alkyl group having 1 to 5 carbon atoms. Examples of the alkyl group having 1 to 5 carbon atoms include methyl group, ethyl group, propyl group, isopropyl group, butyl group, isobutyl group, tert-butyl group, and pentyl group. R ¹ and R ² are bonded to a carbon atom bonded to the aromatic ring (or benzyl if the aromatic ring is a benzene ring). If these are alkyl groups, they are hyperconjugated with the aromatic ring. As a result, the positive charge on the nitrogen atom can be stabilized in the same manner as the electron donating group. For this reason, if at least one of R ¹ and R ² is an alkyl group having 1 to 5 carbon atoms, the deprotection reaction is promoted even if R which is an electron donating group is not bonded to the aromatic ring. The above “n may be 0 on condition that at least one of R ¹ and R ² is an alkyl group having 1 to 5 carbon atoms” means that at least one of R ¹ and R ² has 1 to 5 carbon atoms. This means that the deprotection reaction is promoted by hyperconjugation, so that R does not necessarily have to be bonded to the aromatic ring. The same applies to general formulas (P2) and (P3) described below.

  Specific examples of the general formula (P1) include the following general formulas (P1a) and (P1b). Of course, the general formula (P1) is not limited to these general formulas (P1a) and (P1b).

(In the general formula (P1a), each R, R ¹ and R ² is the same as that in the general formula (P1), and n is an integer of 1 or more and 4 or less, but at least of R ¹ and R ² On the condition that one is an alkyl group having 1 to 5 carbon atoms, n may be 0. In the general formula (P1b), each R, R ¹ and R ² is the same as that in the general formula (P1). Similarly, n is an integer of 1 to 3, but n may be 0 on condition that at least one of R ¹ and R ² is an alkyl group having 1 to 5 carbon atoms.

In the general formula (P2), each R independently represents an alkyl group having 1 to 5 carbon atoms, an alkoxy group having 1 to 5 carbon atoms, an alkylthio group having 1 to 5 carbon atoms, -Si (OR ⁶ ) ₃ , _{^{-SiR 6 (oR 6) 2,}} -SiR 6 2 (oR 6), - SiR 6 3, or an ^-NR _{6 2.} Examples of the alkyl group having 1 to 5 carbon atoms include methyl group, ethyl group, propyl group, isopropyl group, butyl group, isobutyl group, tert-butyl group, and pentyl group. Examples of the alkoxy group having 1 to 5 carbon atoms include a methoxy group, an ethoxy group, a propoxy group, an isopropoxy group, a butoxy group, a tert-butoxy group, and a pentoxy group. Examples of the alkylthio group having 1 to 5 carbon atoms include methylthio group, ethylthio group, propylthio group, isopropylthio group, butylthio group, isobutylthio group, tert-butylthio group, and pentylthio group. Each R ⁶ is independently an alkyl group having 1 to 5 carbon atoms. Examples of the alkyl group having 1 to 5 carbon atoms are the same as those exemplified above. Among these, as R, a methoxy group is preferable. p and q are each independently 0 or 1, and (p + q) is 1 or more. In other words, in the above five-membered ring, at least one nitro group is present, and bonded to the methylene carbon is a carbon atom one atom away (ie adjacent) or three atoms away from the carbon atom on the five-membered ring. Will be combined. n is an integer of 1 to (3-pq), but n may be 0 on condition that at least one of R ¹ and R ² described later is an alkyl group having 1 to 5 carbon atoms.

In the general formula (P2), R ¹ and R ² are each independently a hydrogen atom or an alkyl group having 1 to 5 carbon atoms. Examples of the alkyl group having 1 to 5 carbon atoms include methyl group, ethyl group, propyl group, isopropyl group, butyl group, isobutyl group, tert-butyl group, and pentyl group. X is O or S. By X being these heteroatoms, the five-membered ring in the general formula (P2) comes to have aromaticity. By having such aromaticity, the deprotection when the nitro group in the general formula (P2) is reduced is the same as in the case of the general formula (P1).

  Specific examples of the general formula (P2) include the following general formulas (P2a) and (P2b). Of course, the general formula (P2) is not limited to these general formulas (P2a) and (P2b).

(In the general formula (P2a), each of R, X, R ¹ and R ² is the same as that in the above (P2), n is 1 or 2, but at least one of R ¹ and R ² is carbon. N may be 0 on the condition that it is an alkyl group of formula 1-5.

In the above general formula (P3), R is an alkyl group having 1 to 5 carbon atoms, an alkoxy group having 1 to 5 carbon atoms, an alkylthio group having 1 to 5 carbon atoms, -Si (OR ⁶ ) ₃ , -SiR ⁶ (OR _{^{6) 2, -SiR 6 2 (}} oR 6), - SiR 6 3, or an ^-NR _{6 2.} Examples of the alkyl group having 1 to 5 carbon atoms include methyl group, ethyl group, propyl group, isopropyl group, butyl group, isobutyl group, tert-butyl group, and pentyl group. Examples of the alkoxy group having 1 to 5 carbon atoms include a methoxy group, an ethoxy group, a propoxy group, an isopropoxy group, a butoxy group, a tert-butoxy group, and a pentoxy group. Examples of the alkylthio group having 1 to 5 carbon atoms include methylthio group, ethylthio group, propylthio group, isopropylthio group, butylthio group, isobutylthio group, tert-butylthio group, and pentylthio group. Each R ⁶ is independently an alkyl group having 1 to 5 carbon atoms. Examples of the alkyl group having 1 to 5 carbon atoms are the same as those exemplified above. Among these, as R, a methoxy group is preferable. p and q are each independently 0 or 1, and (p + q) is 1. In other words, in the above imidazole ring, there is one nitro group, which is bonded to a carbon atom bonded to the methylene carbon, one atom away from the carbon atom on the imidazole ring (that is, adjacent) or three atoms away. Become. n is 1, but n may be 0 on condition that at least one of R ¹ and R ² described later is an alkyl group having 1 to 5 carbon atoms. Further, when n is 0, (p + q), that is, the number of nitro groups in the imidazole ring may be two.

In the general formula (P3), R ¹ and R ² are each independently a hydrogen atom or an alkyl group having 1 to 5 carbon atoms. Examples of the alkyl group having 1 to 5 carbon atoms include methyl group, ethyl group, propyl group, isopropyl group, butyl group, isobutyl group, tert-butyl group, and pentyl group. R ⁷ is an alkyl group having 1 to 5 carbon atoms. Examples of the alkyl group having 1 to 5 carbon atoms include methyl group, ethyl group, propyl group, isopropyl group, butyl group, isobutyl group, tert-butyl group, and pentyl group. Since the imidazole ring in the general formula (P3) also has aromaticity, the deprotection when the nitro group in the general formula (P3) is reduced is the same as in the case of the general formula (P1). .

  The following general formula (P3a) can be given as a more specific example of the general formula (P3). Of course, the general formula (P3) is not limited to the general formula (P3a).

(In the general formula (P3a), R, R ¹ and R ² are the same as those in the general formula (P3), but at least one of R ¹ and R ² is an alkyl group having 1 to 5 carbon atoms. R may be a hydrogen atom, provided that it is present.)

In the general formula (1), X ¹ and X ² are each independently a hydrogen atom, an optionally substituted silyl group, a 4-methoxytrityl group, a 4,4′-dimethoxytrityl group, or the following general formula Formula (2) is represented. X ¹ and X ² are respectively bonded to the 5′-position oxygen atom and the 3′-position oxygen atom in the nucleoside derivative of the present invention. These substituents, excluding the hydrogen atom and the substituent represented by the following general formula (2), are often used as protecting groups for the hydroxyl group at the 5 ′ position, and the substituent represented by the following general formula (2) is a phospho group. Loamidite. When the substituent represented by the general formula (2) is bonded to the 3 ′ position, it becomes a material used for a chain extension reaction in solid phase synthesis of RNA.

In the general formula (2), R ³ and R ⁴ each independently represents an alkyl group having 1 to 7 carbon atoms. Examples of such a substituent include a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, an isobutyl group, a tert-butyl group, a pentyl group, a hexyl group, and a hepsyl group. Among these, an isopropyl group is preferable, and it is particularly preferable that both R ³ and R ⁴ are isopropyl groups. R ³ and R ⁴ may be connected to each other to form a ring structure.

R ⁵ is a protecting group for phosphoric acid. As such a protecting group, one that is not detached by each chemical treatment in the chain elongation reaction of RNA synthesis but is easily removed by the deprotection process at the time of RNA excision after the completion of the chain elongation reaction is selected. The As such a protecting group, a cyanoethyl group is preferably used, but is not limited thereto.

In the general formula (1), B ^a represents a nucleic acid base which may have been modified. Here, “nucleobase” refers to all nucleobases found in naturally occurring nucleic acids, such as chromosomal DNA, plasmid DNA, messenger RNA, ribosomal RNA, transfer RNA, small nuclear RNA, etc. Includes all heteroaromatic rings that may be substituted and that may be used in the synthesis. This includes those not found in nature. Representative nucleobases include, but are not limited to, adenine, guanine, cytosine, uracil, thymine and the like. Nucleobase includes those having a protecting group.

Among the nucleoside derivatives of the present invention represented by the above general formula (1), the following general formula (1a) can be given as an example of more specific R ^p . Of course, the present invention is not limited to this.

In the general formula (1a), X ¹ , X ² and ^Ba are the same as those in the general formula (1).

Next, in order to explain the method for synthesizing the nucleoside derivative of the present invention, a method for introducing a protecting group, which is a feature of the present invention, into the 2′-position hydroxyl group of uridine will be described as an example. An example of the synthetic route is shown in Scheme 3 below. As shown in Scheme 3, first, Compound 2 is obtained by using commercially available uridine 1 as a starting material and protecting the hydroxyl groups at the 3 ′ and 5 ′ positions with a silyl group. Next, compound 2 is reacted in the presence of acetic anhydride and acetic acid to give compound 3. Thereafter, a benzyl alcohol compound (2-methoxy-4-nitrobenzyl alcohol or compound 8) is added to compound 3 together with trifluoromethanesulfonic acid (TfOH) and N-iodosuccinimide (NIS), and triethylamine trihydrofluoride is added. The silyl protecting groups at the 3 ′ and 5 ′ positions are removed using (Et ₃ N-3HF) to obtain uridines 4 and 9 in which the hydroxyl groups at the 2 ′ position are protected. The uridine of compound 4 is a compound in which one methoxy group is introduced into the benzene ring in the above general formula (P1), and the uridine of compound 9 is a compound having 2 methoxy groups in the benzene ring in the above general formula (P1). It was introduced.

  An example of a synthetic route for obtaining a monomer compound for RNA synthesis from the nucleoside derivatives of the compounds 4 and 9 is shown in the following scheme 4. Scheme 4 is an example of a route for obtaining phosphoramidites (compounds 13 and 15) by tritylating the 5′-position hydroxyl group of the nucleoside derivatives of the compounds 4 and 9 and subphosphorylating the 3′-position hydroxyl group.

  In the nucleoside derivative of the present invention, although the hydroxyl group at the 2′-position is protected, in the chain elongation reaction during RNA synthesis, the efficiency does not decrease due to steric hindrance as seen conventionally, and the reaction proceeds with high efficiency. It is possible to make it. Further, the protecting group introduced at the 2′-position can be easily removed in a weakly acidic to neutral aqueous solution by performing a reduction operation to reduce the nitro group in the protecting group to an amino group. In addition, it is characterized in that the eliminated protecting group is easily removed by treatment with reverse phase column chromatography or the like. This deprotection can be performed in the state of a nucleoside or its nucleotide derivative, as well as in the state of an RNA derivative obtained by RNA synthesis, and can be performed at a desired timing as required. It is.

  As the reducing agent used in the reduction operation for deprotection, a reducing agent usually used in the reduction of the nitro group can be used. Examples of such a reducing agent include titanium trichloride, adithionic acid, samarium iodide, Zn dust, and the like. Among these, titanium trichloride is preferably exemplified. This reduction treatment can be advanced not only by the reducing agents listed above but also by intracellular enzymes such as nitroreductase in the presence of NADH (reduced nicotinamide adenine dinucleotide). Therefore, an RNA derivative in which the hydroxyl group at the 2′-position is protected is synthesized from the nucleoside derivative of the present invention, and this is administered to the human body as a nucleic acid drug and delivered to a desired cell, followed by deprotection with an intracellular enzyme. It is possible to use it to express its function. In particular, the above deprotection proceeds rapidly in cells placed in a reducing environment such as cancer cells.

[RNA derivatives]
An RNA derivative containing a ribonucleoside derivative represented by the following general formula (3) as a structural unit is also one aspect of the present invention. This RNA derivative is obtained by RNA synthesis based on the nucleoside derivative of the present invention described above, and the hydroxyl group at the 2 ′ position is the above-mentioned protecting group in at least a part of the nucleotides constituting the structural unit. Protected. The RNA derivative of the present invention may have the above-described protecting group at the 2 ′ position in all the structural units, or may have the above-described protecting group at the 2 ′ position only in some of the structural units. In addition, the structural unit of RNA derivative means the fragment | piece part (namely, part represented by following General formula (3)) remove | excluding the phosphate part from each nucleotide contained in the polymer chain which comprises RNA derivative.

  In the general formula (3), each single bond with a wavy line is bonded to a phosphorus atom that forms a phosphodiester bond of an RNA derivative. In addition, when the structural unit represented by the said General formula (3) exists in the terminal of RNA derivative, the single bond attached | subjected the wavy line in General formula (3) couple | bonds with the hydrogen atom which exists in a terminal.

In the general formula (3), B ^a and R ^p are the same as B ^a and R ^p in the general formula (1). Therefore, description here about B ^a and R ^p in the general formula (3) is omitted.

  In the RNA derivative of the present invention, the hydroxyl group at the 2 ′ position is protected with a protecting group represented by the above general formulas (P1) to (P3), and this protecting group is represented by the general formulas (P1) to (P3). Reduction of the nitro group facilitates deprotection at a temperature around 37 ° C. in a weakly acidic to neutral aqueous solution. Then, the aqueous solution subjected to the deprotection reaction is directly subjected to reverse phase column chromatography, whereby the compound derived from the removed protecting group and the target RNA can be easily separated. This deprotection proceeds by reducing the RNA derivative. As already described, the reducing agent used for the reduction operation can be advanced by an intracellular enzyme such as nitroreductase in the presence of NADH in addition to the reducing agent usually used in the reduction of the nitro group.

  Therefore, the RNA derivative of the present invention is useful not only as an intermediate in RNA synthesis but also as a nucleic acid drug. That is, the RNA derivative of the present invention in which the hydroxyl group at the 2 ′ position is protected is administered to the human body as a nucleic acid drug and delivered to a desired cell, and then deprotected by an intracellular enzyme to express the function. Is also possible. In particular, the above deprotection proceeds rapidly in cells placed in a reducing environment such as cancer cells.

[Nucleic acid medicine]
A nucleic acid pharmaceutical comprising the above RNA derivative of the present invention is also one aspect of the present invention. Since this is the same as described in the above RNA derivative, description thereof is omitted here.

[Method for producing RNA derivative]
A method for synthesizing an RNA derivative characterized by comprising a step of extending an RNA chain using the nucleotide derivative of the present invention is also one aspect of the present invention. As already explained, the nucleoside or nucleotide derivative thereof of the present invention is an RNA having a specific protecting group R ^p (that is, a group represented by the above general formulas (P1) to (P3)) at the 2′-position hydroxyl group. It can be used for the synthesis of derivatives. At this time, a process of extending the RNA strand is carried out. As such a process, various methods including a solid phase synthesis method using phosphoramidite are known, so a method suitable for the purpose is appropriately selected. You can do it.

[Method for producing RNA]
RNA, characterized in that it comprises the step of deprotecting against RNA derivative obtained by the production method of the RNA derivative of the present invention, the protecting group R ^p subjected to 2 'position hydroxyl group of RNA by performing reduction operations This manufacturing method is also one aspect of the present invention. As already explained, the RNA derivative of the present invention comprises a protecting group R ^p (that is, a group represented by the above general formulas (P1) to (P3)) introduced into the 2′-position hydroxyl group, and this protecting group R ^p can be easily desorbed by performing a reduction operation in a weakly acidic to neutral aqueous solution. Then, by treating the aqueous solution subjected to this reduction operation with reverse phase column chromatography or the like, it is possible to easily separate the compound resulting from elimination of the protecting group and the target RNA. Since these are as already described, description thereof is omitted here.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to the following examples. In addition, the compound number in the following synthesis example respond | corresponds to the number attached | subjected to the compound in the said scheme 3 and 4.

・ Synthesis of Compound 2

Uridine (10.49 g, 42.59 mmol) was subjected to azeotropic dehydration three times with anhydrous pyridine, then placed in a reaction vessel, purged with argon, and dissolved in pyridine (200 mL). To this, 1,3-dichloro-1,1,3,3, -tetraisopropyldisiloxane (TIPDSCl ₂ , 16.35 μL, 51.11 mmol) was added and stirred on an ice bath for 7 hours. The disappearance of the raw materials was confirmed by thin layer chromatography (TLC), water (20 mL) was added, and the mixture was concentrated under reduced pressure. The residue was dissolved in ethyl acetate and washed once with water and once with saturated brine. The organic layer was collected, dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel chromatography (development with hexane: ethyl acetate = 100: 0 to 0: 100, elution with 87.5: 12.5 to 75:25), and compound 2 was purified as a white foamy solid (18.39 g). 37.78 mmol, yield 89%).

Synthesis of compound 3

Compound 2 (4.02 g, 8.26 mmol) was azeotropically dehydrated three times with anhydrous pyridine and azeotropically removed three times with anhydrous toluene, then placed in a reaction vessel and purged with argon, and dimethyl sulfoxide (DMSO, 7 mL) was added. Dissolved. Acetic acid (AcOH, 11 mL, 189.98 mmol) and acetic anhydride (Ac ₂ O, 7 mL, 74.34 mmol) were sequentially added, and the mixture was stirred at room temperature for 3 days. The disappearance of the raw materials was confirmed by TLC, and a saturated aqueous sodium carbonate solution was added on an ice bath. Then, it was washed twice with a saturated aqueous sodium hydrogen carbonate solution and once with a saturated saline solution. The organic layer was collected, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel chromatography (development with hexane: ethyl acetate = 100: 0 to 0: 100, elution with 75:25), and compound 3 was obtained as a white powder solid (3.42 g, 6.25 mmol, yield 75.). 7%).

Synthesis of compound 4

Compound 3 (276 mg, 0.5 mmol) and 2-methoxy-4-nitrobenzyl alcohol (103 mg, 0.55 mmol) were azeotropically dehydrated three times with anhydrous pyridine, and azeotropically removed three times with anhydrous toluene. The solution was purged with argon and dissolved in tetrahydrofuran (THF, 1.7 mL). N-iodosuccinimide (NIS, 126 mg, 0.55 mmol), molecular sieves 4A (497 mg), and trifluoromethanesulfonic acid (TfOH, 50 μL, 0.55 mmol) were sequentially added, and the mixture was stirred on dry ice + acetonitrile for 40 minutes. After confirming disappearance of the raw material by TLC, triethylamine (Et ₃ N, 285 μL) was added, filtered through Celite, and concentrated under reduced pressure. Dissolve in ethyl acetate and add saturated aqueous sodium thiosulfate solution. Then, it was washed once with a saturated aqueous sodium hydrogen carbonate solution and once with a saturated saline solution. The organic layer was collected, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. This was azeotropically dehydrated three times with anhydrous pyridine, azeotropically removed three times with anhydrous toluene, then placed in a reaction vessel, purged with argon, and dissolved in THF (5 mL). Triethylamine trihydrofluoride (Et ₃ N-3HF, 285 μL, 1.75 mmol) was added, and the mixture was stirred at room temperature for 3 hours. The disappearance of the raw material was confirmed by TLC and concentrated under reduced pressure. The residue was purified by silica gel chromatography (developed with chloroform: methanol = 100: 0 to 97: 3, eluted with 98: 2), and compound 4 was a yellow powder solid (0.129 g, 0.294 mmol, yield 58.6). %). Compound 4 corresponds to the nucleoside derivative of the present invention, and is one in which one methoxy group is introduced into the aromatic ring contained in the protecting group for the 2′-position hydroxyl group.

Synthesis of compound 6

3-hydroxy-4-nitrobenzoic acid (Compound 5, 5 g, 27.3 mmol) was dissolved in 2M aqueous sodium hydroxide solution (100 mL). A solution obtained by dissolving K ₂ S ₂ O ₈ (7.4 g) in water (150 mL) was added, and the mixture was stirred at room temperature for 1 week. Thereafter, concentrated sulfuric acid was added until pH 1-2, and suction filtration was performed. The filtrate was heated to reflux for 1 hour and allowed to cool. Then, it filtered by suction and obtained compound 6 as a brown solid (1.52 g, 7.64 mmol, yield 28%).

Synthesis of compound 7

Compound 6 (1.079 g, 5.42 mmol) was placed in a reaction vessel, purged with argon, and dissolved in dimethylformamide (DMF, 20 mL). CH ₃ I (934 μL, 81.3 mmol) and K ₂ CO ₃ (2.07 g, 108.4 mmol) were sequentially added, and the mixture was stirred at room temperature for 3 days. The disappearance of the raw material was confirmed by TLC and washed twice with water. The organic layer was collected, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel chromatography (hexane: ethyl acetate = developed with 100: 0 to 70:30, eluted with 90:10), and compound 7 was yellow powdered solid (0.778 g, 3.23 mmol, yield 60%). ).

Synthesis of compound 8

  Compound 7 (502 mg, 2.08 mmol) was placed in a reaction vessel, purged with argon, and dissolved in THF (5 mL). 1.0 M diisobutylaluminum hydride (DIBAL-H, 6.24 mL, 6.24 mmol) was added on the ice bath and stirred for 2 hours on the ice bath. After confirming disappearance of the raw materials by TLC, acetone (4 mL) and a saturated aqueous ammonium chloride solution (12 mL) were sequentially added, followed by Celite filtration. Then, it concentrated under pressure reduction and wash | cleaned once with saturated sodium hydrogencarbonate aqueous solution and once with saturated salt solution. The organic layer was collected, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel chromatography (hexane: ethyl acetate = 100: 0 to 40:60, eluted at 60:40), and compound 8 was yellow powdered solid (0.307 g, 1.44 mmol, yield 69.). 2%).

Synthesis of compound 9

Synthesis of Compound 9 Compound 3 (0.672 g, 1.2 mmol) and Compound 8 (283 mg, 1.32 mmol) were azeotropically dehydrated three times with anhydrous pyridine and azeotropically removed twice with anhydrous toluene. The solution was purged with argon and dissolved in THF (4 mL). N-iodosuccinimide (NIS, 326 mg, 1.45 mmol), molecular sieves 4A (496 mg), and trifluoromethanesulfonic acid (TfOH, 120 μL, 1.36 mmol) were sequentially added, and the mixture was stirred on dry ice + acetonitrile for 3.5 hours. did. The disappearance of the raw material was confirmed by TLC, triethylamine (Et ₃ N, 1 mL) was added, filtered through celite, and concentrated under reduced pressure. Then, it was washed once with a saturated aqueous sodium carbonate solution and once with a saturated saline solution. The organic layer was collected, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. This was azeotropically dehydrated three times with anhydrous pyridine, azeotropically removed twice with anhydrous toluene, then placed in a reaction vessel, purged with argon, and dissolved in THF (15 mL). Triethylamine trihydrofluoride (Et ₃ N-3HF, 855 μL, 4.2 mmol) was added and stirred at room temperature for 20 hours. The disappearance of the raw material was confirmed by TLC and concentrated under reduced pressure. The residue was purified by silica gel chromatography (developed with chloroform: methanol = 100: 0 to 94: 6, eluted with 98: 2), and compound 9 was a yellow powder solid (0.332 g, 0.71 mmol, yield 59%). Got as. Compound 9 corresponds to the nucleoside derivative of the present invention, and is obtained by introducing two methoxy groups into the aromatic ring contained in the protecting group for the 2′-position hydroxyl group.

Synthesis of compound 11

  Compound 10 was synthesized according to the procedure described in Non-Patent Document 1 above. Compound 10 does not correspond to the nucleoside derivative of the present invention because the electron donating group is not introduced into the aromatic ring in the protecting group introduced into the hydroxyl group at the 2'-position. Note that the hydroxyl group at the 5 'position of Compound 10 is protected with a dimethoxytrityl group (DMTr). Compound 10 (300 mg, 0.42 mmol) was azeotropically dehydrated 5 times with anhydrous pyridine, azeotropically removed 3 times with anhydrous toluene, then placed in a reaction vessel, purged with argon, and dissolved in dichloromethane (4.0 mL). N, N-diisopropylethylamine (DIPEA, 219 μL, 1.26 mmol) and 2-cyanoethyldiisopropylchlorophosphoramidite (206 μL, 0.92 mmol) were sequentially added, and the mixture was stirred at room temperature for 1 hour. The progress of the reaction was confirmed by TLC and washed once with a saturated aqueous sodium hydrogen carbonate solution and once with a saturated saline solution. The organic layer was collected, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel chromatography (hexane: ethyl acetate = developed with 100: 0-20: 80, eluted with 50:50), and compound 11 was white foamy solid (185 mg, 0.20 mmol, 48.3%). Got as. Compound 11 does not fall under the present invention and is a comparative phosphoramidite.

Synthesis of compound 12

  Compound 4 (316 mg, 0.719 mmol) was azeotropically dehydrated three times with anhydrous pyridine, purged with argon in a reaction vessel, and dissolved in pyridine (7.0 mL). 4,4'-Dimethoxytrityl chloride (DMTr-Cl, 268 mg, 0.791 mmol) was added and stirred at room temperature for 3 hours. The disappearance of the raw materials was confirmed by TLC, a small amount of water was added, and the solution was concentrated under reduced pressure. The residue was dissolved in ethyl acetate and washed once with saturated aqueous sodium hydrogen carbonate solution and once with saturated brine. The organic layer was collected, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel chromatography (development with chloroform: methanol = 100: 0 to 97: 3, elution with 98: 2), and the compound 12 was a white powder solid (0.404 g, 0.639 mmol, yield 89.0). %).

Synthesis of compound 13

  Compound 12 (352 mg, 0.47 mmol) was azeotropically dehydrated 5 times with anhydrous pyridine, azeotropically removed 3 times with anhydrous toluene, then placed in a reaction vessel, purged with argon, and dissolved in dichloromethane (4.0 mL). N, N-diisopropylethylamine (DIPEA, 246 μL, 1.41 mmol) and 2-cyanoethyldiisopropylchlorophosphoramidite (230 μL, 1.03 mmol) were sequentially added, and the mixture was stirred at room temperature for 1 hour. The progress of the reaction was confirmed by TLC and washed once with a saturated aqueous sodium hydrogen carbonate solution and once with a saturated saline solution. The organic layer was collected, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel chromatography (hexane: ethyl acetate = 100: 0 to 40:60, eluted with 50:50 to 40:60), and compound 13 was obtained as a white foamy solid (287 mg, 0.305 mmol, yield). Rate was 64.8%).

Synthesis of compound 14

  Compound 9 (272 mg, 0.58 mmol) was azeotropically dehydrated three times with anhydrous pyridine, placed in a reaction vessel and purged with argon, and then dissolved in pyridine (6.0 mL). 4,4'-Dimethoxytrityl chloride (DMTr-Cl, 266 mg, 0.754 mmol) was added, and the mixture was allowed to stand at room temperature for 4 hours. The disappearance of the raw materials was confirmed by TLC, a small amount of water was added, and the solution was concentrated under reduced pressure. The residue was dissolved in ethyl acetate and washed once with saturated aqueous sodium hydrogen carbonate solution and once with saturated brine. The organic layer was collected, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel chromatography (developed with chloroform: methanol = 100: 0 to 96: 4 and eluted with 99: 1) to give compound 14 as a yellow foamy solid (0.394 g, 0.51 mmol, 88% yield). ).

Synthesis of compound 15

  Compound 14 (344 mg, 0.45 mmol) was azeotropically dehydrated 7 times with anhydrous pyridine and azeotropically removed 5 times with anhydrous toluene, then placed in a reaction vessel, purged with argon, and dissolved in dichloromethane (4.0 mL). N, N-diisopropylethylamine (DIPEA, 235 μL, 1.35 mmol) and 2-cyanoethyldiisopropylchlorophosphoramidite (221 μL, 0.99 mmol) were sequentially added, and the mixture was stirred at room temperature for 1 hour. The progress of the reaction was confirmed by TLC and washed once with a saturated aqueous sodium hydrogen carbonate solution and once with a saturated saline solution. Since the target product was contained in the aqueous layer, extraction was performed using dichloromethane. The organic layer was collected, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel chromatography (elution with hexane: ethyl acetate = 100: 0 to 40:60, elution with 40:60), and Compound 15 was a yellow foamy solid (267 mg, 0.27 mmol, 61% yield). Got as.

[Chemical reduction of compound 4]
Compound 4 (10 mg, 0.0228 mmol) in which one methoxy group was introduced into the protecting group for the 2′-position hydroxyl group was dissolved in methanol (1 mL), and NH ₄ Cl (3 mg, 0.0912 mmol) and Zn (5 mg, 0 1596 mmol) was added sequentially, and the mixture was stirred at room temperature for 2 hours. When the progress of the reaction was confirmed by TLC, the disappearance of the raw material spots and the appearance of new spots were confirmed. The new spot was thought to be an intermediate formed by reduction of the nitro group, and no free (ie deprotected) uridine spot appeared. Celite filtration was performed, and the filtrate was concentrated under reduced pressure. The residue was dissolved in methanol (1 mL), 50 μL was taken therefrom, 50 μL of 100 mM phosphate buffer having pH 6.0 was added, and the mixture was allowed to stand at 37 ° C. TLC confirmed that a new spot with the same Rf value as free uridine appeared. From this, it can be seen that Compound 4 in which one methoxy group is introduced into the protecting group for the 2′-position hydroxyl group can be deprotected by performing a reduction operation and maintaining weak acidity by adding a buffer solution. It was. In addition, in a compound in which a methoxy group is not introduced into the protecting group for the 2′-position hydroxyl group (a compound described in Non-Patent Document 1), free uridine is not generated even if the same operation is performed, and is not deprotected. It was.

[Chemical reduction of compound 9]
Compound 9 (10 mg, 0.0213 mmol) was dissolved in methanol (1 mL), NH ₄ Cl (8.4 mg, 0.256 mmol) and Zn (14 mg, 0.447 mmol) were sequentially added, and the mixture was stirred at room temperature. TLC confirmed that a new spot having the same Rf value as uridine appeared. From this, it was found that the compound 9 in which two methoxy groups were introduced into the protecting group for the 2′-position hydroxyl group was deprotected only by carrying out the reduction operation.

  From the above results, both Compound 4 having 1 methoxy group and Compound 9 having 2 methoxy groups were able to be deprotected at room temperature by performing a reduction operation. Each of the compounds 9 was more easily deprotected than the compound 4 having one methoxy group. When the number of methoxy groups was 0, deprotection was not performed at room temperature even when the reduction operation was performed.

[Synthesis of oligonucleotides]
Compound 11 in which the methoxy group introduced into the protecting group at the 2′-position hydroxyl group is 0 phosphoramidite, Compound 13 in which the methoxy group is one phosphoramidite, and Phosphoroyl having two methoxy groups Using each compound 15 which is an amidite, oligonucleotides (ON1 to ON4) having the sequences shown in Table 1 were synthesized by the solid phase phosphoramidite method using a DNA / RNA synthesizer. In the sequence shown in Table 1, T is thymine, U (MeO) ₀ has a protecting group at the 2′-position hydroxyl group, and the methoxy group introduced into this protecting group has 0 uracil, U (MeO) ₁ Means uracil having one methoxy group, and U (MeO) ₂ also means uracil having two methoxy groups.

[Deprotection of ON2 with titanium trichloride]
The ON2 solution (5.0 μL) was measured in a microtube, and 1 mM thymidine solution (4.8 μL) was added as an internal standard, and dried. 2M citrate buffer (20 μL), pure water (179.6 μL), and 1M titanium trichloride (0.2 μL) were sequentially added to perform a deprotection reaction. 10 μL was sampled every time and analyzed by HPLC using a reverse phase silica gel column. The result is shown in FIG. FIG. 1 is an HPLC chart showing the change over time when ON2 deprotection reaction is carried out with titanium trichloride.

[Deprotection of ON3 with titanium trichloride]
The ON3 solution (4.3 μL) was weighed into a microtube, and 1 mM thymidine solution (2.4 μL) was added as an internal standard and dried. 2M citrate buffer (20 μL), pure water (172.9 μL), and 1M titanium trichloride (0.4 μL) were sequentially added to perform a deprotection reaction. 10 μL was sampled every time and analyzed by HPLC using a reverse phase silica gel column. The result is shown in FIG. FIG. 2 is an HPLC chart showing a change with time when ON3 deprotection reaction is performed with titanium trichloride.

  As shown in FIG. 1, in ON2, the peak of the starting material disappears after 1 hour, and an intermediate in which the nitro group is reduced to an amino group and a peak of 5′-TTTTU-3 ′ that is a deprotected form are present. confirmed. As shown in FIG. 2, in ON3, the peak of the starting material disappears after 1 hour, and the peak at a retention time of about 24 minutes, which seems to be an intermediate, and the peak of 5′-TTTTTU-3 ′, similar to the result of ON2. Was confirmed. Comparing the results after 12 hours for each of FIG. 1 and FIG. 2, after 12 hours, the ON3 with two methoxy groups introduced progressed more than the ON2 with one methoxy group introduced. You can see that

[Deprotection of ON1 by nitroreductase]
Measure ON1 solution (10.8 μL) in a microtube and use 500 μM N ⁶ -Benzoyl-2′-dA-hydrate solution (7.2 μL), 100 mM phosphate buffer (pH 6.0, 90 μL), 100 mM as an internal standard. A NADH aqueous solution (30 μL) and pure water (42 μL) were sequentially added, and the mixture was allowed to stand in a thermostatic bath at 37 ° C. for 20 minutes. From there, 60 μL was taken into another microtube, 100 mM phosphate buffer (pH 6.0, 20 μL) and 20 μL of pure water were added, and this was taken as before the reaction. A 100 mM phosphate buffer (pH 6.0, 40 μL), pure water 30 μL, and an aqueous nitroreductase solution (10 μL) were added to the stock solution to carry out an enzyme reaction. 10 μL was taken every time and analyzed by HPLC using a reverse phase silica gel column. The result is shown in FIG. FIG. 3 is an HPLC chart showing the change with time when the deprotection reaction of ON1 by nitroreductase is performed.

[Deprotection of ON2 by nitroreductase]
Measure ON2 solution (3.75 μL) in a microtube, and use 500 μM N ⁶ -Benzoyl-2′-dA-hydrate solution (7.2 μL), 100 mM phosphate buffer (pH 6.0, 90 μL), 100 mM as an internal standard. A NADH aqueous solution (30 μL) and pure water (49.05 μL) were sequentially added, and the mixture was allowed to stand in a 37 ° C. constant temperature bath for 20 minutes. From there, 60 μL was taken into another microtube, 100 mM phosphate buffer (pH 6.0, 20 μL) and 20 μL of pure water were added, and this was taken as before the reaction. A 100 mM phosphate buffer (pH 6.0, 40 μL), pure water 30 μL, and an aqueous nitroreductase solution (10 μL) were added to the stock solution to carry out an enzyme reaction. 10 μL was taken every time and analyzed by HPLC using a reverse phase silica gel column. The result is shown in FIG. FIG. 4 is an HPLC chart showing the change over time when the deprotection reaction of ON2 by nitroreductase is performed.

[Deprotection of ON3 by nitroreductase]
Measure ON3 solution (6.5 μL) in a microtube and use 500 μM N ⁶ -Benzoyl-2′-dA-hydrate solution (7.2 μL), 100 mM phosphate buffer (pH 6.0, 90 μL), 100 mM as an internal standard. An NADH aqueous solution (30 μL) and pure water (46.3 μL) were sequentially added, and the mixture was allowed to stand in a thermostatic bath at 37 ° C. for 20 minutes. From there, 60 μL was taken into another microtube, 100 mM phosphate buffer (pH 6.0, 20 μL) and 20 μL of pure water were added, and this was taken as before the reaction. A 100 mM phosphate buffer (pH 6.0, 40 μL), pure water 30 μL, and an aqueous nitroreductase solution (10 μL) were added to the stock solution to carry out an enzyme reaction. 10 μL was taken every time and analyzed by HPLC using a reverse phase silica gel column. The result is shown in FIG. FIG. 5 is an HPLC chart showing the change over time when ON3 deprotection reaction with nitroreductase was performed.

[Deprotection of ON4 by nitroreductase]
Measure ON4 solution (4.0 μL) in a microtube and use 500 μM N ⁶ -Benzoyl-2′-dA-hydrate solution (7.2 μL), 100 mM phosphate buffer (pH 6.0, 90 μL), 100 mM as an internal standard. An NADH aqueous solution (30 μL) and pure water (48.8 μL) were sequentially added, and the mixture was allowed to stand in a 37 ° C. constant temperature bath for 20 minutes. From there, 60 μL was taken into another microtube, 100 mM phosphate buffer (pH 6.0, 20 μL) and 20 μL of pure water were added, and this was taken as before the reaction. A 100 mM phosphate buffer (pH 6.0, 40 μL), pure water 30 μL, and an aqueous nitroreductase solution (10 μL) were added to the stock solution to carry out an enzyme reaction. 10 μL was taken every time and analyzed by HPLC using a reverse phase silica gel column. The result is shown in FIG. FIG. 6 is an HPLC chart showing a change with time when ON4 deprotection reaction is carried out with nitroreductase.

  As shown in FIG. 3, in the case of ON1 in which the protecting group at the 2′-position hydroxyl group does not have a methoxy group, the peak of the deprotected 5′-TTTTU-3 ′ is observed although the peak of the starting material disappears. There was no. Moreover, as shown in FIG.4 and FIG.5, both ON2 (one methoxy group) and ON3 (two methoxy group) lose | disappear a starting material, and the peak of 5'-TTTTTU-3 'increases gradually. However, ON3 (2 methoxy groups) was confirmed to have a higher deprotection rate. From the results of these three types (ON1 to ON3) of deprotection experiments, it was confirmed that the reduction reaction of the nitro group on the protecting group to the amino group occurred in any protecting group. However, when there was no methoxy group on the protecting group, it was confirmed that the subsequent deprotection reaction would not proceed even if the nitro group was reduced and converted to an amino group. It was also confirmed that as the methoxy group on the protecting group increased, the rate of the deprotection reaction after the reduction increased.

Furthermore, as shown in FIG. 6, even when the deprotection reaction was performed using ON4 (5′-TTU (MeO) ₂ TT-3 ′) in which a 2′-position hydroxyl-protected uridine was introduced at the center, ON3 As in, the deprotection reaction progressed with time and the 5′-TTUTT-3 ′ peak increased, and the reaction was completed after 48 hours. From this result, it was shown that the nucleoside derivative of the present invention can quickly perform deprotection after RNA synthesis under mild conditions.

Claims (7)

A nucleoside represented by the following general formula (1) or a nucleotide derivative thereof.
(In the above general formula (1), X ¹ and X ² are each independently a hydrogen atom, an optionally substituted silyl group, a 4-methoxytrityl group, a 4,4′-dimethoxytrityl group, or represents the general formula (2), ^{B a} represents a nucleic acid base which may have been modified, ^{R p} is represented by the following general formula (P1), representing the (P2) or (P3).)
(In the general formula (2), R ³ and R ⁴ each independently represents an alkyl group having 1 to 7 carbon atoms, R ⁵ represents a protecting group for phosphoric acid, and R ³ and R ⁴ are linked to each other. To form a ring structure.)
(In the general formula (P1), each R independently represents an alkyl group having 1 to 5 carbon atoms, an alkoxy group having 1 to 5 carbon atoms, an alkylthio group having 1 to 5 carbon atoms, or —Si (OR ⁶ ) _{3. ^{, -SiR 6 (oR 6) 2}} , -SiR 6 2 (oR 6), - SiR 6 3, or an ^-NR _{6 2,} ^{R 1} and ^{R 2} each independently represent a hydrogen atom, or a carbon number 1 to 5 represents an alkyl group, each R ⁶ is independently an alkyl group having 1 to 5 carbon atoms, p, q, and r are each independently 0 or 1, and (p + q + r) is 1 or more, and n is (It may be an integer of 1 to (5-pqr), but n may be 0, provided that at least one of R ¹ and R ² is an alkyl group having 1 to 5 carbon atoms.)
(In the general formula (P2), each R independently represents an alkyl group having 1 to 5 carbon atoms, an alkoxy group having 1 to 5 carbon atoms, an alkylthio group having 1 to 5 carbon atoms, or —Si (OR ⁶ ) _{3. ^{, -SiR 6 (oR 6) 2}} , -SiR 6 2 (oR 6), - SiR 6 3, or an ^-NR _{6 2,} ^{R 1} and ^{R 2} each independently represent a hydrogen atom, or a carbon number 1 to 5 represents an alkyl group, each R ⁶ is independently an alkyl group having 1 to 5 carbon atoms, X is O or S, p and q are each independently 0 or 1, and (p + q) is 1 N is an integer of 1 to (3-pq), but n may be 0 on condition that at least one of R ¹ and R ² is an alkyl group having 1 to 5 carbon atoms. )
(In the above general formula (P3), R is an alkyl group having 1 to 5 carbon atoms, an alkoxy group having 1 to 5 carbon atoms, an alkylthio group having 1 to 5 carbon atoms, -Si (OR ⁶ ) ₃ , -SiR ⁶ ( _{^{oR 6) 2, -SiR 6 2}} (oR 6), - SiR 6 3, or an ^-NR _{6 2,} ^{R 1} and ^{R 2} each independently represent a hydrogen atom, or an alkyl group having 1 to 5 carbon atoms Each R ⁶ is independently an alkyl group having 1 to 5 carbon atoms, R ⁷ is an alkyl group having 1 to 5 carbon atoms, p and q are each independently 0 or 1, and (p + q) is 1 and n is 1, provided that at least one of R ¹ and R ² is an alkyl group having 1 to 5 carbon atoms, n may be 0, and when n is 0 (p + q ) May be 2.) The nucleoside or nucleotide derivative thereof according to claim 1, represented by the following general formula (1a).
(In the general formula (1a), X ¹ and X ² are each independently a hydrogen atom, an optionally substituted silyl group, a 4-methoxytrityl group, a 4,4′-dimethoxytrityl group, or the following general formula Represents formula (2), Ba represents an ^optionally modified nucleobase.)
(In the general formula (2), R ³ and R ⁴ each independently represents an alkyl group having 1 to 7 carbon atoms, R ⁵ represents a protecting group for phosphoric acid, and R ³ and R ⁴ are linked to each other. To form a ring structure.) An RNA derivative containing a nucleoside derivative represented by the following general formula (3) as a structural unit.
(In the above general formula (3), each single bond with a wavy line is bonded to a phosphorus atom that forms a phosphodiester bond of RNA (or a hydrogen atom at the terminal when it is the terminal of RNA), and ^Ba is modified. And R ^p represents the following general formula (P1), (P2) or (P3).
(In the general formula (P1), each R independently represents an alkyl group having 1 to 5 carbon atoms, an alkoxy group having 1 to 5 carbon atoms, an alkylthio group having 1 to 5 carbon atoms, or —Si (OR ⁶ ) _{3. ^{, -SiR 6 (oR 6) 2}} , -SiR 6 2 (oR 6), - SiR 6 3, or an ^-NR _{6 2,} ^{R 1} and ^{R 2} each independently represent a hydrogen atom, or a carbon number 1 to 5 represents an alkyl group, each R ⁶ is independently an alkyl group having 1 to 5 carbon atoms, p, q, and r are each independently 0 or 1, and (p + q + r) is 1 or more, and n is (It may be an integer of 1 to (5-pqr), but n may be 0, provided that at least one of R ¹ and R ² is an alkyl group having 1 to 5 carbon atoms.)
(In the general formula (P2), each R independently represents an alkyl group having 1 to 5 carbon atoms, an alkoxy group having 1 to 5 carbon atoms, an alkylthio group having 1 to 5 carbon atoms, or —Si (OR ⁶ ) _{3. ^{, -SiR 6 (oR 6) 2}} , -SiR 6 2 (oR 6), - SiR 6 3, or an ^-NR _{6 2,} ^{R 1} and ^{R 2} each independently represent a hydrogen atom, or a carbon number 1 to 5 represents an alkyl group, each R ⁶ is independently an alkyl group having 1 to 5 carbon atoms, X is O or S, p and q are each independently 0 or 1, and (p + q) is 1 N is an integer of 1 to (3-pq), but n may be 0 on condition that at least one of R ¹ and R ² is an alkyl group having 1 to 5 carbon atoms. )
(In the above general formula (P3), R is an alkyl group having 1 to 5 carbon atoms, an alkoxy group having 1 to 5 carbon atoms, an alkylthio group having 1 to 5 carbon atoms, -Si (OR ⁶ ) ₃ , -SiR ⁶ ( _{^{oR 6) 2, -SiR 6 2}} (oR 6), - SiR 6 3, or an ^-NR _{6 2,} ^{R 1} and ^{R 2} each independently represent a hydrogen atom, or an alkyl group having 1 to 5 carbon atoms Each R ⁶ is independently an alkyl group having 1 to 5 carbon atoms, R ⁷ is an alkyl group having 1 to 5 carbon atoms, p and q are each independently 0 or 1, and (p + q) is 1 and n is 1, provided that at least one of R ¹ and R ² is an alkyl group having 1 to 5 carbon atoms, n may be 0, and when n is 0 (p + q ) May be 2.) The RNA derivative according to claim 3, comprising a ribonucleoside derivative represented by the following general formula (3a) as a structural unit.
(In the above general formula (3a), each single bond with a wavy line is bonded to a phosphorus atom that forms a phosphodiester bond of RNA (or a hydrogen atom at the terminal if it is the terminal of RNA), and ^Ba is modified. Represents a nucleobase that may be present.)   A nucleic acid pharmaceutical comprising the RNA derivative according to claim 3 or 4.   A method for producing an RNA derivative, comprising a step of extending an RNA chain using the nucleotide derivative according to claim 1 or 2. Against RNA derivative obtained in RNA derivative production method of claim 6, wherein, RNA characterized in that it comprises the step of deprotecting the protecting group R ^p subjected to 2 'position hydroxyl group of RNA by performing reduction operations Manufacturing method.