RNA interference agents and multiple chemically modified oligonucleotides and their uses

Abstract

The present invention comprises a modified oligonucleotide having, in any one or both of a guide strand and a passenger strand of a double-stranded oligonucleotide having an RNA interference activity, at least one modification selected from the following (a)-(c): (a) a modification by a basic group through an alkylene group to the 4'-position carbon atom; (b) an alkyl oxy modification or alkenyl oxy modification to the 2'-position carbon atom; and (c) a halogen atom modification to the 2'-position carbon atom. Also, the present invention comprises an oligospermine region having two or more spermines in tandem in any one or both of the 3'-end and 5'-end of the passenger strand.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims priority based on Japanese patent application No. 2019-148061, filed on August 9, 2019, the entire contents of which are incorporated herein by reference. The present specification relates to RNA interfering agents and multiply chemically modified oligonucleotides and their uses. [Background technology]

[0002] RNA drugs such as siRNA are drugs that act directly on intracellular mRNA. After siRNA penetrates into a cell, it has the advantage of being highly specific to the target mRNA and acting in the cytoplasm, so it is not incorporated into intranuclear genomic DNA and does not become a mutagen. In addition, siRNA has the ideal characteristic of acting in a pinpoint manner, cutting only specific mRNA and leading to its degradation, thereby suppressing (also known as silencing) the expression of the protein that the mRNA should originally produce. This type of action is known as RNA interference.

[0003] In general, siRNA is effective at ultra-low concentrations at the nanomolar level, and its mechanism of action is a defense system that cells naturally possess, so it is thought to be harmless to cells. In addition, after working within the cell for a certain period of time, it is metabolized and broken down into safe nucleotide components. Due to these excellent properties, RNA interference drugs, represented by siRNA, are expected to have safe pharmaceutical effects that could not be expected with conventional low-molecular-weight compounds. In other words, because RNA interference drugs can silence the expression of specific genes with pinpoint accuracy, they have been widely used as reagents in basic biology research, and they have long been expected to be used as gene-targeted drugs with few side effects.

[0004] In particular, it is expected to be a treatment for intractable diseases that have been difficult to treat with previous drugs based on low molecular weight compounds or antibody proteins and have no alternative treatments, making them unmet needs diseases.

[0005] However, although siRNA itself has great potential as a drug, its structural characteristics, that is, since it is an oligonucleotide itself, contain many phosphate groups, and the negative charge of the entire molecule makes it difficult to permeate the cell membrane. That is, since the surface of the cell is weakly negatively charged, natural siRNA, which is rich in negative charges, has limited binding to the cell. In addition, in order to permeate the oily cell membrane consisting of a lipid bilayer, siRNA needs lipophilicity to fuse with the cell membrane, but siRNA composed of natural nucleotides lacks lipophilicity, and such low lipophilicity also impedes cell membrane permeability.

[0006] In addition, siRNAs composed of natural nucleotides have the weakness of being easily hydrolyzed by nucleases, and are unstable because they are degraded by nucleases contained in body fluids, blood, and cells.

[0007] Although siRNA has outstanding pharmacological advantages, its inherent structural characteristics, namely, its difficulty in penetrating into cells and its instability to nucleases, pose major obstacles to its development as a pharmaceutical drug.

[0008] To overcome these shortcomings, a drug delivery system (DDS) has been devised that combines siRNA with a lipid-soluble and positively charged (also called cationic) compound to protect siRNA from nucleases and allow it to penetrate into cells. For example, cationic liposomes using positively charged lipids have been tried as a means of transporting siRNA into cells (Non-Patent Documents 1 and 2). On the other hand, the toxicity of such cationic delivery systems has also been a concern (Non-Patent Document 3). [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] J Control Release, 2012,158(2),:269-276 [Non-Patent Document 2] Biol. Phram. Bull. 34(8), 1338-1342(2011) [Non-Patent Document 3] Expert Opin Drug Metab Toxicol. 2010;6:1347-1362

[0010] Although liposomes have a certain effect on protecting siRNA against nucleases, liposome formulations with particle sizes of 150 nm or more may be trapped in the hepatic capillaries and unable to circulate systemically when injected intravenously, and so the particle size must be controlled depending on the intended site of delivery. However, it has been difficult to prepare liposome formulations while controlling their particle size. Therefore, DDS using liposome formulations has inherent problems. In addition, liposomes are dispersed as particles in aqueous buffer solutions and used as liposome formulations, but in some cases they are difficult to disperse stably, as they may precipitate. In other words, there is a risk that the liposomes themselves may reduce the homogeneity of the formulation.

[0011] Furthermore, due to the non-uniformity of particle size, the dispersion liquid is often subject to aggregation or accelerated precipitation due to freezing / thawing, salting out, etc., and when it is made into a lyophilized formulation, the homogeneity of the formulation is more likely to be a problem, and there is also a risk of the stability of the formulation and reproducibility of its efficacy being reduced. For this reason, liposome formulations have no choice but to be stored at low temperatures as suspensions, and there is a quality limit in that the storage period is limited to less than one year.

[0012] In view of the above circumstances, the present specification provides a multiply chemically modified oligonucleotide that can simultaneously improve cell membrane permeability, nuclease resistance, and silencing ability as an RNA drug, and use thereof. Summary of the Invention

[0013] As a method for overcoming these problems and utilizing the advantages of siRNA, the present inventors have prepared novel nucleotides (amino alkyl modified nucleotides (hereinafter also referred to as AAmNt (amino alkyl modified nucleotide)) in which an aminoalkyl group having both a cationic amino group and an alkyl group that is a lipophilic residue is bound to the 4' position of the ribose of a nucleotide. These nucleotides were then used as amidite reagents for RNA synthesis. The present inventors have found that by introducing such AAmNt into the sites corresponding to cytosine and uridine in natural siRNA and complexing the siRNA with oligospermine, it is possible to construct siRNAs that can exhibit nuclease resistance, intracellular permeability and silencing properties. Based on these findings, the disclosure of the present specification provides the following means.

[0014] [1] An RNA interference agent, Either or both of the guide strand and the passenger strand of the double-stranded oligonucleotide may have the following (a) to (c): (a) Modification of the 4' carbon atom with a basic group via an alkylene group (b) Alkyloxy or alkenyloxy modification to the 2' carbon atom (c) Halogen atom modification of the 2' carbon atom A modified nucleotide having at least one modification selected from the following: An RNA interference agent comprising an oligospermine region having two or more spermines in tandem at either or both of the 3' and 5' ends of the passenger strand. [2] The RNA interference agent described in [1], which comprises a modified nucleotide having the modification (a) within 10 nucleotides from the 3' end to the 5' end of the passenger strand, and a modified nucleotide having the modification (c) within 8 nucleotides from the 5' end to the 3' end of the passenger strand. [3] The RNA interference agent according to [2], wherein the modified nucleotide having the modification (a) further has the modification (b) or (c). [4] The RNA interference agent according to any one of [1] to [3], wherein only the passenger strand comprises one or more modified nucleotides having the modification (a). [5] The RNA interference agent according to any one of [2] to [4], wherein the passenger strand further comprises a modified nucleotide having either the modification (b) or the modification (c). [6] The RNA interference agent according to any one of [1] to [5], comprising a phosphorothioate phosphodiester bond linking adjacent nucleotides at the 3' end of either or both of the guide strand and the passenger strand. [7] The RNA interference agent described in any of [1] to [6], wherein the guide strand comprises a plurality of modified nucleotides having one or more types of modifications selected from (b) and (c). [8] The RNA interference agent according to any one of [1] to [7], wherein the guide strand comprises five or more of the modified nucleotides. [9] The RNA interference agent according to any one of [1] to [8], wherein the passenger strand comprises five or more modified nucleotides.

[10] The RNA interference agent according to any one of [1] to [9], wherein the passenger strand is provided with the oligospermine region at the 5' end and the 3' end.

[11] The RNA interference agent according to any one of [1] to

[10] , wherein the guide strand comprises a region capable of hybridizing to RECQL1-mRNA or WRN-mRNA.

[12] The passenger strand comprises nucleotides having the modification (c) only at its 3'-end and 5'-end; The RNA interference agent according to any of [1] to

[11] , wherein the guide strand does not contain modified nucleotides having the modification (c), but contains modified nucleotides having the modification (a) or the modification (b).

[13] The RNA interference agent according to

[12] , wherein the modification (c) in the passenger strand is the introduction of an aminoethyl group to the carbon atom at the 4' position.

[14] The RNA interference agent according to any one of [1] to

[13] , wherein the oligospermine region has 10 to 20 spermines in tandem.

[15] An oligonucleotide comprising: The following (a) to (c): (a) Modification of the 4' carbon atom with a basic group via an alkylene group (b) Alkyloxy or alkenyloxy modification to the 2' carbon atom (c) Halogen atom modification of the 2' carbon atom a first modified nucleotide having at least the modification (a) among the above, a second modified nucleotide having either the modification (b) or the modification (c); Equipped with An oligonucleotide comprising an oligospermine region having two or more spermines in tandem at either or both of the 3' and 5' ends.

[16] Either or both of the guide strand and the passenger strand of the double-stranded oligonucleotide may contain the following (a) to (c): (a) Modification of the 4' carbon atom with a basic group via an alkylene group (b) Alkyloxy or alkenyloxy modification to the 2' carbon atom (c) Halogen atom modification of the 2' carbon atom A modified nucleotide having at least one modification selected from the following: A spermine region having 10 to 30 spermines in tandem at either or both of the 3' end and the 5' end of the passenger strand, A therapeutic agent for suppressing the action of a target RNA, wherein the guide strand has a region that hybridizes to the target RNA.

[17] The therapeutic agent according to

[16] , wherein the target RNA is associated with cancer and the therapeutic agent is an anti-cancer agent.

[18] The therapeutic agent according to

[16] or

[17] , wherein the target RNA is mRNA of the RECQL1 gene or the WRN gene.

[19] The therapeutic agent according to

[18] , which is a therapeutic agent for ovarian cancer.

[20] The therapeutic agent according to any one of

[17] to

[19] for reducing ascites. [Brief description of the drawings]

[0015] [Figure 1] FIG. 1 shows an example of the present RNA interference agent. [Diagram 2] This figure shows the improvement of nuclease resistance by aminoalkyl groups. siRNA with aminoethyluridine was incubated in 20% bovine serum, and after a certain period of time, the mobility of chemically modified siRNA and natural siRNA were compared by gel electrophoresis using polyacrylamide as a carrier, and the integrity of the molecular structure (resistance to nuclease) was compared. The blue line and block show the behavior of 2'-O-methylaminoethyluridine, the red line and triangle show the behavior of siRNA with 2'-OF aminoethyluridine, and the black line and circle show the behavior of siRNA with natural uridine. [Diagram 3] This figure shows the cell permeability effect of siRQ238 in 5% serum. siRQ238 (200nM, 250nM) was added to monolayer culture (containing 5% calf serum) of ovarian cancer cell ES-2, and the amount of RECQL1 mRNA was measured by PCR 48 hours later. As a control, the results of an experiment in which siRQ238 was introduced in the presence of lipofectamine are shown (hatched columns in the figure). [Figure 4] FIG. 13 shows the effect of serum on the cell permeability of siRQ238, and shows the results of evaluating the effect of serum proteins on the RNA interference effect of siRQ238 by measuring the amount of RECQL1 mRNA using PCR. [Diagram 5] This figure shows the RNA interference effect of DDS-free siRQ238 in various types of cells, including ovarian cancer cells. The figure shows the results of adding siRQ238 (250 nM) to a monolayer culture (containing 5% calf serum) of ovarian cancer cells, a type of human clear cell carcinoma known to be a typical malignant form of cancer, and measuring the silencing of RECQL1 mRNA. [Figure 6] This figure shows the DDS-free cell killing effect of siRQ238. siRQ238 (100 nM-2000 nM) was added to a monolayer culture of ovarian cancer cell ES-2 (containing 5% calf serum), and the cell viability was measured by WST assay after 96 hours. [Figure 7] This figure shows the anti-cancer effect of siRQ238 on mice with intraperitoneal seeding of ovarian cancer. Human ovarian cancer cells ES-2 were transplanted into the abdominal cavity of female nude mice to create a mouse model of "intraperitoneal seeding of human ovarian cancer." 144ug of siRQ238 dissolved in 400ul of phosphate buffer was administered intraperitoneally to these model mice every two days. The mouse on the left is an untreated mouse that received only phosphate buffer, while the mouse on the right is a mouse that received siRQ238. The results of observing the mice 10 days after transplantation using a fluorescent observation camera are shown. [Figure 8] FIG. 8 shows the morphology of mice on day 17 of the mouse model. [Figure 9] This figure shows the anti-cancer effect and life-prolonging effect of siRQ238, showing the life-prolonging effect of siRQ238 administration to mice with intraperitoneal dissemination of ovarian cancer. Two out of three untreated mice (control) died within 18 days. Mice administered siRQ238 were still alive and healthy after 31 days. [Figure 10]The effect of siRQ238 on cancer-bearing mice is shown in the following diagram: (Top) Mice (untreated) with intraperitoneal seeding of ovarian cancer died on the 18th day, and immediately after death, laparotomy was performed to examine the presence or absence of cancer. (Bottom) Laparotomy findings on the 31st day of mice with intraperitoneal seeding of ovarian cancer administered siRQ238. [Figure 11] This figure shows the effect of siRQ238 in preventing ascites accumulation. Abdominal circumference was measured before ascites was removed with an injection solution (17 days). The average value for three mice is shown. Note that Pre-treat: abdominal circumference (cm) before ovarian cancer was transplanted into the abdominal cavity, PBS: abdominal circumference of control mice 17 days after ovarian cancer was transplanted into the abdominal cavity, and siQL#238: abdominal circumference of mice 17 days after administration of siRQ238. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] The disclosure of this specification relates to an RNA interference agent and a multi-chemically modified oligonucleotide, and the use thereof. The RNA interference agent disclosed in this specification (hereinafter, also simply referred to as the agent) can satisfy the cell permeability, nuclease resistance, and silencing ability of a double-stranded oligonucleotide. The passenger strand of the agent has an oligospermine region, and the agent has an amino-modified 4'-position of the ribose of the nucleotide, so that even as a double-stranded oligonucleotide having a guide strand, it has good cell membrane permeability and improves ribonuclease resistance, and as a result, it can synergistically improve silencing ability.

[0017] Although the disclosure herein is not limited thereto, according to the present oligonucleotide derivative, by providing a basic group such as an N-containing group at the 4' position of ribose via a linking group such as an alkylene group, it is possible to adjust the net charge amount of RNA while maintaining RNA functions in vivo such as RNA interference ability, thereby enhancing lipophilicity (van der Waals intermolecular forces) and lowering the melting temperature of dsRNA. This makes it possible to improve nuclease resistance. Furthermore, the basic group can neutralize negative charges due to phosphate groups and the like and adjust the overall charge, so that the cell membrane permeability can be improved by the synergistic effect with the oligospermine region added to the passenger strand.

[0018] This RNA interference agent has excellent nuclease resistance and cell membrane permeability while maintaining its silencing ability.

[0019] Below, we will explain this agent and the multiple chemically modified oligonucleotides that can be used as the passenger strand of this agent, as well as their uses. First, we will explain the nucleoside derivatives (or nucleotide derivatives) that are common to the guide strand and passenger strand of this agent, and then we will explain the RNA interference agent and the oligonucleotides as the guide strand and passenger strand.

[0020] (Modified nucleotides used in this drug) The modified nucleotides used in the present agent (hereinafter also simply referred to as the present modified nucleotides) are the following (a) to (c): (a) Modification of the 4' carbon atom with a basic group via an alkylene group (b) Alkyloxy or alkenyloxy modification to the 2' carbon atom (c) Halogen atom modification of the 2' carbon atom The modified nucleotide of the present invention has one or more of the above. The modified nucleotide of the present invention can be, for example, a nucleoside having a basic structure represented by the following formula (1) (i.e., including an embodiment having no phosphate group) that is converted into an embodiment having a 5'-position phosphate group by a known method and used as the modified nucleotide of the present invention. Hereinafter, the modified nucleotide of the present invention will be described as the modified nucleotide of the present invention. In the following description, all of the above modifications (a) to (c) at the 4'-position and 2'-position of ribose are described for the modified nucleotide of the present invention represented by formula (1), but the modified nucleotide of the present invention merely describes all possible embodiments of the modifications (a) to (c) that may be possessed. Therefore, in reality, the modified nucleotide of the present invention will be described with the intention that it may contain only one type of modification, may contain modifications (a) and (b), or may contain modifications (a) and (c).

[0021] [ka]

[0022] In this specification, the meaning of "lower" in a substituent in a compound represented by a formula or the like means that the number of carbon atoms constituting the substituent is up to 10. For example, typically, the number of carbon atoms is 1 to 6 or 1 to 5, and more preferably, the number of carbon atoms is 1 to 4 or 1 to 3.

[0023] [R 1 Regarding formula (1), R 1 represents a hydroxyl group, a hydroxyl group in which the hydrogen atom is substituted with an alkyl group or an alkenyl group, a protected hydroxyl group, or a halogen atom. 1 When is a halogen atom, the bonding direction to the 2' carbon atom is not limited to the direction represented by formula (1).

[0024] (Alkyl group) In the present specification, examples of the alkyl group include saturated hydrocarbon groups that are linear, branched, cyclic, or a combination thereof. In general, lower alkyl groups are preferred, and more preferred examples include lower alkyl groups having 1 to 6 carbon atoms or lower alkyl groups having 1 to 5 carbon atoms, and particularly preferred examples include lower alkyl groups having 1 to 4 carbon atoms or lower alkyl groups having 1 to 3 carbon atoms. Suitable examples of the linear alkyl group having 1 to 4 carbon atoms include methyl, ethyl, n-propyl, and n-butyl groups, and among these, methyl, ethyl, and n-propyl groups are preferred, and also, for example, methyl groups are preferred. Examples of the branched alkyl group having 1 to 4 carbon atoms include isopropyl, isobutyl, s-butyl, and t-butyl groups, and among these, isopropyl is a particularly preferred example. Examples of the cyclic alkyl group having 1 to 4 carbon atoms include cyclopropyl, cyclobutyl, and cyclopropylmethyl groups.

[0025] (Alkenyl group) In the present specification, the alkenyl group includes a saturated hydrocarbon group which is linear, branched, cyclic, or a combination thereof. In general, a lower alkenyl group is preferred, and 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.

[0026] (Hydroxyl-protecting group or protected hydroxyl group) In this specification, the protecting group for the hydroxyl group is well known to those skilled in the art, and can be referred to, for example, Protective Groups in Organic Synthesis (John Wiley and Sons, 2007 edition). Representative examples of the protecting group for the hydroxyl group include, for example, an aliphatic acyl group, an aromatic acyl group, a lower alkoxymethyl group, an oxycarbonyl group which may have an appropriate substituent, a tetrahydropyranyl group which may have an appropriate substituent, a tetrathiopyranyl group which may have an appropriate substituent, a methyl group substituted with a total of 1 to 3 substituted or unsubstituted aryl groups (wherein the substituent in the substituted aryl group means a lower alkyl, a lower alkoxy, a halogen atom, or a cyano group), or a silyl group.

[0027] In the present specification, the alkoxy group includes a saturated alkyl ether group which is linear, branched, cyclic, or a combination thereof. A lower alkoxy group is preferred, and examples of the lower alkoxy group include a lower alkoxy group having 1 to 6 carbon atoms or a lower alkoxy group having 1 to 5 carbon atoms, and an alkoxy group having 1 to 4 carbon atoms or a lower alkoxy group having 1 to 3 carbon atoms is preferred, and an alkoxy group having 1 to 4 carbon atoms is particularly preferred. Preferred examples of the alkoxy group having 1 to 4 carbon atoms include a methoxy group, an ethoxy group, an n-propoxy group, and an n-butoxy group. Preferred examples include an isopropoxy group, an isobutoxy group, an s-butoxy group, and a t-butoxy group. Preferred examples include a cyclopropoxy group and a cyclobutoxy group, and a cyclopropylmethoxy group.

[0028] In the present specification, the alkylthio group includes a saturated alkylthio group which is linear, branched, cyclic, or a combination thereof. A lower alkylthio group is preferred, and the lower alkylthio group is preferably, 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. Preferred examples of the saturated alkylthio group having 1 to 4 carbon atoms include, for example, a methylthio group, an ethiothio group, an n-propylthio group, an n-butylthio group, and the like. Preferred examples include an isopropylthio group, an isobutylthio group, an s-butylthio group, and a t-butylthio group. Preferred examples include a cyclopropylthio group or a cyclobutylthio group, and a more preferred example is a cyclopropylmethylthio group.

[0029] Among these, particularly preferred examples include an aliphatic acyl group, an aromatic acyl group, and a silyl group. Also preferred examples include a methyl group substituted with a total of 1 to 3 substituted or unsubstituted aryl groups (however, the substituents in the substituted aryl groups are as described above).

[0030] Examples of the aliphatic acyl group include an alkylcarbonyl group, a carboxyalkylcarbonyl group, a halogeno lower alkylcarbonyl group, and a lower alkoxy lower alkylcarbonyl group.

[0031] The alkyl in the alkylcarbonyl group is as defined 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 eicosanoyl group, and a henycosyl group. Among these, acetyl group, propionyl group, butyryl group, isobutyryl group, pentanoyl group, and pivaloyl group are preferred, and acetyl group is particularly preferred. The alkyl in the carboxylated alkylcarbonyl group is as described above. The carboxylated substitution position can also be appropriately selected. That is, examples of the carboxylated alkylcarbonyl group include succinoyl group, glutaroyl group, and adipoyl group.

[0032] The halogen, lower, and alkyl in the halogeno lower alkylcarbonyl group are as described above. The position of the halogen can 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.

[0033] The alkoxy, alkyl, and lower groups in the lower alkoxy-lower alkylcarbonyl group are as described above. The position at which the lower alkoxy is substituted can also be appropriately selected. That is, an example of the lower alkoxy-lower alkylcarbonyl group is a methoxyacetyl group.

[0034] 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, and an arylated arylcarbonyl group.

[0035] The arylcarbonyl group includes, for example, a benzoyl group, an α-naphthoyl group, and a β-naphthoyl group, and more preferably, a benzoyl group. The halogenoarylcarbonyl group includes, for example, a 2-bromobenzoyl group and a 4-chlorobenzoyl group. The lower alkylated arylcarbonyl group includes, for example, a 2,4,6-trimethylbenzoyl group, a 4-toluoyl group, a 3-toluoyl group, and a 2-toluoyl group. The lower alkoxylated arylcarbonyl group includes, for example, a 4-anisoyl group, a 3-anisoyl group, and a 2-anisoyl group.

[0036] The carboxylated arylcarbonyl group may be, for example, a 2-carboxybenzoyl group, a 3-carboxybenzoyl group, or a 4-carboxybenzoyl group. The nitrated arylcarbonyl group may be, for example, a 4-nitrobenzoyl group, a 3-nitrobenzoyl group, or a 2-nitrobenzoyl group. The arylated arylcarbonyl group may be, for example, a 4-phenylbenzoyl group.

[0037] 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. A particularly preferred example is a methoxymethyl group.

[0038] The optionally substituted oxycarbonyl group includes a lower alkoxycarbonyl group, a lower alkoxycarbonyl group substituted with a halogen or a silyl group, and an alkenyloxycarbonyl group.

[0039] The lower alkoxycarbonyl group includes, for example, a methoxycarbonyl group, an ethoxycarbonyl group, and a t-butoxycarbonylisobutoxycarbonyl group. The lower alkoxycarbonyl group substituted with a halogen or a silyl group includes, for example, a 2,2-trichloroethoxycarbonyl group and a 2-(trimethylsilyl)ethoxycarbonyl group.

[0040] The alkenyloxycarbonyl group may be a vinyloxycarbonyl group. The tetrahydropyranyl group which may have an appropriate substituent may be, for example, a tetrahydropyran-2-yl group or a 3-bromotetrahydropyran-2-yl group, and more preferably, a tetrahydropyran-2-yl group.

[0041] Examples of the tetrathiopyranyl group which may have an appropriate substituent include a tetrahydrothiopyran-2-yl group and a 4-methoxytetrahydrothiopyran-4-yl group, and more preferably a tetrahydrothiopyran-2-yl group. In the case of a methyl group substituted with a total of 1 to 3 substituted or unsubstituted aryl groups, the substituents in the substituted aryl groups are lower alkyl, lower alkoxy, halogen, or cyano groups.

[0042] Examples of the methyl group substituted with a total of 1 to 3 substituted or unsubstituted aryl groups include benzyl, α-naphthylmethyl, β-naphthylmethyl, diphenylmethyl, triphenylmethyl, and α-naphthyldiphenylmethyl, preferably benzyl and triphenylmethyl. Other examples include 9-anthrylmethyl 4-methylbenzyl, 2,4,6-trimethylbenzyl, and 3,4,5-trimethylbenzyl, preferably 2,4,6-trimethylbenzyl and 3,4,5-trimethylbenzyl. Other types include 4-methoxybenzyl, 4-methoxyphenyldiphenylmethyl, and 4,4'-dimethoxytriphenylmethyl, preferably 4-methoxybenzyl, 4-methoxyphenyldiphenylmethyl, and 4,4'-dimethoxytriphenylmethyl. Other examples include 4-chlorobenzyl and 4-bromobenzyl. Additionally, another preferred example is a 4-cyanobenzyl group.

[0043] In the present specification, 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, and a diphenylisopropylsilylphenyldiisopropylsilyl group. Among these, more preferred are a trimethylsilyl group, a t-butyldimethylsilyl group, a triisopropylsilyl group, and a diphenylmethylsilyl group, and particularly preferred are a trimethylsilyl group, a t-butyldimethylsilyl group, and a diphenylmethylsilyl group.

[0044] In this specification, the term "protective group for a hydroxyl group" may refer to a substituent that is cleaved and eliminated by either a chemical method (e.g., hydrogenolysis, hydrolysis, electrolysis, photolysis, etc.) or a biological method (e.g., hydrolysis in the human body, etc., and conceivably induction in a microorganism, etc.). As the protective group for a hydroxyl group, particularly preferred examples include substituents that are eliminated by hydrogenolysis or hydrolysis. The protected hydroxyl group can be said to be a hydroxyl group in which a hydrogen atom has been substituted with such a protective group.

[0045] (Halogen atom) Examples of halogen atoms include, but are not limited to, chlorine, iodine, fluorine, and bromine atoms. 1 is a halogen atom (X), as shown in the following formula instead of formula (1), the bonding direction of the halogen atom (X) to the carbon atom at the 2'-position of ribose is not particularly limited, and it may be in the direction corresponding to the hydroxyl group of natural ribose as shown in formula (1) or the opposite direction, but it is preferable that the halogen atom is bonded so as to correspond to the hydroxyl group of natural ribose as shown in formula (1).

[0046] [ka]

[0047] [R 2 and R 4 Regarding formula (1), R 2 and R 4 may be the same or different, and each represents a hydrogen atom, a protecting group for a hydroxyl group, a phosphate group, a protected phosphate group, or -P(=O) n (R 5 )R 6 The protecting group for the hydroxyl group is as explained above.

[0048] (protected phosphate group) Protecting groups for the protected phosphate group are known to those skilled in the art and may be found in the above references and explanations.

[0049] Examples of protecting groups for phosphate groups include lower alkyl groups, lower alkyl groups substituted with a cyano group, ethyl groups substituted with a silyl group, lower alkyl groups substituted with a halogen, lower alkenyl groups, lower alkenyl groups substituted with a cyano group, cycloalkyl groups, lower alkenyl groups substituted with a cyano group, aralkyl groups, aralkyl groups in which the aryl ring is substituted with a nitro group, aralkyl groups in which the aryl ring is substituted with a halogen, aryl groups substituted with a lower alkyl group, aryl groups substituted with a halogen, and aryl groups substituted with a nitro group.

[0050] The lower alkyl group is as described above. The lower alkyl group substituted with the cyano group includes, for example, 2-cyanoethyl group and 2-cyano-1,1-dimethylethyl group, and more preferably, 2-cyanoethyl group. The ethyl group substituted with the silyl group includes, for example, 2-methyldiphenylsilylethyl group, 2-trimethylsilylethyl group and 2-triphenylsilylethyl group.

[0051] Examples of the lower alkyl group substituted with halogen include 2,2,2-trichloroethyl, 2,2,2-tribromoethyl, 2,2,2-trifluoroethyl, and 2,2,2-trichloroethyl groups, and particularly preferably 2,2,2-trichloroethyl groups. Examples of the lower alkenyl group include ethenyl, 1-propenyl, 2-propenyl, 1-methyl-2-propenyl, 1-methyl-1-propenyl, 2-methyl-1-propenyl, 1-butenyl, and 2-butenyl groups.

[0052] Examples of the lower alkenyl group substituted with the 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 group, a 2-naphthylpropyl group, a 3-naphthylpropyl group, 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, and particularly preferably a benzyl group.

[0053] Examples of the aralkyl group in which an aryl ring is substituted with a nitro group include a 2-(4-nitrophenyl)ethyl group, a 0-nitrobenzyl group, a 4-nitrobenzyl group, a 2,4-dinitrobenzyl group, and a 4-chloro-2-nitrobenzyl group.

[0054] In this specification, the term "protective group for phosphate" may refer to a substituent that is cleaved and eliminated by either a chemical method (e.g., hydrogenolysis, hydrolysis, electrolysis, photolysis, etc.) or a biological method (e.g., hydrolysis in the human body, etc., conceivably induction in a microorganism, etc.). As the protective group for phosphate, particularly preferred examples include substituents that are eliminated by hydrogenolysis or hydrolysis.

[0055] (-P(=O) n (R 5 )R 6 ) R of the nucleoside analogues of the present invention 2 and R 4 is -P(=O) n (R 5 )R 6 n is 0 or 1, and R 5and R 6 may be 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 , , and ...

[0056] (protected mercapto group) Protected mercapto groups are well known to those skilled in the art. Examples of the protected mercapto group include those exemplified as the above-mentioned hydroxyl group-protecting groups, as well as alkylthio groups, arylthio groups, aliphatic acyl groups, and aromatic acyl groups. Preferred are aliphatic acyl groups and aromatic acyl groups, and particularly preferred are aromatic acyl groups. Preferred examples of the alkylthio group include lower alkylthio groups, such as methylthio, ethylthio, and t-butylthio groups. Preferred examples of the arylthio group include benzylthio. Examples of the aromatic acyl group include benzoyl groups.

[0057] Preferred examples of the cyano lower alkoxy group include linear, branched, cyclic, or combinations thereof alkoxy groups having 1 to 5 carbon atoms (when counting without including the number of carbon atoms in the cyano group) substituted with a cyano group. Specific examples include cyanomethoxy, 2-cyanoethoxy, 3-cyanopropoxy, 4-cyanobutoxy, 3-cyano-2-methylpropoxy, and 1-cyanomethyl-1,1-dimethylmethoxy, and a particularly preferred example is 2-cyanoethoxy.

[0058] R 5 and R 6 As R, a substituted amino group can be selected. The substituent of the amino group is either a lower alkoxy group, a lower alkylthio group, a cyano lower alkoxy group, or a lower alkyl group. 5 and R6 In the case where both are substituted amino groups, the substituted amino groups may be different from each other. The lower alkoxy group, the lower alkylthio group, the cyano lower alkoxy group, and the lower alkyl group are as described above.

[0059] -P(=O) n (R 5 )R 6 More specifically, preferred examples of the alkyl group include a phosphoramidite group, an H-phosphonate group, and a phosphonyl group, and a particularly preferred example of the alkyl group is a phosphoramidite group.

[0060] -P(=O) n (R 5 )R 6 In the formula, n is 0 and R 5 and R 6 When at least one of R is a substituted amino group and the other is not important, it becomes a phosphoramidite group. 5 and R 6 A phosphoramidite group in which one of R is a substituted amino group and the other is a lower alkoxy group or a cyano lower alkoxy group is particularly preferred because it has good reaction efficiency in the condensation reaction. Preferred examples of the substituted amino group include a diethylamino group, a diisopropylamino group, and a dimethylamino group, and a particularly preferred example is a diisopropylamino group. 5 and R 6 A preferred example of the lower alkoxy group in the other substituent is a methoxy group. A preferred example of the cyano lower alkoxy group is a 2-cyanoethyl group. Specific preferred examples of the phosphoramidite group include -P(OC2H4CN)(N(CH(CH3)2) or -P(OCH3)(N(CH(CH3)2).

[0061] -P(=O) n (R 5 )R 6 In the formula, n is 1 and R5 and R 6 When at least one of the groups is a hydrogen atom and the other is any group other than a hydrogen atom, the resulting group is an H-phosphonate group. Examples of the substituent other than hydrogen include a hydroxyl group, a methyl group, a methoxy group, a thiol group, and the like, and a particularly preferred example is a hydroxyl group.

[0062] Also, -P(=O) n (R 5 )R 6 In the formula, n is 1 and R 5 and R 6 When both R are lower alkoxy groups, the group becomes a phosphonyl group. 5 and R 6 The lower alkoxy groups in may be the same or different. Preferred examples of the lower alkoxy group include a methoxy group and an ethoxy group. A specific example of the phosphonyl group is -P(=O)(OCH3)2.

[0063] R in the nucleoside derivative 2 For example, -P(=O) n (R 5 )R 6 It is particularly preferred that -P(=O) n (R 5 )R 6 Preferred examples of R include a phosphoramidite group, an H-phosphonate group, and a phosphonyl group. 2 In addition, R is preferably a phosphate group or a protected phosphate group. 2 is also preferably a hydrogen atom or a protecting group for a hydroxyl group.

[0064] R 2 Preferred specific examples of the alkyl group 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(OCHCN)(N(CH(CH)), -P(OCH)(N(CH(CH)), or a phosphonyl group.

[0065] R in the nucleoside derivative 4 is preferably, for example, a protecting group for a hydrogen atom or a hydroxyl group, or, for example, a phosphate group, a protected phosphate group, or -P(=O) n (R 5 )R 6 It is also preferable that R 4 Preferred specific examples of include a hydrogen atom, an acetyl group, a benzoyl group, a benzyl group, a p-methoxybenzyl group, a dimethoxytrityl group, a monomethoxytrityl group, a tert-butyldiphenylsilyl group, and a trimethylsilyl group.

[0066] [R 3 Regarding formula (1), R 3 can each represent a basic group having a linking group. The present modified nucleotide having the modification (a) (hereinafter also referred to as the present modified nucleotide (a)) has a basic substituent at the 4'-position of ribose, and thus can have a charge adjusting ability to neutralize at least a part of the negative charge caused by the phosphate group of the oligonucleotide in an oligonucleotide having a partial structure derived from the present modified nucleotide. This can improve the cell membrane permeability of the oligonucleotide having the partial structure. Furthermore, the ribonuclease resistance can be improved in an oligonucleotide having a partial structure derived from the present modified nucleotide (a).

[0067] The linking group can be, for example, a divalent hydrocarbon group having one or more carbon atoms. That is, examples of the divalent hydrocarbon group include an alkylene group having 1 to 8 carbon atoms and an alkenylene group having 2 to 8 carbon atoms.

[0068] The alkylene group as the linking group may be linear or branched, but is preferably linear. For example, a lower alkyl group is preferred, for example a lower alkyl group having 1 to 6 carbon atoms, or for example a lower alkyl group having 2 to 6 carbon atoms, or for example a lower alkyl group having 2 to 4 carbon atoms or 2 to 3 carbon atoms. Examples of the linear alkyl group having 1 to 4 carbon atoms include a methylene group, an ethylene group, a propane-1,3-diyl group, an n-butane-1,1-diyl group, an n-pentyl-1,5,-diyl group, and an n-hexyl-1,6-diyl group. Examples of the butane-1,2-diyl group are also preferred. For example, an ethylene group, a propane-1,3-diyl group, and an n-butane-1,1-diyl group are particularly preferred.

[0069] The alkenylene group as the linking group may be linear or branched, and is preferably linear. For example, a lower alkenylene group is preferred, and examples of the lower alkenylene group include: Examples include an ethene-1,2-diyl group, a propene-1,3-diyl group, and a butene-1,4-diyl group.

[0070] In the nucleoside derivative represented by formula (1), a divalent hydrocarbon group such as an alkylene group having 2 or more carbon atoms, for example an ethylene group, is preferred from the viewpoint of nuclease resistance and cell membrane permeability of the oligonucleotide derivative.

[0071] The basic group is, for example, NHR 7 (R 7 represents a hydrogen atom, an alkyl group, an alkenyl group, or a protecting group for an amino group. R represents an azido group, an amidino group, or a guanidino group. 7 Examples of R include a hydrogen atom, an alkyl group, an alkenyl group, or a protecting group for an amino group. In addition to the alkyl groups already described, preferred examples of the alkyl group include lower alkyl groups. In addition to the alkenyl groups already described, preferred examples of the alkenyl group include lower alkenyl groups. 7When is a hydrogen atom or any of these groups, the linking group is preferably an alkylene group having 2 or more carbon atoms, such as 3 or more, such as 4 or more, and such as 6 or less, such as 5 or less, such as 4 or less. More preferably, the linking group is a linking group having 2 or more carbon atoms, and is an alkylene group having 2 or more carbon atoms.

[0072] Also, R 7 When is a hydrogen atom, R 3 is an NH2 (amino group) having a linking group, that is, when the linking group is an alkylene group or an alkenylene group, it is an aminoalkyl group or an aminoalkenyl group. In formula (1), R 3 is an aminoalkyl group, the modified nucleotide and the oligonucleotide having a monomer unit derived from the modified nucleotide can exhibit a charge-donating property with the characteristic that the charge changes depending on the surrounding pH environment. For example, it is cationic under acidic conditions, and the positive charge can be reduced to zero under neutral physiological conditions. In other words, such a charge-adjusting ability allows the charge of the nucleoside derivative to be dynamically changed or a desired charge to be given when necessary by changing the pH environment. Therefore, such a nucleoside derivative allows the charge of the oligonucleotide to be adjusted in a manner different from the conventional one or with a higher degree of freedom than the conventional one.

[0073] R 3 Examples of R include an azide group, an amidino group, i.e., CH3(NH)C(NH)- (an amino group of amidine with one hydrogen atom removed), and a guanidino group, i.e., NH2(NH)C(NH)- (an amino group of guanidine with one hydrogen atom removed), each of which has a linking group. Among these, a guanidino group is particularly preferred. R 3 When R has these groups, the linking group may be an alkylene group or an alkenylene group having 1 or more carbon atoms, for example, 2 or more carbon atoms. 3 However, when the nucleoside derivative is an amidino group or a guanidino group having a linking group, it is always cationic, unlike the aminoalkyl group described above.3 is useful in combination with the present nucleoside derivatives in which the nucleoside group is an aminoalkyl group or the like.

[0074] Protective groups for amino groups are well known to those skilled in the art, and the above-mentioned references can be referred to.Specific examples of protective groups for hydroxyl groups include, in addition to those listed above, benzyl, methylbenzyl, chlorobenzyl, dichlorobenzyl, fluorobenzyl, trifluoromethylbenzyl, nitrobenzyl, methoxyphenyl, methoxymethyl (MOM), N-methylaminobenzyl, N,N-dimethylaminobenzyl, phenacyl, acetyl, trifluoroacetyl, pivaloyl, benzoyl, phthalimide, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, benzyloxycarbonyl, t-butoxycarbonyl (Boc), 1-methyl-1-(4-biphenyl)ethoxycarbonyl (Bpoc), 9-fluorenylmethoxycarbonyl, benzyloxymethyl (BOM), and 2-(trimethylsilyl)ethoxymethyl (SEM). More preferred are benzyl, methoxyphenyl, acetyl, trifluoroacetyl (TFA), pivaloyl, benzoyl, t-butoxycarbonyl (Boc), 1-methyl-1-(4-biphenyl)ethoxycarbonyl (Bpoc), 9-fluorenylmethoxycarbonyl, benzyloxymethyl (BOM), and 2-(trimethylsilyl)ethoxymethyl (SEM), and particularly preferred are benzyl, methoxyphenyl, acetyl, benzoyl, and benzyloxymethyl.

[0075] In the present invention, the protecting group for an amino group may refer to a substituent that is cleaved and eliminated by either a chemical method (e.g., hydrogenolysis, hydrolysis, electrolysis, photolysis, etc.) or a biological method (e.g., hydrolysis in the human body, etc., conceivably induction in a microorganism, etc.). In particular, a substituent that is eliminated by hydrogenolysis or hydrolysis is preferred as a protecting group for an amino group.

[0076] [B: About bases] The base B in the present nucleoside derivative may be a known natural base or an artificial base. For example, B may be a purine-9-yl group, a 2-oxo-pyrimidin-1-yl group, a substituted purine-9-yl group, or a substituted 2-oxo-pyrimidin-1-yl group.

[0077] That is, B may be a purin-9-yl group or a 2-oxo-pyrimidin-1-yl group, as well as 2,6-dichloropurin-9-yl or 2-oxo-pyrimidin-1-yl, and may further be 2-oxo-4-methoxy-pyrimidin-1-yl, 4-(1H-1,2,4-triazol-1-yl)-pyrimidin-1-yl, or 2,6-dimethoxypurin-9-yl.

[0078] Further examples include 2-oxo-4-amino-pyrimidin-1-yl with a protected amino group, 2-amino-6-bromopurin-9-yl with a protected amino group, 2-amino-6-hydroxypurin-9-yl with a protected amino group, 2-amino-6-hydroxypurin-9-yl with a protected amino group and / or hydroxyl group, 2-amino-6-chloropurin-9-yl with a protected amino group, 6-aminopurin-9-yl with a protected amino group, and 4-amino-5-methyl-2-oxo-pyrimidin-1-yl with a protected amino group. The protecting groups for the hydroxyl group and amino group are as described above.

[0079] Further examples include 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).

[0080] Further examples include 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.

[0081] Further examples include 6-amino-2-methoxypurin-9-yl, 6-amino-2-chloropurin-9-yl, 2-amino-6-chloropurin-9-yl, and 2-amino-6-bromopurin-9-yl.

[0082] The substituents in the substituted purin-9-yl group or the substituted 2-oxo-pyrimidin-1-yl group are 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, and a halogen atom, or any combination of a plurality of these. These substituents are as described above.

[0083] As for B in the present nucleoside derivative, the substituents in the substituted purine-9-yl group or substituted 2-oxo-pyrimidin-1-yl group are preferably the above-mentioned substituents, and in addition, it is also preferable that a triazole group or a lower alkoxymethyl group is added.

[0084] Preferred 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, and 6-mercaptopurin-9-yl. If the above-mentioned substituents contain an amino group or a hydroxyl group, preferred examples include substituents in which the amino group and / or the hydroxyl group are protected.

[0085] Substituted 2-oxo-pyrimidin-1-yls include, for example, 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. Further preferred examples include 2-oxo-4-methoxy-pyrimidin-1-yl and 4-(1H-1,2,4-triazol-1-yl)-pyrimidin-1-yl.

[0086] Among these B groups, when the substituent contains an amino group or a hydroxyl group, preferred examples include substituents in which the amino group or the hydroxyl group is protected.

[0087] The modified nucleotide may be a salt. The form of the salt is not particularly limited, but generally, an acid addition salt is exemplified, and may be in the form of an intramolecular counter ion. Alternatively, a base addition salt may be formed depending on the type of the substituent. As the salt, a pharma- ceutically acceptable salt is preferred. The types of acids and bases that form pharma- ceutical acceptable salts are well known to those skilled in the art, and can be referred to, for example, those described in J.Pharm.Sci., 1-19 (1977). For example, examples of the acid addition salt include mineral acid salts and organic acid salts. In addition, when one or more substituents contain an acidic moiety, a base addition salt is also a preferred example.

[0088] Examples of mineral acid salts include hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, hydrogen sulfate, phosphate, and hydrogen phosphate. Usually, hydrochloride and phosphate are preferred. Examples of organic acid salts include acetate, trifluoroacetate, gluconate, lactate, salicylate, citrate, tartrate, ascorbate, succinate, maleate, fumarate, formate, benzoate, methanesulfonate, ethanesulfonate, and p-toluenesulfonate. Usually, acetate and the like are preferred. Examples of base addition salts include alkali metal salts, alkaline earth metal salts, organic amine salts, and amino acid addition salts.

[0089] 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, aspartic acid salts, and glutamic acid salts.

[0090] The modified nucleotide or a salt thereof may exist as a hydrate or a solvate, and these substances are also included in the scope of the disclosure of the present specification. The modified nucleotide or a salt thereof can be easily produced by those skilled in the art according to the synthesis examples described below or known methods.

[0091] The present modified nucleotide, when introduced into an oligoribonucleotide as a nucleotide at least as a part of an oligonucleotide, can improve the ribonuclease resistance of a single-stranded oligoribonucleotide and a double-stranded oligoribonucleotide, and can also improve the cell membrane permeability of mammalian cells and the like. The present modified nucleotide can have a basic substituent at 4'. This adjusts the negative charge derived from a phosphate group or the like in the oligoribonucleotide, and further functions as a positive charge imparting agent through a synergistic effect with the oligospermine region to further improve the cell membrane permeability.

[0092] The modified nucleotide may appropriately include modifications (a) to (c) based on the above description. For example, without being particularly limited, when the modified nucleotide has modification (a), the modified nucleotide may have modification (b) or (c) in addition to modification (a).

[0093] (RNA interference agent) The RNA interference agent (the agent) disclosed in the present specification is a double-stranded oligonucleotide, and can have a guide strand and a passenger strand. The guide strand has a complementary region capable of hybridizing to a target RNA, and the passenger strand can hybridize with the guide strand to form a double strand.

[0094] A double-stranded oligonucleotide as an RNA interference agent generally has a TT region at the 3' end of the guide strand and a TT region at the 3' end of the passenger strand, and these have a structure (overhang) that protrudes from the 5' end of the other strand, but the number of overhanging nucleotides and the overhanging region are not limited to TT, and various known modifications can be applied. In addition, one or both ends of the double-stranded oligonucleotide may not have an overhang structure as long as it exerts an RNA interference effect.

[0095] Furthermore, a double-stranded oligonucleotide as an RNA interference agent generally comprises, for example, a 19-mer nucleotide in addition to an overhang at the 3' end of each strand, but is not limited thereto and can be, for example, about 10 to 30 nucleotides.

[0096] In addition, double-stranded oligonucleotides serving as RNA interference agents are generally composed of ribonucleotides containing ribose, but may contain deoxyribose from deoxynucleotides as the sugar if necessary, and may also be configured to contain thymidine as the base instead of uridine.

[0097] (Guide strand) The guide strand of the double-stranded oligonucleotide as an RNA interference agent will be described. The single-stranded oligonucleotide as the guide strand can have any of the above modified nucleotides (a) to (c), but preferably has either or both of a nucleotide having modification (b) and a nucleotide having modification (c).

[0098] The site of introduction of the modified nucleotide in the guide strand is not particularly limited, but may be the 5'-end, 3'-end, or central portion, taking into consideration the resistance to nucleases such as ribonuclease and the functionality of the guide strand. The 5'-end of the guide strand refers to a region within 8 nucleotides from the 5'-end to the 3'-end. The 3'-end of the guide strand refers to a region within 10 nucleotides from the 3'-end to the 5'-end. Furthermore, the central portion of the guide strand refers to a portion other than the 5'-end and 3'-end.

[0099] The modified nucleotides may be present in only a portion of these sites, or may be present only at the 5'-end and 3'-end sites, or the modified nucleotides may be introduced only at the 5'-end and central portion, or the modified nucleotides may be introduced at the 3'-end and central portion, or the modified nucleotides may be introduced at all sites.

[0100] On the 5'-end side of the guide strand, for example, the modified nucleotide is provided at the 5'-end. Also, for example, the modified nucleotide is also introduced next to the 5'-end. Also, for example, three or more modified nucleotides are provided within 5 nucleotides from the 5'-end to the 3'-end.

[0101] The 3'-end of the guide strand may or may not have the modified nucleotide within 2 nucleotides from the 3'-end including the overhang. For example, the modified nucleotide may be present adjacent to the overhang on the 5'-end side from the 3'-end of the guide strand, and one or more (for example, 5 or less, for example, 4 or less, for example, 3 or less) modified nucleotides may be introduced within 10 nucleotides from the 3'-end to the 5'-end.

[0102] The central portion of the guide strand, i.e., the region excluding the region within 8 nucleotides from the 5'-end and the region within 10 nucleotides from the 3'-end, may or may not contain the present modified nucleotide having modification (b) and / or the present modified nucleotide having modification (c). The number of such present modified nucleotides in this region is not particularly limited, but may be, for example, 1 to 5, or, for example, 1 to 4, or, for example, 1 to 3, or, for example, 1 to 2.

[0103] The number of modified nucleotides in the entire guide strand is preferably 2 or more, and they may not be consecutive, or 2 or 3 or more may be consecutive. The number of modified nucleotides in the guide strand is, for example, 4 or more, for example, 5 or more, for example, 6 or more, for example, 7 or more, or for example, 8 or more. For example, 12 or less, for example, 11 or less, for example, 10 or less, for example, 9 or less, or for example, 8 or less.

[0104] In addition, the modified nucleotide in the guide strand can be appropriately selected from modification (b) and modification (c), and a modified nucleotide having only modification (b) may be used, or a modified nucleotide having only modification (c) may be used, or a modified nucleotide having both modifications (b) and (c) may be used. It is understood that even a modified nucleotide having only modification (b), for example, has multiple types of modified nucleotide forms, and modification (c) also has multiple types of forms. The same applies to modification (c).

[0105] The guide strand may not have the modified nucleotide of modification (a) because the guide strand forms a complex with the AGO protein, and therefore changing the charge of the guide strand may not be desirable.

[0106] In order to improve nuclease resistance in the guide strand, for example, a phosphorothioate diester bond in which one oxygen atom of the phosphate ester not involved in the phosphodiester bond is replaced with a sulfur atom can be used in place of the natural phosphodiester bond for the bond between nucleotides.

[0107] Such bonds can be provided within, for example, 10 nucleotides (or, for example, within, 9 nucleotides, or, for example, within, 8 nucleotides) including the overhang on the 3'-end side of the guide strand, and such bonds can be provided continuously or intermittently. The overhangs may be connected to each other by such bonds. Furthermore, in the above region, the number of such bonds can be, for example, 2 or more, or, for example, 3 or more, or, for example, 4 or more, or, for example, 5 or more. Note that, in the above region, the number of such bonds is at most 6.

[0108] (Passenger strand) The passenger strand of the double-stranded oligonucleotide as an RNA interference agent will be described. The single-stranded oligonucleotide as the passenger strand can have any of the above modified nucleotides (a) to (c), but preferably has the modified nucleotide having modification (a), and further has the modified nucleotide having modification (b) and / or the modified nucleotide having modification (c) in addition to the modified nucleotide.

[0109] The modified nucleotide having modification (a) may be present at one or more positions, for example, within 8 nucleotides, for example, within 7 nucleotides, for example, within 6 nucleotides, for example, within 5 nucleotides, from the 5' end to the 3' end of the passenger strand. For example, the modified nucleotide may be present at one or more positions, up to four positions, for example, up to one position, up to three positions, for example, up to one position, up to two positions, in the region.

[0110] The modified nucleotide having modification (a) may be present at one or more positions within, for example, 10 nucleotides, for example, 9 nucleotides, for example, 8 nucleotides, for example, 7 nucleotides, for example, 6 nucleotides, for example, 5 nucleotides from the 3' end to the 5' end of the passenger strand. For example, one to four, for example, one to three, may be present in the region. The modified nucleotide may be present at either the 5' end or the 3' end of the passenger strand, or may be present at both ends.

[0111] The modified nucleotide having the modification (a) in the passenger strand can further have either the modification (b) or the modification (c) at its 2' position. In this way, the nuclease resistance can be further increased at the site having the modified nucleotide. In consideration of the nuclease resistance of the modified nucleotide, it may be advantageous to adopt the modification (b).

[0112] In the passenger strand, either or both of the modified nucleotide having modification (b) and the modified nucleotide having modification (c) may be provided adjacent to the modified nucleotide having modification (a). This allows the modified nucleotide having modification (a) to be stably held in the passenger strand. For example, these other modified nucleotides may be provided immediately adjacent to the 3'-end and / or 5'-end of the modified nucleotide having modification (a). For example, when the modified nucleotide having modification (a) also has modification (b) or modification (c), these other modified nucleotides may be the modified nucleotide having modification (c) or modification (b), respectively. Providing nucleotides with different modified forms may be effective for nuclease resistance.

[0113] In the passenger strand, it may be effective not to have the modified nucleotide having the modification (a) at any site other than the 5'-end and 3'-end. Changing the charge in such a region may affect the effect of the siRNA, which is a double-stranded oligonucleotide.

[0114] Furthermore, the passenger strand, like the guide strand, may have continuous or intermittent phosphodiester bonds by phosphorothioate within 10 nucleotides, for example, within 8 nucleotides, or for example, within 6 nucleotides from the 3' end including the overhang. This contributes to the cell membrane permeability of the passenger strand. The overhangs may also be linked to each other by such bonds. The number of phosphodiester bonds by phosphorothioate is not particularly limited, but may be, for example, 2 or more, 3 or more, 4 or more, or 5 or more. Note that the number of such bonds in the above region is at most 6.

[0115] The central portion of the passenger strand, i.e., the region excluding the region within 8 nucleotides from the 5'-end and the region within 10 nucleotides from the 3'-end, may not contain the present modified nucleotide, but may contain the present modified nucleotide having modification (b) and / or the present modified nucleotide having modification (c). The number of such present modified nucleotides in this region is not particularly limited, but may be, for example, 1 to 4, or, for example, 1 to 3, or, for example, 1 to 2.

[0116] The present modified nucleotide having any of the modifications (a) to (c) in the entire passenger strand is preferably 2 or more, and may not be consecutive, or may be 2 or 3 or more consecutive. The present modified nucleotide in the passenger strand is, for example, 4 or more, for example, 5 or more, for example, 6 or more, for example, 7 or more, for example, 8 or more, or for example, 9 or more. For example, 12 or less, for example, 11 or less, for example, 10 or less, or for example, 9 or less.

[0117] (Oligospermine region) The passenger strand may have an oligospermine region at its 5'-end and / or 3'-end. By having the oligospermine region in the passenger strand, the cell membrane permeability is improved by a synergistic effect with the present modified nucleotide having modification (a) in the double-stranded oligonucleotide. For example, by having the present modified nucleotide having modification (a) in the passenger strand, it further contributes to the cell membrane permeability of the double-stranded oligonucleotide.

[0118] The oligospermine region comprises at least two spermines, for example, 5 or more, for example, 10 or more, for example, 15 or more, for example, 20 or more, or for example, 25 or more, for example, 40 or less, for example, 35 or less, for example, 30 or less, or for example, 25 or less. The range of the number of spermines in the oligospermine region is not particularly limited, and can be, for example, 10 to 30, or for example, 15 to 25.

[0119] It may be preferred to provide an oligospermine region at the 5' end of the passenger strand, which may terminate in an OH group.

[0120] Various guide strands and passenger strands constituting the present agent have been described above, but for example, the following combinations of guide strands and passenger strands may be suitable: That is, the guide strand may have three modified nucleotides with modifications (b) and / or (c) at the 5'-end, one modified nucleotide with modifications (b) and / or (c) at the 3'-end, and a phosphodiester bond with phosphorothioate between the 16th and 21st nucleotides from the 5'-end. In addition, the passenger strand may have an oligospermine region at the 5'-end having 15 spermines in tandem, a modified nucleotide having modification (a) and modification (c) at the third position from the 5'-end, and a modified nucleotide having modification (b) on both sides of that, a modified nucleotide having modification (a) and modification (c) at the 18th position from the 5'-end of the 3'-end, and a modified nucleotide having modification (b) on both sides of that, and a phosphodiester bond by phosphorothioate between the 20th and 21st overhangs from the 5'-end.

[0121] In particular, for the single-stranded oligonucleotide serving as the passenger strand, the oligospermine region and the present modified nucleotide having modification (a) can contribute to the excellent cell membrane permeability of the double-stranded oligonucleotide.

[0122] That is, according to the present specification, an oligonucleotide comprising: The following (a) to (c): (a) Alkyloxy or alkenyloxy modification to the 2' carbon atom (b) Halogen atom modification of the 2' carbon atom (c) Modification of the 4' carbon atom with an ami-, azide, or azo group via an alkylene group a first modified nucleotide having at least the modification (c) among the above, a second modified nucleotide having either the modification (a) or the modification (b); Equipped with Oligonucleotides are also provided that comprise an oligospermine region having two or more spermines in tandem at either or both of the 3' and 5' ends.

[0123] The guide strand and passenger strand in the double-stranded oligonucleotide are synthesized so as to introduce a partial structure represented by the following formula (2) according to the purpose. Such a partial structure can be obtained based on a nucleoside derivative represented by formula (1) or a salt thereof.

[0124] [ka]

[0125] R in the partial structure represented by formula (2) 1 , R 3 and B have the same meanings as in formula (1).

[0126] In addition, both the guide strand and the passenger strand may be oligoribonucleotides or chimeras of ribonucleotides and deoxyribonucleotides. Both the guide strand and the passenger strand may be modified by known methods. For example, PEG, RGD peptide, folic acid, cholesterol, BNA, LNA, PNA, and sugar chains may be added. In addition, the bond between each nucleotide in the guide strand and the passenger strand is not particularly limited, and may be bonded by, for example, a phosphodiester bond or a phosphodiester bond by phosphothioate.

[0127] The oligonucleotide constituting this agent may have one or more asymmetric centers in the partial structure represented by formula (2) or other partial structures, and the same applies when stereoisomers exist, and any mixture of stereoisomers or racemates are all included in the scope of the present invention. In addition, it may exist as a tautomer.

[0128] The oligonucleotide may be in the form of a salt. The form of the salt is not particularly limited, but a pharmacy acceptable salt is a preferred example. The salt can be the same as the salt form of the present nucleoside derivative described above. The oligonucleotide or its salt may be a hydrate or solvate, which are also included in the scope of the present invention.

[0129] (Production of the Modified Nucleotides and Guide and Passenger Strands) The present modified nucleotide and the guide strand and passenger strand of the present agent can be easily synthesized by a person skilled in the art based on the specific synthesis examples described below as well as on the synthesis techniques for nucleosides and oligonucleotides publicly known at the time of filing this application.

[0130] The present modified nucleotide and the present oligonucleotide derivative can be produced, for example, by the following method, but the method for producing the nucleoside analog or oligonucleotide analog of the present invention is not limited to the following method.

[0131] In each reaction, the reaction time is not particularly limited, but since the progress of the reaction can be easily tracked by the analytical means described below, it is sufficient to terminate the reaction at the time when the yield of the target product is maximized. In addition, each reaction can be carried out, if necessary, under an inert gas atmosphere such as a nitrogen or argon stream. In each reaction, when protection with a protecting group and subsequent deprotection are required, the reaction can be appropriately carried out by utilizing the method described below.

[0132] In this specification, Bn represents a benzyl group, Ac represents an acetyl group, Bz represents a benzoyl group, PMB represents a p-methoxybenzyl group, Tr represents a triphenylmethyl group, THA represents a trifluoroacetyl group, TsO represents a tosyloxy group, MMTr represents a 4-methoxytriphenylmethyl group, DMTr represents a 4,4'-dimethoxytriphenylmethyl group, TMS represents a trimethylsilyl group, TBDMS represents a tert-butyldimethylsilyl group, TBDPS represents a tert-butyldiphenylsilyl group, MOM represents a methoxymethyl group, BOM represents a benzyloxymethyl group, and SEM represents a 2-(trimethylsilyl)ethoxymethyl group.

[0133] For example, one example of the present nucleoside derivative can be synthesized according to the following scheme. Note that the following scheme is an example of a scheme for synthesizing a thymine ribonucleoside derivative using glucose as a starting material, and synthesizing a phosphoramidite agent for synthesizing the present oligonucleotide derivative.

[0134] [ka]

[0135] [ka]

[0136] According to a conventional method, the above-mentioned compound 2 was obtained from glucose 1. From compound 2, compounds 3 to 20 can be obtained based on the description in Bioorganic & Medical Chemistry 11(2003)211-2226, Bioorganic & Chemistry letters(1999)2667-2672, The Journal of Organic Chemistry 2013, 78, 9956-9962, HELVATICA CHIMICA ACTA Vol. 83 (2000) 128-151, etc., as well as Bioorganic & Medical Chemistry 11(2003)211-2226, Bioorganic & Chemistry letters(1999)2667-2672.

[0137] An oligonucleotide having a partial structure represented by formula (2) can be easily produced by using various modified nucleotides represented by formula (1) as an amidite agent or the like. That is, by using such modified nucleotides, it can be synthesized using a known DNA synthesizer, and the obtained oligonucleotide derivative can be purified using a column, and the purity of the product can be analyzed by reverse phase HPLC or MALDI-TOF-MS to obtain a purified oligonucleotide. Note that methods for converting oligonucleotides into acid addition salts are well known to those skilled in the art.

[0138] Furthermore, the oligospermine region can be added to the 3'-end and / or 5'-end of an oligonucleotide or any other site other than the end of an oligonucleotide chain, for example, by synthesizing an oligospermine amidite having oligospermine instead of a nucleotide and using this amidite in a known method for synthesizing DNA, etc. Alternatively, oligospermine can be added separately to the 5'-end or 3'-end of a synthesized oligonucleotide.

[0139] The double-stranded oligonucleotide disclosed herein can be used as an siRNA for suppressing the action of a target mRNA, that is, for suppressing the expression of a protein encoded by the target mRNA. That is, when it has a structure essential for siRNA, it forms a complex with an in vivo component (RISC protein) and cleaves the mRNA in a sequence-specific manner, thereby preventing the information on the mRNA from being translated into a specific protein by ribosomes. It is also considered that the double-stranded oligonucleotide can be used as a component constituting miRNA, and the single-stranded oligonucleotide can be used as an antisense oligonucleotide, while taking advantage of the characteristics of nuclease resistance and improved cell membrane permeability. Furthermore, it can be linked to other compounds to form a conjugate. Furthermore, the single-stranded oligonucleotide can also be used as a component of a ribozyme. In addition, the double-stranded oligonucleotide is also useful as a functional structure for RNA chips and other reagents due to its stability.

[0140] For these reasons, the double-stranded oligonucleotides containing multiple chemical modifications disclosed in this specification are expected to be more useful than conventional natural siRNAs, taking advantage of characteristics not found in natural nucleotides, as components of various RNA pharmaceuticals, including antitumor and antiviral agents, that treat diseases by inhibiting gene activity.

[0141] In the following, examples are given as specific examples to more specifically explain the disclosure of the present specification. The following examples are intended to explain the disclosure of the present specification, but are not intended to limit the scope thereof. EXAMPLES

[0142] (Creation of double-stranded oligonucleotides) The double-stranded oligonucleotide (siRQ238) shown in Figure 1 was synthesized. This double-stranded oligonucleotide has a passenger strand with a base sequence identical to a part of the exon of human RECQL1 messenger RNA and a guide strand with a base sequence complementary to the above base sequence, and is an siRNA that suppresses the expression of the RECQL1 gene. Specifically, it has the following structure.

[0143] (Passenger strand: Sense strand) The passenger strand has a base sequence (5'-GUUCAGACCACUUCAGCUU-3': SEQ ID NO: 1) identical to a part of an exon of human RECQL1 messenger RNA, and has the following multiple chemical modifications. These modifications in the passenger strand and the guide strand described below were synthesized by synthesizing phosphoramidites having nucleoside derivatives corresponding to the modifications, and using the phosphoramidites to synthesize the modifications using an RNA synthesizer. Spermine was bound to the 5' end of the passenger strand at the end of its synthesis via a phosphodiester bond.

[0144] (a) 15 spermine residues are linked to the 5'-terminus via phosphodiester bonds. (b) Thymidine is incorporated at the 20th and 21st nucleosides from the 5'-end. (c) The internucleotide phosphate bond between the 19th to 21st nucleosides at the 5'-terminus is modified with phosphorothioate (S-modification). (d) 4'-aminoethyl-2'-fluorouridine is introduced into the third and 18th nucleosides from the 5'-terminus. (e) The 2nd, 4th, 12th, 13th, 14th, 17th, and 19th nucleosides from the 5'-end are modified with 2'-O-methyl (OMe).

[0145] (Guide strand: antisense strand) The guide strand has a base sequence (5'-AAGCUGAAGUGGUCUGAAC-3': SEQ ID NO: 2) complementary to a part of the base sequence of an exon of human RECQL1 messenger RNA, and has the following multiple chemical modifications.

[0146] (a) The 1st, 13th, 15th, and 19th nucleotides from the 5'-end are modified with 2'-O-methyl (OMe). (b) The second and fourth nucleotides from the 5'-end are modified with 2'-fluoro (F). (c) The internucleotide phosphate bond between the 16th to 21st nucleotides at the 5'-terminus is modified with phosphorothioate (S-modification). (d) Thymidine is incorporated at the 20th and 21st nucleotides from the 5'-end.

[0147] The synthesis of oligonucleotides was carried out on a 0.2 μmol scale using a DNA automatic synthesizer using the phosphoramidite method. The natural nucleoside amidites and chemically modified nucleotides were diluted with MeCN to 0.1 M, and the oligospermine amidites were diluted with MeCN to 0.15 M. The CPG resin was packed into a column at 0.2 μmol equivalent based on its activity, and synthesis was started. The synthesis was terminated with the 5-hydroxyl group protected with DMTr. After the synthesis was completed, the CPG resin was transferred to a sampling tube, and 900 μL of MeCN and 100 μL of diethylamine were added to the CPG resin and incubated at room temperature for 10 minutes. The supernatant was discarded, and the CPG resin was washed twice with 1 mL of MeCN. 500 μL of methylamine and 500 μL of NH3aq. were added to the mixture and incubated at 65°C for 10 minutes. After incubation, the sample was transferred to a sampling tube, and the CPG resin was washed twice with 1 mL of H2O. The mixture was dried under reduced pressure, and then the sample was dissolved in 100 μL of DMSO to deprotect the TBDMS group. 25 μL of TEA and 125 μL of TEA·3HF were added, stirred, and then incubated at 65°C for 90 minutes.

[0148] (Glen-Pak purification) After washing the cartridge with 1 mL of MeCN and 1 mL of 2MTEAA, 1 mL of RNA / Quenching Buffer was added to the incubated sample and passed through the cartridge, and then the following solutions were passed through the cartridge in the following order: 1.10%MeCN, 90%2M TEAA(pH7.0) 1mL 2. H2O 1mL 3.2% TFA 1mL x 2 4. H2O 1mL x 2 times

[0149] Finally, the oligonucleotide was eluted with 1 mL of 1 M ammonium carbonate / 30% MeCN. The oligonucleotide was dissolved in 1 mL of 0.1% NH3aq, and the yield was calculated from the absorbance at 260 nm of the diluted solution. In addition, 30 pmol of oligonucleotide was dried under reduced pressure, thoroughly mixed with 3 μl of sterilized water and 3 μl of matrix solution, and dried on a plate. The molecular weight was then measured by MALDI-TOF / MS to confirm the target product. EXAMPLES

[0150] (Evaluation of nuclease resistance by basic group at 4' position) In this example, the following double-stranded oligonucleotides were prepared. In addition to synthesizing unmodified oligonucleotides composed entirely of natural nucleotides, double-stranded oligonucleotides were synthesized in which a specific U (italicized) in the following base sequence targeted at Renilla luciferase mRNA had an aminoethyl group at the 4' position and a halogen atom or an alkyl (O-CH3) group substituted for the hydroxyl group at the 2' position. These oligonucleotides were incubated at a concentration of 5.4 μM at 37° C. in D-MEM medium containing 20% ​​bovine serum, and RNAi activity was evaluated from the fluorescence intensity ratio of Renilla luciferase and Firefly luciferase, and nuclease resistance was determined. The control was one in which siRNA was not introduced. The results are shown in FIG. 2.

[0151] [ka]

[0152] As shown in Figure 2, regardless of the chemical modification at the 2' position, these chemically modified oligonucleotides having an alkylamino group at the 4' position were found to be significantly more stable against nucleases than natural, unmodified oligonucleotides. EXAMPLES

[0153] (Evaluation of gene silencing by double-stranded oligonucleotides) In this example, the RNA interference activity was evaluated for the double-stranded oligonucleotide (siRQ238) having the sequence of RECQL1-siRNA synthesized in Example 1. That is, the RNA interference effect of this double-stranded oligonucleotide in ES-2 cells was evaluated in the presence of 5% FBS serum by the following method.

[0154] As shown in Figure 3, double-stranded oligonucleotide (siRQ238) was added at concentrations of 200nM and 250nM without DDS to monolayer culture (3000 cells / well) containing 5% calf serum of ES-2 cells that had been cultured one day before, and the cells were cultured for 48 hours. After that, the amount of RECQL1 mRNA in the ES-2 cells was measured by PCR. As a control, siRQ238 (20nM) was introduced into the cells by transfection using lipofectamine as DDS, and the amount of RECQL1 mRNA was measured by PCR in the same way. Furthermore, in both cases, the amount of RECQL1 mRNA in cells not treated with siRNA is shown as 100% (white histogram). The results are shown in Figure 3.

[0155] As shown in Figure 3, this double-stranded oligonucleotide was found to degrade approximately 60% of intracellular RECQL1 mRNA within 48 hours at a final concentration of 200 nM without DDS. It was also found that approximately 90% of RECQL1 mRNA was degraded at a final concentration of 250 nM. In terms of concentration, approximately 10 times the amount of siRNA was required compared to when conventional lipofectamine was used as DDS. However, considering that unmodified, for example, natural siRNA did not degrade RECQL1 messenger in cells at all (0%) even at a high concentration of 500 nM, obtaining an oligonucleotide with such high activity without DDS was an unexpected effect. In addition, siRQ238 can cause RNA interference at a low concentration of about 20 nM when transfected using lipofectamine, so it can be seen that as long as siRQ238 itself enters the cells, it exerts RNA interference activity equivalent to that of natural siRNA. In fact, siRQ238 overcomes the obstacle to passing through the cell membrane and entering the cell, and for example, in the double-stranded oligonucleotide prepared in Example 1, it was thought that this obstacle was reduced. EXAMPLES

[0156] (Evaluation of cell membrane permeability of double-stranded oligonucleotides) In this example, the cell membrane permeability of the double-stranded oligonucleotide synthesized in Example 3 was evaluated. That is, to determine whether siRQ238 can enter cells free of DDS and cause an RNA interference reaction in the presence of 2.5, 5, and 10% serum, a siRQ238 concentration of 250 nM was selected and the evaluation was performed under the same conditions as those shown in Example 3. The results are shown in FIG.

[0157] As shown in Figure 4, it is generally known that the cell permeability of double-stranded oligonucleotides is hindered by binding to proteins in the blood, but the double-stranded oligonucleotides synthesized in Example 3 showed the greatest RNA interference (80%) at a serum concentration of 0% when measured at various serum concentrations, and as the serum concentration was increased, the RNA interference decreased, but it was found to show RNA interference activity even in the presence of 10% serum. This suggests that siRQ238 is effective in living organisms without the use of DDS. EXAMPLES

[0158] (Evaluation of gene expression suppression activity of double-stranded oligonucleotides) In this example, we investigated whether chemically modified siRNAs, such as the double-stranded oligonucleotide siRQ238 synthesized in Example 3, can exhibit the "activity of penetrating into cells without DDS and catalyzing RNA interference" in various ovarian cancer cells other than HeLa cells and ovarian cancer ES-2 cells. Human ovarian cancers are divided into four types: serous carcinoma, clear cell carcinoma, endometrioid carcinoma, and mucinous carcinoma. The cell penetration and intracellular RNA interference effects of the double-stranded oligonucleotide siRQ238 in these various ovarian cancers were evaluated by the following method.

[0159] That is, whether the RNA interference effect can be exerted similarly to that shown in ES-2 clear cell carcinoma was evaluated by the same method as shown in Example 3. That is, siRQ238 (250nM) was added to monolayer culture of five types of cells, namely three types of human ovarian cancer clear cell (ES-2, KOC-5C, SKOV3), endometrioid carcinoma (TOV-112), and serous carcinoma (OVCAR-3), in the presence of 5% fetal bovine serum without DDS, and after 48 hours, the decrease due to degradation of RECQL1 mRNA in the cells was measured. It has been found from the research of Sanada et al. (reference) that the ovarian cancer cells used here die during culture if the RECQL1 mRNA is decreased (S. Sanada et al., RECQL1 DNA repair helicase: a potential therapeutic target and a proliferative marker against ovarian cancer: PLoS One. (2013) 9:e72820. doi: 10.1371 / journal.pone.0072820). The results are shown in Figure 5.

[0160] As shown in Figure 5, siRQ238 was found to have an inhibitory effect of 40% to 60% in all ovarian cancer cells examined, indicating that chemically modified RECQL1-siRNA, such as siRQ238, can suppress the proliferation of most ovarian cancer cells. EXAMPLES

[0161] (In vitro evaluation of ovarian cancer cell killing activity of double-stranded oligonucleotides) In this example, the killing activity of the double-stranded oligonucleotide synthesized in Example 3 on ovarian cancer cells was confirmed as to whether or not the double-stranded oligonucleotide penetrated into the cells, induced mitotic death during cell division, caused apoptosis-like suicide action, and caused a killing action when administered to ovarian cancer cells under DDS-free conditions. That is, in Examples 3 to 5, it was measured that siRQ238 entered cells free of DDS, catalyzed an RNA interference reaction, and degraded and reduced RECQL1 mRNA, but an experiment was conducted to confirm whether siRQ238 actually killed cancer cells. Specifically, it was evaluated by the following method.

[0162] Different concentrations of siRQ238 were added to monolayer culture of ovarian cancer cell ES-2 in the presence of 5% serum without DDS, and after 96 hours, the viability of the cancer cells was measured by WST assay. This experiment was also an exploratory experiment to find out how much siRQ238 should be given to cancer-bearing mice in the next animal experiment. The experimental conditions were basically the same as in Example 3 unless otherwise stated. The results for ES-2 clear cell carcinoma are shown in Figure 6.

[0163] As shown in Figure 6, it was found that the double-stranded oligonucleotides caused cell death in a concentration-dependent manner 96 hours after contact with cancer cells. Futami et al. have reported that RECQL1-siRNA and WRN-siRNA cause mitotic death during cell division (M phase) in many cancer cells with incomplete checkpoint function due to mRNA silence (References 3-8), and the action of siRQ238 is not inconsistent with these reports. In other words, (i) RNA interference suppresses the synthesis of RECQL1 helicase protein, (ii) this inhibits DNA repair in cancer cells during proliferation, (iii) the replicated genome contains a lot of incomplete DNA, (iv) it is captured by the strict DNA checkpoint (QOL check) that the cells have, and (v) it undergoes apoptosis-like suicide death (References 3, 4).

[0164] In addition, the inventors observed the appearance of ES-2 cells treated with siRQ238 under a microscope in a time-lapse experiment using ES-2 cells labeled with GFP (green fluorescent protein) to observe the scene of double-stranded oligonucleotides causing death of ES-2 cells without DDS. It was found that the M phase of the cell cycle of ES-2 cells became abnormally long, and the cells floated up and died by apoptosis (Data not shown). Therefore, it can be concluded that siRQ238 enters cells without DDS, causes genomic instability in the cells, and leads to apoptotic death. EXAMPLES

[0165] (Evaluation of anticancer effects of intraperitoneal administration of double-stranded oligonucleotides) In this example, the double-stranded oligonucleotide (siRQ238) synthesized in Example 3 was used in mouse animal experiments to examine three effects of intraperitoneal administration: anticancer effect, life-prolonging effect, and ascites accumulation inhibition effect. That is, human ovarian cancer cells ES-2 were transplanted into the abdominal cavity of female nude mice to create intraperitoneal seeding model mice resembling terminal cancer symptoms, and siRQ238 was administered intraperitoneally once every two days to examine the morphology of the cancer-bearing mice, the life-prolonging effect on the cancer-bearing mice, as well as the cancer growth inhibition effect and ascites accumulation inhibition effect. The procedure is shown below, and the results are shown in Figures 7 to 10.

[0166] As shown in Figures 7-8, cancer cells were transplanted (human ovarian cancer cells ES-2: 1x10 6The cells / mouse were transplanted into immunodeficient nude mice by intraperitoneal injection), and the morphology of the mice was observed with a fluorescent camera 10 days after transplantation (Figure 7) and 17 days after transplantation (Figure 8). As shown on the left side of Figure 7, in the mice that received intraperitoneal administration of phosphate buffer as a control, cancer cells were allowed to grow, and the progression of cancer cells accompanied by GFP green fluorescence was observed in the abdominal cavity with a fluorescent camera. As shown on the left side of Figure 8, morphologically, the abdomen of the tumor-bearing mice was greatly swollen on the 17th day after transplantation, and findings suggesting ascites were observed. On the other hand, as shown on the right side of Figure 7, in the mice that received double-stranded oligonucleotide siRQ238 dissolved in phosphate buffer (intraperitoneal injection of 144 micrograms / mouse / mouse), no GFP green fluorescence was observed in the abdomen (intraperitoneal cavity) on the 10th day after transplantation, and as shown on the right side of Figure 8, even on the 17th day, the mice had a healthy appearance similar to that before the cancer cell transplantation, and significant morphological changes were observed depending on whether or not they received siRQ238. These results, both by GFP fluorescence observation and morphological observation, suggested that administration of siRQ238 had a significant inhibitory effect on cancer cell progression.

[0167] Furthermore, as shown in Figure 9, the double-stranded oligonucleotide had a remarkable effect on extending the lifespan of the cancer-bearing mice. That is, while two out of three control mice died by the 18th day, the siRQ238-administered mice continued to survive thereafter, and after siRQ238 administration was discontinued from the 24th day onwards, no mice died even up to the 31st day.

[0168] Furthermore, as shown in Figure 10, the cancer growth inhibitory effect was examined by laparotomy of control mice and mice administered siRQ238. In the control mice, ES-2 tumors had spread throughout the abdominal cavity (around the pancreas and bile duct, around the mesentery, and around the ovaries and retroperitoneum) by the 17th day, forming numerous solid tumor masses (upper mouse in Figure 10). On the other hand, in mice administered with double-stranded oligonucleotide siRQ238 until the 23rd day, and then artificially killed (also called sacrificed) on the 31st day, and laparotomy was performed, no macroscopic tumor masses were detected even in detailed observations of various parts of the abdominal cavity (lower mouse in Figure 10). During this experiment, there was no change in the behavior or weight of the mice administered with double-stranded oligonucleotide, and no serious side effects were observed.

[0169] In general, the proliferation of cancer cells in the peritoneal cavity impairs the ascites management function of the peritoneal cavity and promotes ascites accumulation, which is a phenomenon seen in many solid cancers that spread into the peritoneal cavity. These are seen in solid cancers that develop in organs present in the peritoneum, such as ovarian cancer, pancreatic cancer, gastric cancer, colon cancer, and liver cancer. The inventors evaluated the effect of siRQ238 in preventing ascites accumulation by measuring the abdominal circumference of control mice (PBS) and siRQ238-administered mice (denoted as siQL#238 in the figure) before the start of administration of the double-stranded oligonucleotide siRQ238 (Pre-treat) and on the 17th day after cancer cell transplantation. The results are shown in Figure 11.

[0170] As shown in Figure 11, it was revealed that the double-stranded oligonucleotide has a strong effect of preventing ascites accumulation. That is, in mice administered with double-stranded oligonucleotide (siRQ238) that silences RECQL1 messenger RNA, the abdominal circumference was about 6.5 cm before and after the administration period, whereas the abdominal circumference of the control mice gradually increased, reaching 7.5 cm on the 17th day. For the control mice, the ascites was removed with a syringe just before laparotomy on the 18th day, and the amount of ascites was found to be large, more than 3.4 ml, which is approximately 1.7 times the circulating blood volume (2 ml). On the other hand, no ascites was accumulated at all in the siRQ238-administered mice, and it could not be drawn out with a syringe.

[0171] From the above considerations, the inventors have found that the structural portion of the present oligonucleotide confers nuclease resistance to the double-stranded oligonucleotide, and furthermore, confers the ability to penetrate into cells without DDS, and silences the expression of the RECQL1 gene by RNA interference. Furthermore, it has been revealed that the present oligonucleotide has excellent abilities to suppress the proliferation of human ovarian cancer cells and prevent ascites accumulation. Such siRNA molecules have not been developed or reported so far.

[0172] References 1. Fire A. et al. Nature (1998) 391, 806-811 2. Hartmut Jahns1, Martina Roos1, Jochen Imig1, Fabienne Baumann1, Yuluan Wang1, Ryan Gilmour2 & Jonathan Hall1 (2014) Stereochemical bias introduced during RNA synthesis modulates the activity of phosphorothioate siRNAs Nature communication (DOI: 10.1038 / ncomms7317 3. K. Futami, E. Kumagai, H. Makino, H. Goto, M. Takagi, A. Shimamoto and Y. Furuichi: Induction of mitotic cell death in cancer cells by small interference RNA suppressing the expression of RecQL1 helicase: Cancer Sci. 99, 71-80 (2008). 4. Y. Furuichi: Induction of Cancer Cell-specific Mitotic Catastrophe by Silencing the DNA Repair RecQ Helicases and Application of the Findings for Anticancer siRNA Drugs: DNA REPAIR AND HUMAN CANCERS in the 40 thInternational Symposium of the Princess Takamatsu Cancer Research Fund. pp.154-161 (2009).5.K. Futami, S. Ogasawara, H. Goto, H. Yano and Y. Furuichi: RecQL1 DNA repair helicase: A potential tumor marker and therapeutic target against hepato cellular carcinoma: International Journal of Molecular Medicine 25, 537-545 (2010).6.A. Arai, T. K. Chano, K. Futami, Y. Furuichi, K. Ikebuchi, T. Inui, H.Tameno, Y. Ochi, T. Shimada, Y. Hisa and H. Okabe: RECQL1 and WRN proteins are potential therapeutic targets in head and neck squamous cell carcinoma: Cancer Res. 71, 4598-4607 (2011).7.S. Sanada, K. Futami, A. Terada, K. Yonemoto, S. Ogasawara, J.Akiba, M. Yasumoto, A. Sumi, K. Ushijima, T. Kamura, Y. Furuichi and H. Yano: RECQL1 DNA repair helicase: a potential therapeutic target and a proliferative marker against ovarian cancer: PLoS One. (2013) 9:e72820. doi: 10.1371 / journal.pone.0072820.8.K. Futami and Y.Furuichi: RECQL1 and WRN DNA repair helicases: Potential therapeutic targets and proliferative markers against cancers. Fronties in Genetics. (2015) doi:10.3389 / fgene,8.Marc Nothisen, JeBagilet, Jean-Paul Behr, Jean-Serge Remy,* and Mitsuharu Kotera*. Structure Tuning of Cationic OligospermineConjugates for Carrier-Free Gene Silencing. Laboratoire V-SAT, CAMB, UMR 7199, Universitede Strasbourg and CNRS, Facultede Pharmacie, F-67401 Illkirch, France9.Synthesis of 4-C-aminoalkyl-2-O-methyl modiRNA and their biological properties K. Koizumi, Y. Maeda,f, T. Kano, H. Yoshida, T. Sakamoto, K. Yamagishi, Y. Ueno. Bioorganic & Medicinal Chemistry (2018) 26, 3521-3534.10.Synthesis and properties of 4-C-aminoalkyl-2-RNA oligomers T. Kano, Y. Katsuragi, Y. Maeda, Y. Ueno. Bioorganic & Medicinal Chemistry (2018) 26, 4574-4582.11.Enhanced Intercellular Delivery of cRGDConjugates by an Additional Oligospermine ModiK. Nakamoto, Y. Akao, Y. Furuichi and Y. Ueno. ACS Omega (2018) 3, 8226-823312.Synthesis and Biophysical Characterization of RNAs Containing (R)2 and (S)5C Aminopropyl-2O Methyluridines R.Kajino,Y. Maeda,H. Yoshida, K. Yamagishi and Y. Ueno JOC (in press as of Feb. 2019) DOI:10.1021 / acs.joc.8b03277.

【Dragon-Currnet.

[0173] Residues 1~4:siRNA

Claims

1. 1. An RNA interference agent comprising: The double-stranded oligonucleotide may have the following (a) to (c) in both the guide strand and the passenger strand: (a) Modification of the 4' carbon atom with a basic group via an alkylene group (b) Alkyloxy or alkenyloxy modification to the 2' carbon atom (c) Modification of the 2' carbon atom with a halogen atom The modified nucleotide has at least one modification selected from the group consisting of: an oligospermine region having 10 or more spermines in tandem only at the 5' end of the passenger strand; The passenger strand has the modification (a) only within 5 nucleotides from the 3' end and within 5 nucleotides from the 5' end of the passenger strand, The passenger strand and the guide strand each have an internucleoside bond having a phosphothiol structure at their 3'-ends, a first modified nucleotide having the modifications (a) and (c) within 5 nucleotides from the 3' end of the passenger strand, and a second modified nucleotide having the modification (b) on each side of the first modified nucleotide; a first modified nucleotide having the modifications (a) and (c) within 5 nucleotides from the 5' end of the passenger strand, and a second modified nucleotide having the modification (b) on each side of the first modified nucleotide; An RNA interference agent comprising a third modified nucleotide having the modification (c) within 8 nucleotides from the 5' end of the guide strand.

2. The RNA interference agent of claim 1, further comprising a second modified nucleotide having the modification (b) within 5 nucleotides from the 3' end and within 5 nucleotides from the 5' end of the guide strand.

3. The RNA interference agent of claim 1 or 2, further comprising the second modified nucleotide in a region excluding within 8 nucleotides from the 5' end of the passenger strand and within 10 nucleotides from the 3' end of the passenger strand.

4. The RNA interference agent according to any one of claims 1 to 3, wherein the oligospermine region has 15 to 30 spermines.

5. The RNA interference agent according to any one of claims 1 to 4, A therapeutic agent, wherein the guide strand has a region that hybridizes to the mRNA of the RECQL1 gene or the WRN gene.

6. The therapeutic agent according to claim 5 , wherein the guide strand comprises a region that hybridizes to the mRNA of the RECQL1 gene.

7. The therapeutic agent according to claim 6, which is a therapeutic agent for any cancer selected from the group consisting of ovarian cancer, pancreatic cancer, gastric cancer, colon cancer and liver cancer.

8. The RNA interference agent according to any one of claims 1 to 4, A therapeutic agent for ascites caused by cancer, wherein the guide strand has a region that hybridizes to the mRNA of the RECQL1 gene or the WRN gene.

Images