JP2019010035A - Rna introduction agent and use thereof - Google Patents

Abstract

To provide a RNA introduction agent capable of contributing to delivery property and cell membrane permeability of a RNA medicament or the like.SOLUTION: The RNA introduction agent comprises two or more cellular adhesion motif units and one or more spacer units, in which the one or more spacer units separate the two or more cellular adhesion motif units.SELECTED DRAWING: None

Description

  The present specification relates to an RNA introduction reagent and use thereof.

Many gene-related diseases are known, including cancer, which are caused or related to gene mutations and gene expression abnormalities. Various cells including cancer cells express cell adhesion factors on their surfaces. For example, it is known that integrin α _v β ₃ is strongly expressed as a cell adhesion factor on the surface of cancer cells.

  RNA drugs such as siRNA are drugs that directly act on mRNA. After penetrating into cells, they have high specificity for the target molecule, work in the cytoplasm, have little effect on nuclear genomic DNA, and have a clear mechanism of action. It is. For this reason, RNA medicine is expected to be applied to gene-related diseases.

  On the other hand, RNA drugs have high target selectivity, but are difficult to selectively transport to the target tissue and permeabilize the cell membrane and are easily degraded. Therefore, RNA using Lipid Nano Particles (LNP) such as liposomes is used. Developments have been made so far to prepare complexes, and to make RNA drugs have cell selectivity, delivery function, cell membrane permeation function and protection function, and modification to the ribose ring to enhance effectiveness. (Patent Documents 1 to 4).

HELVATICA CHIMICA ACTA Vol. 83 (2000) 128-151 The Journal of Organic Chemistry 2012, 77, 3233-3245 Bioorganic & Chemistry letters (1999) 2667-2672 The Journal of Organic Chemistry 2013, 78, 9956-9962

  However, despite these attempts, there is a need for further improvement in the effectiveness of RNA drugs. In particular, with regard to the cell selectivity, delivery property and cell membrane permeability of RNA pharmaceuticals, in the delivery using LNP, it has been a problem to make the particle size of liposomes uniform and miniaturized, but still sufficient cell selectivity and delivery property. There was a problem that cell membrane permeability was not achieved.

  The present specification discloses an RNA introduction reagent that can contribute to the deliverability and cell membrane permeability of an RNA drug and the like and use thereof.

  The present inventors paid attention to the use of cell adhesion motifs for improving RNA delivery and cell membrane permeability. As a result of various studies by the present inventors, when a plurality of such cell adhesion motifs are used, RNA is permeated through the cell membrane by modifying the RNA with a compound having a plurality of the motifs separated from each other by a plurality of spacers. The knowledge that the property increases is obtained. According to this specification, the following means are provided based on this knowledge.

[1] An RNA introduction reagent,
Two or more cellular adhesion motif units;
One or more spacer units;
With
The RNA introduction reagent, wherein the one or more spacer units separate the two or more cellular adhesion motif units from each other.
[2] The reagent according to [1], wherein the cellular adhesion motif unit contains a peptide.
[3] The reagent according to [1] or [2], wherein the cell adhesion motif unit includes an RGD peptide.
[4] The reagent according to any one of [1] to [3], wherein the spacer unit includes a polyalkyleneoxy group.
[5] The reagent according to any one of [1] to [4], wherein the spacer unit separates the two or more cell adhesion motif units by at least 2 nm.
[6] A peptide derivative,
Two or more peptide units;
One or more non-peptidic units;
With
The peptide unit has any two or more amino acid residues,
The derivative, wherein the one or more non-peptide units separate the two or more peptide units from each other.
[7] The derivative according to [5], wherein the peptide unit includes an RGD motif, and the non-peptide unit includes a polyoxyalkylene group.
[8] an oligoribonucleotide derivative,
Oligoribonucleotides;
A cell adhesion region comprising two or more cell adhesion motif units and one or more spacer units, wherein the one or more spacer units separate the two or more cell adhesion motif units from each other; A derivative comprising.
[9] The derivative according to [8], wherein the oligoribonucleotide includes a nucleoside derivative represented by the following formula (1).
(In Formula (1), R ¹ represents a hydroxyl group, a hydroxyl group in which a hydrogen atom is substituted with an alkyl group or an alkenyl group, or a protected group, and in Formula (2), X represents a halogen atom. In 1) and formula (2), R ² and R ⁴ may be the same or different from each other, and are a hydrogen atom, a hydroxyl protecting group, a phosphate group, a protected phosphate group, or —P (═O). _n R ⁵ R ⁶ (n represents 0 or 1, and R ⁵ and R ⁶ may be the same or different from each other, and represent a hydrogen atom, a hydroxyl group, a protected hydroxyl group, a mercapto group, a protected mercapto group, a lower group, An alkoxy group, a cyano lower alkoxy group, an amino group, or a substituted amino group, provided that when n is 1, both R ⁵ and R ⁶ are not hydrogen atoms. R ³ is an NHR ⁷ having a linking group (R ⁷ is a hydrogen atom) Represents an alkyl group, an alkenyl group or an amino group protecting group.), Represents an azido group, an amidino group or a guanidino group, and B represents a purin-9-yl group, a 2-oxo-pyrimidin-1-yl group or a substituted group. It represents either a purin-9-yl group or a substituted 2-oxo-pyrimidin-1-yl group.)

It is a figure which shows the component of the reagent for RNA introduction | transduction. It is a figure which shows the outline | summary of the reagent for RNA introduction | transduction. It is a figure which shows the scheme which introduce | transduces a cell adhesive motif unit with respect to the monomer for cell adhesive motif unit introduction | transduction. The last compound in the scheme shows a state in which two cell adhesion motif units and three spacer units are introduced. It is a figure which shows the evaluation result of the cell membrane permeability | transmittance by a conjugate. It is a figure which shows the evaluation result of the gene expression suppression by a conjugate.

  The disclosure of the present specification relates to a reagent for introducing RNA into cells, which is suitable for RNA medicine and the like, and use thereof. As illustrated in FIGS. 1 and 2, the RNA introduction reagent disclosed herein (hereinafter also referred to as this reagent) has a structure in which two or more cell adhesion motif units are separated by one or more spacer units. Can have. Although it is an inference and does not necessarily constrain the present disclosure, since it has such a spaced structure, cells such as integrins that are thought to be present in a large number and fluidly on the cell surface of two or more cell adhesion motif units This facilitates interaction with the adhesion molecule, and as a result, the oligoribonucleotide that forms a complex with the reagent easily interacts with the cell surface. As a result, the efficiency of introduction of the oligoribonucleotide into the cell is increased. Is considered to increase.

  The reagent can have a linear structure in which two or more cell adhesion motif units are connected in series with one or more spacer units interposed. According to the present inventors, the design of the separation structure is achieved by having the linear structure described above rather than the branched structure having cell adhesive motif units in parallel at the tip of the branched skeleton. It has been found that the separation structure can be designed easily and with a high degree of freedom, and the probability of interacting with cell adhesion molecules on the cell surface can be ensured or improved. In addition, although it is preferable that this reagent has at least a linear structure, you may have a branched structure provided with such a linear structure in multiple branches or in parallel.

  An oligoribonucleotide is covalently bonded to either end of this reagent to form a complex, or such a linear structure and oligoribonucleotide are complexed by an interaction other than covalent bond to form a complex. Thus, the efficiency of introducing oligoribonucleotides into cells can be increased.

  Hereinafter, various embodiments disclosed in this specification will be described in detail.

(RNA introduction reagent)
The reagent can comprise two or more cellular adhesion motif units and one or more spacer units.

(Cell adhesion motif unit)
The cell adhesion motif unit is a motif having a molecule that can interact and bind to a cell adhesion molecule expressed on the cell surface, and contributes to the interaction with the cell adhesion molecule (hereinafter referred to as a cell). Also referred to as an adhesive motif).

  The cell adhesion motif can be derived, for example, from a ligand that binds to a known cell adhesion molecule. For example, the cell adhesion molecule is generally a protein, and may be an extracellular protein or a cell membrane protein. The cell adhesion molecule may be responsible for cell-cell adhesion or may be responsible for cell-extracellular matrix adhesion.

  Various cell adhesion molecules are known. Examples of cell adhesion molecules responsible for cell-cell adhesion in animal cells include cadherin, immunoglobulin superfamily, and nectin. In addition, examples of cell adhesion molecules (cell side: receptor, protein) responsible for cell-extracellular matrix adhesion include integrins, sarcoglycans, and β-dystroglycans. Protein cell adhesion molecules (extracellular matrix side: ligand) Examples include fibronectin, laminin, vitronectin, collagen, tenascin, fibrinogen, osteopontin, netrin, thrombospondin, entactin, α-dystroglycan and the like as cell adhesion molecules (extracellular matrix side: ligand) that are proteoglycans May include agrin, aggrecan, syndecan, neurocan, versican, brevican and the like.

  Moreover, animals, plants, and insects each use specific proteins, complex polysaccharides, or glycoproteins as intercellular inclusions for cell adhesion. By binding functional peptides and oligosaccharides in these structures to RNA drugs, they can be applied as animal drugs, agricultural chemicals, and insecticides.

  Among these, the cell adhesion molecule from which the cell adhesion motif used in the present specification is derived is not particularly limited, but many or specifically expressed cell adhesion molecules in cells into which RNA drugs are to be introduced. It can be a cell adhesion molecule that is an extracellular matrix ligand for. The cell adhesion functional molecule from which the cell adhesion motif used in the present specification may be a protein, peptide, oligosaccharide, or a glycoprotein such as proteoglycan. Furthermore, lipids such as vitamins, lipids, and cholesterols known to have cell adhesion may be used.

  Suitable cell adhesion motifs include, for example, fibronectin, laminin, and ICAM (Intercellular Adhesion Molecule), which are extracellular matrix ligands responsible for cell adhesion using the integrin, cadherin, and immunoglobulin superfamily (IgSF) as receptors. This is because some integrins and laminins are expressed in large numbers on the surface of cells such as cancer cells, and are useful as cells to which RNA drugs targeting cancer cells are to be applied. In addition, certain integrins and laminins are expressed in large numbers or specifically on the surface of nerve cells and the like, and are useful as cells to which RNA drugs targeting nerve cells should be applied. In addition, the cell adhesion factor from which the cell adhesion motif is derived can be appropriately selected according to the expression state and type of the cell adhesion factor on the cell surface targeted by RNA.

  The cell adhesion motif is preferably a peptide having a small molecular weight because immunological antigenicity can be reduced. Examples of such cell adhesion motifs include RGD (alphabetic characters indicate single letter amino acids; hereinafter the same) sequences in fibronectin, REDV sequences, LDV sequences, NGR sequences, PRAQ sequences, KLAPPT sequences, AGEGIP Examples thereof include a sequence, a KNEEDD sequence, a PHSRN sequence, an EDGIHEL sequence, a PRARI sequence, and an IDAPS sequence. Moreover, the YIGSR arrangement | sequence etc. in laminin are mentioned. In addition, the cell adhesion motif can be determined as appropriate by synthesizing and screening multiple peptide fragments based on the amino acid sequence of the extracellular matrix ligand that binds to the membrane protein receptor expressed in the target cell. Can do. Such screening is well known to those skilled in the art.

  Further, for example, the cell adhesion motif may be cyclized so that the motif is exposed to the outside of the peptide. For example, as for the RGD motif, a peptide cyclized via F (phenylalanine), C (cysteine) or the like is commercially available.

  The number of amino acid residues contained in the cell adhesion motif is not particularly limited, but is generally about 3 to 20, and the cell adhesion based on the amino acid sequence is completely compared to the above amino acid sequence. An amino acid can be added as long as it is not impaired.

  One cell adhesion motif unit can contain one or more of these cell adhesion motifs, preferably about 1 to 5, and for example 1 to 4, and For example, the number is 1 to 3, and the number is, for example, 1 or 2, and the number is 1, for example.

  The reagent can comprise two or more cell adhesion motif units. In addition, for example, the reagent may include three or more cell adhesion motif units, or may include four or more cell adhesion motif units. The upper limit of the cell adhesion motif unit is not particularly limited, but can be, for example, 10 or less.

  Two or more cell adhesion motif units provided in the present reagent may be the same or different from each other, but the cell adhesion motif units adjacent to each other via the spacer unit may have the same cell adhesion. It is preferable that a sex motif is included. This is because the distance between the same cell adhesion motifs in adjacent cell adhesion motif units is generally defined by the spacer unit.

  As shown in FIG. 2, the cell adhesion motif unit can be used in, for example, a solid-phase synthesis method, for example, a peptide linker or non-peptide region with respect to a skeleton such as DNA, RNA, GNA, PNA, etc. Can be used as a side chain for the GNA skeleton.

(Spacer unit)
The reagent can comprise one or more spacer units so as to separate two or more cell adhesion motif units. That is, the spacer unit can interpose between two cell adhesion motif units and can separate the cell adhesion motifs of the motif unit based on the length of the spacer unit.

  The length of the spacer unit is appropriately determined according to the cell adhesion motif unit or the distance at which the cell adhesion motif is to be separated in this reagent. For example, when the distance information between cell membrane receptors expressed in the target cell can be acquired, the length of the spacer unit can be determined with reference to the distance. Also, for example, the length of the spacer unit can be determined by screening a suitable spacer unit by assigning a spacer unit of various lengths to a cell adhesion motif that is known to adhere to a target receptor. Can also be determined.

  The distance between the cell adhesion motif units separated by the spacer unit is not particularly limited, but can be separated by at least 2 nm or more, for example. For example, it can be 3 nm or more, can be 4 nm or more, for example, can be 5 nm or more. The distance between cell adhesive motif units separated by the spacer unit, or the length of the spacer unit can be obtained by those skilled in the art based on the molecular structure constituting the spacer unit.

  The structure and substance constituting the spacer unit are not particularly limited, but other than general linkers, linkers that link various known proteins and proteins, linkers that link proteins and nucleotides (nucleosides), nucleosides, A linker or the like that links the nucleoside can be appropriately used.

The spacer unit is not particularly limited, but is preferably non-peptidic. This is because, since the cell adhesion receptor is a protein, unintentional adhesion may occur when the spacer unit contains a peptide. In order to prevent degradation by proteolytic enzymes such as protease, it is desirable that it is non-peptidic.
In addition, the spacer is preferably made of a material that does not indiscriminately bind to substances present on the cell surface, such as proteins and fat, such as polyethylene glycol chains.

  The spacer unit is not particularly limited, but it is also preferable to have a linking group that can freely rotate. For example, the spacer unit preferably has a linear structure containing a single bond. Examples of the single bond include a carbon-carbon single bond, a carbon-oxygen single bond, a carbon-nitrogen single bond, an SS single bond, and the like. The spacer unit is preferably mainly composed of such a single bond. The spacer unit may partially contain an aromatic ring or cycloalkane as long as it contains a single bond.

  The spacer unit preferably includes an optionally substituted alkylene chain or polyoxyalkylene chain. This is because such a chain-like connecting structure is easy to design the distance between the cell adhesion motif units and can rotate as a whole.

Examples of such a spacer unit include a spacer unit represented by the following formula (1). In addition, in the following formula | equation (1) and (2), as an example, it shows as an aspect with which the spacer unit is connected through the phosphodiester bond.
5′-O—C _m H _2m —O-3 ′ Formula (1)
(In the formula, 5 ′ represents an oxygen atom of a phosphodiester bond on the 5 ′ side, 3 ′ represents a phosphate atom of a phosphodiester bond on the 3 ′ side, and m represents an integer of 2 or more. )

  In the formula (1), m is preferably 2 or more and 40 or less, more preferably 3 or more and 20 or less. Typical examples of the substituent of H in formula (1) include an alkyl group, an alkoxy group, and a hydroxyl group. It is preferable that carbon number of an alkyl group and an alkoxy group is 1-8, More preferably, it is 1-4. Moreover, when it has two or more substituents, the substituents may be the same or different. Furthermore, it is also preferable that it does not have a substituent.

Moreover, as another spacer unit, the unit represented by the following formula | equation (2) is mentioned.
_{5 '- (OC n H 2n} ) l -3' formula (2)
(In the formula, 5 ′ represents an oxygen atom of a phosphodiester bond on the 5 ′ side, 3 ′ represents a phosphate atom of a phosphodiester bond on the 3 ′ side, and n represents an integer of 2 or more and 4 or less. And l represents an integer of 2 or more.)

  In the formula (2), (n + 1) × l is preferably 2 or more and 40 or less, and more preferably 3 or more and 20 or less. The same aspect as the substituent in Formula (1) is applied to the substituent of H in Formula (2).

  The spacer unit can be provided between the cell adhesive motif units as many as necessary to separate two or more cell adhesive motif units. Therefore, necessary spacer units can be provided according to the number of cell adhesion motif units provided in the present reagent. For example, when the present reagent comprises three cell adhesion motif units, at least two spacer units are required to separate them, and similarly, when four cell adhesion motif units are provided. In order to separate these, at least three are required, and when five cell adhesion motif units are provided, at least four are necessary to separate them.

  As shown in FIG. 2, the spacer unit can be provided as a skeleton rather than as a side chain, for example, using a solid phase synthesis method such as DNA or RNA.

  As shown in FIGS. 1 and 2, in this reagent, one or more spacer units separate the two or more cellular adhesion motif units from each other. For example, the present reagent can have a linear structure in which two cell adhesion motif units are connected in series with a structural unit in which one spacer unit is interposed.

  Although this reagent is not specifically limited, For example, it is preferable to provide 3 or more cell adhesion motif units, for example, it is preferable to provide 4 or more of the same units, and 5 or more of the same units. For example, it is preferable to provide 6 or more of the same units. All of these are preferably separated by a spacer unit. By providing a large number of cell-adhesive motif units separated by spacer units, the probability of interaction with the receptor on the cell surface can be improved, and uptake into cells is improved.

  The present reagent may have only one such linear structure portion, or may have two or more. For example, such a linear structure moiety may be linked to each linker using a branched linker. Linker branches can be linked by a crosslinking reaction or the like to a portion where the cell adhesiveness other than the terminal or non-terminal of the linear structure portion of the reagent is not impaired. As such a branched linker compound, various compounds are well known to those skilled in the art. Examples of such branched linkers include various known bifunctional or trifunctional or higher functional crosslinking agents.

  In order to synthesize this reagent having such a structure, it can be synthesized by various known methods without any particular limitation. For example, the degree of freedom in the selection and linkage between the cell adhesion motif unit and the spacer unit can be used. Considering the degree of freedom of form, a solid phase synthesis method of oligoribonucleotides can be used. In addition, although the manufacturing method of this reagent is explained in full detail behind, the method with easy industrial application in pharmaceutical manufacture etc. is desirable.

(Manufacture of this reagent)
This reagent can be appropriately produced according to the type and sequence of the cell adhesion motif unit and spacer unit. Although not particularly limited, the present reagent can be synthesized using, for example, a solid phase synthesis method of DNA or RNA. Specifically, the present reagent can be produced in any combination by preparing a cell adhesion motif unit, a spacer unit, etc. as a derivative for solid phase synthesis, for example, a phosphoramidite derivative.

  A general oligoribonucleotide solid phase synthesis method is generally a ribonucleotide unit on the 3 ′ end side of an oligoribonucleotide, in which a ribonucleotide unit in which a base, a phosphate group, a hydroxyl group and the like are appropriately protected includes a linker or the like. A solid phase carrier such as beads (solid phase carrier preparation step (A)), then deprotecting the 5′-terminal hydroxyl group (deprotection step (B)), and then appropriately protecting the desired ribonucleic acid A nucleotide amidite derivative is supplied, and a desired ribonucleotide is coupled to the hydroxyl group at the 5 ′ end (coupling step (C)). After step (A), (B) and (C) are repeated, and finally the desired oligoribonucleotide can be obtained by deprotecting the base moiety and separating it from the solid phase carrier. This reagent utilizes steps (A), (B) and (C) of this solid phase synthesis method, and instead of ribonucleotides, various known nucleic acid analogs (glycol nucleic acid (GNA), lock nucleic acid (LNA) , Etc.) can be obtained by linking a cell-adhesive motif unit or spacer unit to other similar skeletons in an arbitrary sequence.

  In addition, in the amidite method using a phosphoramidite or the like applied to the production of this reagent, for example, one of the oxygen atoms of the phosphoric acid diester moiety is a sulfur atom, a lower alkyl group such as a methyl group, or a borano group In addition to substitution, known modification modes such as morpholino phosphorodiamidate modification, N3′-P5 phosphoramidate type modification in which the oxygen atom at the 3 ′ position is replaced with a nitrogen atom can be applied.

  Hereinafter, a synthesis example of this reagent introduced into the 3 'terminal side of the oligoribonucleotide will be exemplified. For example, when obtaining this reagent having the sequence of cell adhesive motif unit / spacer unit / cell adhesive motif unit / spacer unit from the 3 ′ end side, the following three types of monomers that are precursors of each unit: Prepare.

(Monomer for cell adhesion motif unit introduction)
First, a phosphoramidite derivative monomer having a trifluoroacetyl group at the terminal is prepared by the method shown in Scheme 1 below. This monomer can introduce a cell adhesion motif unit through a covalent bond through a terminal trifluoroacetyl group. As shown in the following scheme, a trifluoroacetyl group is introduced into the amino group of aminohexanoic acid [2] (compound [3]), and then a dimethoxytrityl group (DMTr) is used to protect the hydroxyl group of the carboxyl group. Group) is introduced (compound [4]), and 2-cyanoethyl N, N-diisopropylaminochlorophosphoramidite is added to obtain compound [5] as a monomer for introducing a cell adhesion motif unit.

  As will be described later, a maleimide group is introduced into the trifluoroacetyl group by a bifunctional cross-linking agent, and a cell adhesion motif unit is introduced through the maleimide group, but if necessary, as described above The cell-adhesive motif unit can be separated to some extent from the GNA that is the skeleton part in the solid-phase synthesis method.

(Solid phase carrier for cell adhesion motif unit introduction)
Next, in the scheme 2 shown below, a solid phase carrier for introducing a cell adhesion motif unit at the 3 ′ end of the oligoribonucleotide is obtained. The compound [4] obtained in scheme 1, succinic anhydride and N, N-dimethyl-4-aminopyridine (DMAP) are dissolved in pyridine, stirred at room temperature, extracted with ethyl acetate and water, and the organic layer is extracted. After dehydration, a succinyl compound was finally obtained, and [1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC · HCl) and CPG resin were added to the succinyl compound under an inert atmosphere. In addition, a solid phase for introducing a cell-adhesive motif unit for introducing a cell-adhesive motif unit at the 3 ′ end, comprising a marine group protected by a DMTr group, a trifluoroacetyl group, and CPG A carrier can be obtained.

(Monomer for introducing spacer unit)
Further, a phosphoramidite derivative monomer for introducing a spacer unit is prepared in the following scheme 3. This monomer can already contain spacer units. In scheme 3 shown below, a hexaethylene glycol chain is used as a spacer unit. Under an inert atmosphere, hexaethylene glycolic acid is dissolved in pyridine, DMTrCl is added, and the mixture is stirred at room temperature, and the organic layer extracted with ethyl acetate and water is dehydrated and concentrated under reduced pressure to obtain spacer units and DMTr groups. A monomer for introducing a spacer unit comprising a hydroxyl group protected with a phosphoramidite group can be obtained.

  As shown in FIG. 3, for example, the cell adhesion motif unit has a maleimide group introduced via a trifluoroacetyl group or the like, and the maleimide group and a thiol group imparted to the cell adhesion motif unit. The reaction is caused to occur and introduced into the monomer or solid phase carrier. Such cell-adhesive motif units may be introduced in advance to the monomer or solid phase carrier before the coupling step, or all monomers may be introduced at once after coupling. When introducing the same cell adhesion motif unit, it is advantageous to introduce them in a batch by post-modification.

  By using these two types of monomers and one type of solid phase carrier, the precursor polymer of the present reagent and the present reagent can be obtained.

  Hereinafter, synthesis examples of the present reagent having cell adhesion motif units by post-modification will be described. First, the DMTr group of the solid phase carrier obtained in Scheme 2 was removed and the 5′-OH group was activated, and then the phosphoramidite group of the monomer for introducing a spacer unit obtained in Scheme 3 was coupled, The monomer is linked to the 5′-OH group of the solid support.

  The remaining 5'-OH group is then capped and oxidized from trivalent phosphorus to form a phosphodiester bond with pentavalent phosphorus.

  Subsequently, the DMTr group of the coupled spacer unit introduction monomer is removed, and the cell adhesion motif unit introduction monomer synthesized in Scheme 1 is coupled. Furthermore, after coupling the spacer unit introduction monomer synthesized in Scheme 3 to remove the DMTr group of the introduced monomer, it was oxidized, and after removing the DMTr group, the cell adhesion motif unit was removed from the solid phase carrier. Monomer for introduction / monomer for introduction of spacer unit / cell adhesion motif unit introduction monomer / spacer unit is polymerized polymer, and the precursor of this reagent in which the cell adhesion motif unit is not introduced is separated and purified.

  Next, for example, as shown in FIG. 3, N- (6-maleimidocaproxyoxy) succinimide (EMCS) or the like is applied to the polymer thus obtained, and cell adhesion properties are obtained via a trifluoroacetyl group. A maleimide group is introduced at the end of the monomer for monomer unit introduction. By reacting the maleimidated polymer with a compound having a thiol group and containing a cell adhesive motif unit such as a commercially available cyclic RGD peptide, a cell adhesive motif unit / spacer unit / This reagent comprising a cell adhesion motif unit / spacer unit can be obtained.

  In the above description, the cell adhesion motif unit was introduced after the precursor of the reagent was separated from the solid phase carrier, but the cell precursor motif was not separated on the solid phase carrier. It is also possible to introduce units.

  In the above description, when a cell-adhesive motif unit-introducing monomer having a cell-adhesive motif unit in advance is used, post-modification with the cell-adhesive motif unit is not required. In addition, when two or more types of cell adhesion motif units are used, it is preferable to adopt a pre-introduction form using a monomer having a cell adhesion motif unit in advance, but combining pre-introduction and post-modification You can also

  Furthermore, in the above description, it was assumed that the reagent is provided on the 3 ′ end side of the oligoribonucleotide. However, the reagent is provided so that the reagent is provided on the 5 ′ end side of the oligoribonucleotide. Those skilled in the art can also appropriately synthesize.

  Furthermore, in the above description, each monomer is linked by a phosphodiester bond, but the linked form of the monomer is not limited to this, and based on known techniques as described above, Various connection forms can be appropriately selected.

  As described above, by using a solid phase synthesis method such as the amidite method, the present reagent having any cell adhesion motif unit and spacer unit can be easily synthesized. Moreover, the present reagent can be synthesized simultaneously with the synthesis of the oligoribonucleotide to which the present reagent is applied.

  In addition to the amidite solid phase synthesis method widely used for the synthesis of DNA and RNA as described above, this reagent can be synthesized by appropriately applying known DNA and RNA synthesis techniques as appropriate. . In addition to the method described above for introducing the cell adhesion motif unit into the introduction monomer, the monomer and the cell adhesion motif unit can be linked using various known divalent reagents. Further, those skilled in the art can appropriately impart a functional group capable of introducing such a divalent reagent to the monomer. Furthermore, those skilled in the art can appropriately select a spacer in the monomer for introducing a spacer unit from known spacers.

  According to this reagent, the cell adhesion motif unit can be separated by an arbitrary distance with the spacer unit interposed. Since the cell adhesion motif unit is previously separated by a predetermined distance by the spacer unit, it can be effectively bound to the cell adhesion receptor existing on the cell surface. That is, even if the individual interaction is weak, since the plurality of cell adhesion motifs can bind to a plurality of receptors as a whole, they can interact more strongly and adhere to the cell surface. Moreover, the distance between the cell adhesion motif units can be made to correspond to the distance between the receptors to which the cell adhesion motif binds on the cell surface, for example. Furthermore, since the separation distance of the cell adhesion motif unit is given by the interposition of the spacer unit, it is surely and easily given, and the cell adhesion motif unit is connected in series via the spacer unit. Due to the separation, the degree of freedom of access to the receptor of the cell adhesion motif unit is also high. Therefore, compared with the case where the cell adhesion motif unit is provided in a branched shape, the present reagent can adhere to the surface of the target cell easily and effectively. In addition, spacer units and cell-adhesive motif units arranged in series are considered to be able to search for receptors floating on the cell membrane, to combine, hug and cluster them.

  In addition, the reagent is a cell-adhesive motif in series via spacer units, for example, preferably in a side chain direction with respect to the direction in which the spacer units continue through a linker and / or the chain length direction in oligoribonucleotides. Units can be provided. For this reason, even if it is a comparatively bulky cell adhesive motif unit compared with the case where the cell adhesive motif unit is provided in a branched shape, it is easy to approach the target cell surface. Also in this respect, the present reagent can adhere to the surface of the target cell easily and effectively compared to the case where the cell adhesion motif unit is provided in a branched shape.

  This reagent can take a state where spacer units such as polyethylene glycol chains used in the above examples and cell adhesion motif units are arranged in tandem (alternately), etc., and these units can be arbitrarily selected. It has a structure that can be repeated several times. Therefore, the reagent may have a repeating structure in which the cell adhesion motif unit is separated by a spacer unit capable of separating a certain distance, or two or more kinds of spacers may be separated at two or more different distances. The cell adhesion motif unit may be separated by a unit or a plurality of spacer units. Thus, the present reagent can easily optimize the arrangement of cell adhesion motif units.

  Furthermore, the present reagent can be provided with a compound unit that exhibits a certain cell selectivity, such as a polyethylene glycol chain, as a spacer unit as a linear part that separates the cell adhesion motif unit. For this reason, targeting by this reagent, such as functions such as cell selectivity, can be assisted for such spacer units.

  As described above, this reagent retains the cell adhesion motif unit more flexibly and can interact with the cell surface receptor independently of the bulkiness of the cell adhesion motif unit. The oligoribonucleotide complexed with the present reagent can be brought close to the cell surface efficiently, and the intracellular introduction of the oligoribonucleotide can be promoted.

  When the cell adhesion motif unit of this reagent has a peptide motif as the cell adhesion motif, this reagent is a peptide in which two peptide motif units are separated by at least one non-peptide spacer unit It can be understood as a derivative. Such peptide derivatives are novel compounds.

(Cell transduction conjugate)
The cell transfer conjugate disclosed in the present specification (hereinafter, also simply referred to as the present conjugate) includes two or more cellular adhesion motif units and one or more spacer units, A spacer unit comprising a cell adhesion region (hereinafter referred to as the present reagent) that separates the two or more cell adhesion motif units from each other, and an oligoribonucleotide, wherein the reagent and the oligoribonucleotide are covalently bonded. Are conjugates. According to the present conjugate, this reagent makes it easy for oligoribonucleotides to come close to and interact with the cell surface while avoiding trapping from nonspecific blood proteins. For this reason, it becomes easy to introduce oligoribonucleotides into cells.

  In the conjugate, a cell adhesion region, that is, the reagent and an oligoribonucleotide may be complexed by a covalent bond. In addition, it can be said that this form of this conjugate is an oligoribonucleotide derivative. As for the covalent bond between the reagent and the oligoribonucleotide, the reagent is preferably bound to the 5 'end and / or the 3' end of the oligoribonucleotide. For example, when used as siRNA, it is the antisense strand that forms a RISC complex in the cytoplasm and acts on RNA interference. For example, for the 3 ′ end and / or the 5 ′ end of the sense strand It is preferable to provide this reagent.

  As for the binding between this reagent and oligoribonucleotide, oligoribonucleotides prepared separately and this reagent can be covalently bound using a so-called bioconjugate or the like. For example, by introducing a highly reactive crosslinking reactive group on one side and a crosslinking functional thiol on the other side, the two can be linked. As such a binding form, in addition to this, known as bioconjugation, a primary amino group, carboxy group, sulfhydryl group, carbonyl group, carbodiimide group, maleimide, NHS, which can be provided in the present reagent. A cross-linking reaction with an ester or an imide ester can be used.

  In addition, as already explained, when this reagent is synthesized according to the solid phase synthesis method of oligoribonucleotide, the solid phase synthesis method is used to introduce the cell adhesion motif unit that becomes the precursor of this reagent. This conjugate can be synthesized by synthesizing an oligoribonucleotide with synthesis of a polymer chain containing a monomer and a monomer for introducing a spacer unit. Also in the case of solid phase synthesis, this reagent can be added to the 3 'end of the oligoribonucleotide or can be added to the 5' end.

  The oligoribonucleotide used in the conjugate can contain a nucleoside derivative represented by the following formula (1) or (2) (hereinafter also simply referred to as the nucleoside derivative) or a salt thereof. This nucleoside derivative can be included in the partial structure of the oligonucleotide by a method well known to those skilled in the art.

(In Formula (1), R ¹ represents a hydroxyl group, a hydroxyl group in which a hydrogen atom is substituted with an alkyl group or an alkenyl group, or a protected group, and in Formula (2), X represents a halogen atom. In 1) and formula (2), R ² and R ⁴ may be the same or different from each other, and are a hydrogen atom, a hydroxyl protecting group, a phosphate group, a protected phosphate group, or —P (═O). _n R ⁵ R ⁶ (n represents 0 or 1, and R ⁵ and R ⁶ may be the same or different from each other, and represent a hydrogen atom, a hydroxyl group, a protected hydroxyl group, a mercapto group, a protected mercapto group, a lower group, An alkoxy group, a cyano lower alkoxy group, an amino group, or a substituted amino group, provided that when n is 1, both R ⁵ and R ⁶ are not hydrogen atoms. R ³ is an NHR ⁷ having a linking group (R ⁷ is a hydrogen atom) Represents an alkyl group, an alkenyl group or an amino group protecting group.), Represents an azido group, an amidino group or a guanidino group, and B represents a purin-9-yl group, a 2-oxo-pyrimidin-1-yl group or a substituted group. It represents either a purin-9-yl group or a substituted 2-oxo-pyrimidin-1-yl group.)

  This nucleoside derivative is provided with a basic substituent at the 4'-position of ribose, resulting in the oligoribonucleotide having a partial structure derived from this nucleoside derivative resulting from the phosphate group and the like of the oligoribonucleotide. It is possible to provide a charge adjusting ability capable of neutralizing at least a part of the negative charge.

  Moreover, this nucleoside derivative can improve the cell membrane permeability | transmittance of oligoribonucleotide provided with the said partial structure. Furthermore, ribonuclease resistance can be improved in oligoribonucleotides having a partial structure derived from the present nucleoside derivative.

  According to the conjugate of such an oligoribonucleotide and the present reagent, the oligoribonucleotide can be easily brought close to the cell surface by the present reagent, and the nuclease resistance and cell membrane permeability are improved. It is easy to introduce in.

  In the present specification, the meaning of “lower” in a substituent in a compound represented by a structural formula or the like means that the number of carbon atoms constituting the substituent is up to 10. For example, usually 1 to 6 carbon atoms or 1 to 5 carbon atoms are exemplified, and further preferred examples include 1 to 4 carbon atoms or 1 to 3 carbon atoms.

  Hereinafter, the present nucleoside derivative or a salt thereof and use thereof will be described.

(Nucleoside derivatives and salts thereof)
One embodiment of the present nucleoside derivative or a salt thereof is a nucleoside derivative represented by the following formula (1) or a salt thereof.

  Another embodiment of the present nucleoside derivative or a salt thereof is a nucleoside derivative represented by the following formula (2) or a salt thereof.

[About R ¹ ]
In Formula (1), R ¹ represents a hydroxyl group, a hydroxyl group in which a hydrogen atom is substituted with an alkyl group or an alkenyl group, or a protected hydroxyl group.

[About X]
In formula (2), X represents a halogen atom. Although it does not specifically limit as a halogen atom, A chlorine atom, an iodine atom, a fluorine atom, a bromine atom, etc. are mentioned. When R ¹ is a halogen atom, the nucleoside derivative is a deoxyribonucleoside derivative. As is clear from the formula (2), the bonding direction of the halogen atom with respect to the carbon atom at the 2′-position of ribose is not particularly limited. Bonding is preferred.

(Alkyl group)
In the present specification, examples of the alkyl group include a saturated hydrocarbon group that is linear, branched, cyclic, or a combination thereof. Usually, a lower alkyl group is preferable, and for example, a lower alkyl group having 1 to 6 carbon atoms or a lower alkyl group having 1 to 5 carbon atoms is more preferable, and further, 1 to 4 carbon atoms or carbon number is further exemplified. 1 to 3 lower alkyl groups are particularly preferred examples. Examples of the linear alkyl group having 1 to 4 carbon atoms include a methyl group, an ethyl group, an n-propyl group, and an n-butyl group. Among these, a methyl group, an ethyl group, An n-propyl group is preferable, and a methyl group and an ethyl group are preferable, and a methyl group is preferable. Examples of the branched alkyl group having 1 to 4 carbon atoms include isopropyl group, isobutyl group, s-butyl group, t-butyl group, etc. Among them, isopropyl group is particularly preferable. Examples of the cyclic alkyl group having 1 to 4 carbon atoms include a cyclopropyl group, a cyclobutyl group, and a cyclopropylmethyl group.

(Alkenyl group)
In the present specification, examples of the alkenyl group include saturated hydrocarbon groups that are linear, branched, cyclic, or combinations thereof. Usually, a lower alkenyl group is preferable, and examples of the lower alkenyl group include ethenyl group, 1-propenyl group, 2-propenyl group, 1-methyl-2-propenyl group, 1-methyl-1-propenyl group, 2-methyl Examples include a -1-propenyl group, a 1-butenyl group, and a 2-butenyl group.

(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 for example, Protective Groups in Organic Synthesis (John Wiley and Sons, 2007 edition) can be referred to. Typical examples of the hydroxyl-protecting group include an aliphatic acyl group, an aromatic acyl group, a lower alkoxymethyl group, an oxycarbonyl group which may have an appropriate substituent, and an appropriate substituent. A tetrahydropyranyl group which may be optionally substituted, a tetrathiopyranyl group which may be optionally substituted, and a methyl group substituted with 1 to 3 substituted or unsubstituted aryl groups in total (provided that Examples of the substituent for aryl include lower alkyl, lower alkoxy, a halogen atom, or a cyano group.), A silyl group, and the like.

  In the present specification, examples of the alkoxy group include a saturated alkyl ether group that is linear, branched, cyclic, or a combination thereof. A lower alkoxy group is preferable. 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 further 1 to 4 carbon atoms or carbon atoms. A C1-C3 alkoxy group is preferable and a C1-C4 alkoxy group is especially preferable. 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. In addition, isopropoxy group, isobutoxy group, s-butoxy group, t-butoxy group and the like are also preferable examples. Moreover, a cyclopropoxy group and a cyclobutoxy group are also preferable, and a cyclopropylmethoxy group is also mentioned as a preferable example.

In the present specification, examples of the alkylthio group include a saturated alkylthio group that is linear, branched, cyclic, or a combination thereof. A lower alkylthio group is preferred, and the lower alkylthio group is, for example, preferably 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 is a particularly preferable example. Preferred examples of the saturated alkylthio group having 1 to 4 carbon atoms include a methylthio group, an ethylthio group, an n-propylthio group, and an n-butylthio group. In addition, isopropylthio group, isobutylthio group, s-butylthio group, t-butylthio group and the like are also exemplified as preferable examples. Moreover, a cyclopropylthio group or a cyclobutylthio group is mentioned as a preferred example, and a cyclopropylmethylthio group is further exemplified as a more preferred example.
The )

  Of these, aliphatic acyl groups, aromatic acyl groups, and silyl groups are particularly preferred examples. A preferred example is a methyl group substituted with 1 to 3 substituted or unsubstituted aryl groups in total (wherein the substituents in the substituted aryl are as described above).

  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.

  The alkyl in the alkylcarbonyl group is as described above. That is, as the alkylcarbonyl group, for example, formyl group, acetyl group, propionyl group, butyryl group, isobutyryl group, pentanoyl group, pivaloyl group, valeryl group, isovaleryl group, octanoyl group, nonanoyl group, decanoyl group, 3-methylnonanoyl group, 8-methylnonanoyl group, 3-ethyloctanoyl group, 3,7-dimethyloctanoyl group, undecanoyl group, dodecanoyl group, tridecanoyl group, tetradecanoyl group, pentadecanoyl group, hexadecanoyl group, 1-methylpentadecanyl group Noyl group, 14-methylpentadecanoyl group, 13,13-dimethyltetradecanoyl group, heptadecanoyl group, 15-methylhexadecanoyl group, octadecanoyl group, 1-methylheptadecanoyl group, nonadecanoyl group, eicosano Group, or include Henaikosairu group. Of these, acetyl, propionyl, butyryl, isobutyryl, pentanoyl, and pivaloyl are preferred examples, and acetyl is particularly preferred. The alkyl in the carboxylated alkylcarbonyl group is as described above. The substitution position for carboxylation can also be selected as appropriate. That is, examples of the carboxylated alkylcarbonyl group include a succinoyl group, a glutaroyl group, and an adipoyl group.

  The halogen, lower, and alkyl in the halogeno lower alkylcarbonyl group are as described above. The halogen substitution position and the like 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.

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

  Examples of the aromatic acyl group include an arylcarbonyl group, a halogenoarylcarbonyl group, a lower alkylated arylcarbonyl group, a lower alkoxylated arylcarbonyl group, a carboxylated arylcarbonyl group, a nitrated arylcarbonyl group, or an arylated aryl. A carbonyl group is mentioned.

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

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

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

  Examples of the oxycarbonyl group which may have an appropriate substituent include a lower alkoxycarbonyl group, a lower alkoxycarbonyl group substituted with a halogen or a silyl group, or an alkenyloxycarbonyl group.

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

  Examples of the alkenyloxycarbonyl group include a vinyloxycarbonyl group. Examples of the tetrahydropyranyl group, which may have an appropriate substituent, include, for example, a tetrahydropyran-2-yl group or a 3-bromotetrahydropyran-2-yl group, particularly preferably. A tetrahydropyran-2-yl group may be mentioned.

  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 tetrahydrothiopyran-2. -An yl group is mentioned. In the methyl group substituted by 1 to 3 substituted or unsubstituted aryl groups in total, the substituent in the above substituted aryl means a lower alkyl, lower alkoxy, halogen, or cyano group.

  Examples of the methyl group substituted by 1 to 3 substituted or unsubstituted aryl groups in total include, for example, benzyl group, α-naphthylmethyl group, β-naphthylmethyl group, diphenylmethyl group, triphenylmethyl group, α -A naphthyl diphenylmethyl group is mentioned, Preferably a benzyl group and a triphenylmethyl group are mentioned. Other examples include 9-anthrylmethyl 4-methylbenzyl group, 2,4,6-trimethylbenzyl group, 3,4,5-trimethylbenzyl group, and preferably 2,4,6-trimethylbenzyl group. 3,4,5-trimethylbenzyl group. Other types include, for example, 4-methoxybenzyl group, 4-methoxyphenyldiphenylmethyl group, 4,4′-dimethoxytriphenylmethyl group, preferably 4-methoxybenzyl group, 4-methoxyphenyldiphenylmethyl group, A 4,4′-dimethoxytriphenylmethyl group may be mentioned. Furthermore, a 4-chlorobenzyl group and 4-bromobenzyl group are mentioned, for example. In addition, for example, a 4-cyanobenzyl group is also a preferred example.

  In the present specification, as the silyl group, trimethylsilyl group, triethylsilyl group, isopropyldimethylsilyl group, t-butyldimethylsilyl group, methyldiisopropylsilyl group, methyldi-t-butylsilyl group, triisopropylsilyl group, diphenylmethylsilyl group , Diphenylbutylsilyl group, and diphenylisopropylsilylphenyldiisopropylsilyl group. Among these, a trimethylsilyl group, a t-butyldimethylsilyl group, a triisopropylsilyl group, and a diphenylmethylsilyl group are more preferable, and a trimethylsilyl group, a t-butyldimethylsilyl group, and a diphenylmethylsilyl group are particularly preferable. It is done.

  As the hydroxyl-protecting group in the present specification, a chemical method (for example, hydrogenolysis, hydrolysis, electrolysis, or photolysis) or a biological method (for example, hydrolysis in the human body) is imagined. In some cases, it means a substituent that is cleaved and released by any of the following methods. Preferred examples of the hydroxyl-protecting group include a substituent that is eliminated by hydrogenolysis or hydrolysis. The protected hydroxyl group can be said to be a hydroxyl group in which a hydrogen atom is substituted with such a protecting group.

[About R ² and R ⁴ ]
In the formula (1) and the formula (2), R ² and R ⁴ may be the same or different from each other, and are a hydrogen atom, a hydroxyl protecting group, a phosphate group, a protected phosphate group, or —P ( = O) represents the _n (R ⁵⁾ R ^6. The protecting group for the hydroxyl group is as described above.

(Protected phosphate group)
The protecting group in the protected phosphate group is known to those skilled in the art, and the above-mentioned references and explanations can be referred to.

  Examples of the protecting group for the phosphate group include a lower alkyl group, a lower alkyl group substituted with a cyano group, an ethyl group substituted with a silyl group, a lower alkyl group substituted with a halogen, a lower alkenyl group, and a cyano group. Substituted lower alkenyl group, cycloalkyl group, lower alkenyl group substituted with cyano group, aralkyl group, aralkyl group with aryl ring substituted with nitro group, aralkyl group with aryl ring substituted with halogen, lower alkyl group An aryl group substituted with, an aryl group substituted with a halogen, or an aryl group substituted with a nitro group.

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

  Examples of the lower alkyl group substituted with halogen include 2,2,2-trichloroethyl group, 2,2,2-tribromoethyl group, 2,2,2-trifluoroethyl group, 2,2, A 2-trichloroethyl group is exemplified, and a 2,2,2-trichloroethyl group is particularly preferred. Examples of the lower alkenyl group include ethenyl group, 1-propenyl group, 2-propenyl group, 1-methyl-2-propenyl group, 1-methyl-1-propenyl group, 2-methyl-1-propenyl group, 1 -Butenyl group, 2-butenyl group, etc. are mentioned.

  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 benzyl group, α-naphthylmethyl group, β-naphthylmethyl group, indenylmethyl group, phenanthrenylmethyl group, anthracenylmethyl group, diphenylmethyl group, triphenylmethyl group, 1-phenethyl group, 2-phenethyl group, 1-naphthylethyl group, 2-naphthylethyl group, 1-phenylpropyl group, 2-phenylpropyl group, 3-phenylpropyl group, 1-naphthylpropyl, 2-naphthylpropyl, 3-naphthylpropyl, 1-phenylbutyl group, 2-phenylbutyl group, 3-phenylbutyl group, 4-phenylbutyl group, and more preferably benzyl group, diphenylmethyl group, triphenylmethyl group, 1- Examples include phenethyl group and 2-phenethyl group, particularly preferably benzyl group. It is done.

  Examples of the aralkyl group in which the aryl ring is substituted with the nitro group include 2- (4-nitrophenyl) ethyl group, 0-nitrobenzyl group, 4-nitrobenzyl group, 2,4-dinitrobenzyl group, 4-chloro And 2-nitrobenzyl group.

  In this specification, the protecting group for phosphoric acid includes a chemical method (for example, hydrogenolysis, hydrolysis, electrolysis, or photolysis), or a biological method (for example, hydrolysis in the human body). In other words, it may mean a substituent that is cleaved and eliminated by any of the following methods. Preferred examples of the protecting group for phosphoric acid include a substituent that is eliminated by hydrogenolysis or hydrolysis.

_{(-P (= O) n (} R 5) R 6)
R ² and R ⁴ of the nucleoside analog of the present invention may be —P (═O) _n (R ⁵ ) R ⁶ . n represents 0 or 1, and R ⁵ and R ⁶ may be the same or different from each other, and are a hydrogen atom, a hydroxyl group, a protected hydroxyl group, a mercapto group, a protected mercapto group, a lower alkoxy group, or a cyano lower alkoxy. It represents either a group, an amino group, or a substituted amino group. However, when n is 1, R ⁵ and R ⁶ are not both hydrogen atoms. The protected hydroxyl group and lower alkoxy group are as described above.

(Protected mercapto group)
Protected mercapto groups are well known to those skilled in the art. Examples of the protected mercapto group include, for example, alkylthio groups, arylthio groups, aliphatic acyl groups, and aromatic acyl groups in addition to those mentioned as the protective group for the hydroxyl group. An aliphatic acyl group and an aromatic acyl group are preferable, and an aromatic acyl group is particularly preferable. As the alkylthio group, a lower alkylthio group is preferable, and for example, methylthio, ethylthio, and t-butylthio groups are preferable examples. Examples of the arylthio group include benzylthio. Examples of the aromatic acyl group include a benzoyl group.

  Examples of the cyano lower alkoxy group include, for example, a linear, branched, cyclic, or a combination thereof having 1 to 5 carbon atoms substituted by a cyano group (in addition, carbon atoms in the cyano group). (When counting is not included) is a preferred example, specifically, for example, cyanomethoxy, 2-cyanoethoxy, 3-cyanopropoxy, 4-cyanobutoxy, 3-cyano-2-methylpropoxy, or Examples include 1-cyanomethyl-1,1-dimethylmethoxy, and particularly preferably a 2-cyanoethoxy group.

As R ⁵ and R ⁶ , a substituted amino group can be selected. The substituent of the amino group is any one of a lower alkoxy group, a lower alkylthio group, a cyano lower alkoxy group, or a lower alkyl group. When both R ⁵ and R ⁶ are substituted amino groups, the substituted amino groups may be different substituted amino groups. The lower alkoxy group, lower alkylthio group, cyano lower alkoxy group, and lower alkyl group are as described above.

More specific examples of —P (═O) _n (R ⁵ ) R ⁶ include a phosphoramidide group, an H-phosphonate group, or a phosphonyl group. A preferred example is given.

—P (═O) _n (R ⁵ ) In R ⁶ , when n is 0, at least one of R ⁵ and R ⁶ is a substituted amino group, and the other can be anything, It becomes loamidide group. As the phosphoramidide group, a phosphoramidide group in which one of R ⁵ and R ⁶ is a substituted amino group and the other is a lower alkoxy group or a cyano lower alkoxy group has a reaction efficiency of the condensation reaction. Good and particularly preferred. Examples of the substituted amino group include a diethylamino group, a diisopropylamino group, a dimethylamino group, and the like, and a diisopropylamino group is particularly preferable. A preferred example of the lower alkoxy group for the other substituent of R ⁵ and R ⁶ is a methoxy group. Moreover, as a cyano lower alkoxy group, a 2-cyanoethyl group is mentioned as a preferable example. The phosphoramidite group, _{specifically, -P (OC 2 H 4 CN} ) (N (CH (CH 3) 2), or _{-P (OCH 3) (N (} CH (CH 3) 2) Is a preferred example.

-P (= O) _n (R ⁵ ) In R ⁶ , when n is 1, at least one of R ⁵ and R ⁶ is a hydrogen atom, and the other may be anything other than a hydrogen atom Becomes an H-phosphonate group. Examples of the substituent other than hydrogen include a hydroxyl group, a methyl group, a methoxy group, and a thiol group, and a hydroxyl group is particularly preferable.

Further, in _{-P (= O) n (R} 5) R 6, n is 1, if R ⁵ and R ⁶ are both a lower alkoxy group, a phosphonyl group. The lower alkoxy groups for R ⁵ and R ⁶ may be the same as or different from each other. As the lower alkoxy group, for example, a methoxy group, an ethoxy group and the like are preferable examples. Specific examples of the phosphonyl group include —P (═O) (OCH ₃ ) ₂ .

As R ² in the present nucleoside derivative, for example, —P (═O) _n (R ⁵ ) R ⁶ is particularly preferable. Preferred examples of —P (═O) _n (R ⁵ ) R ⁶ include a phosphoramidide group, an H-phosphonate group, or a phosphonyl group. R ² is also preferably a phosphate group or a protected phosphate group. R ² is preferably a hydrogen atom or a hydroxyl-protecting group.

Other specific examples of R ² 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 (OC ₂ H ₄ CN) ( Preferred examples include N (CH (CH ₃ ) ₂ ), —P (OCH ₃ ) (N (CH (CH ₃ ) ₂ ), or a phosphonyl group.

As R ⁴ in the present nucleoside derivative, for example, a hydrogen atom or a hydroxyl-protecting group is preferable. Further, for example, phosphate groups, protected phosphate group, or _{-P (= O) n (R} 5) It is also preferred that R ^6. Specific examples of R ⁴ include a hydrogen atom, acetyl group, benzoyl group, benzyl group, p-methoxybenzyl group, dimethoxytrityl group, monomethoxytrityl group, tert-butyldiphenylsilyl group, or trimethylsilyl group. A preferred example is given.

[About R ³ ]
In formula (1) and formula (2), R ³ may represent NHR ⁷ , an azido group, an amidino group or a guanidino group each having a linking group. That is, each of NHR ⁷ , azido group, amidino group or guanidino group is bonded to the 4′-position carbon atom via a linking group.

  As the linking group, for example, a divalent hydrocarbon group having 1 or more carbon atoms can be represented. 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.

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

  The alkenylene group as the linking group is linear or branched, and preferably linear. For example, a lower alkenylene group is preferable, and examples of the lower alkenylene group include an ethene-1,2-diyl group, a propene-1,3-diyl group, a butene-1,4-diyl group, and the like.

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

R ⁷ includes a hydrogen atom, an alkyl group, an alkenyl group, or an amino protecting group. The alkyl group is preferably a lower alkyl group in addition to the alkyl group already described. Preferred examples of the alkenyl group include lower alkenyl groups in addition to the alkenyl groups already described. When R ⁷ is a group such as a hydrogen atom, the linking group is an alkylene group having 2 or more carbon atoms, for example 3 or more, for example 4 or more, for example 6 or less, for example 5 or less, for example 4 or less. Preferably it is.

When R ⁷ is a hydrogen atom, R ³ is NH ₂ (amino group) having a linking group, that is, when the linking group is an alkylene group or an alkenylene group, it becomes an aminoalkyl group or an aminoalkenyl group. In Formula (1) and Formula (2), when R ³ is an aminoalkyl group or the like, the nucleoside derivative and the oligonucleotide derivative having a monomer unit derived from the nucleoside derivative change in charge in the surrounding pH environment. It is possible to exhibit charge imparting properties with the feature of. For example, it is cationic under acidic conditions, and the neutral charge under physiological conditions can reduce the positive charge to zero. That is, according to such charge control ability, by changing the pH environment, the charge of the nucleoside derivative can be dynamically changed or a desired charge can be imparted when necessary. Therefore, according to the present nucleoside derivative, the charge of the oligonucleotide can be adjusted in a mode different from the prior art or with a higher degree of freedom than in the past. Based on the above, the present nucleoside derivative in which R ³ is an aminoalkyl group or the like is useful as a charge (positive charge) imparting agent or charge control agent for oligonucleotides and the like.

R ³ includes an azido group and an amidino group each having a linking group, that is, CH ₃ (NH) C (NH)-(one obtained by removing one hydrogen atom from the amino group of amidine), a guanidino group, NH ₂ (NH) C (NH)-(in which one hydrogen atom is removed from the amino group of guanidine). Among them, a guanidino group can be mentioned. When R ³ has these groups, the linking group can be an alkylene group having 1 or more carbon atoms, for example, 2 or more, or an alkenylene group. When R ³ is an amidino group or guanidino group having a linking group, it is always cationic, unlike the aminoalkyl group described above. Such a nucleoside derivative is useful in combination with the present nucleoside derivative in which R ³ is an aminoalkyl group or the like.

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

  In the present invention, the amino-protecting group may be a chemical method (for example, hydrogenolysis, hydrolysis, electrolysis, or photolysis) or a biological method (for example, hydrolysis in the human body). In some cases, it means a substituent that is cleaved and released by any of the following methods. In particular, a substituent capable of leaving by hydrogenolysis or hydrolysis is preferred as an amino-protecting group.

[B: About the base]
Examples of B: base in the nucleoside derivative include known natural bases and artificial bases. For example, as B, a purin-9-yl group, a 2-oxo-pyrimidin-1-yl group, a substituted purin-9-yl group, or a substituted 2-oxo-pyrimidin-1-yl group can be selected.

  That is, examples of B include a purin-9-yl group or a 2-oxo-pyrimidin-1-yl group, and 2,6-dichloropurin-9-yl or 2-oxo-pyrimidin-1-yl. Is mentioned. Further, 2-oxo-4-methoxy-pyrimidin-1-yl, 4- (1H-1,2,4-triazol-1-yl) -pyrimidin-1-yl, or 2,6-dimethoxypurine-9- Ill.

  Furthermore, 2-oxo-4-amino-pyrimidin-1-yl with amino group protected, 2-amino-6-bromopurin-9-yl with amino group protected, 2-amino with amino group protected -6-hydroxypurin-9-yl, amino group and / or hydroxyl group protected 2-amino-6-hydroxypurin-9-yl, amino group protected 2-amino-6-chloropurine-9- And amino group-protected 6-aminopurin-9-yl, or amino-protected 4-amino-5-methyl-2-oxo-pyrimidin-1-yl group. The protecting groups for hydroxyl group and amino group are as described above.

  Further, 6-aminopurin-9-yl (adenine), 2-amino-6-hydroxypurin-9-yl (guanidine), 2-oxo-4-amino-pyrimidin-1-yl (cytosine), 2-oxo Examples include -4-hydroxy-pyrimidin-1-yl (uracil) or 2-oxo-4-hydroxy-5-methylpyrimidin-1-yl (thymine).

  Furthermore, 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.

  6-amino-2-methoxypurin-9-yl, 6-amino-2-chloropurin-9-yl, 2-amino-6-chloropurin-9-yl, or 2-amino-6-bromopurine -9-yl is mentioned.

The substituent in each of the substituted purin-9-yl group or the substituted 2-oxo-pyrimidin-1-yl group is a hydroxyl group, a protected hydroxyl group, a lower alkoxy group, a mercapto group, a protected mercapto group, a lower alkylthio group, It is any one of an amino group, a protected amino group, an amino group substituted with a lower alkyl group, a lower alkyl group, a lower alkoxymethyl group, a halogen atom, or a combination thereof. These substituents are as described above.

  As B in the present nucleoside derivative, the substituent in the substituted purin-9-yl group or the substituted 2-oxo-pyrimidin-1-yl group is preferably each of the above-described substituents, but in addition to this, a triazole group It is also preferred that a lower alkoxymethyl group is added.

  Preferable examples of the substituted purin-9-yl group include, for example, 6-aminopurin-9-yl, 2,6-diaminopurin-9-yl, 2-amino-6-chloropurin-9-yl, 2- Amino-6-bromopurin-9-yl, 2-amino-6-hydroxypurin-9-yl, 6-amino-2-methoxypurin-9-yl, 6-amino-2-chloropurin-9-yl, Examples include 6-amino-2-fluoropurin-9-yl, 2,6-dimethoxypurin-9-yl, 2,6-dichloropurin-9-yl, and 6-mercaptopurin-9-yl. If an amino group or a hydroxyl group is present in the above-described substituents, preferred examples include those in which the amino group and / or hydroxyl group is protected.

Examples of substituted 2-oxo-pyrimidin-1-yl include 2-oxo-4-amino-pyrimidin-1-yl, 1H- (1,2,4-triazol-1-yl) -pyrimidin-1-yl, 4-1H-1,4-amino-2-oxo-5-chloro-pyrimidin-1-yl, 2-oxo-4-methoxy-pyrimidin-1-yl, 2-oxo-4-mercapto-pyrimidine-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- Ir etc. are mentioned.
In addition, preferred examples include 2-oxo-4-methoxy-pyrimidin-1-yl or 4- (1H-1,2,4-triazol-1-yl) -pyrimidin-1-yl.

  Among these B, when an amino group or a hydroxyl group is present in the substituent, a substituent in which the amino group or the hydroxyl group is protected is a preferred example.

  The nucleoside derivative may be a salt. Although the form of a salt is not specifically limited, Generally, an acid addition salt is illustrated and may take the form of an intramolecular counter ion. Alternatively, a base addition salt may be formed depending on the type of substituent. As the salt, a pharmaceutically acceptable salt is preferable. The types of acids and bases that form pharmaceutically acceptable salts are well known to those skilled in the art, for example, see J. Org. Pharm. Sci. 1-19 (1977) can be referred to. For example, the acid addition salt includes a mineral acid salt and an organic acid salt. In addition, when one or more substituents contain an acidic moiety, a base addition salt is also a preferred example.

  Examples of the mineral acid salt include hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, hydrosulfate, phosphate, and hydrophosphate. Usually, preferred examples include hydrochloride and phosphate. Examples of organic acid salts include acetate, trifluoroacetate, gluconate, lactate, salicylate, citrate, tartrate, ascorbate, succinate, maleate, fumarate, formic acid Examples thereof include salts, benzoates, methanesulfonates, ethanesulfonates, and p-toluenesulfonates. Usually, acetate and the like are preferable examples. Base addition salts include alkali metal salts, alkaline earth metal salts, organic amine salts, and amino acid addition salts.

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

The present nucleoside derivatives or salts thereof may exist as hydrates or solvates, and these substances are also included in the scope of the disclosure herein. The present nucleoside derivative or a salt thereof can be easily produced by those skilled in the art according to the following synthesis examples and known methods.

  This nucleoside derivative can be introduced into oligoribonucleotides as at least a part of oligoribonucleotides to improve the nuclease resistance of single-stranded oligoribonucleotides and double-stranded oligoribonucleotides. Cell membrane permeability of animal cells and the like can be improved. That is, the nucleoside derivative itself is useful as a nuclease resistance improving agent and / or a cell membrane permeability imparting agent. Further, the present nucleoside derivative can have a basic substituent at 4 '. Thereby, it can function as a charge control agent or a positive charge imparting agent capable of adjusting a negative charge derived from a phosphate group or the like in an oligoribonucleotide or the like.

  In addition, the oligoribonucleotide disclosed in the present specification can contain at least one partial structure represented by Formula (3) and Formula (4). The partial structures represented by Formula (3) and Formula (4) can be obtained based on the nucleoside derivative represented by Formula (1) and Formula (2) or a salt thereof, respectively.

R ¹ , X, R ³ and B in the partial structures represented by formula (3) and formula (4) have the same meanings as in formula (1) and formula (2), respectively.

  Two or more partial structures represented by formula (3) and formula (4) may be included in the oligoribonucleotide. In that case, these partial structures may be the same or different from each other. Further, the entire partial structure contained in the oligoribonucleotide may be composed only of the partial structure represented by the formula (3), or may be composed only of the partial structure represented by the formula (4). Also good. Moreover, you may have 1 or 2 or more of partial structures represented by Formula (3), and you may have 1 or 2 or more of partial structures represented by Formula (4).

  Moreover, as arrangement | positioning of the partial structure represented by Formula (3) and Formula (4), you may mutually adjoin and may exist separately. For example, an oligoribonucleotide can have at least three of the partial structures. In this case, each partial structure can be provided approximately equally on the 5 ′ end side, the central portion and the 3 ′ end side of the oligoribonucleotide. The provision of partial structures substantially uniformly in these parts of the oligoribonucleotide does not necessarily limit that the same number of parts is provided in each part, but that each part has at least one each. It is sufficient if it is at least satisfied. For example, when each part is provided with about 1-3 partial structures, it can be said that it is substantially equal. In the oligoribonucleotide, at least 6 partial structures can be provided.

  In the partial structure represented by the formula (3), since the sugar chain part is derived from ribose or deoxyribose, the oligoribonucleotide may be an oligoribonucleotide or an oligodeoxyribonucleotide. Good. The oligoribonucleotide may be a chimera of ribonucleotide and deoxyribonucleotide.

  Oligoribonucleotides are themselves single-stranded, but can also take the form of oligoribonucleotides and hybrids with oligodeoxyribo / ribonucleotides (chimeric strands), ie double-stranded.

  The oligoribonucleotide has a partial structure other than the partial structures represented by the formulas (3) and (4) having a partial structure corresponding to other natural nucleotides or known nucleoside derivatives and / or nucleotide derivatives. Can be provided. The partial structures defined in the present specification and other partial structures can be bonded to each other by, for example, a phosphodiester bond, a phosphomonoester bond, a thiophosphate bond, and the like.

  The number of oligoribonucleotides is preferably at least 2 or more, more preferably 8 or more, and particularly preferably 15 or more, based on the number of partial structures and other nucleoside derivatives. The upper limit is not particularly limited, but is, for example, 100 or less, for example, 80 or less, for example, 60 or less, for example, 50 or less, or, for example, 40 or less. Also, for example, it is 30 or less, and for example, it may be 20 or less.

  Oligoribonucleotides may have one or more asymmetric centers in the partial structures represented by formulas (3) and (4) as well as other partial structures, and the same applies when stereoisomers exist. Any mixture of stereoisomers, racemates, and the like are included in the scope of the present invention. It may also exist as a tautomer.

  The oligoribonucleotide may be a salt. Although the form of a salt is not specifically limited, A pharmaceutically acceptable salt is mentioned as a preferable example. Regarding the salt, the embodiment of the salt in the present nucleoside derivative described above can be applied. The oligoribonucleotide or a salt thereof may be a hydrate or a solvate, and these are also included in the scope of the present invention.

(Production of the present nucleoside derivative and oligoribonucleotide)
This nucleoside derivative and oligoribonucleotide can be easily synthesized by those skilled in the art based on synthesis techniques for nucleosides and oligonucleotides known at the time of filing of the present application, in addition to specific examples of synthesis in the latter stage.

  The present nucleoside derivative and oligoribonucleotide can be produced, for example, by the following method, but the production method of the nucleoside analog or oligoribonucleotide of the present invention is not limited to the following method.

  In each reaction, the reaction time is not particularly limited, but the progress of the reaction can be easily traced by an analysis means described later, and therefore it may be terminated when the yield of the target product is maximized. Moreover, each reaction can be performed in inert gas atmospheres, such as under nitrogen stream or argon stream, as needed. In each reaction, when protection by a protecting group and subsequent deprotection are required, the reaction can be appropriately performed by using the method described later.

  In the present 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, and THA represents , 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. Show.

  For example, an example of the present nucleoside derivative can be synthesized according to the following scheme. The following scheme is an example of a scheme from the synthesis of a thymine ribonucleoside derivative using glucose as a starting material to the synthesis of a phosphoramidite agent for the synthesis of oligoribonucleotides.

  The compound 2 was obtained from glucose 1 according to a conventional method. From Compound 2, 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 In addition to -151 etc., compounds 3 to 20 can be obtained based on the description of Bioorganic & Medical Chemistry 11 (2003) 211-2226, Bioorganic & Chemistry letters (1999) 2667-2672.

  Oligoribonucleotides having a partial structure represented by formulas (3) and (4) can be easily obtained by using various nucleoside derivatives represented by formula (1) or (2) as amidite agents and the like. Can be manufactured. That is, by using such a nucleoside derivative, it can be synthesized using a known DNA synthesizer, and the resulting oligonucleotide derivative is purified using a column, and the purity of the product is determined by reverse-phase HPLC or MALDI-TOF. -Purified oligoribonucleotides can be obtained by analysis with MS. The method of converting oligoribonucleotides into acid addition salts is well known to those skilled in the art.

  According to the oligoribonucleotide, by providing a predetermined N-containing group via a linking group at the ribose 4 ′ position, it is possible to adjust the substantial charge amount of RNA while maintaining the RNA function in the living body such as RNA interference ability, It is possible to enhance fat solubility (van der Waals intermolecular force) and reduce the dsRNA melting temperature. Thereby, in addition to improving ribonuclease resistance, cell membrane permeability can be improved. Furthermore, the negative charge due to the phosphate group or the like can be neutralized to adjust the overall charge.

  An oligoribonucleotide can have at least two of this partial structure. By providing a plurality of the partial structures, cell membrane permeability, ribonuclease resistance and the like can be reliably improved and adjusted. The oligoribonucleotide can also have at least three of this partial structure.

  In the oligoribonucleotide, the site of one or more of the partial structures is not particularly limited. For example, it can be provided on either the 5 ′ terminal side or the 3 ′ terminal side or both. The 5′-end side and the 3′-end side refer to regions in an appropriate number of ranges from each end of the polymer chain of the oligonucleotide, and do not exceed, for example, 30% of the total constituent units of the polymer chain. An area composed of structural units. The ratio of the range from the end varies depending on the total length of the polymer chain, but may be, for example, 25% or less, or, for example, 20% or less, or, for example, 10% or less, or, for example, 5% or less. More specifically, the 5 ′ terminal side and the 3 ′ terminal side are, for example, 1 to 30 from each terminal, for example, 1 to 25, for example 1 to 20, and for example 1 to 15, for example 1-10, for example 1-8, for example 1-6, for example 1-5, for example 1-4, for example 1-3, For example, it can be set as the area | region of the structural unit derived from 1-2 nucleoside derivatives. Oligoribonucleotides can be provided with one or more of this partial structure in any of these terminal regions. Preferably, two or more can be provided. In addition, the oligoribonucleotide may have this partial structure at either or both of the 5 'end and the 3' end (that is, the first constituent unit from each end).

  In the oligoribonucleotide, one or two or more of the partial structures can be provided in the central portion other than the 5 'terminal side and the 3' terminal side. Since the oligoribonucleotide has this partial structure in the central part, the ribonuclease resistance and cell membrane permeability can be further improved and adjusted. In addition, the charge of the whole oligonucleotide can be adjusted more easily.

  Oligoribonucleotides can also be provided with this partial structure in the central portion of either or both of the 5 'end and the 3' end. Preferably, one or two or more of the partial structures can be provided at each of the 5 'terminal side, 3' terminal side, and central part. Thus, as a whole, by providing this partial structure approximately evenly or dispersedly, ribonuclease resistance, cell membrane permeability, and charge regulation can be improved. From the viewpoint of improving characteristics, it is useful to provide two or more of this partial structure at the center of the oligoribonucleotide.

  As this partial structure in the oligoribonucleotide, a partial structure derived from the ribonucleoside derivative represented by the formula (3) and a partial structure derived from the deoxyribonucleotide derivative represented by the formula (4) can be used. In addition, the ribonucleoside derivative represented by the formula (3) and the partial structure of the formula (4) include uracil (U), which is a base in RNA, as a base of B, so that the ribonucleoside derivative can be used as an alternative to the ribonucleoside derivative. Can be used.

Further, in the present partial structure, R ³ in formulas (3) and (4) has NHR ⁷ with an alkylene group having 1 or 2 carbon atoms as a linking group, so that ribonuclease resistance, cell membrane permeability, and charge It is preferable from the viewpoint of adjustability. In this case, R ⁷ may be a hydrogen atom or an acyl group having an alkyl group having about 1 to 6 carbon atoms. The alkylene group can be an ethylene group, a propylene group, a butylene group, a pentylene group, a hexylene group, or the like. For example, it can be an ethylene group, a propylene group, a butylene group, or the like. For example, it can be an ethylene group, a propylene group, or the like. By using an ethylene group or a propylene group as a linking group, it is possible to obtain higher ribonuclease resistance, cell membrane permeability, and charge regulation as compared with the case of using a methylene group.

  Moreover, this partial structure may be an amidino group, an azide group, and a guanidino group having a linking group. Even with such a functional group, high ribonuclease resistance and cell membrane permeability can be obtained. In this case, the linking group may be an alkylene group having 1 or more carbon atoms.

Further, in this partial structure, R ^{3 in} the formula (3) and formula (4) is an alkyl group having about 1 to 6 carbon atoms, and further, for example, the lower limit of the carbon number is 2 or more, for example, 3 or more. Preferably it is. Such a structure is effective for ribonuclease resistance and cell membrane permeability.

The oligoribonucleotide preferably has at least 6 partial structures. By providing six or more, it is advantageous in terms of ribonuclease resistance, cell membrane permeability, and charge control.

  The oligoribonucleotide in this conjugate preferably has this partial structure in an RNA chain that is not an RNA chain that substantially functions in the cytoplasm, or a portion thereof. For example, when this conjugate is intended to be an siRNA having an oligoribonucleotide duplex, expect the cell permeability and nuclease resistance of this conjugate by having this partial structure in the sense strand. Can do. In addition, for introduction into short RNA other than siRNA, this partial structure is provided at a position located on the surface of the three-dimensional structure of RNA, and this reagent is provided at the 5 ′ end and / or 3 ′ end. It is preferable to do so.

  This conjugate can be used, for example, as siRNA. That is, this conjugate comprising an oligoribonucleotide duplex forms a complex with an in vivo component (RISC protein) and cleaves mRNA in a sequence-specific manner, so that information on the mRNA is specified by the ribosome. Make it impossible to translate into protein. It is also considered that it can be used as a constituent of miRNA or also as a constituent of aptamer RNA, taking advantage of ribonuclease resistance and improved cell membrane permeability. Furthermore, it can be linked to other compounds to form a conjugate. Furthermore, the conjugate can be used as a constituent of a ribozyme.

  Based on these facts, this conjugate is utilized as a component of various RNA pharmaceuticals for treating diseases by inhibiting the function of genes such as antitumor agents and antiviral agents, taking advantage of the characteristics not found in natural nucleotides. Expected utility over natural nucleotides. That is, the conjugate is useful as a raw material or intermediate reagent in addition to such RNA pharmaceuticals.

  Those skilled in the art can appropriately determine the charge controllability, ribonuclease resistance, cell membrane permeability, charge control ability of oligoribonucleotides in this conjugate, and biological activities of various RNAs including oligoribonucleotides. It can be easily evaluated by referring to a well-known method of those skilled in the art at the time of filing this application.

  Hereinafter, in order to explain the disclosure of the present specification more specifically, specific examples will be described. The following examples are intended to illustrate the disclosure herein and are not intended to limit the scope thereof.

(Synthesis of amino-modified amidites)
In this example, according to the following scheme, the following compound 5 was synthesized as a monomer for cell adhesion motif (RGD motif) unit introduction.

6-trifluoroacetamido-hexanoic acid (3)
Under an argon atmosphere, 6-aminohexanoic acid (2) (0.60 g, 4.57 mmol) was dissolved in MeOH (15 mL). Subsequently, TEA (1.0 mL, 6.86 mmol) and CF ₃ COOEt (0.8 mL, 6.86 mmol) were added and stirred overnight at room temperature. The resulting reaction solution was concentrated under reduced pressure to obtain Compound 3 (0.45 g, 1.98 mmol, 43%).
¹ H NMR (400 MHz, CDCl ₃ ) δ 6.36 (s, 1H), 3.39 (dd, 2H, J = 6.9), 2.39 (t, 2H, J = 7.3), 1.70-1.60 (m, 4H), 1.16 -1.09 (m, 2H).

(S) -3-[(6-trifluoroacetamido) hexanamido] -1- (4,4'-dimethoxytrityloxy) -2-propanol (4)
Compound 3 (0.45 g, 1.98 mmol) was dissolved in DMF (5 mL) under an argon atmosphere. Subsequently, TEA (0.4 mL, 1.98 mmol), Compound 1 ^1,2 (0.65 g, 1.65 mmol) dissolved in DMF (5 mL), EDCl (0.38 g, 1.98 mmol), DMAP (0 .24 g, 1.98 mmol) was added and stirred at room temperature overnight. The product was extracted with ethyl acetate and H ₂ O, and the organic layer was washed with saturated aqueous sodium hydrogen carbonate solution and saturated brine, and dried over anhydrous sodium sulfate. The volatile solvent was concentrated under reduced pressure using an evaporator, and the residue was purified by silica gel column chromatography (chloroform: methanol = 30: 1) to obtain Compound 4 (0.78 g, 1.28 mmol, 78%).

(S) -3-[(6-trifluoroacetamido) hexanamido] -1- (4,4'-dimethoxytrityloxy) propyl-2-O-[(2-cyanoethyl) (N, N-diisopropylamino)] phosphoramidite (5)
Compound 4 (0.64 g, 1.06 mmol) was dissolved in CH ₂ Cl ₂ under an argon atmosphere. Subsequently, DIPEA (0.7 mL, 4.24 mmol), 2-CyanoethylN, N-diisopropylchlorophosphoramidite (0.5 mL, 2.12 mmol) were added, and the mixture was stirred at room temperature for 1 hour. The product was extracted with chloroform and H ₂ O, and the organic layer was washed with saturated aqueous sodium hydrogen carbonate solution and saturated brine, and dried over anhydrous sodium sulfate. The volatile solvent was concentrated under reduced pressure using an evaporator, and the residue was purified by silica gel column chromatography (hexane: ethyl acetate = 1: 2) to obtain Compound 5 (0.43 g, 0.54 mmol, 51%).
³¹ P NMR (242 MHz, CDCl ₃ ) δ 149.7, 149.0.

(Synthesis of solid support)
In this example, according to the following scheme, the following compounds were synthesized as solid phase carriers to which a monomer for introducing a cell adhesion motif unit was bound.

Compound 4 (0.10 g, 0.17 mmol), succinic anhydride (0.10 g, 1.00 mmol), and DMAP (61 mg, 0.50 mmol) synthesized in Example 1 were dissolved in pyridine (2 mL) overnight at room temperature. Stir. The product was extracted with ethyl acetate and H ₂ O, and the organic layer was washed with saturated aqueous sodium hydrogen carbonate solution and saturated brine, and dried over anhydrous sodium sulfate. The volatile solvent was concentrated under reduced pressure using an evaporator to obtain a succinyl compound. Subsequently, the succinyl compound was dissolved in DMF (1.7 mL) under an Ar atmosphere, EDC (32 mg, 0.17 mmol) and CPG resin (0.26 g, 0.04 mmol) were added, and the mixture was allowed to stand at room temperature for 3 days. . The CPG resin was transferred to a filter, washed with pyridine, and then added with pyridine (5 mL), DMAP (0.18 g, 1.47 mmol) and acetamide hydride (1.5 mL, 15.87 mmol) under an Ar atmosphere and allowed to stand overnight. . The CPG resin was transferred to a filter, washed with pyridine, EtOH, and MeCN, and then dried to obtain the desired solid phase carrier with an introduction efficiency of 25.8 μmol / g.

(Synthesis of spacer amidite)
In this example, according to the following scheme, the following compound 8 was synthesized as a monomer for introducing a spacer unit.

18-O-Dimethoxytritylhexaethyleneglycol (7)
Under an Ar atmosphere, Hexaethyleneglycol (6) (1.7 mL, 5.31 mmol) was dissolved in pyridine (30 mL), DMTrCl (1.36 g, 4.00 mmol) was added, and the mixture was stirred overnight at room temperature. The product was extracted with ethyl acetate and H ₂ O, and the organic layer was washed with saturated aqueous sodium hydrogen carbonate solution and saturated brine, and then dried over anhydrous sodium sulfate. The volatile solvent was concentrated under reduced pressure using an evaporator, and the residue was purified by silica gel column chromatography (chloroform: methanol = 20: 1) to obtain Compound 7 (1.49 g, 2.55 mmol, 48%).
¹ H NMR (600 MHz, CDCl ₃ ) δ 7.46 (d, 2H, J = 7.56), 7.35-7.33 (m, 4H), 7.28 (d, 2H, J = 7.56), 7.20 (t, 1H, J = 7.56), 6.83-6.80 (m, 4H), 3.78 (s, 6H), 3.70 (m, 2H,), 3.69-3.63 (m, 17H), 3.59 (t, 2H, J = 4.80), 3.22 (t , 2H, J = 5.46), 1.99 (s, 1H).

18-O-Dimethoxytritylhexaethyleneglycol-1-[(2-cyanoethyl) (N, N-diisopropylamino)] phosphoramidite (8)
Compound 7 (1.49 g, 2.55 mmol) was dissolved in CH ₂ Cl ₂ under an argon atmosphere. Subsequently, DIPEA (1.8 mL, 10.20 mmol), 2-CyanoethylN, N-diisopropylchlorophosphoramide (1.1 mL, 5.10 mmol) were added, and the mixture was stirred at room temperature for 1 hour. The product was extracted with chloroform and H ₂ O, and the organic layer was washed with saturated aqueous sodium hydrogen carbonate solution and saturated brine, and dried over anhydrous sodium sulfate. The volatile solvent was concentrated under reduced pressure using an evaporator, and the residue was purified by silica gel column chromatography (hexane: ethyl acetate = 1: 2) to obtain Compound 8 (1.08 g, 1.38 mmol, 54%).
³¹ P NMR (242 MHz, CDCl ₃ ) δ 149.9, 148.9.

(Synthesis of oligonucleotide derivatives)
Using a nucleic acid automatic synthesizer according to the phosphoramidite method, a cell adhesion motif unit-introducing monomer, a solid phase carrier, a spacer unit-introducing monomer and various nucleotides synthesized in Examples 1 to 3, and a 0.2 μmol scale The oligonucleotide derivative was synthesized with Base deprotection and cleaving from the resin were carried out by incubation at 55 ° C. for 4 hours in 1 mL of NH ₄ OH: EtOH = 3: 1 solution. The TBDMS group at the sugar moiety 2′-position was deprotected by incubation at 65 ° C. for 1.5 hours in a solution of 100 μL of DMSO and 125 μL of TEA · 3HF. Furthermore, purification was performed using 20% PAGE containing 7M urea to synthesize an oligonucleotide having the target sequence.

  In the following table, 0N1, 3 and 7 are chemically modified guide strands of RecQL1RNA, ON2, 4, 5 and 6 are natural and chemically modified passenger strands of RecQL1RNA, and ON8 is a natural guide strand of luciferase RNA. ON9 is the RGD-modified passenger chain of luciferase.

  In the table, a lower case base represents a 2′-OMe nucleotide, an underbar base represents a 2′-F nucleotide of ribose, p represents a monomer for introducing a PEG spacer unit, and X represents a cell adhesion motif unit. Represents a monomer for introducing a cell adhesion motif unit having cRGD as “•”, and “•” represents a phosphorothioate bond.

(Synthesis of cRGD conjugate oligonucleotide)
The oligonucleotide derivative (20 nmol) synthesized in Example 4 was dissolved in 320 μL of 0.1 M phosphate buffer (pH = 7.4), and 80 μL (400 nmol) of 50 mM 6-maleidohexaneicacid N-hydroxysuccinimideester (EMCS) / DMSO solution was dissolved. In addition, it was incubated overnight at room temperature. Maleimidated oligonucleotide was fractionated by HPLC, and 16 μL (80 nmol) of 50 mM cRGD (manufactured by Funakoshi Co., Ltd., structural formula is shown below) / DMSO solution was directly added to the collected solution. After incubating overnight at room temperature and linking the maleimide group of the oligoribonucleotide derivative and the thiol group of cRGD by Michael addition reaction, the reaction solution is purified by HPLC to obtain the desired cRGD oligonucleotide conjugate. It was.

(Observation with a fluorescence microscope)
Melanoma cells A2058 were seeded on a 35 mm glass bottom dish at 0.3 × 10 ⁵ cells / mL and cultured in a CO ₂ incubator for 24 hours. After removing the medium, the dish was washed with OPTI-MEM, and siRNA-conjugated M / OPTI-MEM solution was added using 250 nM of the oligoribonucleotide derivative prepared in Examples 4 and 5. The siRNA used was the following combination.

siRNA (1): ON3 / ON2: siRNA without cRGD motif unit and spacer unit (2): ON3 / ON4: siRNA without three spacer units with three cRGD motifs (3): ON3 / ON5: cRGD motif 3 SiRNA with 4 spacer units (4): ON3 / ON6: 3 cRGD motifs and 3 spacer units alternately

After siRNA was added to the dish, it was cultured in a CO ₂ incubator for 18 hours, and then Hoechst 33342 was added to a final concentration of 25 μg / mL. After culturing for 10 minutes in a CO ₂ incubator, the staining reagent was removed, and the dish was washed with PBS. PBS was added to the dish and the cells were observed with a fluorescence microscope. The results are shown in FIG.

  As shown in FIG. 4, it was found that the cellular uptake was improved by combining the cRGD motif and the spacer unit. In particular, siRNA (4) comprising three cRGD motifs and three spacer units alternately and having a structure in which the cRGD motifs are separated by spacer units showed a high intracellular uptake rate.

(Western blotting)
Melanoma cell A2058 was seeded on a 6-well plate at 0.3 × 10 ⁵ cells / mL and cultured in a CO ₂ incubator for 24 hours. After removing the medium, the 6-well plate was washed with OPTI-MEM, and a 50 nM or 250 nM siRNA conjugate / OPTI-MEM solution using the oligoribonucleotide derivatives synthesized in Examples 4 and 5 was added. After culturing for 18 hours in a CO ₂ incubator, whole cells were collected and lysis buffer (10 mM Tris-HCl (pH 7.4), 1% NP-40, 0.1% deoxycholicacid, 0.1% SDS, 150 mM NaCl, 1 mM EDTA, 1% Protease Inhibitor Cocktail) was added and allowed to stand on ice for 20 minutes.

siRNA (4): ON7 / ON2: siRNA without cRGD motif unit and spacer unit (5): ON8 / ON9: siRNA with three cRGD motifs and three spacer units alternately (6): ON7 / ON6: cRGD Alternating 3 motifs and 3 spacer units

  The obtained cell suspension was centrifuged at 4 ° C, 13000 rpm for 20 minutes, and the supernatant was recovered as a protein solution. The concentration in the solution was measured with DC protein assay kit (Biorad, Hercules). 10 μg of protein solution was separated by 12.5% SDS-PAGE and blotted on a PVDF membrane. The membrane was blocked with a 5% skim milk solution and allowed to stand overnight at 4 ° C. in a RecQL1 antibody solution. The membrane was washed, and an HRP-conjugated secondary antibody was added and permeated for 1 hour, and then Amersham ECL Plus Western Blotting Detection Reagents (GE Healthcare) was added directly on the membrane to detect RecQL1 protein. The results are shown in FIG.

  As shown in FIG. 5, when siRNA (4) (si-RecQL1) and siRNA (6) (si-RecQL1 + RGD) were compared, a decrease in protein expression level was observed only at + RGD. This indicates that siRNA cannot be taken into cells without RGD modification, whereas siRNA was efficiently taken into cells by RGD modification with this reagent. Furthermore, when siRNA (5) (si-Luciferase + cRGD) and siRNA (6) (si-RecQL1 + RGD) are compared, there is no change in the expression level of the protein in Luciferase. This revealed that si-RecQL1 + RGD acts selectively on RecQL1 mRNA, and the expression of protein was suppressed by the RNAi activity of si-RecQL1. From the above, it was shown that the RGD modification mode by this reagent enables efficient intracellular uptake of siRNA and suppression of target gene expression by RNAi activity through RISC formation.

Claims (9)

An RNA introduction reagent,
Two or more cellular adhesion motif units;
One or more spacer units;
With
The RNA introduction reagent, wherein the one or more spacer units separate the two or more cellular adhesion motif units from each other.   The reagent according to claim 1, wherein the cellular adhesion motif unit comprises a peptide.   The reagent according to claim 1 or 2, wherein the cell adhesion motif unit comprises an RGD peptide.   The reagent according to any one of claims 1 to 3, wherein the spacer unit includes a polyoxyalkylene group.     The reagent according to any one of claims 1 to 4, wherein the spacer unit separates the two or more cell adhesion motif units by at least 2 nm or more. A peptide derivative comprising:
Two or more peptide units;
One or more non-peptidic units;
With
The peptide unit has any two or more amino acid residues,
The derivative, wherein the one or more non-peptide units separate the two or more peptide units from each other.   6. The derivative according to claim 5, wherein the peptide unit comprises an RGD motif and the non-peptide unit comprises a polyalkyleneoxy group. An oligoribonucleotide derivative comprising:
Oligoribonucleotides;
A cell adhesion region comprising two or more cell adhesion motif units and one or more spacer units, wherein the one or more spacer units separate the two or more cell adhesion motif units from each other;
A derivative comprising. The derivative according to claim 8, wherein the oligoribonucleotide includes a nucleoside derivative represented by the following formula (1) or (2).
(In Formula (1), R ¹ represents a hydroxyl group, a hydroxyl group in which a hydrogen atom is substituted with an alkyl group or an alkenyl group, or a protected group, and in Formula (2), X represents a halogen atom. In 1) and formula (2), R ² and R ⁴ may be the same or different from each other, and are a hydrogen atom, a hydroxyl protecting group, a phosphate group, a protected phosphate group, or —P (═O). _n R ⁵ R ⁶ (n represents 0 or 1, and R ⁵ and R ⁶ may be the same or different from each other, and represent a hydrogen atom, a hydroxyl group, a protected hydroxyl group, a mercapto group, a protected mercapto group, a lower group, An alkoxy group, a cyano lower alkoxy group, an amino group, or a substituted amino group, provided that when n is 1, both R ⁵ and R ⁶ are not hydrogen atoms. R ³ is an NHR ⁷ having a linking group (R ⁷ is a hydrogen atom) Represents an alkyl group, an alkenyl group or an amino group protecting group.), Represents an azido group, an amidino group or a guanidino group, and B represents a purin-9-yl group, a 2-oxo-pyrimidin-1-yl group or a substituted group. It represents either a purin-9-yl group or a substituted 2-oxo-pyrimidin-1-yl group.)