Sirna capable of suppressing expression of α-synuclein

Abstract

The purpose of the present invention is, for example, to provide novel siRNA that has a gene expression suppressing effect and / or an RNA interference effect with respect to mRNA encoding SNCA.　Provided are an oligonucleotide having a knockdown effect on SNCA mRNA, the oligonucleotide comprising an antisense strand region containing a target corresponding sequence complementary to a target sequence in mRNA encoding SNCA, and a sense strand region containing a nucleotide sequence complementary to the target corresponding sequence in the antisense strand region, or a pharmaceutically acceptable salt of the oligonucleotide etc.

Description

siRNA that inhibits the expression of alpha-synuclein

[0001] The present invention relates to siRNAs having an RNA interference effect and / or a gene expression-suppressing effect on mRNA encoding α-synuclein (SNCA), uses of the siRNAs, methods for suppressing SNCA gene expression using the siRNAs, and pharmaceuticals containing the siRNAs.

[0002] siRNA against mRNA encoding α-synuclein protein can suppress SNCA mRNA expression and inhibit the accumulation of intracellular insoluble inclusions containing α-synuclein. It is known that insoluble and phosphorylated intracellular insoluble inclusions containing α-synuclein protein are expressed in the neural tissues of patients with α-synucleinopathies such as Parkinson's disease, Parkinson's disease dementia, Lewy body dementia, multiple system atrophy, and pure autonomic failure. Therefore, it is expected that these diseases can be treated by suppressing the expression of α-synuclein protein, which serves as a substrate for the insoluble inclusions, using siRNA.

[0003] α-Synuclein is a soluble protein belonging to the synuclein protein family (Non-Patent Document 1). It is primarily expressed in neurons and plays a role in regulating synaptic transmission at presynaptic terminals (Non-Patent Document 2). α-Synuclein forms insoluble inclusions called Lewy bodies in neurons, which are a pathological characteristic of Parkinson's disease, Parkinson's disease dementia, and dementia with Lewy bodies. α-Synuclein also forms insoluble inclusions called glial cytoplasmic inclusions in oligodendrocytes, which are a pathological characteristic of multiple system atrophy. Therefore, it has been suggested that α-synuclein protein that has become oligomerized, phosphorylated, or insolubilized for some reason may be the cause of α-synucleinopathies.

[0004] Neurodegenerative diseases (α-synucleinopathies) in which the expression of insoluble inclusions containing α-synuclein is observed include, for example, Parkinson's disease, Parkinson's disease dementia, dementia with Lewy bodies, multiple system atrophy, and pure autonomic failure. It is known that α-synuclein gene mutations or gene duplications increase the risk of Parkinson's disease (Non-Patent Document 3). It is also known that conditional knockout of α-synuclein in adulthood does not result in significant changes (Non-Patent Document 4). Therefore, suppressing α-synuclein expression using siRNA is expected to be useful in treating α-synucleinopathies.

[0005] Patent Document 1 discloses that administration of SNCA siRNA AD-21822 into the brain parenchyma of mice resulted in a decrease in SNCA mRNA expression near the site of administration. However, Patent Document 1 shows the results of continuous administration of siRNA using an osmotic pump, and also shows that the degree of decrease in SNCA mRNA expression is lower 1 mm caudal to the administration site.

[0006] Patent Document 2 discloses antisense oligonucleotide compounds that target SNCA mRNA. It discloses that intracerebroventricular administration of Compound No. 763391 and Compound No. 827599 to human SNCA PAC transgenic mice or intrathecal administration to non-human primates resulted in a decrease in SNCA mRNA expression in the brain.

[0007] One method for inhibiting the expression of a target gene in a cell, tissue, or individual involves introducing double-stranded RNA into the cell, tissue, or individual. The introduction of double-stranded RNA degrades mRNA that is homologous to the double-stranded RNA, inhibiting the expression of the target gene. This effect is called "RNA interference" or "RNAi." Small interfering RNA (siRNA), a double-stranded RNA consisting of 21 nucleotides in each of the sense and antisense strands and with a 2-nucleotide overhang at the 3' end, has been reported to have RNA interference effects in cultured vertebrate cells (see, for example, Non-Patent Document 5).

[0008] Although siRNA is useful for identifying gene functions, screening cell lines suitable for producing useful substances, and regulating genes involved in diseases, it has the drawbacks of being easily degraded by RNases, making it difficult to maintain its activity in the body and requiring high synthesis costs. Therefore, various chemical modifications have been attempted to develop oligonucleotides that are highly stable against RNases, can be produced inexpensively, and retain RNAi activity.

[0009] Phosphorothioate (PS) bonds, in which the non-bridging oxygen atom of the phosphate group of a phosphodiester bond in an oligonucleotide is replaced with a sulfur atom, are known to be resistant to nucleases. However, it has been reported that increasing the number of PS bonds in an oligonucleotide is undesirable because it leads to thermodynamic instability of the double-stranded RNA and binding to nonspecific proteins (e.g., Non-Patent Document 6).

[0010] Furthermore, siRNAs employing sugar-modified nucleosides such as DNA, 2'-O-methylnucleosides, 2'-deoxy-2'-fluoronucleosides, and 2'-O,4'-C bridged nucleosides as nucleosides are disclosed in, for example, Patent Documents 3, 4, and 5.

[0011] Attempts have also been made to obtain stable siRNA by replacing natural RNA with modified RNA. For example, numerous derivatives have been reported in which the 2'-OH group of RNA is alkylated to form 2'-O-alkylnucleosides, which prevent the siRNA from becoming a substrate for RNase. 2'-O-methyl nucleotides are naturally occurring modified nucleotides found in tRNA and have been studied since the early days of antisense research (e.g., Non-Patent Document 7).

[0012] It has been reported that RNAi activity is weakened or completely lost when all RNA in either or both of the sense and antisense strands of siRNA is substituted with 2'-O-methyl nucleotides (e.g., Non-Patent Document 8, Non-Patent Document 9, Non-Patent Document 10, Non-Patent Document 11, etc.). It has also been reported that when three consecutive 2'-O-methyl nucleotides are introduced into the sense strand, no decrease in activity is observed, but when introduced into the antisense strand, a decrease in activity is observed, and that the activity is particularly marked when introduced into the 5' end of the sense strand (e.g., Non-Patent Document 12).

[0013] Oligonucleotides containing 2'-deoxy-2'-fluoronucleotides (2'-F or 2'-F RNA), which are artificially synthesized modified RNAs, preferentially form the same N-type conformation as ribonucleotides and have high affinity with RNA. However, oligonucleotides containing phosphodiester bonds are not nuclease resistant, so they must be made nuclease resistant by using phosphorothioate bonds (e.g., Non-Patent Document 13).

[0014] ENA (2'-O,4'-C-ethylene-bridged nucleic acids) is a modified nucleic acid that is stable against nucleases. However, it has been reported that when ENA is introduced into the two nucleotides in the overhanging region at the 3' end of one or both of the sense strand and antisense strand of siRNA, RNAi activity is reduced (e.g., Non-Patent Document 14).

[0015] There have been many reports on siRNAs that combine multiple modified nucleic acids. For example, it has been reported that by alternately introducing 2'-O-methyl nucleotides and 2'-F into the sense and antisense strands of siRNA, RNAi activity comparable to or greater than that of unmodified siRNA was obtained, and the resulting siRNA was relatively stable in serum (e.g., Non-Patent Document 15).

[0016] Double-stranded oligonucleotides combining DNA, 2'-O-methyl RNA, and 2'-F have been reported, and in particular, siRNAs in which the 14th position from the 5' end of the antisense strand is 2'-F have been reported (e.g., Patent Documents 6, 7, 8, and 9). Double-stranded oligonucleotides combining DNA, 2'-O-methyl RNA, 2'-F RNA, and LNA (2'-O,4'-C-methylene-bridged nucleic acids) have also been reported, and in particular, the activity of siRNAs in which the 2nd or 14th position from the 5' end of the antisense strand is 2'-F, DNA, or LNA has been reported (e.g., Patent Documents 4, 10, and 11).

[0017] International Publication No. WO 2012 / 027713 International Publication No. WO 2019 / 164562 U.S. Patent No. US9,399,775 U.S. Patent No. US9,796,974 U.S. Patent No. US10,612,024 International Publication No. WO 2012 / 058210 Pamphlet International Publication No. WO 2013 / 074974 Pamphlet International Publication No. WO 2016 / 028649 Pamphlet International Publication No. WO 2018 / 185241

[0018] Cell Death Dis. 3, e350 (2012). J Parkinson's Dis. 2015; 5(4): 699-713 Cold Spring Herb Perspective Med. 2018 Jun 1; 8(6): a024109. Neurobiol Aging. 2020 Jul; 91: 76-87 Nature, 2001, Vol. 411, pp. 494-498 Antisense Nucleic Acid Drug Development, 2000, Vol. 10, p. 117-121 Nucleic Acids Research, 1987, Vol. 15, pp. 6131-6148 EMBO Journal, 2001, Vol. 20, pp. 6877-6888 RNA, Vol. 9, 2003, pp. 1034-1048 Biochemistry, 2003, Vol. 42, pp. 7967-7975 Nucleic Acids Research, 2003, Vol. 31, pp. 2705-2716 Journal of Medicinal Chemistry, 2005, Vol. 48, p. 4247-4253 Journal of Medicinal Chemistry, 1993, Vol. 36, pp. 831-841 Antisense and Nucleic Acid Drug Development, 2002, Vol. 12, pp. 301-309 Journal of Medicinal Chemistry, 2005, Vol. 48, pp. 901-904

[0019] There is a need for pharmacologically active SNCA siRNA that can suppress the accumulation of intracellular insoluble inclusion bodies containing α-synuclein and prevent, ameliorate, and / or treat α-synucleinopathies such as Parkinson's disease, Parkinson's disease dementia, dementia with Lewy bodies, multiple system atrophy, pure autonomic failure, etc. There is also a need for SNCA siRNA that has low toxicity to cells and excellent stability in the blood and drug delivery properties.

[0020] An object of the present invention is to provide a novel siRNA that has RNA interference activity and / or gene expression inhibitory activity (these activities are also referred to as knockdown activity) against mRNA encoding SNCA. Another object of the present invention is to provide a novel SNCA siRNA with improved cytotoxicity, stability, and / or drug delivery. A further object of the present invention is to provide a novel pharmaceutical composition for treating or preventing diseases that can be treated or prevented by inhibiting the expression of SNCA mRNA.

[0021] The present inventors conducted extensive research and found that SNCA siRNA targeting a base sequence at a specific position in SNCA mRNA has RNA interference and / or gene expression-inhibiting activity, and that the cytotoxicity observed with the target sequence of Patent Document 1 was not observed with the target sequence of the present invention. Furthermore, the present inventors found that siRNAs containing oligonucleotides appropriately combining DNA, RNA, 2'-O-methyl RNA, 2'-F RNA, LNA, ENA, and 3'-O-methyl RNA have RNA interference and / or gene expression-inhibiting activity and are stable in vivo. Furthermore, the present inventors found that drug delivery can be improved by adding a fatty acid unit for transport derived from stearic acid or the like to siRNA. Furthermore, the present inventors discovered that SNCA mRNA expression can be inhibited using SNCA siRNA, and completed the present invention.

[0022] That is, the present invention provides the following: [1] An oligonucleotide or a pharmaceutically acceptable salt thereof, which has an effect of knocking down α-synuclein mRNA and comprises the following antisense strand region (A) and sense strand region (B): (A) an antisense strand region consisting of a target corresponding sequence substantially complementary to a target sequence consisting of 18 to 21 consecutive bases in a region between nucleotide numbers 676 and 708 of SEQ ID NO: 1, wherein the 5'-end of the target corresponding sequence is a nucleoside or phosphonate unit, the base of the nucleoside or phosphonate unit at the 5'-end of the target corresponding sequence being adenine, thymine, or uracil, the base of a nucleoside substantially complementary to the nucleoside at the 3'-end of the target sequence, and wherein an overhang structure of 5 bases or less may be added to the 5'-end and / or 3'-end of the target corresponding sequence; (B) A sense strand region consisting of 18 to 31 bases in length, which contains a nucleotide sequence substantially complementary to the target-corresponding sequence of the antisense strand region, and which may further comprise an overhang structure of 5 bases or less at the 5'-end and / or 3'-end of the nucleotide sequence. [2] The oligonucleotide or a pharmaceutically acceptable salt thereof according to [1], wherein the target sequence is a 19-base nucleotide sequence consisting of nucleotides 676 to 698 or 686 to 708 of SEQ ID NO: 1. [3] The oligonucleotide or a pharmaceutically acceptable salt thereof according to [1], wherein the target sequence is a 19-base nucleotide sequence consisting of nucleotides 678 to 696 or 688 to 706 of SEQ ID NO: 1. [4] The oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of [1] to [3], wherein the nucleoside or phosphonic acid base at the 5'-end of the target-corresponding sequence of the antisense strand region is adenine or uracil. [5] The oligonucleotide or pharmaceutically acceptable salt thereof according to any one of [1] to [4], wherein the target-corresponding sequence of the antisense strand region is a nucleotide sequence that is completely complementary to a target sequence consisting of 19 bases from nucleotide numbers 678 to 696 or nucleotide numbers 688 to 706 of SEQ ID NO: 1.[6] The oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of [1] to [5], wherein the 5'-end of the target-corresponding sequence in the antisense strand region is a phosphonate unit, and the phosphonate unit is any one of the following formulas:

[0023]

[0024] (In the formula, the end of the dashed line represents a phosphodiester bond which may be chemically modified with the 5'-phosphate group of the adjacent nucleotide.) [7] The oligonucleotide according to any one of [1] to [6], wherein at least one of the sugars and / or phosphodiester bonds constituting the oligonucleotide is modified, or a pharmaceutically acceptable salt thereof. [8] The oligonucleotide according to [7], wherein the sugar constituting the oligonucleotide is D-ribofuranose, and the sugar modification is modification of the hydroxyl group at the 2'-position of D-ribofuranose, or a pharmaceutically acceptable salt thereof. [9] The oligonucleotide according to [8], wherein the sugar modification is 2'-deoxylation, 2'-O-alkylation, 2'-O-alkoxyalkylation, 2'-halogenation, and / or 2'-O,4'-C-alkylenation of D-ribofuranose, or a pharmaceutically acceptable salt thereof.

[10] The oligonucleotide or pharmaceutically acceptable salt thereof according to [9], wherein the 2'-O-alkylation is 2'-O-methylation, the 2'-O-alkoxyalkylation is 2'-O-methoxyethylation, the 2'-halogenation is 2'-fluorolation, and the 2'-O,4'-C-alkylenation is 2'-O,4'-C-methylenation and / or 2'-O,4'-C-ethylenation.

[11] The oligonucleotide or pharmaceutically acceptable salt thereof according to any one of [7] to

[10] , wherein the phosphodiester bond is modified by phosphorothioate.

[12] The oligonucleotide or pharmaceutically acceptable salt thereof according to any one of [7] to

[11] , wherein D-ribofuranose in the 3'-terminal nucleoside in the sense strand region and / or antisense strand region is 3'-O-alkylated.

[13] The oligonucleotide or pharmaceutically acceptable salt thereof according to

[12] , wherein the 3'-O-alkylation is 3'-O-methylation.

[14] The oligonucleotide or pharmaceutically acceptable salt thereof according to any one of [1] to

[13] , wherein an overhang structure of 3 bases or less is added to the 5'-end and / or 3'-end of the sense strand region and / or the antisense strand region.

[15] The oligonucleotide or pharmaceutically acceptable salt thereof according to

[14] , wherein the overhang structure consists of 2 bases.

[16] The oligonucleotide or a pharmaceutically acceptable salt thereof according to

[15] , wherein the overhang structure is a nucleoside having two consecutive thymines or a nucleoside having two consecutive uracils.

[17] The oligonucleotide or a pharmaceutically acceptable salt thereof according to

[16] , wherein the overhang structure is two consecutive U (M) added to the 3'-end of the antisense strand region.

[18] The oligonucleotide or a pharmaceutically acceptable salt thereof according to [1], wherein the sense strand region consists of an oligonucleotide represented by formula (I) below, the antisense strand region consists of an oligonucleotide represented by formula (II) below, and further has the characteristics shown in (a) to (g) below: Sense strand region: 5' S. Ｏ５ -S ａ -S １９ -S １８ -S １７ -S １６ -S １５ -S １４ -S １３ -S １２ -S １１ -S １０ -S ９ -S ８ -S ７ -S ６ -S ５ -S ４ -S ３ -S ２ -S １ -S Ｏ３ 3'(I) Antisense strand region: 5' A Ｏ５ -A １ -A ２ -A ３ -A ４ -A ５ -A ６ -A ７ -A ８ -A ９ -A １０ -A １１ -A １２ -A １３ -A １４ -A １５ -A １６ -A １７ -A １８ - A １９ -A ａ -A Ｏ３3'(II) (a)S １ is a 3'-modified nucleoside, and S ２ ~S １９ represents one nucleoside, each independently being 2'-OMe RNA, 2'-F RNA or DNA; S ａ represents 0 to 3 nucleosides, each nucleoside independently representing 2'-OMe RNA, 2'-F RNA, or DNA; S Ｏ３ and S Ｏ５ each independently represent 0 to 3 nucleosides, each of which independently represents 2'-OMe RNA, 2'-F RNA, DNA, or a 2'-O,4'-C-bridged modified nucleoside. The bond between each nucleoside represents a phosphodiester bond which may be chemically modified; (b) A １１ , A １２ , A １３ , A １４ and A １５ represents one nucleoside, at least one of which is DNA, RNA, 2'-O,4'-C-bridged modified nucleoside or 2'-MOE RNA, and the others are 2'-OMe RNA or 2'-F RNA; A １ represents one nucleoside or phosphonic acid unit, A １ is a nucleoside, it represents 2'-OMe RNA, 2'-F RNA or DNA, and A ２ ~A １０ and A １６ ~A １９ represents one nucleoside, each independently representing 2'-OMe RNA, 2'-F RNA, or DNA; A ａ represents 0 to 3 nucleosides, each nucleoside independently representing 2'-OMe RNA, 2'-F RNA, or DNA; A Ｏ３ and A Ｏ５ each independently represent 0 to 3 nucleosides, each of which independently represents 2'-OMe RNA, 2'-F RNA, DNA, or a 2'-O,4'-C-bridged modified nucleoside. The bond between each nucleoside represents a phosphodiester bond which may be chemically modified; (c) A ２ ~Aａ The nucleotide sequence between A and B consists of a nucleotide sequence substantially complementary to the target sequence, １ is a nucleoside or phosphonate unit, the base of the nucleoside or phosphonate unit is a nucleoside base, adenine, thymine, or uracil, that is substantially complementary to the corresponding nucleoside of the target sequence; A Ｏ３ and A Ｏ５ (d) S １ ~S ａ and the nucleotide sequence between A １ ~A ａ The nucleotide sequences between are substantially complementary nucleotide sequences and form a double-stranded structure; (e) S Ｏ５ and A Ｏ３ If both exist, S Ｏ５ and A Ｏ３ are nucleotide sequences that are not complementary to each other; (f) S Ｏ３ and A Ｏ５ If both exist, S Ｏ３ and A Ｏ５ are nucleotide sequences that are not complementary to each other; and (g) the 5'-position of the 5'-terminal nucleoside and / or the 3'-position of the 3'-terminal nucleoside in the sense strand region and / or the 2'-position of the 3'-terminal nucleoside when the 3'-terminal nucleoside is a 3'-modified nucleoside in the sense strand region and / or the antisense strand region may be chemically modified.

[19] In formula (II), S １

[20] The oligonucleotide or a pharmaceutically acceptable salt thereof according to

[18] or

[19] , wherein the 2'-O,4'-C-bridged modified nucleoside is LNA or ENA.

[21] The oligonucleotide or a pharmaceutically acceptable salt thereof according to

[18] or

[19] , wherein in formula (II), A １１ , A １２ , A １３ , A １４ and A １５

[22] The oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of

[18] to

[20] , wherein two or more of A may be the same or different, and are DNA, RNA, a 2'-O,4'-C-bridged modified nucleoside, or a 2'-MOE RNA. １４

[23] The oligonucleotide or a pharmaceutically acceptable salt thereof according to

[21] , wherein A is an RNA or a 2'-O,4'-C-bridged modified nucleoside. １３ is a 2'-O,4'-C-bridged modified nucleoside or a 2'-OMe RNA, and A １４

[24] The oligonucleotide or a pharmaceutically acceptable salt thereof according to

[22] , wherein A is RNA. １１ -A １２ -A １３ -A １４ -A １５ is one of the following: A １１ (2'-OMe RNA)-A １２ (2'-OMe RNA)-A １３ (ENA)-A １４ (RNA)-A １５ (2'-OMe RNA), A １１ (2'-F RNA)-A １２ (2'-OMe RNA)-A １３ (ENA)-A １４ (RNA)-A １５ (2'-OMe RNA), A １１ (2'-OMe RNA)-A １２ (2'-F RNA)-A １３ (ENA)-A １４ (RNA)-A １５ (2'-OMe RNA), A １１ (2'-OMe RNA)-A １２ (2'-OMe RNA)-A １３ (LNA)-A １４ (RNA)-A １５ (2'-OMe RNA), A １１ (2'-F RNA)-A １２ (2'-OMe RNA)-A １３ (LNA)-A １４ (RNA)-A１５ (2'-OMe RNA), A １１ (2'-OMe RNA)-A １２ (2'-F RNA)-A １３ (LNA)-A １４ (RNA)-A １５ (2'-OMe RNA), A １１ (2'-F RNA)-A １２ (2'-OMe RNA)-A １３ (2'-OMe RNA)-A １４ (RNA)-A １５ (2'-OMe RNA), or A １１ (2'-OMe RNA)-A １２ (2'-F RNA)-A １３ (2'-OMe RNA)-A １４ (RNA)-A １５ (2'-OMe RNA), or a pharmaceutically acceptable salt thereof.

[25] The oligonucleotide according to

[23] , wherein A １２ is DNA, and A １４

[26] The oligonucleotide or a pharmaceutically acceptable salt thereof according to

[22] , wherein A is a 2'-O,4'-C-bridged modified nucleoside. １１ -A １２ -A １３ -A １４ -A １５ is one of the following: A １１ (2'-F RNA)-A １２ (DNA)-A １３ (2'-OMe RNA)-A １４ (ENA)-A １５ (2'-OMe RNA), A １１ (2'-OMe RNA)-A １２ (DNA)-A １３ (2'-OMe RNA)-A １４ (ENA)-A １５ (2'-OMe RNA), A １１ (2'-F RNA)-A １２ (DNA)-A １３ (2'-OMe RNA)-A １４ (LNA)-A １５(2'-OMe RNA), or A １１ (2'-OMe RNA)-A １２ (DNA)-A １３ (2'-OMe RNA)-A １４ (LNA)-A １５ (2'-OMe RNA), or a pharmaceutically acceptable salt thereof.

[27] In formula (II), A ２ , A ６ and A １６

[28] The oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of

[18] to

[26] , wherein A is 2'-F RNA. ８ , A ９ and A １０ one or two nucleosides selected from the group consisting of: ３ ~A ５ , A ７ , A １７ ~A １９ and A ａ

[29] The oligonucleotide or a pharmaceutically acceptable salt thereof according to

[27] , wherein the nucleoside represented by the formula (II) is 2'-OMe RNA. ２ , A ６ , A ８ , A １０ and A １６ is 2'-F RNA, and A ３ ~A ５ , A ７ , A ９ , A １７ ~A １９ and A ａ

[30] The oligonucleotide or a pharmaceutically acceptable salt thereof according to

[28] , wherein the nucleoside represented by the formula (II) is 2'-OMe RNA. １

[31] The oligonucleotide or a pharmaceutically acceptable salt thereof according to

[29] , wherein A is 2'-OMe RNA. １

[29] The oligonucleotide or a pharmaceutically acceptable salt thereof according to

[29] , wherein:

[0025]

[0026] (In the formula, the end of the dashed line represents a phosphodiester bond which may be chemically modified with the 5'-phosphate group of the adjacent nucleotide.)

[32] In formula (I), S １１ , S １２ , S １３ and S １５

[33] The oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of

[18] to

[31] , wherein S is 2'-F RNA. ２ ~S １０ , S １４ , S １６ ~S １９ and S ａ is 2'-OMe RNA.

[34] The oligonucleotide or pharmaceutically acceptable salt thereof according to any one of

[18] to

[33] , wherein when RNA is used in formula (II), the bond between the RNA and the nucleoside adjacent to it on the 3' side is a phosphorothioate bond.

[35] The oligonucleotide or pharmaceutically acceptable salt thereof according to any one of

[18] to

[34] , wherein each internucleoside bond in 2 to 5 nucleotides from the 5' end and 3' end of the oligonucleotide is a phosphorothioate bond.

[36] S Ｏ３ , S Ｏ５ , A Ｏ５ and A Ｏ３

[37] The oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of

[18] to

[35] , wherein the number of nucleosides is 0 to 2. Ｏ３

[38] The oligonucleotide or a pharmaceutically acceptable salt thereof according to

[36] , wherein the number of nucleosides is 0. Ｏ３ , S Ｏ５ and A Ｏ５ The number of nucleosides in A is 0, Ｏ３

[39] The oligonucleotide or pharmaceutically acceptable salt thereof according to any one of [1] to

[38] , wherein the antisense strand region and the sense strand region each form a double-stranded oligonucleotide as independent oligonucleotides.

[40] The oligonucleotide or pharmaceutically acceptable salt thereof according to any one of [1] to

[38] , wherein the 5'-terminal nucleoside of the antisense strand region and the 3'-terminal nucleoside of the sense strand region are linked by a linker structure.

[41] The oligonucleotide or pharmaceutically acceptable salt thereof according to any one of [1] to

[38] , wherein the linker structure is represented by the following formula:

[0027]

[0028] [In the formula, the end of the dashed line represents a phosphodiester or phosphorothioate bond between the oxygen atom bonded to the phenyl group and the 5'-phosphate group of a nucleotide other than the phosphonate unit at the 5'-end of the adjacent antisense strand region, and the methylene group at the other end represents a phosphodiester or phosphorothioate bond between the 3'-phosphate group (the 2'-phosphate group in a nucleotide modified at the 3'-position) of a nucleotide at the 3'-end of the adjacent sense strand region.]

[42] The oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of [1] to

[41] , wherein the 5'-position of the 5'-terminal nucleoside of the oligonucleotide and / or the 3'-position of the 3'-terminal nucleoside, or the 2'-position of the 3'-terminal nucleoside when the 3'-terminal nucleoside is a 3'-modified nucleoside, is chemically modified with a fatty acid unit.

[43] The oligonucleotide or a pharmaceutically acceptable salt thereof according to

[42] , wherein the fatty acid unit is any one of units represented by the following formulae:

[0029]

[0030] [In the formula, the tip of the dashed line represents a phosphodiester bond with the 5'-terminal phosphate group and / or the 3'-terminal phosphate group (the 2'-position phosphate group in the case of a nucleotide modified at the 3'-position) of an adjacent nucleotide.]

[44] The oligonucleotide or a pharmaceutically acceptable salt thereof according to

[43] , wherein the fatty acid unit is a unit represented by the following formula:

[0031]

[0032] [In the formula, the tip of the dashed line represents a phosphodiester bond with the 5'-terminal phosphate group and / or the 3'-terminal phosphate group (the 2'-terminal phosphate group in a nucleotide modified at the 3'-position) of an adjacent nucleotide.]

[45] The oligonucleotide or a pharmaceutically acceptable salt thereof according to [1], wherein the sense strand region excluding the overhang structure and the antisense strand region excluding the overhang structure are represented by any of the following formulae (I-1) to (I-6), the sense strand region and the antisense strand region each form a double-stranded oligonucleotide as independent oligonucleotides, and the sense strand region and the antisense strand region each independently may contain an overhang structure, and the 3'-terminal and / or 5'-terminal nucleotide of the sense strand region and / or the antisense strand region may be chemically modified. (Formula) (I-1) Sense strand region 5' U(M)^C(M)^U(M)G(M)C(F)U(M)G(F)A(F)C(F)A(M)G(M)A(M)U(M)G(M)U(M)U(M)C(M)^C(M)^A(3M) 3 Antisense strand region 5' VPu^G(F)^G(M)A(F)A(M)C(F)A(M)U(M)C(F)U(M)G(M)T(D)C(M)A(E)G(M)C(F)A(M)G(M)^A(M) 3' (I-2) Sense strand region 5' G(M)^A(M)^U(M)G(M)U(F)U(M)C(F)C(F)A(F)U(M)C(M)C(M)U(M)G(M)U(M)A(M)C(M)^A(M)^A(3M) 3' Antisense strand region 5' VPu^U(F)^G(M)U(F)A(M)C(F)A(M)G(M)G(F)A(M)U(M)G(D)G(M)A(E)A(M)C(F)A(M)U(M)^C(M) 3' (I-3) Sense strand region 5' U(M)^C(M)^U(M)G(M)C(F)U(M)G(F)A(F)C(F)A(M)G(M)A(M)U(M)G(M)U(M)U(M)C(M)^C(M)^A(3M) 3' Antisense strand region 5' VPu^G(F)^G(M)A(M)A(M)C(F)A(M)U(M)C(F)U(M)G(F)U(M)C(M)A(R)^G(M)C(F)A(M)G(M)^A(M) 3' (I-4)Sense strand region 5' G(M)^A(M)^U(M)G(M)U(F)U(M)C(F)C(F)A(F)U(M)C(M)C(M)U(M)G(M)U(M)A(M)C(M)^A(M)^A(3M) 3' Antisense strand region 5' VPu^U(F)^G(M)U(M)A(M)C(F)A(M)G(M)G(F)A(M)U(F)G(M)G(M)A(R)^A(M)C(F)A(M)U(M)^C(M) 3' (I-5) Sense strand region 5' G(M)^A(M)^U(M)G(M)U(F)U(M)C(F)C(F)A(F)U(M)C(M)C(M)U(M)G(M)U(M)A(M)C(M)^A(M)^A(3M) 3' Antisense strand region 5' EPu^U(F)^G(M)U(F)A(M)C(F)A(M)G(M)G(F)A(M)U(M)G(D)G(M)A(E)A(M)C(F)A(M)U(M)^C(M) 3' (I-6) Sense strand region 5' G(M)^A(M)^U(M)G(M)U(F)U(M)C(F)C(F)A(F)U(M)C(M)C(M)U(M)G(M)U(M)A(M)C(M)^A(M)^A(3M) 3' Antisense strand region 5' Vpuam^U(F)^G(M)U(F)A(M)C(F)A(M)G(M)G(F)A(M)U(M)G(D)G(M)A(E)A(M)C(F)A(M)U(M)^C(M) 3' [In the formula, for the bases of the nucleic acid, A represents adenine, U represents uracil, G represents guanine, and C represents cytosine (however, when C is ENA, it represents 2'-O,4'-C-ethylene-bridged-5-methylcytidine), for the nucleic acid, (R) represents RNA, (D) represents DNA, (M) represents 2'-OMe RNA, (3M) represents 3'-OMe RNA, (F) represents 2'-F RNA, and (E) represents ENA, and for the phosphonic acid unit, VPu, EPu, and VPuam represent the following formulae, respectively.

[0033]

[0034] (In the formula, the end of the dashed line indicates a phosphorothioate bond (-P(=S)(OH)-) with the 5'-phosphate group of the 5'-terminal nucleotide of the adjacent sense strand region.) In addition, in the formula, "^" indicates that the internucleoside bond is a phosphorothioate bond (-P(=S)(OH)-), and unless otherwise specified, indicates that the internucleoside bond is a phosphodiester bond (-P(=O)(OH)-). However, even if not otherwise specified, two internucleoside bonds in the three nucleosides from the 5'-end and 3'-end of the sense strand region, and two internucleoside bonds in the three nucleosides from the 5'-end and three internucleoside bonds in the four nucleosides from the 3'-end of the antisense strand region are phosphorothioate bonds.

[46] The oligonucleotide or pharmaceutically acceptable salt thereof according to

[45] , wherein an overhang structure of 3 bases or less is added to the 5'-end and / or 3'-end of the sense strand region and / or the antisense strand region.

[47] The oligonucleotide or pharmaceutically acceptable salt thereof according to

[46] , wherein the overhang structure is 2 bases.

[48] The oligonucleotide or pharmaceutically acceptable salt thereof according to

[47] , wherein the overhang structure is a nucleoside containing two consecutive thymines or a nucleoside containing two consecutive uracils.

[49] The oligonucleotide or pharmaceutically acceptable salt thereof according to

[48] , wherein the overhang structure is two consecutive U (M) added to the 3'-end of the antisense strand region.

[50] The oligonucleotide or pharmaceutically acceptable salt thereof according to any one of

[45] to

[49] , wherein the 5'-end of the sense strand region is chemically modified with a fatty acid unit represented by any unit represented by the following formula:

[0035]

[0036] (In the formula, the end of the dashed line represents a phosphodiester bond which may be chemically modified with the 5'-phosphate group of the adjacent nucleotide.)

[51] The oligonucleotide or a pharmaceutically acceptable salt thereof according to [1], wherein the sense strand region and the antisense strand region are represented by any of the following formulae (I-7) to (I-12), and the sense strand region and the antisense strand region each serve as independent oligonucleotides to form a double-stranded oligonucleotide. (Formula) (I-7) Sense strand region 5' SPhU(M)^C(M)^U(M)G(M)C(F)U(M)G(F)A(F)C(F)A(M)G(M)A(M)U(M)G(M)U(M)U(M)C(M)^C(M)^A(3M) 3 Antisense strand region 5' VPu^G(F)^G(M)A(F)A(M)C(F)A(M)U(M)C(F)U(M)G(M)T(D)C(M)A(E)G(M)C(F)A(M)G(M)^A(M)^U(M)^U(M) 3' (I-8) Sense strand region 5' SPhG(M)^A(M)^U(M)G(M)U(F)U(M)C(F)C(F)A(F)U(M)C(M)C(M)U(M)G(M)U(M)A(M)C(M)^A(M)^A(3M) 3' Antisense strand region 5' VPu^U(F)^G(M)U(F)A(M)C(F)A(M)G(M)G(F)A(M)U(M)G(D)G(M)A(E)A(M)C(F)A(M)U(M)^C(M)^U(M)^U(M) 3' (I-9) Sense strand region 5' SPhU(M)^C(M)^U(M)G(M)C(F)U(M)G(F)A(F)C(F)A(M)G(M)A(M)U(M)G(M)U(M)U(M)C(M)^C(M)^A(3M) 3' Antisense strand region 5' VPu^G(F)^G(M)A(M)A(M)C(F)A(M)U(M)C(F)U(M)G(F)U(M)C(M)A(R)^G(M)C(F)A(M)G(M)^A(M)^U(M)^U(M) 3' (I-10) Sense strand region 5' SPhG(M)^A(M)^U(M)G(M)U(F)U(M)C(F)C(F)A(F)U(M)C(M)C(M)U(M)G(M)U(M)A(M)C(M)^A(M)^A(3M) 3'Antisense strand region 5' VPu^U(F)^G(M)U(M)A(M)C(F)A(M)G(M)G(F)A(M)U(F)G(M)G(M)A(R)^A(M)C(F)A(M)U(M)^C(M)^U(M)^U(M) 3' (I-11) Sense strand region 5' SPhG(M)^A(M)^U(M)G(M)U(F)U(M)C(F)C(F)A(F)U(M)C(M)C(M)U(M)G(M)U(M)A(M)C(M)^A(M)^A(3M) 3' Antisense strand region 5' EPu^G(F)^G(M)A(F)A(M)C(F)A(M)U(M)C(F)U(M)G(M)T(D)C(M)A(E)G(M)C(F)A(M)G(M)^A(M)^U(M)^U(M) 3' (I-12) Sense strand region 5' SPhG(M)^A(M)^U(M)G(M)U(F)U(M)C(F)C(F)A(F)U(M)C(M)C(M)U(M)G(M)U(M)A(M)C(M)^A(M)^A(3M) 3' Antisense strand region 5' VPuam^G(F)^G(M)A(F)A(M)C(F)A(M)U(M)C(F)U(M)G(M)T(D)C(M)A(E)G(M)C(F)A(M)G(M)^A(M)^U(M)^U(M)) 3' [Wherein, SPh is the following formula

[0037]

[0038] (wherein the end of the dashed line indicates a phosphodiester bond with the 5'-phosphate group of the 5'-terminal nucleotide of the adjacent sense strand region), A represents adenine, U represents uracil, G represents guanine, and C represents cytosine (however, when C is ENA, it represents 2'-O,4'-C-ethylene-bridged-5-methylcytidine) as bases of nucleic acids, (R) represents RNA, (D) represents DNA, (M) represents 2'-OMe RNA, (3M) represents 3'-OMe RNA, (F) represents 2'-F RNA, and (E) represents ENA as nucleic acids, and VPu, EPu, and VPuam as phosphonic acid units are represented by the following formulae, respectively:

[0039]

[0040] (In the formula, the end of the dashed line indicates a phosphonate unit formed by a phosphorothioate bond (-P(=S)(OH)-) with the 5'-phosphate group of the 5'-terminal nucleotide of the adjacent sense strand region.) In addition, in the formula, "^" indicates that the internucleoside bond is a phosphorothioate bond (-P(=S)(OH)-), and unless otherwise specified, indicates that the internucleoside bond is a phosphodiester bond (-P(=O)(OH)-). The 3'-position of the 3'-terminal nucleotide of the sense strand region and the 3'-position of the 3'-terminal nucleotide of the antisense strand region are each a hydroxyl group.)

[52] A pharmaceutical composition comprising, as an active ingredient, the oligonucleotide according to any one of [1] to

[51] or a pharmaceutically acceptable salt thereof.

[53] The pharmaceutical composition according to

[52] , for treating or preventing a disease that can be treated or prevented by suppressing expression of α-synuclein.

[54] The pharmaceutical composition according to

[53] for the prevention or treatment of an α-synucleinopathy.

[55] The pharmaceutical composition according to

[54] , wherein the α-synucleinopathy is Parkinson's disease, Parkinson's disease dementia, dementia with Lewy bodies, multiple system atrophy, or pure autonomic failure.

[56] A method for treating or preventing an α-synucleinopathy, comprising administering to a subject an effective amount of the oligonucleotide according to any one of [1] to

[51] or a pharmaceutically acceptable salt thereof.

[57] The method according to

[56] , wherein the α-synucleinopathy is Parkinson's disease, Parkinson's disease dementia, dementia with Lewy bodies, multiple system atrophy, or pure autonomic failure.

[58] The oligonucleotide according to any one of [1] to

[51] or a pharmaceutically acceptable salt thereof, for use in the treatment or prevention of an α-synucleinopathy.

[59] Use of the oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of [1] to

[51] in the manufacture of the pharmaceutical composition according to any one of

[52] to

[55] .

[0041] The siRNA of the present invention can suppress the expression of SNCA mRNA. Furthermore, the siRNA of the present invention can suppress the expression of SNCA mRNA in the brain in vivo, thereby suppressing the accumulation of intracellular insoluble inclusions containing α-synuclein and preventing, ameliorating, and / or treating α-synucleinopathies such as Parkinson's disease, Parkinson's disease dementia, dementia with Lewy bodies, multiple system atrophy, and pure autonomic failure.

[0042] [Correction based on Rule 91, 27.09.2024] This figure shows the modification patterns of double-stranded siRNA. In the figure, filled circles represent 2'-O-methyl RNA, open circles represent 2'-deoxy-2'-fluoro RNA, filled triangles represent DNA, open triangles represent 3'-O-methyl RNA, open diamonds represent ENA, filled diamonds represent RNA, open circles with vertical bars represent LNA, open circles with horizontal bars represent 2'-O-methoxyethyl RNA, the lines connecting the marks represent phosphodiester bonds, and lines connecting the marks with an "s" represent phosphorothioate bonds. In the figure, the top strand represents the sense strand (or passenger strand), with the left end of the sense strand representing the 5' end and the right end representing the 3' end. The bottom strand represents the antisense strand (or guide strand), with the right end of the antisense strand representing the 5' end and the left end representing the 3' end. Each siRNA is represented by NNN-xxx, and the numbers and letters following NNN-xxx represent the abbreviations for the modification patterns shown in Figures 1 to 6. １４ This figure shows the modification patterns when the A of the antisense strand in the double-stranded siRNA is DNA or RNA. Each symbol, position, modification, etc. is displayed in the same format as in Figure 1. ８ and A ９ This figure shows the modification pattern when A of the antisense strand in the double-stranded siRNA is 2'-deoxy-2'-fluoro RNA. Each symbol, position, modification, etc. are displayed in the same format as in Figure 1. １４ This figure shows the modification pattern when A of the antisense strand in the double-stranded siRNA is ENA. Each symbol, position, modification, etc. are displayed in the same format as in Figure 1. ８ and A ９is 2'-deoxy-2'-fluoro RNA, and A of the antisense strand １３ This figure shows the modification pattern when A of the antisense strand in the double-stranded siRNA is ENA. Each symbol, position, modification, etc. are displayed in the same format as in Figure 1. ８ is 2'-O-methyl RNA, and A ９This figure shows the modification pattern when is 2'-deoxy-2'-fluoro RNA. Symbols, positions, modifications, etc. are indicated in the same format as in Figure 1. This figure shows the base sequence (SEQ ID NO: 1) of the mRNA of the gene encoding human α-synuclein (hSNCA). The underlined sequence (sequence 678-696) indicates the target sequence of hSNCA-023, and the boxed sequence (sequence 688-706) indicates the target sequence of hSNCA-012. This figure is a continuation of Figure 7-1. Figure 8A shows the knockdown activity of SNCA mRNA in H4 cells treated with SNCA siRNA. Figure 8B shows the cytotoxicity of H4 cells treated with SNCA siRNA. This figure shows the knockdown activity of siRNA against SNCA mRNA in human SNCA BAC transgenic mice administered SNCA siRNA intracerebroventricularly. Figures 9A-C show knockdown activity in the cerebral cortex, midbrain, and cerebellum, respectively. Figures 10A-D show knockdown activity in the cerebral cortex, midbrain, cerebellum, and diencephalon / striatum, respectively. Figures 11-1A and 11-1B show knockdown activity in the cerebral cortex and midbrain, respectively. Figures 11-2C and 11-2D show knockdown activity in the cerebellum and diencephalon / striatum, respectively. 12A and 12B show the knockdown activity of SNCA siRNA or ASO against SNCA mRNA in human SNCA BAC transgenic mice. Figures 12A and 12B show the knockdown activity in the diencephalon / striatum and brainstem, respectively. Figure 12A shows the knockdown activity of SNCA siRNA or ASO against SNCA protein in human SNCA BAC transgenic mice administered intracerebroventricularly.13A and 13B show the knockdown activity in the diencephalon / striatum and the brainstem, respectively. 13B shows the knockdown activity of SNCA siRNA against SNCA mRNA in H4 cells.

[0043] <1. Explanation of Terms> As used herein, the term "target gene" refers to mRNA for a gene encoding alpha-synuclein (SNCA), and may be immature mRNA (also referred to as pre-mRNA or pre-mRNA) before undergoing RNA processing, or may be mature mRNA after undergoing RNA processing. Examples of RNA processing include RNA splicing, formation of a cap structure at the 5' end, and formation of a poly A tail at the 3' end.

[0044] Alpha-synuclein is a protein expressed primarily in the central nervous system, localized in presynaptic terminals, and has been suggested to be involved in the regulation of synaptic transmission (J Parkinson's Dis. 2015; 5(4): 699-713.). On the other hand, the lack of significant behavioral and pathological changes observed in alpha-synuclein knockout mice and conditional knockout mice after the in vivo stage suggests that alpha-synuclein is unnecessary and / or has compensatory mechanisms in neurodevelopment and central function (Neuron. 2000 Jan; 25(1): 239-52., Neurobiol Aging. 2020 Jul; 91: 76-87.). Abnormal structure, oligomerization, phosphorylation, and insolubilization of α-synuclein have been suggested to be involved in neurodegenerative diseases, including Parkinson's disease, Parkinson's disease dementia, dementia with Lewy bodies, and multiple system atrophy (Cell Death & Disease volume 14, Article number: 176 (2023)).

[0045] Alpha-synucleinopathies refer to neurodegenerative diseases characterized by the appearance of insoluble inclusions containing α-synuclein in the central and / or peripheral nervous system, including Parkinson's disease, Parkinson's disease dementia, dementia with Lewy bodies, multiple system atrophy, and pure autonomic failure. It has been suggested that the appearance of insoluble inclusions containing α-synuclein is accompanied by neuronal cell death, abnormalities in neural activity, and abnormalities in neural function in specific central and / or peripheral nervous regions. Symptoms of α-synucleinopathies include parkinsonism, cognitive impairment, sleep disorders, hallucinations, cerebellar ataxia, and autonomic dysfunction. The cause of α-synucleinopathies is unknown, but risk factors include specific gene mutations or gene duplications involving α-synuclein, exposure to neurotoxins, and aging. The mechanism by which overexpression, abnormal structure, or insolubilization of α-synuclein causes neurodegeneration is unknown. However, it has been reported that in mouse models of α-synucleinopathies, knockout or knockdown of the SNCA gene reduces the expression of insoluble inclusions containing α-synuclein, suppressing neuronal cell death and neuronal dysfunction (Science. 2012 Nov 16; 338(6109):949-53, JCI Insight. 2021; 6(5):e135633.).

[0046] As used herein, nucleic acid or nucleic acid molecule is a general term for nucleosides, nucleotides, oligonucleotides, and polynucleotides.

[0047] "Nucleoside" refers to a chemical structural moiety that combines a base moiety that is important for genetic information with a sugar moiety that is important for the physicochemical properties of the molecule.

[0048] A "nucleotide" is a compound in which the hydroxyl group at the 3'-position (or the 2'-position if the 3'-position is modified) of the sugar moiety of a nucleoside forms an ester with a phosphate group.

[0049] An "oligonucleotide" refers to an oligomer composed of two or more nucleotides, each of which is formed by bonding the phosphate group of one nucleotide to the hydroxyl group at the 5' position of the sugar moiety of another nucleotide (phosphodiester bond). However, the 3' position of the 3'-terminal nucleotide (or the 2' position in a nucleotide modified at the 3' position) and / or the 5' position of the 5'-terminal nucleotide may be a hydroxyl group or a phosphate group, or these may be chemically modified. Furthermore, the phosphodiester bond between nucleosides may be chemically modified. In this specification, the terms "oligonucleotide" and "polynucleotide" are used interchangeably.

[0050] The base moieties of nucleic acids are adenine (A), guanine (G), cytosine (C), uracil (U), and thymine (T). A and U or T, and G and C, respectively, form Watson-Crick base pairs through hydrogen bonds, and the relationship between bases that form a base pair is called "complementary." It is also known that G forms wobble base pairs with U or T, respectively (G:T or G:U). The base moiety may be chemically modified, but even modified bases usually exhibit the same base pairing ability as the original base. Representative modified bases include 5-methylcytosine (5C) and 5-methyluracil (5U), and 5U has the same structure as T. In the sequence listing attached to this specification, 5C is represented as C, and 5U is represented as T or U.

[0051] In this specification, nucleic acids are sometimes represented as "base symbols (sugar moiety symbols)." The sugar moiety symbols are explained in the sections describing various nucleosides below.

[0052] As used herein, the "modification pattern" of an oligonucleotide refers to the arrangement of the modification mode of the sugar moiety of each nucleoside constituting the oligonucleotide, regardless of the base sequence, unless otherwise specified. Furthermore, when specifically mentioned, it also refers to the modification pattern combined with the bond mode between each nucleoside. The structure of the sugar moiety and the internucleoside bond are factors that affect the molecular stability and the binding performance resulting from that stability, independent of the target sequence. Therefore, it is highly likely that the effect of a modification pattern confirmed for a certain target sequence will also be similar when the same modification pattern is applied to another target sequence (see, for example, Mol Ther. (2018) 26:708-717). In this specification, when a modification pattern is referred to as DNA, RNA, ENA, LNA, etc., unless otherwise specified, it refers to the nucleoside portion of the respective nucleic acid, and the "nucleoside" after each term may be omitted.

[0053] As used herein, a "natural nucleoside" refers to a 2'-deoxynucleoside or a ribonucleoside. 2'-deoxynucleosides (also referred to as "DNAs"; the sugar moiety is represented by (D), and the base moiety may be represented by a lowercase letter) corresponding to each base have a structure represented by the following formula, where 2'-deoxyadenosine is sometimes represented as A(D) or a, 2'-deoxyguanosine is sometimes represented as G(D) or g, 2'-deoxycytidine is sometimes represented as C(D) or c, thymidine is sometimes represented as T(D) or t, 2'-deoxy-5-methylcytidine is sometimes represented as 5C(D) or 5c, and 2'-deoxyuridine is sometimes represented as U(D) or u. 2'-deoxynucleosides that do not specify a base may sometimes be represented as N(D).

[0054] (In each of the above formulas, the end of the dashed line represents a bond to another atom, an adjacent nucleotide, a structural unit, a chemically modified structure, a chemical linker, an oligonucleotide linker, or the like.)

[0055] The ribonucleosides (also called "RNA"; the symbol for the sugar moiety is (R) and the base moiety is sometimes written in capital letters) corresponding to each base have the structure shown in the following formula, where adenosine is sometimes written as A(R) or A, guanosine as G(R) or G, cytidine as C(R) or C, and uridine as U(R) or U. Ribonucleosides that do not specify a base are sometimes written as N(R).

[0056] (In each of the above formulas, the end of the dashed line represents a bond to another atom, an adjacent nucleotide, a structural unit, a chemically modified structure, a chemical linker, an oligonucleotide linker, or the like.)

[0057] As used herein, the terms "modified nucleoside" and "modified nucleotide" refer to a nucleoside or nucleotide that contains at least one chemically modified structure in the base moiety, sugar moiety, or phosphate diester moiety of a natural nucleoside.

[0058] As used herein, the term "optionally chemically modified phosphodiester bond" refers to a phosphodiester bond used in natural internucleoside bonds, or a chemically modified phosphodiester bond. A natural internucleoside bond is a phosphodiester bond represented by the following formula (P). In representing base sequences herein, no letters or symbols are inserted between nucleosides, or between a nucleoside and an adjacent structural unit (e.g., a fatty acid unit described below), a chemically modified structure (e.g., a structure represented by "2" described below), or a chemical linker or oligonucleotide linker (e.g., a structure represented by "Z" described below). Furthermore, as used herein, the term "chemically modified phosphodiester bond" refers to a bonding mode in which the phosphorus atom in a phosphodiester bond is chemically modified. Examples of chemically modified phosphodiester bonds include a phosphorothioate bond represented by the following formula (PS), a phosphoramidate bond, which is a modified bond in which a nitrogen atom is added to the phosphorus atom, an alkylphosphonate bond, which is a modified bond in which a substitutable alkyl group is added to the phosphorus atom (for example, in the case of a methyl group, it becomes a methylphosphonate bond), and a phosphotriester bond, which is a modified bond in which a substitutable alkyl group is added to the non-covalently bonded oxygen atom of a phosphate group. When a phosphorothioate bond is used, in the representation of the base sequence herein, a "^" is displayed between nucleosides, or between a nucleoside and its adjacent structural unit (for example, a fatty acid unit described below), a chemically modified structure (for example, a structure represented by "2" described below), or a chemical linker or oligonucleotide linker (for example, a structure represented by "Z" described below).

[0059]

[0060] (In each of the above formulas, the end of one dashed line represents an ester bond between the oxygen atom of the 3'-hydroxyl group of the adjacent nucleoside on the 5' side (the oxygen atom at the 2'-position in a nucleoside modified at the 3'-position), an adjacent structural unit, or a chemical linker or oligonucleotide linker; the end of the other dashed line represents an ester bond between the oxygen atom of the 5'-hydroxyl group of the adjacent nucleoside on the 3' side, a structural unit, or a chemical linker or oligonucleotide linker.)

[0061] As used herein, the term "sugar-modified nucleoside" refers to a nucleoside in which the sugar moiety of the nucleoside has been modified. Sugar-modified nucleosides include all types of sugar modifications known in the technical field to which the present invention pertains. Examples of sugar-modified nucleosides include 2'-modified nucleosides, 3'-modified nucleosides, 4'-thio-modified nucleosides, 4'-thio-2'-modified nucleosides, and 2'-O,4'-C-bridged modified nucleosides. Preferred examples of sugar modifications include 2'-O-alkylation (methylation, ethylation, propylation, isopropylation, butylation, etc.), 2'-O-alkoxyalkylation (methoxyethylation, methoxypropylation, methoxybutylation, etc.), 2'-halogenation (chlorination, fluorination, etc.), 3'-O-alkylation (methylation, ethylation, propylation, isopropylation, butylation, etc.), 3'-O-alkoxyalkylation (methoxyethylation, methoxypropylation, methoxybutylation, etc.), 3'-halogenation (chlorination, fluorination, etc.), and 2'-O,4'-C-bridging (alkylenation bridge, etc.).

[0062] As used herein, the term "2'-modified nucleoside" refers to a nucleoside in which the 2'-position of the ribofuranose moiety is chemically modified. Examples of such modifications include 2'-O-alkylated (methylated, ethylated, propylated, isopropylated, butylated, etc.) nucleosides, 2'-O-alkoxyalkylated (methoxyethylated, methoxypropylated, methoxybutylated, etc.) nucleosides, 2'-halogenated (chlorinated, fluorinated, etc.) nucleosides, 2'-allylated nucleosides, 2'-aminated nucleosides, 2'-azido nucleosides, 2'-O-allylated nucleosides, 2'-OCF ３ Nucleosides, 2'-O(CH ２ ) ２ SCH ３ Nucleosides, 2'-O-(CH ２ ) ２ -O-N(R ｍ ) (R ｎ ) nucleoside, or 2'-O-CH ２ -C(=O)-N(R ｍ ) (R ｎ ) nucleosides (each R ｍ and R ｎ are individually H, an amino-protecting group, or a substituted or unsubstituted C1-C10 alkyl. Preferred 2'-modified nucleosides are 2'-O-alkylated nucleosides, 2'-O-alkoxyalkylated nucleosides, or 2'-halogenated nucleosides.

[0063] Among sugar-modified nucleosides, examples of 2'-O-alkylated modifications include 2'-O-methylnucleosides (sometimes referred to as "2'-OMe RNA" or "2'-O-methyl RNA." The symbol representing the sugar moiety is (M)). 2'-O-methylnucleosides corresponding to each base have the structure represented by the following formula, and are sometimes represented as follows: 2'-O-methyladenosine as A(M), 2'-O-methylguanosine as G(M), 2'-O-methylcytidine as C(M), 2'-O-methyl-5-methylcytidine as 5C(M), 2'-O-methyluridine as U(M), and 2'-O-methyl-5-methyluridine as T(M). 2'-O-methylnucleosides that do not specify a base are sometimes represented as N(M).

[0064] (In each of the above formulas, the end of the dashed line represents a bond to another atom, an adjacent nucleotide, a structural unit, a chemically modified structure, a chemical linker, an oligonucleotide linker, or the like.)

[0065] Examples of 2'-O-alkoxyalkylated modifications include 2'-O-methoxyethyl nucleosides (sometimes referred to as "2'-MOE RNA," "2'-O-methoxyethyl RNA," or "2'-methoxyethoxy RNA." The symbol representing the sugar moiety is (m)). 2'-O-Methoxyethyl nucleosides corresponding to each base have the structures represented by the following formulas, where 2'-O-methoxyethyl adenosine is represented as A(m), 2'-O-methoxyethyl guanosine as G(m), 2'-O-methoxyethyl-5-methylcytidine as C(m) (although the base structure is 5C, it is represented as C(m) for convenience. It can also be used in place of 2'-O-methoxyethylcytidine), 2'-O-methoxyethyluridine as U(m), and 2'-O-methoxyethyl-5-methyluridine as T(m). Furthermore, 2'-O-methoxyethyl nucleosides that do not specify a base may be represented as N(m).

[0066] (In each of the above formulas, the end of the dashed line represents a bond to another atom, an adjacent nucleotide, a structural unit, a chemically modified structure, a chemical linker, an oligonucleotide linker, or the like.)

[0067] Furthermore, an example of a 2'-halogenated modification is 2'-deoxy-2'-fluoronucleoside (sometimes referred to as "2'-F RNA" or "2'-deoxy-2'-fluoro RNA." The symbol representing the sugar moiety is (F)). 2'-Deoxy-2'-fluoronucleosides corresponding to each base have the structure represented by the following formula, and are sometimes represented as follows: 2'-deoxy-2'-fluoroadenosine as A(F), 2'-deoxy-2'-fluoroguanosine as G(F), 2'-deoxy-2'-fluorocytidine as C(F), 2'-deoxy-2'-fluoro-5-methylcytidine as 5mC(F), 2'-deoxy-2'-fluorouridine as U(F), and 2'-deoxy-2'-fluoro-5-methyluridine as T(F). Furthermore, 2'-deoxy-2'-fluoronucleosides that do not specify a base may be represented as N(F).

[0068] (In each of the above formulas, the end of the dashed line represents a bond to another atom, an adjacent nucleotide, a structural unit, a chemically modified structure, a chemical linker, an oligonucleotide linker, or the like.)

[0069] As used herein, the term "3'-modified nucleoside" refers to a nucleoside in which the 3'-position of the ribofuranose moiety contained therein has been chemically modified. The 2'-hydroxyl group in a 3'-modified nucleoside may be modified. Examples of such modifications include 3'-O-alkylated (methylated, ethylated, propylated, isopropylated, butylated, etc.) nucleosides, 3'-O-alkoxyalkylated (methoxyethylated, methoxypropylated, methoxybutylated, etc.) nucleosides, 3'-halogenated (chlorinated, fluorinated, etc.) nucleosides, 3'-allylated nucleosides, 3'-aminated nucleosides, 3'-azido nucleosides, 3'-O-allylated nucleosides, and 3'-OCF ３ Nucleosides, 3'-O(CH ２ )２ SCH ３ Nucleosides, 3'-O-(CH ２ ) ２ -O-N(R ｍ ) (R ｎ ) nucleoside, or 3'-O-CH ２ -C(=O)-N(R ｍ ) (R ｎ ) nucleosides (each R ｍ and R ｎ are each independently H, an amino-protecting group, or a substituted or unsubstituted C1-C10 alkyl. Preferred 3'-modified nucleosides are 3'-O-alkylated nucleosides, 3'-O-alkoxyalkylated nucleosides, or 3'-halogenated nucleosides.

[0070] Among 3'-modified nucleosides, examples of 3'-O-alkylated modifications include 3'-O-methylnucleosides (sometimes referred to as "3'-OMe RNA." The symbol representing the sugar moiety is (3M)). 3'-O-methylnucleosides corresponding to each base have the structure represented by the following formula, and are sometimes represented as follows: 3'-O-methyladenosine as A(3M), 3'-O-methylguanosine as G(3M), 3'-O-methylcytidine as C(3M), 3'-O-methyl-5-methylcytidine as 5mC(3M), 3'-O-methyluridine as U(3M), and 3'-O-methyl-5-methyluridine as T(3M). Furthermore, 3'-O-methylnucleosides that do not specify a base are sometimes represented as N(3M).

[0071] (In each of the above formulas, the end of the dashed line represents a bond to another atom, an adjacent nucleotide, a structural unit, a chemically modified structure, a chemical linker, an oligonucleotide linker, or the like.)

[0072] An example of a 3'-O-alkoxyalkylation modification is 3'-O-methoxyethyl nucleoside (sometimes referred to as "3'-MOE RNA," "3'-O-methoxyethyl RNA," or "3'-methoxyethoxy RNA." The symbol representing the sugar moiety is (3m).) The 3'-O-methoxyethyl nucleoside corresponding to each base has a structure in which a methoxyethyl group is bonded in place of the methyl group bonded to the oxygen atom at the 3' position in the structural diagram of the 3'-O-methyl nucleoside corresponding to each base. The 3'-O-methoxyethyl nucleosides corresponding to each base are sometimes represented as follows: 3'-O-methoxyethyl adenosine as A(3m), 3'-O-methoxyethyl guanosine as G(3m), 3'-O-methoxyethyl-5-methylcytidine as C(3m) (although the base structure is 5C, it is represented as C(3m) for convenience. It can also be used in place of 3'-O-methoxyethyl cytidine), 3'-O-methoxyethyl uridine as U(3m), and 3'-O-methoxyethyl-5-methyluridine as T(3m). Also, 3'-O-methoxyethyl nucleosides that do not specify a base may be represented as N(3m).

[0073] An example of a 3'-halogenated modification is 3'-deoxy-3'-fluoronucleoside (sometimes referred to as "3'-F RNA" or "3'-deoxy-3'-fluoro RNA." The symbol representing the sugar moiety is (3F).) The 3'-deoxy-3'-fluoronucleoside corresponding to each base has a structure in which a fluorine atom is bonded in place of the methoxy group bonded to the carbon atom at the 3' position in the above structural diagram of the 3'-O-methylnucleoside corresponding to each base. The 3'-deoxy-3'-fluoronucleosides corresponding to each base are sometimes represented as follows: 3'-deoxy-3'-fluoroadenosine as A(3F), 3'-deoxy-3'-fluoroguanosine as G(3F), 3'-deoxy-3'-fluorocytidine as C(3F), 3'-deoxy-3'-fluoro-5-methylcytidine as 5mC(3F), 3'-deoxy-3'-fluorouridine as U(3F), and 3'-deoxy-3'-fluoro-5-methyluridine as T(3F). 3'-Deoxy-3'-fluoronucleosides that do not specify a base may be represented as N(3F).

[0074] As used herein, the term "4'-thio-modified nucleoside" refers to a nucleoside in which the oxygen atom at the 4'-position of the ribofuranose moiety is replaced with a sulfur atom. Examples of 4'-thio-modified nucleosides include β-D-ribonucleosides in which the 4'-oxygen atom is replaced with a sulfur atom (Hoshika, S. et al. FEBS Lett. 579, pp. 3115-3118, (2005); Dande, P. et al. J. Med. Chem. 49, pp. 1624-1634 (2006); Hoshika, S. et al. ChemBioChem. 8, pp. 2133-2138, (2007)).

[0075] As used herein, the term "4'-thio-2'-modified nucleoside" refers to a nucleoside in which the 2'-position of the ribofuranose moiety is chemically modified and the oxygen atom at the 4'-position of the ribofuranose moiety is replaced with a sulfur atom. Examples of 4'-thio-2'-modified nucleosides include 4'-thio-2'-modified nucleosides that retain 2'-H or 2'-O-methyl (Matsugami, et al. Nucleic Acids Res. 36, 1805 (2008)).

[0076] Among sugar-modified nucleosides, examples of 2'-O,4'-C-bridged modifications include 2'-O,4'-C-ethylene-bridged nucleosides (sometimes referred to as "ENA" or "ENA unit." The symbol representing the sugar moiety is (E)) (Morita, K. et al. Bioorg. Med. Chem. Lett., 12, p. 73 (2002); Morita, K. et al. Bioorg. Med. Chem., 11, p. 2211 (2003)). The 2'-O,4'-C-ethylene-bridged nucleosides corresponding to each base have the structures represented by the following formulas, where 2'-O,4'-C-ethylene-bridged adenosine is sometimes represented as A(E), 2'-O,4'-C-ethylene-bridged guanosine as G(E), 2'-O,4'-C-ethylene-bridged-5-methylcytidine as C(E) (although the base structure is 5C, it is represented as C(E) for convenience. It can also be used in place of 2'-O,4'-C-ethylene-bridged cytidine), 2'-O,4'-C-ethylene-bridged uridine as U(E), and 2'-O,4'-C-ethylene-bridged-5-methyluridine as T(E). Furthermore, 2'-O,4'-C-ethylene-bridged nucleosides that do not specify a base are sometimes represented as N(E).

[0077] (In each of the above formulas, the end of the dashed line represents a bond to another atom, an adjacent nucleotide, a structural unit, a chemically modified structure, a chemical linker, an oligonucleotide linker, or the like.)

[0078] Another example of the 2'-O,4'-C-bridged modification is, for example, 2'-O,4'-C-methylene-bridged nucleoside (sometimes referred to as "LNA" or "LNA unit", the symbol representing the sugar moiety is (L)) (Obika, S. et al. Tetrahedron Lett., 38, p. 8735- (1997); Obika, S. et al., Tetrahedron Lett., 39, p. 5401- (1998); A. A. Koshkin, A. A. et al. Tetrahedron, 54, p. 3607 (1998); Obika, S., Bioorg. Med. Chem., 9, p. 1001 (2001).) 2'-O,4'-C-methylene-bridged nucleosides corresponding to each base have structures represented by the following formulas, where 2'-O,4'-C-methylene-bridged adenosine is represented as A(L), 2'-O,4'-C-methylene-bridged guanosine as G(L), 2'-O,4'-C-methylene-bridged-5-methylcytidine as C(L) (although the base structure is 5C, it is represented as C(L) for convenience. It can also be used interchangeably with 2'-O,4'-C-methylene-bridged cytidine), 2'-O,4'-C-methylene-bridged uridine as U(L), and 2'-O,4'-C-methylene-bridged-5-methyluridine as T(L). Furthermore, 2'-O,4'-C-methylene-bridged nucleosides that do not specify a base may be represented as N(L).

[0079] (In each of the above formulas, the end of the dashed line represents a bond to another atom, an adjacent nucleotide, a structural unit, a chemically modified structure, a chemical linker, an oligonucleotide linker, or the like.)

[0080] The oligonucleotide or salt thereof of the present invention has an antisense strand region (sometimes simply referred to as the antisense strand) having a nucleotide sequence substantially complementary to the nucleotide sequence of a target sequence, and a sense strand region (sometimes simply referred to as the sense strand) having a nucleotide sequence substantially complementary to the nucleotide sequence in the complementary region of the antisense strand region. The sense strand region and the antisense strand region form a double-stranded structure in the nucleotide sequence of the complementary region, but each may form a double-stranded oligonucleotide as an independent oligonucleotide, or may form a single-stranded oligonucleotide by linking the 5' position of the 5'-terminal nucleotide of one to the 3' position of the 3'-terminal nucleotide of the other (the 2' position in the case of a nucleotide modified at the 3' position). That is, the oligonucleotide of the present invention may be an oligonucleotide of two molecules or an oligonucleotide of one molecule. When the oligonucleotide of the present invention is a single molecule of oligonucleotide, the linking manner of the sense strand region and the antisense strand region is not particularly limited as long as the oligonucleotide of the present invention has RNA interference activity and / or gene expression suppression activity, but for example, the 5' position of one 5'-terminal nucleotide and the 3' position of the other 3'-terminal nucleotide (the 2' position in the case of a nucleotide modified at the 3' position) can be linked by a desired chemical linker or oligonucleotide linker. When a phosphonic acid unit is not used at the 5' end of the antisense strand region, the linker used can be, for example, the linker described in WO2012 / 074038, and preferably the linker represented by the following formula (Z). The linker represented by the following formula (Z) can be appropriately adjusted according to WO2012 / 074038.

[0081]

[0082] (In the above formula, the end of the dashed line indicates that the oxygen atom bonded to the phenyl group is linked by a phosphodiester bond to the 5'-phosphate group of a nucleotide other than the phosphonate unit at the 5'-end of the adjacent antisense strand, and the methylene group at the other end is linked by a phosphodiester bond to the 3'-phosphate group (or the 2'-phosphate group in a nucleotide modified at the 3'-position) of a nucleotide at the 3'-end of the adjacent sense strand, and each phosphodiester bond formed may be a chemically modified phosphodiester bond such as a phosphorothioate bond.)

[0083] In the oligonucleotide of the present invention, the 3'-position of the 3'-terminal nucleotide (2'-position in the case of a nucleotide modified at the 3'-position) and / or the 5'-position of the 5'-terminal nucleotide may be a hydroxyl group, a phosphate group, or a thiophosphate group, or may have a chemically modified structure. Examples of the chemically modified structure include the structure shown in the following formula (2).

[0084]

[0085] (In the above formula, the end of one dashed line represents bonding to an adjacent nucleotide, forming a phosphodiester bond with the 5'-phosphate group when bonding to the 5'-terminal nucleotide, or with the 3'-phosphate group (the 2'-phosphate group in a nucleotide modified at the 3'-position) when bonding to the 3'-terminal nucleotide; the end of the other dashed line represents bonding to a hydrogen atom to form a hydroxyl group, and the phosphodiester bond formed may be a chemically modified phosphodiester bond such as a phosphorothioate bond.)

[0086] As used herein, the term "phosphonate unit" refers to a compound in which any of the compounds having a phosphonate represented by the following formula is bound to the 4'-position (facing upward relative to the five-membered ring of the sugar moiety, and β-position in relation to the base) of the 5'-terminal nucleoside of the antisense strand in the oligonucleotide of the present invention. That is, this is a compound in which the carbon atom at the 5'-position of the nucleoside is increased in carbon atom number and further bound to phosphorus, and there is a single bond, double bond, or triple bond between the 5'-position carbon atom and the increased carbon atom.

[0087]

[0088] (In the formula, R represents a nucleoside and indicates that it is bonded to the 4' carbon atom of the nucleoside.)

[0089] In the phosphonic acid unit, there is a single bond, double bond, or triple bond between the carbon atom at the 5' position and the carbon atom following the addition. The base in the phosphonic acid unit is not particularly limited, and any base can be used. The sugar in the phosphonic acid unit may be natural (i.e., ribose) or modified. Examples of sugar modifications in the phosphonic acid unit include those used in the sugar-modified nucleosides described above, as well as 2'-amination, in which the 2' position of the sugar is substituted with an amino group. Also usable are 2'-acyl amination, in which the amino group at the 2' position of the sugar is acylated, and 2'-formyl amination, in which the amino group at the 2' position of the sugar is formylated (2'-formyl amination). Examples of 2'-acyl amination include acetyl amination (acetamidation), propionyl amination, and butanoyl amination. The acyl group in the 2'-acyl amination modification may be substituted with an alkoxy group, and 2'-alkoxyacyl amination modification (methoxyacetyl amination, methoxypropionyl amination, methoxybutanoyl amination, etc.) can also be employed.

[0090] In the phosphonate unit, the base, the bond (single bond, double bond, or triple bond) between the carbon atom at the 5' position and the carbon atom after the addition, and the sugar modification can be combined as appropriate and are not particularly limited. For example, a specific phosphonate unit using uracil as the base has the structure shown in the following formula. That is, for example, EPu is a compound in which the base is uracil, the bond between the carbon atom at the 5' position and the carbon atom after the addition is a triple bond, and the sugar is modified by 2'-O-methylation.

[0091] (In the above formula, the end of the dashed line represents a bond to an adjacent nucleotide. The bond to the adjacent nucleotide represents the formation of a phosphodiester bond with the 5'-phosphate group of the adjacent nucleotide, and the phosphodiester bond formed may be a chemically modified phosphodiester bond such as a phosphorothioate bond.)

[0092] In this specification, a phosphonate unit is treated as a nucleoside having a base contained in the phosphonate unit. Therefore, in the oligonucleotide of the present invention, a phosphonate unit is treated as one nucleoside.

[0093] As used herein, "substantially identical nucleotide sequences" refers to nucleotide sequences consisting entirely of the same bases as the target nucleotide sequence, as well as nucleotide sequences with 70% or more sequence identity, and nucleotide sequences containing one or more non-identical bases but capable of performing the desired function as an oligonucleotide. Here, "identical bases" include not only bases that are completely identical to the original base, but also bases with base pairing ability equal to or greater than that of the original base. Examples of such bases include chemically modified bases with base pairing ability equal to or greater than that of the original base (e.g., C and 5C, U and T (5U)). Sequence identity refers to a sequence with preferably 80% or more, more preferably 90% or more, and even more preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the target nucleotide sequence, or a sequence with 3-, 2-, or 1-base mismatch. Nucleotide sequence identity can be calculated using known genetic analysis software such as BLAST (registered trademark).

[0094] As used herein, the term "substantially complementary nucleotide sequence" refers not only to a nucleotide sequence consisting entirely of nucleotides complementary to a target nucleotide sequence, but also to a nucleotide sequence in which one or several (e.g., five, four, three, or two) nucleotides are not complementary nucleotides but form base pairs between oligonucleotides. Furthermore, as used herein, the term "complementary base" refers to a base that can form a Watson-Crick base pair with a target base, and may be an unmodified base or a chemically modified base. For example, 5-methyluracil and adenine are complementary bases, and 5-methylcytosine and guanine are complementary bases.

[0095] As used herein, the term "double-stranded structure" of an oligonucleotide refers to a double-stranded structure formed by the formation of Watson-Crick base pairs between substantially complementary nucleotide sequences. The nucleotide sequences forming the double-stranded structure may be substantially complementary nucleotide sequences between two different oligonucleotide molecules, or may be substantially complementary sequences within a single-stranded oligonucleotide.

[0096] As used herein, the term "structural unit" refers to a chemical structural unit that imparts a desired function to an oligonucleotide. Examples of the desired function imparted to an oligonucleotide include the function of transporting the oligonucleotide to a target tissue or target cell (also referred to as a target tissue, etc.), the function of improving the pharmacokinetics of the oligonucleotide, and the like. The structural unit can be attached to an oligonucleotide by known methods. For example, by having an amidite structure in the structural unit, the structural unit can be attached to the 3'-position of the 3'-terminal nucleotide (the 2'-position in a nucleotide modified at the 3'-position) or the 5'-position of the 5'-terminal nucleotide of the oligonucleotide. Furthermore, by having a structure in the structural unit that promotes the transport of a ligand or the like to a target tissue, etc., the oligonucleotide can be imparted with transportability to the target tissue, etc. Examples of such structures include a fatty acid unit for transport to liver or muscle tissue, a GalNAc unit for transport to hepatocytes, etc. In the present specification, such a structural unit for imparting transportability to a target tissue, etc., is sometimes referred to as a "transport unit."

[0097] 2. RNAi Oligonucleotides As used herein, an "RNAi oligonucleotide" refers to an oligonucleotide that includes a complementary region in which substantially complementary nucleotides contained in the sense strand region and the antisense strand region form Watson-Crick base pairs to form a double-stranded structure, and that has an RNA interference effect and / or gene expression suppression effect on a target gene. As long as the RNAi oligonucleotide has an RNA interference effect and / or gene expression suppression effect on a target gene, not all bases in the complementary region contained in the RNAi oligonucleotide need form Watson-Crick base pairs. As used herein, RNAi oligonucleotides may also be referred to as "siRNA."

[0098] As used herein, the term "a nucleotide sequence substantially identical to a target sequence" refers to a nucleotide sequence identical to the target sequence, but includes not only a completely identical sequence but also a substantially identical nucleotide sequence as long as the RNAi-oligonucleotide has an RNA interference effect and / or a gene expression suppression effect on the target gene.

[0099] As used herein, the term "nucleotide sequence substantially complementary to a target sequence" refers to a nucleotide sequence complementary to a target sequence, and includes not only completely complementary sequences but also substantially complementary nucleotide sequences as long as the RNAi-oligonucleotide has an RNA interference effect and / or a gene expression suppression effect on a target gene.

[0100] As used herein, an oligonucleotide that contains a nucleotide sequence substantially complementary to a target sequence and has an RNA interference effect and / or gene expression suppression effect on a target gene is referred to as an RNAi-oligonucleotide for a target gene.

[0101] The nucleotide sequence of an RNAi-oligonucleotide for a target gene is not particularly limited as long as it has an RNA interference effect and / or a gene expression suppressing effect on the target gene, and can be determined, for example, based on a sequence predicted to have an RNA interference effect and / or a gene expression suppressing effect on the target gene using computer software (e.g., GENETYX (registered trademark): manufactured by GENETYX COORPORATION, etc.). Furthermore, the RNA interference effect and / or the gene expression suppressing effect of an RNAi-oligonucleotide prepared based on a selected sequence can also be determined by confirming the effect.

[0102] As used herein, the term "gene expression inhibitory effect" includes not only the effect of completely suppressing gene expression but also the effect of reducing gene expression compared to a control, and gene silencing is also included in the gene expression inhibitory effect. Furthermore, as used herein, the terms "gene expression inhibitory effect" and "gene expression inhibitory activity" are used interchangeably.

[0103] The RNA interference effect and / or gene expression suppression effect can be confirmed by methods commonly used by those skilled in the art, for example, by administering an RNAi-oligonucleotide against the target gene to cells in which the target gene is expressed, quantifying the protein that is the translation product of the target gene after a certain period of time using Western blot analysis, and comparing the expression level of the protein with that of a control. Alternatively, it can be confirmed by measuring the expression level of the target gene in real time after administering an RNAi-oligonucleotide against the target gene using real-time PCR techniques.

[0104] As used herein, the term "target sequence" refers to a sequence of 18 or more nucleotides, which may be any portion of the nucleotide sequence of the mRNA for the target gene, the gene encoding α-synuclein, and which is targeted by the RNAi-oligonucleotide of the present invention. Furthermore, if SNPs or the like are known in the target sequence, sequences containing such mutations are also included in the target sequence. The RNAi-oligonucleotide of the present invention contains a sequence substantially complementary to the target sequence (hereinafter referred to as a "target-corresponding sequence") in at least a portion of the antisense strand region.

[0105] As used herein, the term "complementary region" refers to a region in which the nucleotide sequences of the sense strand region and the antisense strand region contained in an RNAi oligonucleotide are substantially complementary to each other. The sense strand region and the antisense strand region do not necessarily have to be completely complementary in their complementary region, but at least the terminal bases in the complementary region are complementary to each other. In the RNAi oligonucleotide of the present invention, the complementary region of the antisense strand region includes the target-corresponding sequence, but may maintain complementarity with the sense strand region in a region further extended at the 5' end and / or 3' end of the complementary region.

[0106] The length of the target-corresponding sequence in the antisense strand region constituting the RNAi-oligonucleotide of the present invention is not particularly limited as long as it has RNA interference activity and / or gene expression inhibitory activity, and may be any length from 18 nucleotides to the full length of the open reading frame (ORF) of the target gene. For example, the length of the target-corresponding sequence can be 18 to 29 nucleotides, preferably 18 to 24 nucleotides, 18 to 23 nucleotides, or 18 to 22 nucleotides, more preferably 18 to 21 nucleotides, and even more preferably 18 or 19 nucleotides.

[0107] The chain length of each complementary region in the sense strand region and the antisense strand region constituting the RNAi-oligonucleotide of the present invention is not particularly limited as long as it has an RNA interference effect and / or a gene expression inhibitory effect, and may be any length. For example, the chain length of the complementary region can be 19 to 29 nucleotides, preferably 19 to 24 nucleotides, 19 to 23 nucleotides, or 19 to 22 nucleotides, more preferably 19 to 21 nucleotides, and even more preferably 19 nucleotides.

[0108] The chain lengths of the sense strand region and antisense strand region constituting the RNAi-oligonucleotide of the present invention are not particularly limited as long as they have an RNA interference effect and / or a gene expression inhibitory effect, and may be any length. The chain length of the sense strand region can be, for example, 19 to 39 nucleotides, preferably 19 to 29 nucleotides, more preferably 19 to 24 nucleotides, 19 to 23 nucleotides, or 19 to 22 nucleotides, even more preferably 19 to 21 nucleotides, and most preferably 19 nucleotides. The chain length of the antisense strand region can be, for example, 19 to 39 nucleotides, preferably 19 to 29 nucleotides, more preferably 19 to 24 nucleotides, 19 to 23 nucleotides, or 19 to 22 nucleotides, even more preferably 21 to 23 nucleotides, and most preferably 21 nucleotides.

[0109] In the RNAi-oligonucleotide of the present invention, when the sense strand region and the antisense strand region are present in different oligonucleotide molecules, the entire structure does not need to be a double-stranded structure, and the 5'- and / or 3'-terminal nucleosides may be partially protruding, or may have an overhang structure that does not form a double-stranded structure. Examples of such an overhang structure include a structure consisting of 1 to 5 nucleosides, preferably 1 to 3 nucleosides, and more preferably 2 nucleosides. Furthermore, the overhang structure may be present at all ends of the double-stranded oligonucleotide, or may be only a portion thereof. In this specification, the 5'-end of the sense strand region is represented by S Ｏ５ , 3' end is S Ｏ３ , the 5' end of the antisense strand region is A Ｏ５ , 3' end is A Ｏ３ The base sequence of the overhang structure is not particularly limited, but oligo T or oligo U can be used.

[0110] The RNAi-oligonucleotide has at least one property selected from the following (i) to (iv): (i) it has an RNA interference effect and / or a gene expression-inhibiting effect on a target gene; (ii) it is stable against RNases and has an RNA interference effect and / or a gene expression-inhibiting effect on a target gene; (iii) it is stable against exonucleases and has an RNA interference effect and / or a gene expression-inhibiting effect on a target gene; (iv) it is stable against RNases and exonucleases and has an RNA interference effect and / or a gene expression-inhibiting effect on a target gene;

[0111] An example of an RNAi oligonucleotide is derived from a double-stranded oligonucleotide having an antisense strand region for a target gene and a sense strand region having a nucleotide sequence substantially complementary to the antisense strand region, and the 5'-position of the 5'-terminal nucleotide of the antisense strand region and the 3'-position of the 3'-terminal nucleotide of the sense strand region (or the 2'-position in the case of a nucleotide modified at the 3'-position) may each be linked by a linker forming a phosphodiester structure, which may be chemically modified. Such an oligonucleotide may have the structure oligonucleotide 3'-P(=O)(OH)-[linker]-P(=O)(OH)-5'-oligonucleotide (where "oligonucleotide 3'" refers to a structure in which the 3'-terminal nucleotide of the oligonucleotide does not have a hydrogen atom on the hydroxyl group at the 3'-position (or the 2'-position in the case of a nucleotide modified at the 3'-position), and "5'-oligonucleotide" refers to a structure in which the 5'-terminal nucleotide of the oligonucleotide does not have a hydrogen atom on the hydroxyl group at the 5'-position). Such linkers are described, for example, in WO2012 / 074038 and can be synthesized by referring to the descriptions in these documents.

[0112] The nucleosides constituting the RNAi oligonucleotide may be natural nucleosides or sugar-modified nucleosides. The internucleoside bond is a phosphodiester bond which may be chemically modified, such as a phosphodiester bond or a phosphorothioate bond.

[0113] The RNAi-oligonucleotides of the present invention can suppress the expression of SNCA mRNA. Furthermore, the RNAi-oligonucleotides of the present invention have low toxicity to cells and can be used as highly safe pharmaceuticals. Furthermore, by suppressing the expression of SNCA mRNA, the RNAi-oligonucleotides of the present invention can suppress the accumulation of intracellular insoluble inclusions containing α-synuclein. While the mechanism of action is not particularly limited, the RNAi-oligonucleotides of the present invention can be used to treat or prevent diseases that can be treated or prevented by suppressing the expression of SNCA mRNA. Examples of such diseases include α-synucleinopathies.

[0114] The present invention provides pharmaceutical use inventions such as a pharmaceutical composition containing the RNAi-oligonucleotide of the present invention as an active ingredient and used for treating or preventing an α-synucleinopathies, a method for treating or preventing an α-synucleinopathies comprising administering an effective amount of the oligonucleotide, the oligonucleotide for use in treating or preventing an α-synucleinopathies, and use of the RNAi-oligonucleotide of the present invention in the manufacture of a pharmaceutical composition for treating or preventing an α-synucleinopathies. α-Synucleinopathies that can be treated or prevented by the RNAi-oligonucleotide of the present invention include, for example, Parkinson's disease, Parkinson's disease dementia, dementia with Lewy bodies, multiple system atrophy, and pure autonomic failure.

[0115] As used herein, the term "combination" refers to the simultaneous, separate, or sequential administration of two or more different drugs. The present invention provides a method for treating or preventing α-synucleinopathies, comprising the combination of an RNAi-oligonucleotide of the present invention with one or more other drugs. The RNAi-oligonucleotide of the present invention may be used in combination with one or more other drugs, as long as the effects of the present invention are achieved. Preferably, the RNAi-oligonucleotide of the present invention may be used in combination with another drug having a different mechanism of action against α-synucleinopathies to provide greater therapeutic efficacy. For example, for Parkinson's disease, drugs such as levotopa, a dopamine precursor; carbidopa, a dopa decarboxylase inhibitor; dopamine agonists; anticholinergics; MAO-B inhibitors; and COMT inhibitors are sometimes prescribed, but these drugs have different mechanisms of action from SNCA siRNA. Therefore, the combination of these drugs with SNCA siRNA may provide greater therapeutic efficacy against PD than single drugs. In addition, drugs such as cholinesterase inhibitors may be prescribed for Parkinson's disease dementia and dementia with Lewy bodies, but these drugs have different mechanisms of action from SNCA siRNA. Therefore, the combination of these drugs with SNCA siRNA can provide greater therapeutic effects for Parkinson's disease dementia and dementia with Lewy bodies than single drugs. In addition, drugs such as adrenergic α1 receptor agonists and droxidopa, a precursor of noradrenaline and adrenaline, may be prescribed for multiple system atrophy, but these drugs have different mechanisms of action from SNCA siRNA. Therefore, the combination of these drugs with SNCA siRNA can provide greater therapeutic effects for multiple system atrophy than single drugs. Although existing drugs for α-synucleinopathies are all symptomatic treatments, they have shown certain therapeutic effects on motor symptoms, cognitive symptoms, and autonomic symptoms, so the combination of these drugs with SNCA siRNA can provide useful therapeutic effects for α-synucleinopathies.

[0116] When the RNAi-oligonucleotides of the present invention are used in combination with other drugs, the order of administration of the RNAi-oligonucleotides of the present invention is not particularly limited as long as the effects of the present invention are achieved. The RNAi-oligonucleotides of the present invention may be administered simultaneously with the other drugs, or before or after the other drugs. Examples of drugs that can be used in combination with the RNAi-oligonucleotides of the present invention include therapeutic agents for motor symptoms, cognitive symptoms, and autonomic symptoms in α-synucleinopathies. Examples of therapeutic agents for motor symptoms include levotopa, a dopamine precursor; carbidopa, a dopa decarboxylase inhibitor; dopamine agonists; anticholinergics; MAO-B inhibitors; and COMT inhibitors. Examples of therapeutic agents for cognitive symptoms include cholinesterase inhibitors. Examples of therapeutic agents for autonomic symptoms include adrenergic α1 receptor agonists and droxidopa, a precursor of noradrenaline and adrenaline.

[0117] <3. S3M-oligonucleotide> The present invention provides an RNAi-oligonucleotide (hereinafter referred to as "S3M-oligonucleotide") or a pharmaceutically acceptable salt thereof, wherein the sense strand region consists of an oligonucleotide represented by the following formula (I), the antisense strand region consists of an oligonucleotide represented by the following formula (II), and the antisense strand region consists of an oligonucleotide represented by the following formula (a) to (g): Ｏ５ -S ａ -S １９ -S １８ -S １７ -S １６ -S １５ -S １４ -S １３ -S １２ -S １１ -S １０ -S ９ -S ８ -S ７ -S ６ -S ５ -S ４ -S ３ -S ２ -S １ -S Ｏ３ 3' (I) Antisense strand region: 5' A Ｏ５ -A１ -A ２ -A ３ -A ４ -A ５ -A ６ -A ７ -A ８ -A ９ -A １０ -A １１ -A １２ -A １３ -A １４ -A １５ -A １６ -A １７ -A １８ - A １９ -A ａ -A Ｏ３ 3' (II) (a)S １ ~S １９ each represents one nucleoside, and S １ is a 3'-modified nucleoside. ａ is 0 to 10 nucleosides, preferably 0 to 5 or 0 to 4, more preferably 0 to 3, 0 to 2, or 0 to 1 nucleosides. Ｏ３ and S Ｏ５ are each independently 0 to 5, preferably 0 to 4, more preferably 0 to 3, 0 to 2, or 0 to 1 nucleosides. The bond between each nucleoside represents a phosphodiester bond which may be chemically modified; (b) A １ ~A １９ each represents one nucleoside, and A １１ , A １２ , A １３ , A １４ and A １５ At least one of the is DNA, RNA, 2'-O,4'-C-bridged nucleoside, or 2'-MOE RNA. ａ A is 0 to 10 nucleosides, preferably 0 to 5 or 0 to 4, more preferably 0 to 3, 0 to 2, or 0 to 1 nucleosides. Ｏ３ and A Ｏ５are each independently 0 to 5, preferably 0 to 4, more preferably 0 to 3, 0 to 2, or 0 to 1 nucleosides. The bond between each nucleoside represents a phosphodiester bond which may be chemically modified; (c) A ２ ~A ａ The nucleotide sequence between A and B consists of a nucleotide sequence substantially complementary to the target sequence (target-corresponding sequence), １ is a nucleoside having a base complementary to the corresponding nucleoside of the target sequence, a nucleoside having adenine, a nucleoside having thymine, or a nucleoside having uracil; A Ｏ３ and A Ｏ５ (d) S １ ~S ａ The nucleotide sequence between １ ~A ａ The nucleotide sequence between is a complementary region consisting of nucleotide sequences that are substantially complementary to each other and form a double-stranded structure; (e) S Ｏ５ and A Ｏ３ If both exist, S Ｏ５ and A Ｏ３ are nucleosides that are not complementary to each other; (f) S Ｏ３ and A Ｏ５ If both exist, S Ｏ３ and A Ｏ５ are nucleosides that are not complementary to each other; and (g) the 5'-position of the 5'-terminal nucleoside and / or the 3'-position of the 3'-terminal nucleoside in the sense strand region and / or the 2'-position of the 3'-terminal nucleoside when the 3'-terminal nucleoside is a 3'-modified nucleoside may be chemically modified.

[0118] <3-1. Sense strand region> The sense strand region of the S3M-oligonucleotide is the region represented by the above formula (I), i.e., S Ｏ５ ~S Ｏ３ The complementary region in the sense strand region of the S3M-oligonucleotide is numbered S starting from the 3'-terminal nucleoside. １ ~S１９ and S located on the 5' side of the complementary region ａ That is, the complementary region in the sense strand of the S3M-oligonucleotide is represented by S in the above formula (I). １ ~S ａ This is the area of ​​S １ ~S １９ each represents one nucleoside, S ａ represents 0 to 5, preferably 0 to 4, more preferably 0 to 3, 0 to 2, or 0 to 1 nucleosides. Ｏ５ , and those added to the 3' end are S Ｏ３ and each represents 0 to 5, preferably 0 to 4, more preferably 0 to 3, 0 to 2, or 0 to 1 nucleosides.

[0119] The S3M-oligonucleotide is characterized in that the nucleoside located at the 3'-end of the complementary region in the sense strand region (i.e., S1 in the above formula (I)) is a 3'-modified nucleoside. Such a 3'-modified nucleoside is not particularly limited as long as the S3M-oligonucleotide has an RNA interference activity and / or a gene expression inhibitory activity, but is preferably 3'-deoxy-3'-fluoroRNA (3'-F RNA), 3'-OMe RNA, or 3'-MOE RNA, and more preferably 3'-OMe RNA. Furthermore, the 2'-position of this 3'-modified nucleoside may be a hydroxyl group, an optionally modified alkoxy group (preferably a methoxy group, an ethoxy group, or a methoxyethoxy group), or an optionally modified phosphate group (preferably a thiophosphate group, a dithiophosphate group, a methylphosphate group, or an ethylphosphate group), and may have an overhang structure (i.e., S1 in the above formula (I)). Ｏ３ The 2'-position is preferably a hydroxyl group.

[0120] S3M - S of the complementary region in the sense strand region of the oligonucleotide １The nucleosides other than the above are not particularly limited, and various natural nucleosides or sugar-modified nucleosides can be employed, but preferably, they are each independently 2'-OMe RNA, 2'-F RNA or DNA.

[0121] One embodiment of the complementary region in the sense strand region of the S3M-oligonucleotide is the 11th, 12th, 13th and 15th nucleosides from the 3' end (i.e., S in the above formula (I)). １１ , S １２ , S １３ and S １５ ) at least one of the nucleosides is 2'-F RNA, and the other nucleosides are 2'-OMe RNA, 2'-F RNA, DNA, or a combination thereof. １１ , S １２ , S １３ and S １５ are all 2'-F RNAs.

[0122] One preferred embodiment of the complementary region in the sense strand region of the S3M-oligonucleotide is ２ ~S １０ , S １４ , S １６ ~S １９ and S ａ are each independently 2'-OMe RNA, 2'-F RNA or DNA, or a modified pattern in which two types selected from them are alternately arranged, more preferably, are each independently 2'-OMe RNA or DNA, or a modified pattern in which they are alternately arranged, and even more preferably, all of them are 2'-OMe RNA. ａ The number of nucleosides can be appropriately selected from 0 to 10, and if necessary, from the 3' side, ２０ , S ２１ , S ２２ , S ２３ , S ２４ , S ２５ , S ２６ , S ２７ , S ２８ , and S ２９ It may be displayed as.

[0123] The nucleotide sequence of the complementary region in the sense strand region of the S3M-oligonucleotide (i.e., S in the above formula (I) １ ~S ａ ) is a complementary region in the antisense region (i.e., A in the above formula (II) １ ~A ａ ) is a nucleotide sequence substantially complementary to

[0124] The sense strand region of the S3M-oligonucleotide has an overhang structure (i.e., S in the above formula (I)) at the 3' end and / or 5' end of its complementary region. Ｏ３ and S Ｏ５ ) may be included. Ｏ３ and S Ｏ５ each independently represent 0 to 5 (preferably 4, 3, 2, 1 or 0) nucleosides, each of which is independently 2'-OMe RNA, 2'-F RNA, DNA or a 2'-O,4'-C-bridged modified nucleoside. Ｏ３ , and S Ｏ５ does not exist, and

[0125] S Ｏ３ and S Ｏ５ When present, the base is not particularly limited as long as it functions as an overhang, but is preferably oligo-U or oligo-T.

[0126] The modification patterns of the sense strand region of the S3M-oligonucleotide are exemplified by the following formulas (Ia) to (Ic): 5' S Ｏ５ (N(M))-S ａ (N(M)-N(M))-S １９ (N(M))-S １８ (N(M))-S １７ (N(M))-S １６ (N(M))-S １５ (N(F))-S １４ (N(M))-S １３ (N(F))-S １２ (N(F))-S １１ (N(F))-S １０ (N(M))-S ９ (N(M))-S ８(N(M))-S ７ (N(M))-S ６ (N(M))-S ５ (N(M))-S ４ (N(M))-S ３ (N(M))-S ２ (N(M))-S １ (N(3M))-2’H 3’(Ia) 5’ S ａ (N(M))-S １９ (N(M))-S １８ (N(M))-S １７ (N(M))-S １６ (N(M))-S １５ (N(F))-S １４ (N(M))-S １３ (N(F))-S １２ (N(F))-S １１ (N(F))-S １０ (N(M))-S ９ (N(M))-S ８ (N(M))-S ７ (N(M))-S ６ (N(M))-S ５ (N(M))-S ４ (N(M))-S ３ (N(M))-S ２ (N(M))-S １ (N(3M))-2’H 3’(Ib) 5’ S １９ (N(M))-S １８ (N(M))-S １７ (N(M))-S １６ (N(M))-S １５ (N(F))-S １４ (N(M))-S １３ (N(F))-S １２ (N(F))-S １１ (N(F))-S １０ (N(M))-S ９ (N(M))-S ８ (N(M))-S ７ (N(M))-S ６ (N(M))-S ５ (N(M))-S ４ (N(M))-S ３ (N(M))-S ２ (N(M))-S １(N(3M))-2'H 3'(Ic) [In the above formula, (N(M)) represents 2'-OMe RNA, (N(F)) represents 2'-F RNA, and (N(3M)) represents 3'-OMe RNA. S Ｏ５ (N(M)) and S ａ (N(M)) represents 0 to 3 nucleosides consisting of 2'-OMe RNA. Two internucleoside bonds between the three nucleosides from the 5' and 3' ends of each oligonucleotide are phosphorothioate bonds, and the remaining internucleoside bonds are phosphodiester bonds. That is, each oligonucleotide has two phosphorothioate bonds at each of the 3' and 5' ends, for a total of four.]

[0127] <3-2. Antisense strand region> The antisense strand region of the S3M-oligonucleotide is the region represented by the above formula (II), i.e., A Ｏ５ ~A Ｏ３ The complementary region in the antisense strand of the S3M-oligonucleotide is numbered from the 5'-terminal nucleoside to the A １ ~A １９ and A located on the 3' side of the complementary region ａ That is, the complementary region of the antisense strand is represented by A in the above formula (II). １ ~A ａ The nucleotide sequence of the complementary region in the antisense strand region of the S3M-oligonucleotide may be a nucleotide sequence that is substantially complementary to the target sequence, and may contain, for example, 5, 4, 3, 2, or 1 base mismatches with the target sequence, but is preferably completely complementary. １ ~A １９ each represents one nucleoside, A ａ represents 0 to 5 nucleosides. The overhang structure of the antisense strand region is added to the 5' end as A Ｏ５ , and the one added to the 3' end is A Ｏ３ and displays:

[0128] A １may be a base substantially complementary to the corresponding base in the target sequence, or may be adenine, uracil, or thymine, independent of the target sequence. It is described in US Patent No. 10,093,923, etc., that when the base at the 3' end of the complementary region of the sense strand of siRNA is adenine or uracil, regardless of the target sequence, good RNA interference activity is exhibited. Furthermore, a literature (Nature 465, 818-822 (2010)) has shown that the nucleoside at the 5' end of the antisense strand binds to the Ago2 protein, which is involved in RNA interference activity, but does not bind to the target mRNA. It has also been shown that when the nucleoside at the 5' end of the antisense strand is an adenine or uracil nucleoside, it binds more strongly to the Ago2 protein than when it is guanine or cytosine, and therefore it is preferable that the 5' end of the antisense strand is an adenine or uracil nucleoside.

[0129] A of the complementary region in the antisense strand region of the S3M-oligonucleotide １１ , A １２ , A １３ , A １４ and A １５ Among these, at least one (preferably two or more) is any one selected from the group consisting of DNA, RNA, 2'-O,4'-C-bridged modified nucleosides, and 2'-MOE RNA. The 2'-O,4'-C-bridged modified nucleosides used here are not particularly limited, but are preferably LNA or ENA. In the antisense strand complementary region, nucleosides other than the nucleosides identified above are not particularly limited, and various natural nucleosides or sugar-modified nucleosides can be used, but are preferably each independently 2'-OMe RNA, 2'-F RNA, or DNA.

[0130] In one example of the present invention, the A of the complementary region in the antisense strand region of the S3M-oligonucleotide １４However, the oligonucleotide is RNA. Examples of the modification patterns of such oligonucleotides include, but are not limited to, types 56 and 57 in Figure 2, types 61, 62, 66, and 67 in Figure 3, types 75 to 83 in Figure 5, and types 91 to 95 in Figure 6.

[0131] A １４ A preferred embodiment of the antisense strand region of the oligonucleotide is RNA is １３ is a 2'-O,4'-C-bridged modified nucleoside, 2'-OMe RNA or 2'-F RNA, more preferably a 2'-O,4'-C-bridged modified nucleoside, even more preferably ENA or LNA (Types 76 to 83 in Figure 5 and Types 91 to 93 in Figure 6). １１ -A １２ -A １３ -A １４ -A １５ The modification pattern of is preferably any one of the following formulas (IIIa) to (IIIh): １１ (2'-OMe RNA)-A １２ (2'-OMe RNA)-A １３ (ENA)-A １４ (RNA)-A １５ (2'-OMe RNA) (IIIa) A １１ (2'-F RNA)-A １２ (2'-OMe RNA)-A １３ (ENA)-A １４ (RNA)-A １５ (2'-OMe RNA) (IIIb) A １１ (2'-OMe RNA)-A １２ (2'-F RNA)-A １３ (ENA)-A １４ (RNA)-A １５ (2'-OMe RNA) (IIIc) A １１ (2'-OMe RNA)-A １２ (2'-OMe RNA)-A １３ (LNA)-A １４ (RNA)-A １５ (2'-OMe RNA) (IIId) A１１ (2'-F RNA)-A １２ (2'-OMe RNA)-A １３ (LNA)-A １４ (RNA)-A １５ (2'-OMe RNA) (IIIe) A １１ (2'-OMe RNA)-A １２ (2'-F RNA)-A １３ (LNA)-A １４ (RNA)-A １５ (2'-OMe RNA) (IIIf) A １１ (2'-F RNA)-A １２ (2'-OMe RNA)-A １３ (2'-OMe RNA)-A １４ (RNA)-A １５ (2'-OMe RNA) (IIIg) A １１ (2'-OMe RNA)-A １２ (2'-F RNA)-A １３ (2'-OMe RNA)-A １４ (RNA)-A １５ (2'-OMe RNA) (IIIh)

[0132] In one example of the present invention, the A of the complementary region in the antisense strand region of the S3M-oligonucleotide １４ is a 2'-O,4'-C-bridged modified nucleoside. Here, the 2'-O,4'-C-bridged modified nucleoside is preferably ENA or LNA. Modification patterns of such oligonucleotides include, but are not limited to, those shown in types 70 to 74 in Figure 4 and types 88 to 90 in Figure 6.

[0133] A １４ A preferred embodiment of the oligonucleotide in which A is a 2'-O,4'-C-bridged modified nucleoside is １２ In such an S3M-oligonucleotide, the A of the antisense strand complementary region is １１ -A １２ -A １３ -A １４ -A １５The modification pattern of is preferably any one of the following formulae (IIIi) to (IIIl): １１ (2'-F RNA)-A １２ (DNA)-A １３ (2'-OMe RNA)-A １４ (ENA)-A １５ (2'-OMe RNA) (IIIi) A １１ (2'-OMe RNA)-A １２ (DNA)-A １３ (2'-OMe RNA)-A １４ (ENA)-A １５ (2'-OMe RNA) (IIIj) A １１ (2'-F RNA)-A １２ (DNA)-A １３ (2'-OMe RNA)-A １４ (LNA)-A １５ (2'-OMe RNA) (IIIk) A １１ (2'-OMe RNA)-A １２ (DNA)-A １３ (2'-OMe RNA)-A １４ (LNA)-A １５ (2'-OMe RNA) (IIIl)

[0134] In one example of the present invention, the A of the complementary region in the antisense strand region of the S3M-oligonucleotide １４ The present invention provides an oligonucleotide in which the nucleotide sequence is DNA. Examples of the modification patterns of such oligonucleotides include, but are not limited to, those shown in types 58 and 59 in Figure 2, and types 63, 64, 68, and 69 in Figure 3.

[0135] In one example of the present invention, the antisense strand region of the S3M-oligonucleotide is A of formula (II). １５ is 2'-MOE RNA. Examples of the modification pattern of such an oligonucleotide include, but are not limited to, those shown in types 79 and 83 in Figure 5.

[0136] In one example of the present invention, the 11th to 13th nucleosides from the 5' end of the complementary region in the antisense strand region of the S3M-oligonucleotide (A of formula (II)) １１ , A １２ , A １３ ) is a 2'-O,4'-C-bridged modified nucleoside. Examples of modification patterns of such oligonucleotides include, but are not limited to, ENA types 43 and 45 in Figure 1, types 75 to 84 in Figure 5, and types 85, 86, and 91 to 93 in Figure 6, and LNA types 44 and 46 in Figure 1, and types 67 and 69 in Figure 3.

[0137] In one example of the present invention, the 11th to 13th nucleosides from the 5' end of the complementary region in the antisense strand region of the S3M-oligonucleotide (A of formula (II)) １１ , A １２ , A １３ ) is DNA. Examples of modification patterns of such oligonucleotides include, but are not limited to, those shown in types 41 and 42 in Figure 1, type 71 in Figure 4, and types 87 to 90 in Figure 6.

[0138] In one example of the present invention, the 11th to 13th nucleosides from the 5' end of the complementary region in the antisense strand region of the S3M-oligonucleotide (A of formula (II)) １１ , A １２ , A １３ ) is 2'-MOE RNA. Examples of modification patterns of such oligonucleotides include, but are not limited to, those shown in types 47 and 48 in Figure 1 and type 74 in Figure 4.

[0139] In the complementary region of the antisense strand region of the S3M-oligonucleotide of the present invention, various types of nucleosides other than the nucleosides specified above can be appropriately selected. ２ , A ６ and A １６At least one (preferably all) nucleoside of the antisense strand is 2'-F RNA. ２ , A ６ and A １６ In addition, A ８ , A ９ and A １０ One or two nucleosides selected from the group consisting of are 2'-F RNA, and nucleosides other than those specified above (e.g., A １ , A ３ ~A ５ , A ７ , A １７ ~A １９ and A ａ ) is 2'-OMe RNA or DNA.

[0140] In the complementary region of the antisense strand region of the S3M-oligonucleotide of the present invention, A １１ ~A １５ Preferred modification patterns for regions other than the above are exemplified in, for example, Tables A-1 and A-2 below.

[0141]

[0142]

[0143] [In the above table, (M) is 2'-OMe RNA and (F) is 2'-F RNA. ａ A is 0 to 5 nucleosides, and all nucleosides other than 0 are the same. Ｏ３ represents 0 to 5 nucleosides, and all nucleosides other than 0 are the same. The internucleoside bond is appropriately selected from a phosphodiester bond or a phosphorothioate bond.]

[0144] The antisense strand region contained in the S3M-oligonucleotide has an overhang structure (i.e., A in the above formula (II)) at the 3' end and / or 5' end of its complementary region. Ｏ３ and A Ｏ５ ) may be present. Ｏ３ and A Ｏ５each independently represent 0 to 5 (preferably 3, 2, 1 or 0) nucleosides, each of which is independently 2'-OMe RNA, 2'-F RNA, DNA or a 2'-O,4'-C-bridged modified nucleoside. Ｏ３ is an overhang consisting of two or three 2'-OMe RNAs, or an overhang consisting of one or two 2'-OMe RNAs and one or two 2'-O,4'-C-bridged modified nucleosides, and A Ｏ５ does not exist.

[0145] A Ｏ３ and A Ｏ５ When present, the base is not particularly limited as long as it functions as an overhang, but is preferably oligo-U or oligo-T.

[0146] <3-3. Internucleoside Bonds> The bond between each nucleoside of the S3M-oligonucleotide of the present invention can be appropriately selected from optionally chemically modified phosphodiester bonds, and is preferably a phosphodiester bond or a phosphorothioate bond, either alone or in combination. Preferably, a combination of a phosphodiester bond or a phosphorothioate bond is used, and the content of phosphorothioate bonds in the internucleoside bond in each of the sense strand region and the antisense strand region is 50% or less, 40% or less, 30% or less, 29% or less, 28% or less, 27% or less, 26% or less, or 25% or less.

[0147] When RNA and / or 2'-F RNA is used, the bond between the nucleoside and its adjacent nucleoside on the 3' side may be a phosphorothioate bond. Preferably, when RNA is used, the bond between the nucleoside and its adjacent nucleoside on the 3' side is a phosphorothioate bond.

[0148] In the S3M-oligonucleotide of the present invention, the first, second, third, fourth, or fifth internucleoside bond (1, 2, 3, or 4 internucleoside bonds) from the 5'-end and / or 3'-end of the oligonucleotide is preferably a phosphorothioate bond. In the oligonucleotide of the present invention, when the sense strand region and the antisense strand region are contained in different oligonucleotides, the internucleoside bonds between the first, second, third, fourth, and fifth nucleosides from the 5'-end and 3'-end of the sense strand region and the internucleoside bonds between the first, second, third, fourth, and fifth nucleosides from the 5'-end and 3'-end of the antisense strand region may be phosphorothioate bonds, and preferably, the internucleoside bonds between the first, second, and third nucleosides from the 5'-end and 3'-end of the sense strand region (two internucleoside bonds), the internucleoside bonds between the first, second, and third nucleosides from the 5'-end of the antisense strand region (two internucleoside bonds), and the internucleoside bonds between the first, second, and third nucleosides from the 3'-end of the antisense strand region (three internucleoside bonds) are phosphorothioate bonds.

[0149] The oligonucleotide of the present invention may employ a phosphonate unit as the 5'-terminal nucleoside of the antisense strand region. In the phosphonate unit, the bond between the 5'-position carbon atom and the carbon atom following the phosphonate unit may be a single bond, a double bond, or a triple bond, preferably a double bond or a triple bond.

[0150] The base in the phosphonate unit is not particularly limited, but is preferably a base complementary to the corresponding nucleoside in the target sequence, such as adenine, thymine, or uracil, and more preferably uracil. The sugar in the phosphonate unit may be natural (i.e., ribose) or modified. Examples of sugar modifications in the phosphonate unit include those used in the sugar-modified nucleosides described above, as well as 2'-amination, in which the 2'-position of the sugar is substituted with an amino group. Also usable are 2'-acyl amination, in which the amino group at the 2'-position of the sugar is acylated, and 2'-formyl amination, in which the amino group at the 2'-position of the sugar is formylated (2'-formyl amination). Examples of 2'-acyl amination include acetyl amination (acetamidation), propionyl amination, and butanoyl amination. The acyl group in the 2'-acyl amination modification may be substituted with an alkoxy group, and 2'-alkoxy acyl amination modification (methoxyacetyl amination, methoxypropionyl amination, methoxybutanoyl amination, etc.) can also be employed. Preferred sugar modifications include 2'-O-methylation and 2'-acetyl amination.

[0151] In the phosphonic acid unit, the base, the bond (single bond, double bond, or triple bond) between the carbon atom at the 5' position and the carbon atom after the addition, and the sugar modification can be appropriately combined, and are not particularly limited. Preferably, any of the phosphonic acid units shown in the following formulas employing uracil as the base is used.

[0152]

[0153] (In the above formula, the end of the dashed line represents a bond to an adjacent nucleotide. The bond to the adjacent nucleotide represents the formation of a phosphodiester bond with the 5'-phosphate group of the adjacent nucleotide, and the phosphodiester bond formed may be a chemically modified phosphodiester bond such as a phosphorothioate bond.)

[0154] In more preferred phosphonate units, the base is uracil, and the combination of the bond between the 5' carbon atom and the carbon atom following the sugar modification is a double bond and 2'-O-methylation, a triple bond and 2'-O-methylation, or a double bond and acetylamination, i.e., VPu, EPuam, or EPuam as shown in the formula above.

[0155] 4. Oligonucleotide Synthesis Method The method for preparing the oligonucleotide of the present invention is not particularly limited as long as it allows the synthesis of the desired oligonucleotide, and known chemical synthesis methods (such as the phosphate triester method, phosphoramidite method, or H-phosphonate method) can be used. For example, the oligonucleotide can be synthesized using a commercially available nucleic acid synthesizer and commercially available reagents used for DNA / RNA synthesis.

[0156] By the conventional phosphoramidite method, an oligonucleotide having a desired nucleotide sequence can be synthesized using a DNA synthesizer, such as Perkin-Elmer's Model 392 phosphoramidite method, according to the method described in the literature (Nucleic Acids Research, 12, 4539 (1984)).

[0157] Amidite reagents corresponding to various nucleosides may be purchased commercially or may be synthesized according to known methods.

[0158] The oligonucleotides of the present invention can be synthesized using a commercially available synthesizer (e.g., Perkin-Elmer Model 392 using the phosphoramidite method) according to the method described in the literature (Nucleic Acids Research, 12, 4539 (1984)). Regarding the phosphoramidite reagents used, commercially available phosphoramidite reagents can be used for natural nucleosides and 2'-O-methyl nucleosides (i.e., 2'-O-methylguanosine, 2'-O-methyladenosine, 2'-O-methylcytidine, and 2'-O-methyluridine). The following applies to 2'-O-alkylguanosine, 2'-O-alkyladenosine, 2'-O-alkylcytidine, and 2'-O-alkyluridine, each of which has a 2'-O-alkyl group containing 2 to 6 carbon atoms.

[0159] 2'-O-aminoethylguanosine, 2'-O-aminoethyladenosine, 2'-O-aminoethylcytidine, and 2'-O-aminoethyluridine can be synthesized by synthesizing the corresponding phosphoramidite reagents according to the literature (Blommers et al., Biochemistry (1998), 37, 17714-17725).

[0160] 2'-O-propylguanosine, 2'-O-propyladenosine, 2'-O-propylcytidine, and 2'-O-propyluridine can be synthesized by synthesizing the corresponding phosphoramidite reagents according to the literature (Lesnik, EA et al., Biochemistry (1993), 32, 7832-7838).

[0161] Commercially available phosphoramidite reagents can be used for 2'-O-allylguanosine, 2'-O-allyl adenosine, 2'-O-allyl cytidine, and 2'-O-allyl uridine.

[0162] For 2'-MOE RNA, 2'-O-methoxyethylguanosine, 2'-O-methoxyethyladenosine, 2'-O-methoxyethyl-5-methylcytidine, and 2'-O-methoxyethyl-5-methyluridine can be synthesized using corresponding phosphoramidite reagents according to a patent (US6261840) or the literature (Martin, P. Helv. Chim. Acta. (1995) 78, 486-504.), or commercially available phosphoramidite reagents can also be used.

[0163] 2'-O-butylguanosine, 2'-O-butyladenosine, 2'-O-butylcytidine, and 2'-O-butyluridine can be synthesized as corresponding phosphoramidite reagents according to the literature (Lesnik, EA et al., Biochemistry (1993), 32, 7832-7838).

[0164] 2'-O-pentylguanosine, 2'-O-pentyladenosine, 2'-O-pentylcytidine, and 2'-O-pentyluridine can be synthesized by synthesizing the corresponding phosphoramidite reagents according to the literature (Lesnik, EA et al., Biochemistry (1993), 32, 7832-7838).

[0165] Commercially available phosphoramidite reagents can be used for 2'-O-propargylguanosine, 2'-O-propargyladenosine, 2'-O-propargylcytidine, and 2'-O-propargyluridine.

[0166] For 2'-F RNA, 2'-deoxy-2'-fluoroguanosine, 2'-deoxy-2'-fluoroadenosine, 2'-deoxy-2'-fluorocytidine, and 2'-deoxy-2'-fluorouridine can be synthesized using corresponding phosphoramidite reagents according to literature (J. Med. Chem. 36, 831 (1993)), or commercially available phosphoramidite reagents can also be used.

[0167] For the 3'-modified nucleoside 3'-F RNA, the corresponding phosphoramidite reagents can be synthesized according to WO 2017 / 027645 for 3'-deoxy-3'-fluoroguanosine, WO 2018 / 198076 for 3'-deoxy-3'-fluoroadenosine, and US 2011 / 0196141 for 3'-deoxy-3'-fluorouridine. 3'-Deoxy-3'-fluorocytidine can be synthesized by reference to known methods such as those described in the above-mentioned literature.

[0168] Regarding the 3'-modified nucleosides, 3'-OMe-RNA, 3'-O-methylguanosine, 3'-O-methyladenosine, 3'-O-methylcytidine, and 3'-O-methyluridine, can be synthesized using commercially available phosphoramidite reagents.

[0169] For the 3'-modified nucleosides 3'-MOE-RNA, 3'-O-methoxyethylguanosine, 3'-O-methoxyethyladenosine, 3'-O-methoxyethyl-5-methylcytidine, and 3'-O-methoxyethyl-5-methyluridine, the corresponding phosphoramidite reagents can be synthesized according to US 2003 / 0096979.

[0170] For LNAs, 2'-O,4'-C-methyleneguanosine, 2'-O,4'-C-methyleneadenosine, 2'-O,4'-C-methylenecytidine, 2'-O,4'-C-methylene-5-methylcytidine, and 2'-O,4'-C-methylene-5-methyluridine, the corresponding phosphoramidite reagents can be prepared according to the method described in WO99 / 14226.

[0171] For 2'-O,4'-C-alkyleneguanosine, 2'-O,4'-C-alkyleneadenosine, 2'-O,4'-C-alkylenecytidine, 2'-O,4'-C-alkylene-5-methylcytidine, and 2'-O,4'-C-alkylene-5-methyluridine, each of which has 2 to 5 carbon atoms in the alkylene group of the 2'-O,4'-C-bridged moiety, the corresponding phosphoramidite reagent can be prepared according to the method described in WO 00 / 47599.

[0172] The 2'-deoxy-2'-C,4'-C-methyleneoxymethylenated nucleoside of D-ribofuranose can be synthesized into the corresponding phosphoramidite reagent according to the literature (Wang, G. et al. Tetrahedron (1999), 55, 7707-7724).

[0173] For S-cEt (constrained ethyl), the corresponding phosphoramidite reagent can be synthesized according to the literature (Seth, PP et al. J. Org. Chem (2010), 75, 1569-1581).

[0174] AmNA can be synthesized by synthesizing the corresponding phosphoramidite reagent according to the literature (Yahara, A. et al. ChemBioChem (2012), 13, 2513-2516.) or WO2014 / 109384.

[0175] After coupling with a phosphoramidite reagent, antisense oligonucleotides having phosphorothioate bonds can be synthesized by reacting with a reagent such as sulfur, tetraethylthiuram disulfide (TETD, Applied Biosystems), Beaucage reagent (Glen Research), or xanthan hydride (Tetrahedron Letters, 32, 3005 (1991), J. Am. Chem. Soc. 112, 1253 (1990), PCT / WO98 / 54198).

[0176] The solid phase supports used in the synthesizer, such as controlled pore glass (CPG) and polystyrene, to which 2'-O-methylnucleosides and 3'-O-methylnucleosides are bound, can be commercially available. Nucleosides produced according to the method described in WO 99 / 14226 were used for 2'-O,4'-C-methyleneguanosine, 2'-O,4'-C-methyleneadenosine, 2'-O,4'-C-methylene-5-methylcytidine, and 2'-O,4'-C-methylene-5-methyluridine, and those produced according to the method described in WO 00 / 47599 were used for 2'-O,4'-C-alkyleneguanosine, 2'-O,4'-C-alkyleneadenosine, 2'-O,4'-C-alkylene-5-methylcytidine, and 2'-O,4'-C-alkylene-5-methyluridine, each of which has an alkylene group with 2 to 5 carbon atoms. These nucleosides were used as described in the literature (Oligonucleotide Synthesis, Edited by M. J. Gait, The oligonucleotide can be linked to CPG according to the method described in "Chemical Synthesis of Oligonucleotides with 2-hydroxyethyl phosphate groups at the 3' end" (Oxford University Press, 1984). Alternatively, the oligonucleotide can be synthesized by linking the above-mentioned phosphoramidite reagent using a universal resin. Alternatively, the oligonucleotide can be synthesized by using a modified CPG (described in Example 12b of JP-A-7-87982). Furthermore, by using 3'-amino-Modifier C3 CPG, 3'-amino-Modifier C7 CPG, Glyceryl CPG (Glen Research), 3'-spacer C3 SynBase CPG 1000, or 3'-spacer C9 SynBase CPG 1000 (Link Technologies), it is possible to synthesize oligonucleotides having a hydroxyalkyl phosphate group or an aminoalkyl phosphate group bound to the 3' end.

[0177] Furthermore, when thioating an oligonucleotide analogue, a thioate derivative can be obtained in accordance with the method described in the literature (Tetarhedron Letters, 32, 3005 (1991), J. Am. Chem. Soc., 112, 1253 (1990)) using a reagent such as tetraethylthiuram disulfide (TETD, Applied Biosystems), Beaucage reagent, or phenylacetyl disulfide / pyridine-acetonitrile (1:1 v / v) solution (Ravikumar, V.T. et al. Bioorg.Med.Chem.Lett. (2006) 16, pp. 2513-2517) in addition to sulfur.

[0178] When a silyl protecting group such as t-butyldimethylsilyl (TBS) is used as the protecting group for the 2'-hydroxyl group of RNA, the protecting group can be deprotected using tetrabutylammonium fluoride (TBAF), HF-pyridine, HF-triethylamine, or the like.

[0179] The acyl group in the base moiety and the cyanoethyl group in the phosphate moiety can be deprotected using concentrated aqueous ammonia, methanolic ammonia, ethanolic ammonia, a mixture of concentrated aqueous ammonia and ethanol (3:1 V / V), a mixture of concentrated aqueous ammonia and 40% aqueous methylamine (1:1 V / V), methylamine, 0.5 M aqueous LiOH, a mixture of 3.5 M triethylamine / methanol (1:10 V / V), 0.05 M potassium carbonate in methanol, or the like, with concentrated aqueous ammonia and a mixture of concentrated aqueous ammonia and ethanol (3:1 V / V) being preferred.

[0180] The oligonucleotide of the present invention can be obtained by purifying it using a purification procedure typically used for purifying nucleic acids, such as various types of chromatography, such as reverse phase chromatography and ion exchange chromatography (including high performance liquid chromatography).

[0181] The oligonucleotides of the present invention include oligonucleotides conjugated with a transport unit for transporting the oligonucleotide to a target tissue or target cell. Examples of oligonucleotides conjugated with such a transport unit include oligonucleotides in which a unit having an aminoalkyl phosphate group (e.g., an alkyl having 3 to 9 carbon atoms) introduced into a desired chemical structure is conjugated to the 5' position of the 5'-terminal nucleotide and / or the 3' position of the 3'-terminal nucleotide (the 2' position for nucleotides modified at the 3' position). Examples of such transport units include fatty acid units (e.g., myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, etc.) for transport to liver or muscle tissue (see, for example, Nucleic Acids Res. (2020) 47, 6029-6044, WO2017 / 19267, etc.), and GalNAc units that bind to the asialoglycoconjugate receptor (ASGRP) of liver parenchymal cells.

[0182] As used herein, a fatty acid unit refers to a unit in which an aminoalkyl alcohol or 4-aminophenol is bound to a fatty acid via an amide bond. In the oligonucleotide of the present invention, the fatty acid unit may be bound to the 5'-phosphate group of the 5'-terminal nucleotide or the 3'-phosphate group of the 3'-terminal nucleotide (or the 2'-phosphate group in a nucleotide modified at the 3'-position) via a phosphodiester bond, which may be chemically modified. Examples of fatty acids contained in the fatty acid unit include fatty acids having 6 to 30 carbon atoms, and preferred are myristic acid (14 carbon atoms), palmitic acid (16 carbon atoms), stearic acid (18 carbon atoms), arachidic acid (20 carbon atoms), and behenic acid (22 carbon atoms).

[0183] The aminoalkyl alcohol contained in the fatty acid unit in which an aminoalkyl alcohol is bound to a fatty acid via an amide bond includes an aminoalkyl alcohol having 2 to 8 carbon atoms, preferably an aminoalkyl alcohol having 2 to 6 carbon atoms, and more preferably aminoethanol, aminobutanol, or aminohexanol.

[0184] Preferred examples of fatty acid units in which an aminoalkyl alcohol is bound to a fatty acid via an amide bond include myristic acid (C14), palmitic acid (C16), stearic acid (C18 or S), arachidic acid (C20), and behenic acid (C22) of the following formulae, with S (also referred to as C18) being more preferred. These fatty acid units can be appropriately prepared according to the method described in WO2021 / 010301.

[0185]

[0186] (In the above formula, the end of the dashed line indicates that the oxygen atom bonds to the adjacent nucleotide, forming a phosphodiester bond with the 5'-phosphate group when bonding to the 5'-terminal nucleotide, or with the 3'-phosphate group (the 2'-phosphate group in a nucleotide modified at the 3'-position) when bonding to the 3'-terminal nucleotide; the phosphodiester bond formed may be a chemically modified phosphodiester bond such as a phosphorothioate bond.)

[0187] A preferred example of a fatty acid unit in which 4-aminophenol is bound to a fatty acid via an amide bond is SPh shown in the following formula: Such a fatty acid unit can be prepared as appropriate according to a conventional method, but can also be synthesized, for example, by condensing 4-aminophenol and stearic acid via an amide bond and then converting the resulting phenol compound into a phosphoramidite form.

[0188]

[0189] (In the above formula, the end of the dashed line indicates that the oxygen atom bonds to the adjacent nucleotide, forming a phosphodiester bond with the 5'-phosphate group when bonding to the 5'-terminal nucleotide, or with the 3'-phosphate group (the 2'-phosphate group in a nucleotide modified at the 3'-position) when bonding to the 3'-terminal nucleotide; the phosphodiester bond formed may be a chemically modified phosphodiester bond such as a phosphorothioate bond.)

[0190] In the oligonucleotide of the present invention, SPh can be employed as a preferred fatty acid unit.

[0191] The fatty acid unit is involved in the in vivo drug delivery of the oligonucleotide, and can contribute to improving the persistence of the oligonucleotide of the present invention in the body and / or enhancing the cellular uptake of the oligonucleotide of the present invention. Furthermore, the fatty acid unit can be applied regardless of the nucleotide sequence of the oligonucleotide to be added, and can be applied to oligonucleotides targeting various sequences.

[0192] The GalNAc unit is a structural unit containing N-acetyl-D-galactosamine (GalNAc) capable of binding to ASGRP, and can be used to deliver the oligonucleotide of the present invention to the liver. The GalNAc unit may have a linear or branched linker structure, for example, a linear, two or more-branched, three or more-branched, or four or more-branched, four or less-branched, three or less-branched, or two or less-branched linker structure. The GalNAc unit may also contain a phosphate group or a thiophosphate group for binding to an oligonucleotide. As long as the ability to bind to ASGRP is maintained, there is no limit to the number of GalNAc units contained in one GalNAc unit, and the GalNAc structure may be modified. Examples of the GalNAc unit that can be used in the oligonucleotide of the present invention include those described in, for example, WO2021 / 049504, WO2009 / 073809, WO2014 / 076196, WO2014 / 179620, WO2015 / 006740, WO2015 / 105083, WO2016 / 055601, WO2017 / 023817, WO2017 / 084987, and WO2017 / 131236; Methods in Enzymology, 1999, Vol. 313, pp. 297-321; and Bioorganic & Medicinal Chemistry, vol. Letters 26 (2016) 3690-3693, etc., can be appropriately employed.

[0193] The oligonucleotide of the present invention includes oligonucleotides into which a cholesterol unit or a vitamin E unit has been introduced (e.g., Lorenz, C. et al. Bioorg. Med. Chem. Lett., 14, pp. 4975-4977 (2004); Soutschek, J., et al. Nature, 432, pp. 173-178, (2004); Wolfrum, C. et al. Nature Biotech. 25, pp. 1149-1157, (2007)), Kubo, T. et al. Oligonucleotides, 17, pp. 1-20, (2007); Kubo, T., et al. Biochem. Biophys. Res. Comm. 365, pp. 54-61, (2008); Nishina, K., et al., Mol. Ther. 16, pp. 734-740, (2008). ) and oligonucleotides having an aptamer, a nucleic acid molecule that binds to proteins, attached to the end of the oligonucleotide.

[0194] Vpu can be synthesized using the method described in J. Med. Chem. 2018, 61, 3, 734-744.

[0195] Vpuam, Vpuam2, Vpuam3, and Vpuam4 can be synthesized using the following steps A-1 to A-10.

[0196]

[0197] (In the formula, R １ is a general protecting group for a hydroxyl group, and R ２ is an optionally substituted acyl group having 1 to 6 carbon atoms, and R ３ is hydrogen or an alkyl group having 1 to 4 carbon atoms which may be substituted. ４ ) R ５ is [R ４ and R ５ are the same or different and represent an amino group substituted with an alkoxy group having 1 to 4 carbon atoms, an alkylthio group having 1 to 4 carbon atoms, a cyanoalkoxy group having 1 to 5 carbon atoms, or an alkyl group having 1 to 4 carbon atoms.

[0198] Step A-1 is a step of reacting the two hydroxyl groups of compound (1) with a protecting reagent in an inert solvent to produce compound (2).

[0199] Preferred examples of the solvent to be used include aromatic hydrocarbons such as benzene, toluene, and xylene; halogenated hydrocarbons such as methylene chloride, chloroform, carbon tetrachloride, dichloroethane, chlorobenzene, and dichlorobenzene; esters such as ethyl formate, ethyl acetate, propyl acetate, butyl acetate, and diethyl carbonate; ethers such as diethyl ether, diisopropyl ether, tetrahydrofuran, dioxane, dimethoxyethane, and diethylene glycol dimethyl ether; ketones such as acetone, methyl ethyl ketone, methyl isobutyl ketone, isophorone, and cyclohexanone; nitro compounds such as nitroethane and nitrobenzene; nitriles such as acetonitrile and isobutyronitrile; amides such as formamide, dimethylformamide (DMF), dimethylacetamide, and hexamethylphosphorotriamide; and sulfoxides such as dimethyl sulfoxide and sulfolane. More preferred examples include ethers (particularly tetrahydrofuran), halogenated hydrocarbons (particularly methylene chloride), aromatic hydrocarbons (particularly toluene), and amides (particularly DMF). More preferred are amides (particularly DMF). The protecting reagent used is not particularly limited as long as it can normally be used for deprotection, but preferred are silyl halides such as t-butyldimethylsilyl chloride, triisopropylsilyl chloride, and triethylsilyl chloride. More preferred is triethylsilyl chloride.

[0200] When a silyl halide is used as a protecting reagent, a base is usually used. The base to be used is preferably an organic base (particularly, triethylamine, pyridine, N-methylmorpholine, DBU, imidazole, etc.). More preferably, imidazole is used.

[0201] The reaction temperature is usually −20 to 150° C., preferably −10 to 50° C., depending on the reagents, raw materials, solvent, etc. used. The reaction time varies depending on the raw materials, solvent, reaction temperature, etc. used, but is usually 1 to 100 hours, preferably 1 to 24 hours.

[0202] After the reaction is complete, for example, the reaction mixture is poured into water or saturated aqueous sodium bicarbonate, extracted with a water-immiscible solvent such as benzene, ether, or ethyl acetate, and the solvent is distilled off from the extract to obtain a product which is usually used as is in the next step. If desired, the product can be isolated and purified by various chromatography or recrystallization methods.

[0203] Step A-2 is a step of preparing compound (3) by reacting compound (2) prepared by the above-mentioned method with a deprotecting agent in an inert solvent to deprotect the protecting group of the 5'-hydroxyl group.

[0204] The solvent to be used is preferably an aromatic hydrocarbon such as benzene, toluene, or xylene; a halogenated hydrocarbon such as methylene chloride, chloroform, carbon tetrachloride, dichloroethane, chlorobenzene, or dichlorobenzene; an ester such as ethyl formate, ethyl acetate, propyl acetate, butyl acetate, or diethyl carbonate; an ether such as diethyl ether, diisopropyl ether, tetrahydrofuran, dioxane, dimethoxyethane, or diethylene glycol dimethyl ether; methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, t-butanol, isoamyl alcohol, Examples of the solvent include alcohols such as diethylene glycol, glycerin, octanol, cyclohexanol, and methyl cellosolve; ketones such as acetone, methyl ethyl ketone, methyl isobutyl ketone, isophorone, and cyclohexanone; nitro compounds such as nitroethane and nitrobenzene; nitriles such as acetonitrile and isobutyronitrile; amides such as formamide, dimethylformamide, dimethylacetamide, and hexamethylphosphorotriamide; and sulfoxides such as dimethyl sulfoxide and sulfolane, and more preferably, alcohols (particularly methanol and ethanol) and methylene chloride are mentioned.

[0205] The deprotecting reagent used is not particularly limited as long as it is a commonly used one, and examples thereof include Lewis acids such as acetic acid, dichloroacetic acid, trifluoroacetic acid, pyridinium toluenesulfonic acid, hydrochloric acid, and zinc bromide, and preferably includes acetic acid, dichloroacetic acid, trifluoroacetic acid, and pyridinium toluenesulfonic acid, and more preferably includes pyridinium toluenesulfonic acid.

[0206] The reaction temperature varies depending on the reagents, raw materials, solvents, etc. used, but is usually -10 to 100°C, preferably 0 to 50°C.

[0207] The reaction time varies depending on the starting materials, solvent, reaction temperature, etc. used, but is usually from 1 minute to 50 hours, preferably from 1 minute to 24 hours.

[0208] After completion of the reaction, for example, the reaction mixture is neutralized with a base such as pyridine or triethylamine, poured into water or saturated aqueous sodium bicarbonate, and extracted with a water-immiscible solvent such as benzene, ether, or ethyl acetate. The solvent is then distilled off from the extract to give a product which is usually used as is in the next step. If desired, the product can also be isolated and purified by various chromatography or recrystallization methods.

[0209] Step A-3 is a step of producing compound (4) by reacting compound (3) produced by the above-mentioned method with an oxidizing agent in an inert solvent.

[0210] Examples of the solvent to be used include aliphatic hydrocarbons such as hexane, heptane, ligroin, and petroleum ether; aromatic hydrocarbons such as benzene, toluene, and xylene; halogenated hydrocarbons such as methylene chloride, chloroform, carbon tetrachloride, dichloroethane, chlorobenzene, and dichlorobenzene; esters such as ethyl formate, ethyl acetate, propyl acetate, butyl acetate, and diethyl carbonate; ethers such as diethyl ether, diisopropyl ether, tetrahydrofuran, dioxane, dimethoxyethane, and diethylene glycol dimethyl ether; ketones such as acetone, methyl ethyl ketone, methyl isobutyl ketone, isophorone, and cyclohexanone; and nitriles such as acetonitrile and isobutyronitrile. Preferred are halogenated hydrocarbons (particularly methylene chloride); and nitriles such as acetonitrile and isobutyronitrile. Acetonitrile is even more preferred.

[0211] Examples of the oxidizing agent that can be used include a Swern oxidation reagent, a Dess-Martin oxidation reagent, a chromium trioxide complex such as a pyridine hydrochloride-chromium trioxide complex (pyridinium chlorochromate, pyridinium dichromate), and 2-iodoxybenzoic acid, with 2-iodoxybenzoic acid being the preferred reagent.

[0212] The reaction temperature varies depending on the starting compounds, solvent and oxidizing agent used, but is usually -100 to 150°C, preferably -20 to 100°C.

[0213] The reaction time varies depending on the starting compounds, solvent, oxidizing agent and reaction temperature used, but is usually 30 minutes to 12 hours, preferably 1 to 5 hours.

[0214] After completion of the reaction, the target compound (4) can be obtained by, for example, decomposing the oxidizing agent, concentrating the reaction mixture, adding water and an immiscible organic solvent such as ethyl acetate, washing with water, separating the organic layer containing the target compound, drying it over anhydrous magnesium sulfate, etc., and then distilling off the solvent. If necessary, the resulting compound can be further purified by conventional methods such as recrystallization or silica gel column chromatography.

[0215] Step A-4 is a step of producing compound (6) by reacting compound (4) produced by the above-mentioned method with a carbon-increasing reagent (5) in an inert solvent.

[0216] Examples of the solvent to be used include aliphatic hydrocarbons such as hexane, heptane, ligroin, and petroleum ether; aromatic hydrocarbons such as benzene, toluene, and xylene; halogenated hydrocarbons such as methylene chloride, chloroform, carbon tetrachloride, dichloroethane, chlorobenzene, and dichlorobenzene; esters such as ethyl formate, ethyl acetate, propyl acetate, butyl acetate, and diethyl carbonate; ethers such as diethyl ether, diisopropyl ether, tetrahydrofuran, dioxane, dimethoxyethane, and diethylene glycol dimethyl ether; and ketones such as acetone, methyl ethyl ketone, methyl isobutyl ketone, isophorone, and cyclohexanone. Of these, halogenated hydrocarbons (particularly methylene chloride) and ethers (particularly tetrahydrofuran) are preferred. Tetrahydrofuran is even more preferred.

[0217] The reagent (5) used includes tetra(acyloxymethyl)methylenebisphosphonate esters. Examples of acyloxymethyl include acetoxymethyl, propionyloxymethyl, and pivaloyloxymethyl. Pivaloyloxymethyl is preferred. Examples of the reagent include tetra(acetoxymethyl)methylenebisphosphonate, tetra(propionyloxymethyl)methylenebisphosphonate, and tetra(pivaloyloxymethyl)methylenebisphosphonate. Tetra(pivaloyloxymethyl)methylenebisphosphonate is preferred. Sodium hydride is usually added to the reaction.

[0218] The reaction temperature varies depending on the starting compounds, solvent and reaction reagents used, but is usually -50°C to 50°C, preferably 0°C to 30°C.

[0219] The reaction time varies depending on the starting compounds, solvent, reaction reagents and reaction temperature used, but is typically 30 minutes to 12 hours, preferably 1 to 5 hours.

[0220] After completion of the reaction, the target compound (6) can be obtained, for example, by concentrating the reaction mixture, adding an immiscible organic solvent such as water and ethyl acetate, washing with water, separating the organic layer containing the target compound, drying it over anhydrous magnesium sulfate, etc., and then distilling off the solvent. If necessary, the resulting compound can be further purified by a conventional method, for example, recrystallization, silica gel column chromatography, etc.

[0221] Step A-5 is a step of reacting compound (6) produced by the above-mentioned method with a reducing agent in an inert solvent to reduce the azido group to an amino group, and further amidating the amino group with an acylating agent or a formylating agent to produce compound (7).

[0222] Examples of the solvent to be used include alcohols such as methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, t-butanol, isoamyl alcohol, diethylene glycol, glycerin, octanol, cyclohexanol, and methyl cellosolve; and esters such as ethyl formate, ethyl acetate, propyl acetate, butyl acetate, and diethyl carbonate, with esters (particularly ethyl acetate) being preferred.

[0223] Examples of the reducing agent to be used include alkali metal borohydrides such as lithium borohydride; aluminum hydride compounds such as lithium aluminum hydride and lithium aluminum triethoxide hydride; and catalytic hydrogenation using a Lindlar catalyst, with catalytic hydrogenation using a Lindlar catalyst being preferred.

[0224] Examples of the acylating agent or formylating agent to be used include formic acid chloride, formic acid anhydride, acetic acid chloride, acetic acid anhydride, propionic acid chloride, propionic acid anhydride, butyric acid chloride, butyric acid anhydride, etc. Acetic acid anhydride is preferred.

[0225] When an acylating agent or a formylating agent is used, a base is usually used. The base to be used is preferably an organic base (particularly, triethylamine, pyridine, N-methylmorpholine, DBU, imidazole, etc.). More preferably, N-methylmorpholine is used.

[0226] The reaction temperature varies depending on the starting compounds, solvent and reducing agent used, but is usually 0 to 80°C, preferably 10 to 50°C.

[0227] The reaction time varies depending on the starting compounds, solvent, reducing agent and reaction temperature used, but is usually 10 minutes to 24 hours, preferably 30 minutes to 5 hours.

[0228] After completion of the reaction, the target compound (7) of this reaction can be obtained, for example, by decomposing or filtering the reducing agent, concentrating the reaction mixture, adding an immiscible organic solvent such as water and ethyl acetate, washing with water, separating the organic layer containing the target compound, drying it over anhydrous magnesium sulfate, etc., and then distilling off the solvent.

[0229] Step A-6 is a step of reacting compound (7) produced by the above-mentioned method with a deprotecting agent in an inert solvent to deprotect the hydroxyl-protecting group, thereby producing compound (8) or compound (9).

[0230] The solvent to be used is not particularly limited as long as it does not inhibit the reaction and dissolves the starting materials to some extent, but preferred examples include ethers such as diethyl ether, diisopropyl ether, tetrahydrofuran, dioxane, dimethoxyethane, and diethylene glycol dimethyl ether; nitriles such as acetonitrile and isobutyronitrile; water; organic acids such as acetic acid; and mixed solvents thereof. Tetrahydrofuran is even more preferred.

[0231] The reagent used can be removed by treating with a compound that generates fluorine anions, such as tetrabutylammonium fluoride, hydrofluoric acid, hydrofluoric acid-pyridine, or potassium fluoride, or by treating with an organic acid, such as acetic acid, formic acid, methanesulfonic acid, paratoluenesulfonic acid, trifluoroacetic acid, or trifluoromethanesulfonic acid, or an inorganic acid, such as hydrochloric acid. Formic acid is more preferred.

[0232] The reaction temperature varies depending on the starting compounds, solvent and reaction reagents used, but is usually -20 to 100°C, preferably 0 to 50°C.

[0233] The reaction time varies depending on the starting compounds, solvent, reaction reagents and reaction temperature used, but is 5 minutes to 24 hours, preferably 20 minutes to 12 hours.

[0234] After completion of the reaction, the target compound (8) or (9) of this reaction can be obtained, for example, by concentrating the reaction mixture, adding an immiscible organic solvent such as water and ethyl acetate, washing with water, separating the organic layer containing the target compound, drying it over anhydrous magnesium sulfate, etc., and then distilling off the solvent. The resulting compound (8) and compound (9) can be separated and purified by a conventional method, for example, recrystallization, silica gel column chromatography, etc.

[0235] Step A-7 and Step A-9 are steps for producing compound (10) or compound (12) by reacting compound (8) or compound (9) produced by the above-mentioned method with a monosubstituted-chloro(alkoxy)phosphine or disubstituted-alkoxyphosphine that is generally used for amiditization in an inert solvent.

[0236] The solvent to be used is not particularly limited as long as it does not affect the reaction, but preferred examples include ethers such as tetrahydrofuran, diethyl ether, and dioxane, and halogenated hydrocarbons such as methylene chloride, chloroform, carbon tetrachloride, dichloroethane, chlorobenzene, and dichlorobenzene, and more preferred examples include methylene chloride.

[0237] Examples of the mono-substituted chloro(alkoxy)phosphine to be used include phosphines such as chloro(morpholino)methoxyphosphine, chloro(morpholino)cyanoethoxyphosphine, chloro(dimethylamino)methoxyphosphine, chloro(dimethylamino)cyanoethoxyphosphine, chloro(diisopropylamino)methoxyphosphine, and chloro(diisopropylamino)cyanoethoxyphosphine, and preferred are chloro(morpholino)methoxyphosphine, chloro(morpholino)cyanoethoxyphosphine, chloro(diisopropylamino)methoxyphosphine, and chloro(diisopropylamino)cyanoethoxyphosphine.

[0238] When a mono-substituted chloro(alkoxy)phosphine is used, an acid scavenger is used. In this case, examples of the acid scavenger that can be used include heterocyclic amines such as pyridine and dimethylaminopyridine, and aliphatic amines such as trimethylamine, triethylamine and diisopropylamine, and preferably aliphatic amines (particularly triethylamine and diisopropylamine).

[0239] Examples of the di-substituted alkoxyphosphine to be used include phosphines such as bis(diisopropylamino)cyanoethoxyphosphine, bis(diethylamino)methanesulfonylethoxyphosphine, bis(diisopropylamino)(2,2,2-trichloroethoxy)phosphine, and bis(diisopropylamino)(4-chlorophenylmethoxy)phosphine, and preferably bis(diisopropylamino)cyanoethoxyphosphine.

[0240] When a disubstituted alkoxyphosphine is used, an acid is used, and in this case, the acid to be used is preferably tetrazole, acetic acid, dicyanoimidazole or p-toluenesulfonic acid.

[0241] The reaction temperature is not particularly limited, but is usually 0 to 80°C, preferably room temperature.

[0242] The reaction time varies depending on the starting materials, reagents, temperature, etc. used, but is usually 5 minutes to 30 hours, and preferably 30 minutes to 10 hours when the reaction is carried out at room temperature.

[0243] After completion of the reaction, the target compound (10) or (12) of this reaction can be obtained, for example, by neutralizing the reaction mixture appropriately and, if any insoluble matter is present, removing it by filtration, adding an immiscible organic solvent such as water and ethyl acetate, washing with water, separating the organic layer containing the target compound, drying it over anhydrous magnesium sulfate, etc., and then distilling off the solvent. If necessary, the obtained target compound can be further purified by a conventional method, for example, recrystallization, reprecipitation, or chromatography.

[0244] Steps A-8 and A-10 are steps in which compound (8) or compound (9) prepared by the above method is reacted with tris-(1,2,4-triazolyl)phosphite or 2-chloro-1,3,2-benzodioxaphosphorin-4-one (or salicylphosphorochloridite) in an inert solvent (preferably a halogenated hydrocarbon such as methylene chloride), and then water is added to convert the resulting compound to an H-phosphonate, thereby producing compound (11) or compound (13). The reaction temperature is not particularly limited, but is usually −20 to 100° C., and preferably 10 to 40° C.

[0245] The reaction time varies depending on the raw materials, reagents, temperature, etc. used, but is usually 5 minutes to 30 hours, and preferably 30 minutes when the reaction is carried out at room temperature. After completion of the reaction, the target compound (11) or the target compound (13) of this reaction can be obtained, for example, by appropriately neutralizing the reaction mixture and, if any insoluble matter is present, removing it by filtration, adding an immiscible organic solvent such as water and ethyl acetate, washing with water, separating the organic layer containing the target compound, drying it over anhydrous magnesium sulfate, etc., and then distilling off the solvent. If necessary, the obtained target compound can be further purified by a conventional method, for example, recrystallization, reprecipitation, chromatography, etc.

[0246] EPu can be synthesized using the following steps B-1 to B-10.

[0247]

[0248] (In the formula, R1, R ２ is a general protecting group for a hydroxyl group, and R ３ is hydrogen, an optionally substituted acyl group having 1 to 6 carbon atoms, or an optionally substituted aromatic acyl group; R ４ is a protecting group in which an optionally substituted alkyl group having 1 to 4 carbon atoms or an optionally substituted alkyleneoxycarbonyl group having 1 to 4 carbon atoms is bonded to an optionally substituted alkyl group having 1 to 4 carbon atoms. ５ ) R ６ is [R ５ and R ６are the same or different and represent an amino group substituted with an alkoxy group having 1 to 4 carbon atoms, an alkylthio group having 1 to 4 carbon atoms, a cyanoalkoxy group having 1 to 5 carbon atoms, or an alkyl group having 1 to 4 carbon atoms.

[0249] Step B-1 is a step of preparing compound (15) by reacting the 5'-hydroxyl group of compound (14) with a protecting reagent in an inert solvent.

[0250] The solvent to be used is preferably an aromatic hydrocarbon such as benzene, toluene, or xylene; a halogenated hydrocarbon such as methylene chloride, chloroform, carbon tetrachloride, dichloroethane, chlorobenzene, or dichlorobenzene; an ester such as ethyl formate, ethyl acetate, propyl acetate, butyl acetate, or diethyl carbonate; an ether such as diethyl ether, diisopropyl ether, tetrahydrofuran, dioxane, dimethoxyethane, or diethylene glycol dimethyl ether; a ketone such as acetone, methyl ethyl ketone, methyl isobutyl ketone, isophorone, or cyclohexanone; a nitro compound such as nitroethane or nitrobenzene; a nitrile such as acetonitrile or isobutyronitrile; an amide such as formamide, dimethylformamide (DMF), dimethylacetamide, or hexamethylphosphorotriamide; a sulfoxide such as dimethyl sulfoxide or sulfolane; or an amine such as pyridine, triethylamine, or diisopropylethylamine, or more preferably a halogenated hydrocarbon (particularly methylene chloride). More preferred examples include halogenated hydrocarbons (especially methylene chloride).

[0251] The protecting reagent used is not particularly limited as long as it can selectively protect the 5'-hydroxyl group, but preferred examples include trityl chlorides such as trityl chloride, 4-methoxytrityl chloride, 4,4'-dimethoxytrityl chloride, and 4,4'-dimethoxy-4''-methylsulfonyltrityl chloride. More preferred examples include 4,4'-dimethoxytrityl chloride.

[0252] When trityl chlorides are used as the protecting reagent, a base is usually used. The base to be used is preferably an organic base (particularly, triethylamine, pyridine, N-methylmorpholine, DBU, imidazole, etc.). More preferably, imidazole is used.

[0253] The reaction temperature is usually -20 to 80°C, preferably -10 to 50°C, depending on the reagents, raw materials, solvent, etc. used.

[0254] The reaction time varies depending on the starting materials, solvent, reaction temperature, etc., but is usually 1 to 100 hours, preferably 1 to 24 hours. After completion of the reaction, the target compound (15) of this reaction can be obtained, for example, by pouring the reaction mixture into water or saturated aqueous sodium bicarbonate, adding an immiscible organic solvent such as methylene chloride, washing with water, separating the organic layer containing the target compound, drying it over sodium sulfate, etc., and then distilling off the solvent. If necessary, the resulting compound can be further purified by conventional methods, such as recrystallization or silica gel column chromatography.

[0255] Step B-2 is a step of preparing compound (16) by reacting the 3'-hydroxyl group of compound (15) with a protecting reagent in an inert solvent.

[0256] Preferred examples of the solvent to be used include aromatic hydrocarbons such as benzene, toluene, and xylene; halogenated hydrocarbons such as methylene chloride, chloroform, carbon tetrachloride, dichloroethane, chlorobenzene, and dichlorobenzene; esters such as ethyl formate, ethyl acetate, propyl acetate, butyl acetate, and diethyl carbonate; ethers such as diethyl ether, diisopropyl ether, tetrahydrofuran, dioxane, dimethoxyethane, and diethylene glycol dimethyl ether; ketones such as acetone, methyl ethyl ketone, methyl isobutyl ketone, isophorone, and cyclohexanone; nitro compounds such as nitroethane and nitrobenzene; nitriles such as acetonitrile and isobutyronitrile; amides such as formamide, dimethylformamide (DMF), dimethylacetamide, and hexamethylphosphorotriamide; and sulfoxides such as dimethyl sulfoxide and sulfolane. More preferred examples include ethers (particularly tetrahydrofuran), halogenated hydrocarbons (particularly methylene chloride), aromatic hydrocarbons (particularly toluene), and amides (particularly DMF). More preferred examples include amides (particularly DMF).

[0257] The protecting reagent to be used is not particularly limited as long as it can normally be used for deprotection, but preferred examples include silyl halides such as t-butyldimethylsilyl chloride, triethylsilyl chloride, triisopropylsilyl chloride, and triethylsilyl chloride, and more preferred examples include t-butyldiphenylsilyl chloride.

[0258] When a silyl halide is used as a protecting reagent, a base is usually used. The base to be used is preferably an organic base (particularly, triethylamine, pyridine, N-methylmorpholine, DBU, imidazole, etc.). More preferably, imidazole is used.

[0259] The reaction temperature is usually -20 to 150°C, preferably -10 to 50°C, depending on the reagents, raw materials, solvent, etc. used.

[0260] The reaction time varies depending on the starting materials, solvent, reaction temperature, etc. used, but is usually 1 to 100 hours, preferably 1 to 24 hours.

[0261] After the reaction is complete, for example, the reaction mixture is poured into water or saturated aqueous sodium bicarbonate, extracted with a water-immiscible solvent such as benzene, ether, or ethyl acetate, and the solvent is distilled off from the extract to obtain a product which is usually used as is in the next step. If desired, the product can be isolated and purified by various chromatography or recrystallization methods.

[0262] Step B-3 is a step of preparing compound (17) by reacting compound (16) with a protecting reagent in an inert solvent.

[0263] The solvent to be used is preferably an aromatic hydrocarbon such as benzene, toluene, or xylene; a halogenated hydrocarbon such as methylene chloride, chloroform, carbon tetrachloride, dichloroethane, chlorobenzene, or dichlorobenzene; an ester such as ethyl formate, ethyl acetate, propyl acetate, butyl acetate, or diethyl carbonate; an ether such as diethyl ether, diisopropyl ether, tetrahydrofuran, dioxane, dimethoxyethane, or diethylene glycol dimethyl ether; or an alkyl ether such as acetone, methyl ethyl ketone, methyl isobutyl ketone, isophorone, or cyclohexanone. Examples of suitable solvents include ketones, nitro compounds such as nitroethane and nitrobenzene, nitriles such as acetonitrile and isobutyronitrile, amides such as formamide, dimethylformamide (DMF), dimethylacetamide and hexamethylphosphorotriamide, sulfoxides such as dimethyl sulfoxide and sulfolane, and amines such as pyridine, triethylamine and diisopropylethylamine. More preferred solvents include ethers (particularly tetrahydrofuran), halogenated hydrocarbons (particularly methylene chloride), amides (particularly DMF), and amines (particularly pyridine). More preferred solvents include amines (particularly pyridine).

[0264] The protecting reagent used is not particularly limited as long as it can be protected with ammonia, but preferred examples include acyl halides such as benzoyl chloride, p-nitrobenzoyl chloride, and p-methoxybenzoyl chloride; pivaloylmethylene chloride, etc. More preferred is benzoyl chloride.

[0265] When an acyl halide is used as a protecting reagent, a base is usually used. The base to be used is preferably an organic base (particularly, triethylamine, pyridine, N-methylmorpholine, DBU, imidazole, etc.). More preferably, diisopropylethylamine is used.

[0266] The reaction temperature is usually -20 to 150°C, preferably -10 to 80°C, depending on the reagents, raw materials, solvent, etc. used.

[0267] The reaction time varies depending on the starting materials, solvent, reaction temperature, etc. used, but is usually 1 to 100 hours, preferably 1 to 24 hours.

[0268] After the reaction is complete, for example, the reaction mixture is poured into water or saturated aqueous sodium bicarbonate, and extracted with a water-immiscible solvent such as benzene, ether, ethyl acetate, or methylene chloride. The solvent is then distilled off from the extract, and the resulting product is usually used as is in the next step. If desired, the product can be isolated and purified by various chromatography or recrystallization methods.

[0269] Step B-4 is a step of producing compound (18) by selectively deprotecting the 5'-protecting group of compound (17) produced by the above-mentioned method.

[0270] The solvent and deprotecting reagent used are not particularly limited as long as they can selectively deprotect the 5'-protecting group. However, when trityl chlorides are used as the 5'-protecting reagent, an acid is usually used. Preferred acids used include hydrogen chlorides (particularly hydrochloric acid, hydrogen chloride-methanol solution, hydrogen chloride-ethyl acetate solution, hydrogen chloride-1,4-dioxane solution, etc.), acetic acids (acetic acid aqueous solution, chloroacetic acid aqueous solution, dichloroacetic acid aqueous solution, trichloroacetic acid aqueous solution, dichloroacetic acid toluene solution, trichloroacetic acid methylene chloride solution, etc.), and trifluoroacetic acid aqueous solution. A more preferred example is trichloroacetic acid methylene chloride solution. The reaction temperature is usually −20 to 80°C, and preferably −10 to 50°C, depending on the reagents, raw materials, solvent, etc. used.

[0271] The reaction time varies depending on the starting materials, solvent, reaction temperature, etc., but is usually 1 to 100 hours, preferably 1 to 24 hours. After completion of the reaction, the target compound (18) of this reaction can be obtained, for example, by pouring the reaction mixture into water or saturated aqueous sodium bicarbonate, adding an immiscible organic solvent such as methylene chloride, washing with water, separating the organic layer containing the target compound, drying it over sodium sulfate, etc., and then distilling off the solvent. If necessary, the resulting compound can be further purified by conventional methods, such as recrystallization or silica gel column chromatography.

[0272] Step B-5 is a step of producing compound (19) by reacting compound (18) produced by the above-mentioned method with an oxidizing agent in an inert solvent. Examples of the solvent used include aliphatic hydrocarbons such as hexane, heptane, ligroin, and petroleum ether; aromatic hydrocarbons such as benzene, toluene, and xylene; halogenated hydrocarbons such as methylene chloride, chloroform, carbon tetrachloride, dichloroethane, chlorobenzene, and dichlorobenzene; esters such as ethyl formate, ethyl acetate, propyl acetate, butyl acetate, and diethyl carbonate; ethers such as diethyl ether, diisopropyl ether, tetrahydrofuran, dioxane, dimethoxyethane, and diethylene glycol dimethyl ether; ketones such as acetone, methyl ethyl ketone, methyl isobutyl ketone, isophorone, and cyclohexanone; and nitriles such as acetonitrile and isobutyronitrile. Preferred are halogenated hydrocarbons (particularly methylene chloride); and nitriles such as acetonitrile and isobutyronitrile. Acetonitrile is even more preferred.

[0273] Examples of the oxidizing agent that can be used include a Swern oxidation reagent, a Dess-Martin oxidation reagent, a chromium trioxide complex such as a pyridine hydrochloride-chromium trioxide complex (pyridinium chlorochromate, pyridinium dichromate), and 2-iodoxybenzoic acid, with 2-iodoxybenzoic acid being the preferred reagent.

[0274] The reaction temperature varies depending on the starting compounds, solvent and oxidizing agent used, but is usually -100 to 150°C, preferably -20 to 100°C.

[0275] The reaction time varies depending on the starting compounds, solvent, oxidizing agent and reaction temperature used, but is usually 30 minutes to 12 hours, preferably 1 to 5 hours.

[0276] After the reaction is complete, the target compound (19) can be obtained by removing the oxidizing agent and concentrating the reaction mixture. If necessary, it can be purified by a conventional method, for example, by adding an immiscible organic solvent such as water and ethyl acetate, washing with water, separating the organic layer containing the target compound, drying it over anhydrous magnesium sulfate, etc., and then distilling off the solvent. If necessary, it can be further purified by a conventional method, for example, recrystallization, silica gel column chromatography, etc.

[0277] Step B-6 is a step of mixing carbon tetrabromide and triphenylphosphine, and then adding compound (19) to react them to produce a dibromo compound (20) from compound (19) produced by the above-mentioned method.

[0278] The solvent to be used is not particularly limited as long as it does not inhibit the reaction and dissolves the starting materials to some extent, but preferred examples include ethers such as diethyl ether, diisopropyl ether, tetrahydrofuran, dioxane, dimethoxyethane, and diethylene glycol dimethyl ether; and halogenated hydrocarbons such as methylene chloride and chloroform. Methylene chloride is even more preferred.

[0279] The reaction temperature is usually −20° C. to 80° C., preferably −10° C. to 30° C. The reaction time varies depending on the starting compounds, solvent, reaction reagents, and reaction temperature used, but is 5 minutes to 48 hours, preferably 30 minutes to 24 hours.

[0280] After completion of the reaction, the target compound (20) of this reaction can be obtained, for example, by pouring the reaction mixture into water or saturated aqueous sodium bicarbonate, adding an immiscible organic solvent such as methylene chloride, washing with water, separating the organic layer containing the target compound, drying it over sodium sulfate, etc., and then distilling off the solvent. If necessary, the resulting compound can be further purified by a conventional method, for example, recrystallization, silica gel column chromatography, etc.

[0281] Step B-7 is a step of reacting compound (20) produced by the above-mentioned method with a phosphite ester in the presence of a palladium catalyst to produce alkyne (21). The solvent used is not particularly limited as long as it does not inhibit the reaction and can dissolve the starting materials to some extent, but DMF is preferred.

[0282] The additives used include palladium catalysts such as palladium acetate and Pd2(dba)2, phosphine ligands such as dppf, and propylene oxide. The reagents used include phosphites such as diethyl phosphite and dimethyl phosphite. Diethyl phosphite is more preferred.

[0283] The reaction temperature varies depending on the starting compounds, solvent and reaction reagents used, but is usually 10 to 150°C, preferably 40 to 100°C.

[0284] The reaction time varies depending on the starting compounds, solvent, reaction reagents and reaction temperature used, but is 5 minutes to 48 hours, preferably 30 minutes to 24 hours.

[0285] After completion of the reaction, the target compound (21) of this reaction can be obtained, for example, by pouring the reaction mixture into water or saturated aqueous sodium bicarbonate, adding an immiscible organic solvent such as hexane, ether, ethyl acetate, or methylene chloride, washing with water, separating the organic layer containing the target compound, drying it over sodium sulfate, or the like, and then distilling off the solvent. If necessary, the resulting compound can be further purified by a conventional method, for example, recrystallization, silica gel column chromatography, or the like.

[0286] Step B-8 is a step of reacting compound (21) produced by the above-mentioned method with a deprotecting agent in an inert solvent to deprotect the hydroxyl-protecting group, thereby producing compound (22). The solvent used is not particularly limited as long as it does not inhibit the reaction and dissolves the starting materials to some extent. Preferred examples include ethers such as diethyl ether, diisopropyl ether, tetrahydrofuran, dioxane, dimethoxyethane, and diethylene glycol dimethyl ether; nitriles such as acetonitrile and isobutyronitrile; water; organic acids such as acetic acid; and mixed solvents thereof. Tetrahydrofuran is even more preferred.

[0287] The reagent used can be removed by treating with a compound that generates fluorine anions, such as tetrabutylammonium fluoride, hydrofluoric acid, hydrofluoric acid-pyridine, or potassium fluoride, or by treating with an organic acid, such as acetic acid, formic acid, methanesulfonic acid, paratoluenesulfonic acid, trifluoroacetic acid, or trifluoromethanesulfonic acid, or an inorganic acid, such as hydrochloric acid. Formic acid is more preferred.

[0288] The reaction temperature varies depending on the starting compounds, solvent and reaction reagents used, but is usually -20 to 100°C, preferably 0 to 50°C.

[0289] The reaction time varies depending on the starting compounds, solvent, reaction reagents, and reaction temperature used, but is generally 5 minutes to 24 hours, preferably 20 minutes to 12 hours. After completion of the reaction, the target compound (22) of this reaction can be obtained, for example, by concentrating the reaction mixture, adding an immiscible organic solvent such as water and ethyl acetate, washing with water, separating the organic layer containing the target compound, drying it with sodium sulfate, or the like, and then distilling off the solvent. The resulting compound (22) can be separated and purified by conventional methods, such as recrystallization or silica gel column chromatography.

[0290] Step B-9 is a step of producing compound (23) by reacting compound (22) produced by the above-mentioned method with a monosubstituted chloro(alkoxy)phosphine or disubstituted alkoxyphosphine commonly used for amiditization in an inert solvent. The solvent used is not particularly limited as long as it does not affect the reaction, but preferred examples include ethers such as tetrahydrofuran, diethyl ether, and dioxane; and halogenated hydrocarbons such as methylene chloride, chloroform, carbon tetrachloride, dichloroethane, chlorobenzene, and dichlorobenzene. More preferred is methylene chloride.

[0291] Examples of the mono-substituted chloro(alkoxy)phosphine to be used include phosphines such as chloro(morpholino)methoxyphosphine, chloro(morpholino)cyanoethoxyphosphine, chloro(dimethylamino)methoxyphosphine, chloro(dimethylamino)cyanoethoxyphosphine, chloro(diisopropylamino)methoxyphosphine, and chloro(diisopropylamino)cyanoethoxyphosphine, and preferred are chloro(morpholino)methoxyphosphine, chloro(morpholino)cyanoethoxyphosphine, chloro(diisopropylamino)methoxyphosphine, and chloro(diisopropylamino)cyanoethoxyphosphine.

[0292] When a mono-substituted chloro(alkoxy)phosphine is used, an acid scavenger is used. In this case, examples of the acid scavenger that can be used include heterocyclic amines such as pyridine and dimethylaminopyridine, and aliphatic amines such as trimethylamine, triethylamine and diisopropylamine, and preferably aliphatic amines (particularly triethylamine and diisopropylamine).

[0293] Examples of the di-substituted alkoxyphosphine to be used include phosphines such as bis(diisopropylamino)cyanoethoxyphosphine, bis(diethylamino)methanesulfonylethoxyphosphine, bis(diisopropylamino)(2,2,2-trichloroethoxy)phosphine, and bis(diisopropylamino)(4-chlorophenylmethoxy)phosphine, and preferably bis(diisopropylamino)cyanoethoxyphosphine.

[0294] When a disubstituted alkoxyphosphine is used, an acid is used, and in this case, the acid to be used is preferably tetrazole, acetic acid, dicyanoimidazole or p-toluenesulfonic acid.

[0295] The reaction temperature is not particularly limited, but is usually 0 to 80° C., and preferably room temperature. The reaction time varies depending on the raw materials, reagents, temperature, etc. used, but is usually 5 minutes to 30 hours, and preferably 30 minutes to 10 hours when the reaction is carried out at room temperature.

[0296] After completion of the reaction, the target compound (23) of this reaction can be obtained, for example, by neutralizing the reaction mixture appropriately and, if any insoluble matter is present, removing it by filtration, adding an immiscible organic solvent such as water and ethyl acetate, washing with water, separating the organic layer containing the target compound, drying it over anhydrous magnesium sulfate, etc., and then distilling off the solvent. If necessary, the obtained target compound can be further purified by a conventional method, for example, recrystallization, reprecipitation, or chromatography.

[0297] Step B-10 is a step of reacting compound (22) prepared by the above method with tris-(1,2,4-triazolyl)phosphite or 2-chloro-1,3,2-benzodioxaphosphorin-4-one (or salicylphosphorochloridite) in an inert solvent (preferably a halogenated hydrocarbon such as methylene chloride), followed by adding water to convert the compound to an H-phosphonate, thereby preparing compound (24).

[0298] The reaction temperature is not particularly limited, but is usually from -20 to 100°C, preferably from 10 to 40°C.

[0299] The reaction time varies depending on the raw materials, reagents, temperature, etc. used, but is usually 5 minutes to 30 hours, and is preferably 30 minutes when the reaction is carried out at room temperature. After completion of the reaction, the target compound (24) of this reaction can be obtained, for example, by appropriately neutralizing the reaction mixture and, if any insoluble matter is present, removing it by filtration, adding an immiscible organic solvent such as water and ethyl acetate, washing with water, separating the organic layer containing the target compound, drying it over anhydrous magnesium sulfate, etc., and then distilling off the solvent. If necessary, the obtained target compound can be further purified by a conventional method, for example, recrystallization, reprecipitation, chromatography, etc.

[0300] EPuam can be synthesized using the following steps C-1 to C-13.

[0301] (In the formula, R1 and R2 are general protecting groups for a hydroxyl group, R3 and R4 are hydrogen, an optionally substituted acyl group having 1 to 6 carbon atoms, or an optionally substituted aromatic acyl group, and R5 is an optionally substituted alkyl group having 1 to 4 carbon atoms, or a protecting group in which an optionally substituted alkyleneoxycarbonyl group having 1 to 4 carbon atoms is bonded to an optionally substituted alkyl group having 1 to 4 carbon atoms. In the formula, -P(R6)R7 is [R6 and R7 are the same or different and represent an amino group substituted with an alkoxy group having 1 to 4 carbon atoms, an alkylthio group having 1 to 4 carbon atoms, a cyanoalkoxy group having 1 to 5 carbon atoms, or an alkyl group having 1 to 4 carbon atoms].)

[0302] Step C-1 is a step of preparing compound (26) by reacting the 2'-amino group of compound (25) with a protecting reagent in an inert solvent.

[0303] Examples of the solvent to be used include alcohols such as methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, t-butanol, isoamyl alcohol, diethylene glycol, glycerin, octanol, cyclohexanol, and methyl cellosolve; and esters such as ethyl formate, ethyl acetate, propyl acetate, butyl acetate, and diethyl carbonate. However, alcohols (particularly methanol) are preferred.

[0304] Examples of the acylating agent to be used include acetic acid chloride, acetic anhydride, ethyl acetate, ethyl trifluoroacetate, trifluoroacetic anhydride, benzoyl chloride, ethyl benzoate, etc. Ethyl trifluoroacetate is preferred.

[0305] When an acylating agent is used, a base is usually used. The base to be used is preferably an organic base (particularly, triethylamine, pyridine, N-methylmorpholine, DBU, imidazole, etc.). More preferably, triethylamine is used.

[0306] The reaction temperature varies depending on the starting compounds, solvent and reducing agent used, but is usually 0 to 80°C, preferably 10 to 50°C.

[0307] The reaction time varies depending on the starting compounds, solvent, acylating agent and reaction temperature used, but is usually 1 to 72 hours, preferably 6 to 48 hours.

[0308] After completion of the reaction, the target compound (26) of this reaction can be obtained, for example, by decomposing or filtering the acylating agent, concentrating the reaction mixture, adding an immiscible organic solvent such as water and ethyl acetate, washing with water, separating the organic layer containing the target compound, drying it over anhydrous magnesium sulfate, etc., and then distilling off the solvent.

[0309] Step C-2 is a step of preparing compound (27) by reacting the 5'-hydroxyl group of compound (26) with a protecting reagent in an inert solvent.

[0310] The solvent to be used is preferably an aromatic hydrocarbon such as benzene, toluene, xylene, or pyridine; a halogenated hydrocarbon such as methylene chloride, chloroform, carbon tetrachloride, dichloroethane, chlorobenzene, or dichlorobenzene; an ester such as ethyl formate, ethyl acetate, propyl acetate, butyl acetate, or diethyl carbonate; an ether such as diethyl ether, diisopropyl ether, tetrahydrofuran, dioxane, dimethoxyethane, or diethylene glycol dimethyl ether; a ketone such as acetone, methyl ethyl ketone, methyl isobutyl ketone, isophorone, or cyclohexanone; a nitro compound such as nitroethane or nitrobenzene; a nitrile such as acetonitrile or isobutyronitrile; an amide such as formamide, dimethylformamide (DMF), dimethylacetamide, or hexamethylphosphorotriamide; a sulfoxide such as dimethyl sulfoxide or sulfolane; or an amine such as pyridine, triethylamine, or diisopropylethylamine. More preferably, an aromatic hydrocarbon (particularly pyridine) or a halogenated hydrocarbon (particularly methylene chloride) is used. More preferably, pyridine is used.

[0311] The protecting reagent used is not particularly limited as long as it can selectively protect the 5'-hydroxyl group, but preferred examples include trityl chlorides such as trityl chloride, 4-methoxytrityl chloride, 4,4'-dimethoxytrityl chloride, and 4,4'-dimethoxy-4''-methylsulfonyltrityl chloride. More preferred examples include 4,4'-dimethoxytrityl chloride.

[0312] The reaction temperature is usually -20 to 80°C, preferably -10 to 50°C, depending on the reagents, raw materials, solvent, etc. used.

[0313] The reaction time varies depending on the starting materials, solvent, reaction temperature, etc. used, but is usually 1 to 100 hours, preferably 1 to 24 hours.

[0314] After completion of the reaction, the target compound (27) of this reaction can be obtained, for example, by pouring the reaction mixture into water or saturated aqueous sodium bicarbonate, adding an immiscible organic solvent such as ethyl acetate, washing with water, separating the organic layer containing the target compound, drying it over magnesium sulfate, etc., and then distilling off the solvent. If necessary, the resulting compound can be further purified by a conventional method, for example, recrystallization, silica gel column chromatography, etc.

[0315] Step C-3 is a step of preparing compound (28) by selectively deprotecting the 2'-acyl protecting group of compound (27) prepared by the above-mentioned method.

[0316] The solvent and deprotecting reagent used are not particularly limited as long as they can selectively deprotect the 2'-acyl protecting group, but a base is usually used. The base used is preferably an amine (particularly ammonia, methylamine, ethylamine, etc.). Ammonia is more preferred. The reaction temperature is usually −20 to 50°C, preferably −10 to 40°C, depending on the reagents, raw materials, solvent, etc. used.

[0317] The reaction time varies depending on the starting materials, solvent, reaction temperature, etc. used, but is usually 1 to 100 hours, preferably 6 to 48 hours.

[0318] After completion of the reaction, the target compound (28) of this reaction can be obtained, for example, by pouring the reaction mixture into water or saturated aqueous sodium bicarbonate, adding an immiscible organic solvent such as ethyl acetate, washing with water, separating the organic layer containing the target compound, drying it over magnesium sulfate, etc., and then distilling off the solvent. If necessary, the resulting compound can be further purified by a conventional method, for example, recrystallization, silica gel column chromatography, etc.

[0319] Step C-4 is a step of preparing compound (29) by reacting the 3'-hydroxyl group of compound (28) with a protecting reagent in an inert solvent.

[0320] Preferred examples of the solvent to be used include aromatic hydrocarbons such as benzene, toluene, and xylene; halogenated hydrocarbons such as methylene chloride, chloroform, carbon tetrachloride, dichloroethane, chlorobenzene, and dichlorobenzene; esters such as ethyl formate, ethyl acetate, propyl acetate, butyl acetate, and diethyl carbonate; ethers such as diethyl ether, diisopropyl ether, tetrahydrofuran, dioxane, dimethoxyethane, and diethylene glycol dimethyl ether; ketones such as acetone, methyl ethyl ketone, methyl isobutyl ketone, isophorone, and cyclohexanone; nitro compounds such as nitroethane and nitrobenzene; nitriles such as acetonitrile and isobutyronitrile; amides such as formamide, dimethylformamide (DMF), dimethylacetamide, and hexamethylphosphorotriamide; sulfoxides such as dimethyl sulfoxide and sulfolane; and amines such as triethylamine, pyridine, and diisopropylethylamine, and more preferred examples include amines (particularly pyridine).

[0321] The protecting reagent to be used is not particularly limited as long as it can normally be used for deprotection, but preferred examples include silyl halides such as t-butyldimethylsilyl chloride, triethylsilyl chloride, triisopropylsilyl chloride, and triethylsilyl chloride, and more preferred example is t-butyldimethylsilyl chloride.

[0322] When a silyl halide is used as a protecting reagent, a base is usually used. The base to be used is preferably an organic base (particularly, triethylamine, pyridine, N-methylmorpholine, DBU, imidazole, etc.). More preferably, imidazole is used.

[0323] The reaction temperature is usually -20 to 150°C, preferably -10 to 50°C, depending on the reagents, raw materials, solvent, etc. used.

[0324] The reaction time varies depending on the starting materials, solvent, reaction temperature, etc. used, but is usually 1 to 100 hours, preferably 1 to 24 hours.

[0325] After the reaction is complete, for example, the reaction mixture is poured into water or saturated aqueous sodium bicarbonate, extracted with a water-immiscible solvent such as benzene, ether, or ethyl acetate, and the solvent is distilled off from the extract to obtain a product which is usually used as is in the next step. If desired, the product can be isolated and purified by various chromatography or recrystallization methods.

[0326] Step C-5 is a step of preparing compound (30) by reacting the 2'-amino group of compound (29) with an acetylating reagent in an inert solvent.

[0327] Examples of the solvent to be used include halogenated hydrocarbons such as methylene chloride, chloroform, carbon tetrachloride, dichloroethane, chlorobenzene, and dichlorobenzene; esters such as ethyl formate, ethyl acetate, propyl acetate, butyl acetate, and diethyl carbonate; ethers such as diethyl ether, diisopropyl ether, tetrahydrofuran, dioxane, dimethoxyethane, and diethylene glycol dimethyl ether; ketones such as acetone, methyl ethyl ketone, methyl isobutyl ketone, isophorone, and cyclohexanone; nitro compounds such as nitroethane and nitrobenzene; nitriles such as acetonitrile and isobutyronitrile; amides such as formamide, dimethylformamide (DMF), dimethylacetamide, and hexamethylphosphorotriamide; sulfoxides such as dimethyl sulfoxide and sulfolane; and amines such as triethylamine, pyridine, and diisopropylethylamine, more preferably amines such as triethylamine, pyridine, and diisopropylethylamine.

[0328] The acetylating agent to be used includes, for example, acetyl chloride, acetic anhydride, ethyl acetate, etc. Acetic anhydride is preferred.

[0329] The reaction temperature varies depending on the starting compounds, solvent and reducing agent used, but is usually 0 to 80°C, preferably 10 to 50°C.

[0330] The reaction time varies depending on the starting compounds, solvent, acylating agent and reaction temperature used, but is usually from 10 minutes to 48 hours, preferably from 1 hour to 24 hours.

[0331] After completion of the reaction, the target compound (30) of this reaction can be obtained, for example, by decomposing or filtering the acetylating agent, concentrating the reaction mixture, adding an immiscible organic solvent such as water and ethyl acetate, washing with water, separating the organic layer containing the target compound, drying it over anhydrous magnesium sulfate, etc., and then distilling off the solvent.

[0332] Step C-6 is a step of preparing compound (31) by reacting compound (30) with a protecting reagent in an inert solvent.

[0333] The solvent to be used is preferably an aromatic hydrocarbon such as benzene, toluene, or xylene; a halogenated hydrocarbon such as methylene chloride, chloroform, carbon tetrachloride, dichloroethane, chlorobenzene, or dichlorobenzene; an ester such as ethyl formate, ethyl acetate, propyl acetate, butyl acetate, or diethyl carbonate; an ether such as diethyl ether, diisopropyl ether, tetrahydrofuran, dioxane, dimethoxyethane, or diethylene glycol dimethyl ether; or an alkyl ether such as acetone, methyl ethyl ketone, methyl isobutyl ketone, isophorone, or cyclohexanone. Examples of suitable solvents include ketones, nitro compounds such as nitroethane and nitrobenzene, nitriles such as acetonitrile and isobutyronitrile, amides such as formamide, dimethylformamide (DMF), dimethylacetamide and hexamethylphosphorotriamide, sulfoxides such as dimethyl sulfoxide and sulfolane, and amines such as pyridine, triethylamine and diisopropylethylamine. More preferred solvents include ethers (particularly tetrahydrofuran), halogenated hydrocarbons (particularly methylene chloride), amides (particularly DMF), and amines (particularly pyridine). More preferred solvents include amines (particularly pyridine).

[0334] The protecting reagent used is not particularly limited as long as it can be protected with ammonia, but preferred examples include acyl halides such as benzoyl chloride, p-nitrobenzoyl chloride, and p-methoxybenzoyl chloride; pivaloylmethylene chloride, etc. More preferred is benzoyl chloride.

[0335] When an acyl halide is used as a protecting reagent, a base is usually used. The base to be used is preferably an organic base (particularly, triethylamine, pyridine, N-methylmorpholine, DBU, imidazole, etc.). More preferably, diisopropylethylamine is used.

[0336] The reaction temperature is usually -20 to 150°C, preferably -10 to 80°C, depending on the reagents, raw materials, solvent, etc. used.

[0337] The reaction time varies depending on the starting materials, solvent, reaction temperature, etc. used, but is usually from 10 minutes to 100 hours, preferably from 30 minutes to 4 hours.

[0338] After the reaction is complete, for example, the reaction mixture is poured into water or saturated aqueous sodium bicarbonate, and extracted with a water-immiscible solvent such as benzene, ether, ethyl acetate, or methylene chloride. The solvent is then distilled off from the extract, and the resulting product is usually used as is in the next step. If desired, the product can be isolated and purified by various chromatography or recrystallization methods.

[0339] Step C-7 is a step of producing compound (32) by selectively deprotecting the 5'-protecting group of compound (31) produced by the above-mentioned method.

[0340] The solvent and deprotecting reagent used are not particularly limited as long as they can selectively deprotect the 5'-protecting group, but when trityl chlorides are used as the 5'-protecting reagent, an acid is usually used. Preferred acids used include hydrogen chlorides (particularly hydrochloric acid, hydrogen chloride-methanol solution, hydrogen chloride-ethyl acetate solution, hydrogen chloride-1,4-dioxane solution, etc.), acetic acids (acetic acid aqueous solution, chloroacetic acid aqueous solution, dichloroacetic acid aqueous solution, trichloroacetic acid aqueous solution, dichloroacetic acid toluene solution, trichloroacetic acid methylene chloride solution, etc.), and trifluoroacetic acid aqueous solution. More preferred is trifluoroacetic acid solution.

[0341] The reaction temperature is usually -20 to 80°C, preferably -10 to 50°C, depending on the reagents, raw materials, solvent, etc. used.

[0342] The reaction time varies depending on the starting materials, solvent, reaction temperature, etc. used, but is usually from 10 minutes to 100 hours, preferably from 30 minutes to 4 hours.

[0343] After completion of the reaction, the target compound (32) of this reaction can be obtained, for example, by pouring the reaction mixture into water or saturated aqueous sodium bicarbonate, adding an immiscible organic solvent such as methylene chloride, washing with water, separating the organic layer containing the target compound, drying it over sodium sulfate, etc., and then distilling off the solvent. If necessary, the resulting compound can be further purified by a conventional method, for example, recrystallization, silica gel column chromatography, etc.

[0344] Step C-8 is a step of preparing compound (33) by reacting compound (32) prepared by the above-mentioned method with an oxidizing agent in an inert solvent.

[0345] Examples of the solvent to be used include aliphatic hydrocarbons such as hexane, heptane, ligroin, and petroleum ether; aromatic hydrocarbons such as benzene, toluene, and xylene; halogenated hydrocarbons such as methylene chloride, chloroform, carbon tetrachloride, dichloroethane, chlorobenzene, and dichlorobenzene; esters such as ethyl formate, ethyl acetate, propyl acetate, butyl acetate, and diethyl carbonate; ethers such as diethyl ether, diisopropyl ether, tetrahydrofuran, dioxane, dimethoxyethane, and diethylene glycol dimethyl ether; ketones such as acetone, methyl ethyl ketone, methyl isobutyl ketone, isophorone, and cyclohexanone; and nitriles such as acetonitrile and isobutyronitrile. Preferred are halogenated hydrocarbons (particularly methylene chloride); and nitriles such as acetonitrile and isobutyronitrile. Acetonitrile is even more preferred.

[0346] Examples of the oxidizing agent that can be used include a Swern oxidation reagent, a Dess-Martin oxidation reagent, a chromium trioxide complex such as a pyridine hydrochloride-chromium trioxide complex (pyridinium chlorochromate, pyridinium dichromate), and 2-iodoxybenzoic acid, with 2-iodoxybenzoic acid being the preferred reagent.

[0347] The reaction temperature varies depending on the starting compounds, solvent and oxidizing agent used, but is usually -100 to 150°C, preferably -20 to 100°C.

[0348] The reaction time varies depending on the starting compounds, solvent, oxidizing agent and reaction temperature used, but is usually 30 minutes to 12 hours, preferably 1 to 5 hours.

[0349] After the reaction is complete, the target compound (33) can be obtained by removing the oxidizing agent and concentrating the reaction mixture. If necessary, it can be purified by a conventional method, for example, by adding an immiscible organic solvent such as water and ethyl acetate, washing with water, separating the organic layer containing the target compound, drying it over anhydrous magnesium sulfate, etc., and then distilling off the solvent. If necessary, it can be further purified by a conventional method, for example, recrystallization, silica gel column chromatography, etc.

[0350] Step C-9 is a step of mixing carbon tetrabromide and triphenylphosphine, and then adding compound (33) to react them to produce dibromo compound (34) from compound (33) produced by the above-mentioned method.

[0351] The solvent to be used is not particularly limited as long as it does not inhibit the reaction and dissolves the starting materials to some extent, but preferred examples include ethers such as diethyl ether, diisopropyl ether, tetrahydrofuran, dioxane, dimethoxyethane, and diethylene glycol dimethyl ether; and halogenated hydrocarbons such as methylene chloride and chloroform. Methylene chloride is even more preferred.

[0352] The reaction temperature is usually −20° C. to 80° C., preferably −10° C. to 30° C. The reaction time varies depending on the starting compounds, solvent, reaction reagents, and reaction temperature used, but is 5 minutes to 48 hours, preferably 30 minutes to 24 hours.

[0353] After completion of the reaction, the target compound (33) of this reaction can be obtained, for example, by pouring the reaction mixture into water or saturated aqueous sodium bicarbonate, adding an immiscible organic solvent such as methylene chloride, washing with water, separating the organic layer containing the target compound, drying it over sodium sulfate, etc., and then distilling off the solvent. If necessary, the resulting compound can be further purified by a conventional method, for example, recrystallization, silica gel column chromatography, etc.

[0354] Step C-10 is a step in which the compound (34) produced by the above-mentioned method is reacted with a phosphite in the presence of a palladium catalyst to produce an alkyne (35).

[0355] The solvent to be used is not particularly limited as long as it does not inhibit the reaction and can dissolve the starting materials to some extent, but a preferred example is DMF.

[0356] The additives used include palladium catalysts such as palladium acetate and Pd2(dba)2, phosphine ligands such as dppf, and propylene oxide. The reagents used include phosphites such as diethyl phosphite and dimethyl phosphite. Diethyl phosphite is more preferred.

[0357] The reaction temperature varies depending on the starting compounds, solvent and reaction reagents used, but is usually 10 to 150°C, preferably 40 to 100°C.

[0358] The reaction time varies depending on the starting compounds, solvent, reaction reagents and reaction temperature used, but is 5 minutes to 48 hours, preferably 30 minutes to 24 hours.

[0359] After completion of the reaction, the target compound (35) of this reaction can be obtained, for example, by pouring the reaction mixture into water or saturated aqueous sodium bicarbonate, adding an immiscible organic solvent such as hexane, ether, ethyl acetate, or methylene chloride, washing with water, separating the organic layer containing the target compound, drying it over sodium sulfate, or the like, and then distilling off the solvent. If necessary, the resulting compound can be further purified by a conventional method, for example, recrystallization, silica gel column chromatography, or the like.

[0360] Step C-11 is a step of reacting compound (35) prepared by the above-mentioned method with a deprotecting agent in an inert solvent to deprotect the hydroxyl-protecting group, thereby preparing compound (36).

[0361] The solvent to be used is not particularly limited as long as it does not inhibit the reaction and dissolves the starting materials to some extent, but preferred examples include ethers such as diethyl ether, diisopropyl ether, tetrahydrofuran, dioxane, dimethoxyethane, and diethylene glycol dimethyl ether; nitriles such as acetonitrile and isobutyronitrile; water; organic acids such as acetic acid; and mixed solvents thereof. Tetrahydrofuran is even more preferred.

[0362] The reagents used include tetrabutylammonium fluoride, hydrofluoric acid, hydrofluoric acid-pyridine, hydrofluoric acid-triethylamine, and potassium fluoride, which can be removed by treatment with a compound that generates fluorine anions, or with an organic acid such as acetic acid, formic acid, methanesulfonic acid, paratoluenesulfonic acid, trifluoroacetic acid, or trifluoromethanesulfonic acid, or an inorganic acid such as hydrochloric acid. More preferred is hydrofluoric acid-triethylamine.

[0363] The reaction temperature varies depending on the starting compounds, solvent and reaction reagents used, but is usually -20 to 100°C, preferably 0 to 50°C.

[0364] The reaction time varies depending on the starting compounds, solvent, reaction reagents and reaction temperature used, but is 5 minutes to 24 hours, preferably 20 minutes to 12 hours.

[0365] After completion of the reaction, the target compound (36) of this reaction can be obtained, for example, by concentrating the reaction mixture, adding an immiscible organic solvent such as water and methylene chloride, washing with water, separating the organic layer containing the target compound, drying it with sodium sulfate, etc., and then distilling off the solvent. The obtained compound (36) can be separated and purified by a conventional method, for example, recrystallization, silica gel column chromatography, etc.

[0366] Step C-12 is a step of preparing compound (37) by reacting compound (36) prepared by the above-mentioned method with a monosubstituted-chloro(alkoxy)phosphine or disubstituted-alkoxyphosphine that is generally used for amiditization in an inert solvent.

[0367] The solvent to be used is not particularly limited as long as it does not affect the reaction, but preferred examples include ethers such as tetrahydrofuran, diethyl ether, and dioxane, and halogenated hydrocarbons such as methylene chloride, chloroform, carbon tetrachloride, dichloroethane, chlorobenzene, and dichlorobenzene, and more preferred examples include methylene chloride.

[0368] Examples of the mono-substituted chloro(alkoxy)phosphine to be used include phosphines such as chloro(morpholino)methoxyphosphine, chloro(morpholino)cyanoethoxyphosphine, chloro(dimethylamino)methoxyphosphine, chloro(dimethylamino)cyanoethoxyphosphine, chloro(diisopropylamino)methoxyphosphine, and chloro(diisopropylamino)cyanoethoxyphosphine, and preferred are chloro(morpholino)methoxyphosphine, chloro(morpholino)cyanoethoxyphosphine, chloro(diisopropylamino)methoxyphosphine, and chloro(diisopropylamino)cyanoethoxyphosphine.

[0369] When a mono-substituted chloro(alkoxy)phosphine is used, an acid scavenger is used. In this case, examples of the acid scavenger that can be used include heterocyclic amines such as pyridine and dimethylaminopyridine, and aliphatic amines such as trimethylamine, triethylamine and diisopropylamine, and preferably aliphatic amines (particularly triethylamine and diisopropylamine).

[0370] Examples of the di-substituted alkoxyphosphine to be used include phosphines such as bis(diisopropylamino)cyanoethoxyphosphine, bis(diethylamino)methanesulfonylethoxyphosphine, bis(diisopropylamino)(2,2,2-trichloroethoxy)phosphine, and bis(diisopropylamino)(4-chlorophenylmethoxy)phosphine, and preferably bis(diisopropylamino)cyanoethoxyphosphine.

[0371] When a disubstituted alkoxyphosphine is used, an acid is used, and in this case, the acid to be used is preferably tetrazole, acetic acid, dicyanoimidazole or p-toluenesulfonic acid.

[0372] The reaction temperature is not particularly limited, but is usually 0 to 80°C, preferably room temperature.

[0373] The reaction time varies depending on the starting materials, reagents, temperature, etc. used, but is usually 5 minutes to 30 hours, and preferably 30 minutes to 10 hours when the reaction is carried out at room temperature.

[0374] After completion of the reaction, the target compound (37) of this reaction can be obtained, for example, by neutralizing the reaction mixture appropriately and, if any insoluble matter is present, removing it by filtration, adding an immiscible organic solvent such as water and ethyl acetate, washing with water, separating the organic layer containing the target compound, drying it over anhydrous magnesium sulfate, etc., and then distilling off the solvent. If necessary, the obtained target compound can be further purified by a conventional method, for example, recrystallization, reprecipitation, or chromatography.

[0375] Step C-13 is a step of reacting compound (36) prepared by the above method with tris-(1,2,4-triazolyl)phosphite or 2-chloro-1,3,2-benzodioxaphosphorin-4-one (or salicylphosphorochloridite) in an inert solvent (preferably a halogenated hydrocarbon such as methylene chloride), followed by adding water to convert the compound to an H-phosphonate, thereby preparing compound (38).

[0376] The reaction temperature is not particularly limited, but is usually from -20 to 100°C, preferably from 10 to 40°C.

[0377] The reaction time varies depending on the raw materials, reagents, temperature, etc. used, but is usually 5 minutes to 30 hours, and is preferably 30 minutes when the reaction is carried out at room temperature. After completion of the reaction, the target compound (38) of this reaction can be obtained, for example, by appropriately neutralizing the reaction mixture and, if any insoluble matter is present, removing it by filtration, adding an immiscible organic solvent such as water and ethyl acetate, washing with water, separating the organic layer containing the target compound, drying it over anhydrous magnesium sulfate, etc., and then distilling off the solvent. If necessary, the obtained target compound can be further purified by a conventional method, for example, recrystallization, reprecipitation, chromatography, etc.

[0378] The oligonucleotides of the present invention also include those bound to a monoclonal antibody (or a suitable binding portion thereof) or a protein (or a suitable oligopeptide fragment thereof) (see, for example, Song, et al. Nature Biotech. 23, pp. 709-717 (2005); Xia et al. Pharm. Res. 24, pp. 2309-2316 (2007); Kumar, et al. Nature, 448, pp. 39-43 (2007)).

[0379] The oligonucleotides of the present invention also include those which are positively charged complexes formed by adding a cationic polymer to the oligonucleotide (see Leng et al. J. Gen. Med. 7, pp. 977-986 (2005), Baigude et al. 2, pp. 237-241, ACS Chem. Biol. (2007), Yadava et al. Oligonucleotide 17, pp. 213-222 (2007) for examples in which distribution to organs and cells has been achieved).

[0380] The oligonucleotides of the present invention include any pharmaceutically acceptable salts, esters, or salts of such esters of the above-mentioned oligonucleotides. Preferred pharmaceutically acceptable salts of the oligonucleotides of the present invention include metal salts such as alkali metal salts (e.g., sodium salt, potassium salt, and lithium salt), alkaline earth metal salts (e.g., calcium salt and magnesium salt), aluminum salt, iron salt, zinc salt, copper salt, nickel salt, and cobalt salt; inorganic salts (e.g., ammonium salt), t-octylamine salt, dibenzylamine salt, morpholine salt, glucosamine salt, phenylglycine alkyl ester salt, ethylenediamine salt, N-methylglucamine salt, guanidine salt, diethylamine salt, triethylamine salt, dicyclohexylamine salt, N,N'-dibenzylethylenediamine salt, chloroprocaine salt, procaine salt, diethanolamine salt, N-benzyl-phenethylamine salt, pipette salt, and the like. Examples of the salt include amine salts, such as organic salts like azine salts, tetramethylammonium salts, and tris(hydroxymethyl)aminomethane salts; inorganic acid salts like hydrohalogen salts like hydrofluoride, hydrochloride, hydrobromide, and hydroiodide, nitrate, perchlorate, sulfate, and phosphate; organic acid salts like lower alkanesulfonates like methanesulfonate, trifluoromethanesulfonate, and ethanesulfonate, arylsulfonates like benzenesulfonate and p-toluenesulfonate, acetate, malate, fumarate, succinate, citrate, tartrate, oxalate, and maleate; and amino acid salts like glycine salt, lysine salt, arginine salt, ornithine salt, glutamate, and aspartate.

[0381] The oligonucleotide of the present invention or a pharmaceutically acceptable salt thereof can suppress the expression of SNCA mRNA. Furthermore, the oligonucleotide of the present invention or a pharmaceutically acceptable salt thereof can suppress the accumulation of intracellular insoluble inclusions containing α-synuclein by suppressing the expression of SNCA mRNA. The mechanism of action is not particularly limited, and the oligonucleotide of the present invention or a pharmaceutically acceptable salt thereof can be used to treat or prevent diseases that can be treated or prevented by suppressing the expression of SNCA mRNA. In one preferred embodiment, when administered to a patient with α-synucleinopathic disease, the oligonucleotide can suppress the expression of SNCA mRNA, and the symptoms of α-synucleinopathic disease can be expected to be improved. Furthermore, the oligonucleotide of the present invention or a pharmaceutically acceptable salt thereof can also be used as a pharmaceutical composition containing the oligonucleotide or a pharmaceutically acceptable salt thereof as an active ingredient.

[0382] Pharmaceutical compositions containing the oligonucleotides of the present invention may be mixed, encapsulated, or conjugated with other molecules, molecular structures, or compound mixtures, for example, as liposomes, receptor-targeting molecules, orally, rectally, topically, or other formulations to aid uptake, distribution, and / or absorption. When the oligonucleotides of the present invention are used as prophylactic or therapeutic agents for diseases, the oligonucleotides or pharmaceutically acceptable salts thereof may be administered orally as tablets, capsules, granules, powders, syrups, or parenterally as injections, suppositories, patches, or topical preparations.

[0383] These preparations contain excipients (e.g., sugar derivatives such as lactose, sucrose, glucose, mannitol, and sorbitol; starch derivatives such as corn starch, potato starch, alpha starch, and dextrin; cellulose derivatives such as crystalline cellulose; gum arabic; dextran; organic excipients such as pullulan; and silicate derivatives such as light anhydrous silicic acid, synthetic aluminum silicate, calcium silicate, and magnesium aluminometasilicate; phosphates such as calcium hydrogen phosphate; carbonates such as calcium carbonate; and calcium sulfate). Examples of suitable excipients include inorganic excipients such as sulfates, lubricants (e.g., metal stearates such as stearic acid, calcium stearate, and magnesium stearate; talc; colloidal silica; waxes such as beeswax and Gay's wax; boric acid; adipic acid; sulfates such as sodium sulfate; glycol; fumaric acid; sodium benzoate; DL-leucine; lauryl sulfates such as sodium lauryl sulfate and magnesium lauryl sulfate; silicic acids such as anhydrous silicic acid and hydrated silicic acid; and the above-mentioned starch derivatives. Examples of the additives include: binders (for example, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, polyvinylpyrrolidone, macrogol, and compounds similar to the excipients described above); disintegrants (for example, cellulose derivatives such as low-substituted hydroxypropyl cellulose, carboxymethyl cellulose, carboxymethyl cellulose calcium, and internally cross-linked sodium carboxymethyl cellulose; and chemically modified starches and celluloses such as carboxymethyl starch, carboxymethyl starch sodium, and cross-linked polyvinylpyrrolidone); emulsifiers (for example, colloidal clays such as bentonite and Veegum; metal hydroxides such as magnesium hydroxide and aluminum hydroxide; anionic surfactants such as sodium lauryl sulfate and calcium stearate; cationic surfactants such as benzalkonium chloride; and nonionic surfactants such as polyoxyethylene alkyl ethers, polyoxyethylene sorbitan fatty acid esters, and sucrose fatty acid esters.The composition is produced by a well-known method using additives such as stabilizers (including paraoxybenzoic acid esters such as methylparaben and propylparaben; alcohols such as chlorobutanol, benzyl alcohol, and phenylethyl alcohol; benzalkonium chloride; phenols such as phenol and cresol; thimerosal; dehydroacetic acid; and sorbic acid), flavoring agents (including, for example, commonly used sweeteners, acidulants, fragrances, etc.), and diluents.

[0384] The recipient into which the oligonucleotide prepared as described above is introduced is not particularly limited as long as the target gene can be transcribed into RNA within the cell. The recipient refers to a cell, tissue, or individual.

[0385] The cells into which the oligonucleotides of the present invention are introduced may be any of germline cells, somatic cells, totipotent cells, pluripotent cells, dividing cells, non-dividing cells, parenchymal cells, epithelial cells, immortalized cells, transformed cells, neural cells, and immune cells.

[0386] Tissues include single-cell embryos or constitutive cells, or multi-cell embryos, fetal tissues, etc. Furthermore, examples of the differentiated cells include adipocytes, fibroblasts, muscle cells, cardiac muscle cells, endothelial cells, nerve cells, glia, blood cells, megakaryocytes, lymphocytes, macrophages, neutrophils, eosinophils, basophils, mast cells, leukocytes, granulocytes, keratinocytes, chondrocytes, osteoblasts, osteoclasts, hepatocytes, and cells of endocrine or exocrine glands. Examples of such cells that are preferably used include CHO-K1 cells (RIKEN Cell bank), Drosophila S2 cells (Schneider, I. et al., J. Embryol. Exp. Morph., 27, pp. 353-365 (1972)), human HeLa cells (ATCC: CCL-2), and human HEK293 cells (ATCC: CRL-1573).

[0387] Furthermore, specific examples of individuals to be used as recipients of the oligonucleotides of the present invention include plants, animals, protozoa, viruses, bacteria, and fungi. Plants may be monocotyledonous, dicotyledonous, or gymnosperms, and animals may be vertebrates or invertebrates. Preferred vertebrates are mammals, including mice, rats, monkeys, dogs, and humans.

[0388] When the recipient is a cell or tissue, the oligonucleotide of the present invention can be introduced into the recipient by calcium phosphate, electroporation, lipofection, viral infection, immersion in a 3L5-oligonucleotide solution, transformation, or the like. Methods for introducing the oligonucleotide into an embryo include microinjection, electroporation, viral infection, and the like. When the recipient is a plant, injection, perfusion, or spraying into the body cavity or interstitial cells of the plant body can be used. When the recipient is an animal, systemic introduction can be achieved by oral, topical, subcutaneous, intramuscular, or intravenous administration, parenteral, vaginal, rectal, nasal, ocular, or intramembrane administration, or by electroporation or viral infection. Oral introduction can also be achieved by directly mixing the oligonucleotide of the present invention with the food of the organism.

[0389] In addition to the above, a colloidal dispersion system can be used to introduce the oligonucleotide of the present invention into a patient. Colloidal dispersion systems are expected to have the effects of increasing the stability of compounds in vivo and efficiently transporting compounds to specific organs, tissues, or cells. The colloidal dispersion system is not limited to a commonly used one, and examples thereof include lipid-based dispersion systems including polymer complexes, nanocapsules, microspheres, beads, and oil-in-water emulsions, micelles, mixed micelles, and liposomes. Preferably, the colloidal dispersion system is a plurality of liposomes or artificial membrane vesicles, which are effective in efficiently transporting compounds to specific organs, tissues, or cells (Mannino et al., Biotechniques, 1988, 6, p. 682-; Blume and Cevc, Biochem. et Biophys. Acta, 1990, 1029, p. 91-; Lappalainen et al., Antiviral Res., 1994, 23, p. 119-; Chonn and Cullis, Current Op. Biotech., 1995, 6, p. 698-). Unilamellar liposomes with a size range of 0.2-0.4 μm can encapsulate a significant proportion of aqueous buffer containing macromolecules, and the compounds are encapsulated in this aqueous inner membrane and transported to brain cells in a biologically active form (Fraley et al., Trends Biochem. Sci., 1981, 6, p. 77-).

[0390] Liposome compositions are typically lipids, particularly phospholipids, especially high-phase-transition-temperature phospholipids, usually complexed with one or more steroids, especially cholesterol. Examples of lipids useful in liposome production include phosphatidyl compounds such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, sphingolipids, phosphatidylethanolamine, cerebrosides, and gangliosides.

[0391] Particularly useful are diacylphosphatidylglycerols, in which the lipid moiety contains 14-18 carbon atoms, particularly 16-18 carbon atoms, and is saturated (lacking a double bond within the 14-18 carbon atom chain).

[0392] Representative phospholipids include phosphatidylcholine, dipalmitoylphosphatidylcholine, and distearoylphosphatidylcholine.

[0393] Targeting of colloidal dispersion systems, including liposomes, can be either passive or active.

[0394] Passive targeting is achieved by taking advantage of the natural tendency of liposomes to distribute to reticuloendothelial cells of organs containing sinusoidal capillaries.

[0395] On the other hand, active targeting can be achieved by, for example, attaching specific ligands to liposomes, such as viral protein coats (Morishita et al., Proc. Natl. Acad. Sci. (U.S.A.), 1993, 90, p. 8474-), monoclonal antibodies (or suitable binding portions thereof), sugars, glycolipids, or proteins (or suitable oligopeptide fragments thereof), or by modifying liposomes by changing their composition to achieve distribution to organs and cell types other than their naturally occurring localization site.

[0396] The surface of targeted colloidal dispersion systems can be modified in various ways. In liposome-targeted delivery systems, lipid groups can be incorporated into the lipid bilayer of the liposome to maintain the targeting ligand in close association with the lipid bilayer. Various linking groups can be used to link the lipid chains to the targeting ligand.

[0397] The targeting ligand that binds to a specific cell surface molecule found predominantly on cells to which delivery of the oligonucleotides of the invention is desired can be, for example, (1) a hormone, growth factor, or appropriate oligopeptide fragment thereof, that binds to a specific cellular receptor that is predominantly expressed by the cells to which delivery is desired, or (2) a polyclonal or monoclonal antibody, or appropriate fragment thereof (e.g., Fab; F(ab')2), that specifically binds to an antigenic epitope found predominantly on the target cells. Two or more bioactive agents can also be combined within a single liposome and administered.

[0398] Agents that enhance the intracellular stability and / or targeting of the contents can also be added to the colloidal dispersion system.

[0399] The dosage of the oligonucleotide of the present invention or a pharmaceutically acceptable salt thereof varies depending on symptoms, age, etc., but it is desirable to administer the following to an adult 1 to 3 times per day depending on symptoms: a lower limit of 1 mg (preferably 30 mg) and an upper limit of 2000 mg (preferably 1500 mg) per dose for oral administration; a lower limit of 0.5 mg (preferably 5 mg) and an upper limit of 1000 mg (preferably 250 mg) per dose for intravenous or subcutaneous administration; a lower limit of 0.5 mg (preferably 5 mg) and an upper limit of 500 mg (preferably 250 mg) per dose for intratracheal administration; and a lower limit of 0.05 mg (preferably 0.5 mg) and an upper limit of 10 mg (preferably 5 mg) per dose for intraocular administration. In addition, in the case of a drug with enhanced stability, it is desirable to administer it 1 to 3 times a week, and in the case of a drug with even enhanced stability, it is desirable to administer it 1 to 3 times every 1 month, 3 months, 6 months, 12 months, 18 months, or 24 months, depending on the symptoms.

[0400] Pharmaceutical compositions and formulations for topical administration include transdermal patches, ointments, lotions, creams, gels, lozenges, suppositories, sprays, liquids and powders.

[0401] The oligonucleotides of the present invention can suppress the expression of SNCA mRNA. Furthermore, the oligonucleotides of the present invention have low toxicity to cells and can be used as highly safe pharmaceuticals. Furthermore, by suppressing the expression of SNCA mRNA, the oligonucleotides of the present invention can suppress the accumulation of intracellular insoluble inclusions containing α-synuclein. While the mechanism of action is not particularly limited, the oligonucleotides of the present invention can be used to treat or prevent diseases that can be treated or prevented by suppressing the expression of SNCA mRNA. Examples of such diseases include α-synucleinopathies.

[0402] The present invention provides pharmaceutical use inventions such as a pharmaceutical composition containing the oligonucleotide of the present invention as an active ingredient and used for treating or preventing an α-synucleinopathies, a method for treating or preventing an α-synucleinopathies comprising administering an effective amount of the oligonucleotide, the oligonucleotide for use in treating or preventing an α-synucleinopathies, and use of the oligonucleotide of the present invention in the manufacture of a pharmaceutical composition for treating or preventing an α-synucleinopathies. α-Synucleinopathies that can be treated or prevented by the oligonucleotide of the present invention include, for example, Parkinson's disease, Parkinson's disease dementia, dementia with Lewy bodies, multiple system atrophy, pure autonomic failure, etc.

[0403] The oligonucleotide of the present invention can be preferably used for the treatment or prevention of Parkinson's disease, Parkinson's disease dementia, dementia with Lewy bodies, multiple system atrophy, and pure autonomic failure.

[0404] The present invention provides a method for treating or preventing α-synucleinopathies, comprising administering the oligonucleotide of the present invention in combination with one or more other drugs. The oligonucleotide of the present invention may be administered in combination with one or more other drugs, as long as the effects of the present invention are achieved. Preferably, the oligonucleotide of the present invention may be administered in combination with other drugs having a different mechanism of action against α-synucleinopathies to provide greater therapeutic effects. For example, drugs such as levotopa, a dopamine precursor; carbidopa, a dopa decarboxylase inhibitor; dopamine agonists; anticholinergics; MAO-B inhibitors; and COMT inhibitors may be prescribed for Parkinson's disease, but these drugs have a different mechanism of action from SNCA siRNA. Therefore, the combination of these drugs with SNCA siRNA may provide greater therapeutic effects against PD than single drugs. Furthermore, drugs such as cholinesterase inhibitors may be prescribed for Parkinson's disease dementia and dementia with Lewy bodies, but these drugs have a different mechanism of action from SNCA siRNA. Therefore, the combination of these drugs with SNCA siRNA can provide greater therapeutic effects for Parkinson's disease dementia and dementia with Lewy bodies than single drugs. Furthermore, for multiple system atrophy, drugs such as adrenergic α1 receptor agonists and droxidopa, a precursor of noradrenaline and adrenaline, are sometimes prescribed, but these drugs have different mechanisms of action from SNCA siRNA. Therefore, the combination of these drugs with SNCA siRNA can provide greater therapeutic effects for multiple system atrophy than single drugs. Although existing drugs for α-synucleinopathies are all symptomatic treatments, they have demonstrated certain therapeutic effects on motor symptoms, cognitive symptoms, and autonomic symptoms, so the combination of these drugs with SNCA siRNA can provide useful therapeutic effects for α-synucleinopathies.

[0405] When the oligonucleotide of the present invention is used in combination with other drugs, the order of administration of the RNAi-oligonucleotide of the present invention is not particularly limited as long as the effects of the present invention are achieved. The RNAi-oligonucleotide of the present invention may be administered simultaneously with the other drug, or before or after the other drug. Examples of drugs that can be used in combination with the RNAi-oligonucleotide of the present invention include therapeutic agents for motor symptoms, cognitive symptoms, and autonomic symptoms in α-synucleinopathies. Examples of therapeutic agents for motor symptoms include levotopa, a dopamine precursor; carbidopa, a dopa decarboxylase inhibitor; dopamine agonists; anticholinergics; MAO-B inhibitors; and COMT inhibitors. Examples of therapeutic agents for cognitive symptoms include cholinesterase inhibitors. Examples of therapeutic agents for autonomic symptoms include adrenergic α1 receptor agonists and droxidopa, a precursor of noradrenaline and adrenaline.

[0406] The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.

[0407] (Examples 1-2 and Comparative Examples 1-5) Each of the Examples and Comparative Examples in Table 1 is a double-stranded oligonucleotide composed of a strand region and an antisense strand region in the table, and the complementary regions of the sense strand region and the antisense strand region are all composed of natural RNA (i.e., A(R), G(R), C(R), and U(R)). Furthermore, two consecutive T(D) are bound to the 3' end of each complementary region in the sense strand region and the antisense strand region of each compound in Table 1 as an overhang structure. At the 5' position of the 5'-terminal nucleotide and the 3' position of the 3'-terminal nucleotide of each oligonucleotide, a hydrogen atom is a hydroxyl group bound to an oxygen atom in the structural diagram of each nucleoside described above. Furthermore, each internucleoside bond is a chemically unmodified phosphodiester bond.

[0408] The compound of Comparative Example 1 (SNCA-038) is AD-21792 described in WO2012 / 027713, the compound of Comparative Example 2 (SNCA-058) is AD-21797 described in WO2012 / 027713, the compound of Comparative Example 3 (SNCA-059) is AD-21794 described in WO2012 / 027713, and the compound of Comparative Example 4 (SNCA-052) is AD-21758 described in WO2012 / 027713. All of these compounds are made of natural RNA, and further, all of the internucleoside bonds are phosphodiester bonds. The compound of Comparative Example 5 (SNCA-019) is a double-stranded oligonucleotide in which the complementary region in the sense strand is a 19-base sequence described as Seq ID No: 501 in US 2008 / 139799, the complementary region in the antisense strand is a sequence completely complementary to said sequence, and two consecutive Ts (Ds) are further bound to the 3'-end of each of these regions as an overhang structure.

[0409] The target sequence in the compound of Example 1 (SNCA-023) is a sequence consisting of 19 bases from the 678th to the 696th bases in the base sequence of human SNCA mRNA shown in SEQ ID NO: 1 (disclosed as accession number NM_000345.4 in NCBI Genbank). That is, it is 5' UCUGCUGACAGAUGUUCCA 3' (SEQ ID NO: 2). The antisense strand region has a nucleotide sequence completely complementary to the target sequence, with two consecutive Ts (Ds) bound to its 3' end as an overhang structure. The sense strand region has a nucleotide sequence completely complementary to the antisense strand region, with two consecutive Ts (Ds) bound to its 3' end as an overhang structure.

[0410] The target sequence in the compound of Example 2 (SNCA-012) is a sequence consisting of 19 bases from the 688th to 706th bases in the base sequence of human SNCA mRNA shown in SEQ ID NO: 1 (disclosed as accession number NM_000345.4 in NCBI Genbank). That is, it is 5' GAUGUUCCAUCCUGUACAA 3' (SEQ ID NO: 3). The antisense strand region has a nucleotide sequence completely complementary to the target sequence, with two consecutive Ts (Ds) bound to its 3' end as an overhang structure. The sense strand region has a nucleotide sequence completely complementary to the antisense strand region, with two consecutive Ts (Ds) bound to its 3' end as an overhang structure.

[0411] The oligonucleotides in Table 1 were synthesized using the phosphoramidite method (Nucleic Acids Research, 12, 4539 (1984), Nature Communications 6, Article number: 6317 (2015)). Equimolar amounts of the sense strand region and the antisense strand region, each of which had been synthesized individually, were placed in one tube and subjected to annealing treatment. The formation of double-stranded oligonucleotides was confirmed by native gel or HPLC, and these were designated as example compounds and comparative example compounds having a double-stranded structure.

[0412]

[0413] In the sequences in the table, uppercase letters indicate RNA and lowercase letters indicate DNA. The structure of each nucleoside is represented by the structural formula described above. The structure of each nucleoside is represented by the structural formula described above. The 5'-position of the 5'-terminal nucleotide and the 3'-position of the 3'-terminal nucleotide of each oligonucleotide are hydroxyl groups in which a hydrogen atom is bonded to an oxygen atom in the structural diagram of each nucleoside described above.

[0414] (Examples 3 to 8 and Comparative Examples 6 to 7) Each Example and Comparative Example shown in Table 3 is a double-stranded oligonucleotide composed of the sense strand region and antisense strand region in the table. The nucleotide sequences of the sense strand region and antisense strand region constituting each Example and Comparative Example are shown in Table 2. These oligonucleotides were synthesized using the phosphoramidite method (Nucleic Acids Research, 12, 4539 (1984), Nature Communications 6, Article number: 6317 (2015)). Each oligonucleotide was identified by negative ion ESI mass spectrometry, and the results are listed in Table 1. Furthermore, equimolar amounts of the sense strand region and antisense strand region synthesized individually were placed in one tube and annealed, and then the formation of double-stranded oligonucleotides was confirmed using native gel or HPLC, resulting in compounds of the Examples or Comparative Examples having a double-stranded structure. The compounds of each Example and Comparative Example are shown in Table 3.

[0415]

[0416] In the sequences in the table, N(R) represents RNA, N(D) represents DNA, N(M) represents 2'-OMe RNA, N(m) represents 2'-MOE RNA, N(3M) represents 3'-OMe RNA, N(F) represents 2'-F RNA, and N(E) represents ENA. The structure of each nucleoside is represented by the structural formula shown above. "^" represents a phosphorothioate bond (-P(=S)(OH)-, shown in the structural diagram of the internucleoside bond (PS) shown above). Unless otherwise specified, it represents a bond formed by a phosphodiester bond (-P(=O)(OH)-, shown in the structural diagram of the internucleoside bond (P) shown above). VPu, EPu, VPuam, and SPh in the table are each represented by the following structures: The 5'-position of the 5'-terminal nucleotide and the 3'-position of the 3'-terminal nucleotide of each oligonucleotide (the 2'-position in the case of a nucleotide modified at the 3'-position) have hydrogen atoms that are hydroxyl groups bonded to oxygen atoms in the structural diagrams of each nucleoside described above, and when VPu, EPu, VPuam, or SPh is bonded to the 5'-terminal of each oligonucleotide, VPu, EPu, VPuam, or SPh is bonded to the adjacent nucleotide in the bonding mode shown in the table. Furthermore, VPu, EPu, VPuam, or SPh has no bonding site other than to the adjacent oligonucleotide portion.

[0417] Furthermore, two consecutive U (M) are bound as an overhang structure to the 3' end of the antisense strand region in Table 2. The "molecular weight" in the table indicates the value actually measured by negative ion ESI mass spectrometry.

[0418]

[0419] (In the formula, the tip of the dashed line represents bonding to the adjacent nucleotide.)

[0420]

[0421] The compounds of Examples 4 and 6 to 8 are double-stranded oligonucleotides whose target sequence is the same as the target sequence of the compound of Example 2 (SNCA-012) described above (i.e., the sequence of SEQ ID NO: 3). The compounds of Examples 3 and 5 are double-stranded oligonucleotides whose target sequence is the same as the target sequence of the compound of Example 1 (SNCA-023) described above (i.e., the sequence of SEQ ID NO: 2). The compounds of Comparative Examples 6 and 7 are double-stranded oligonucleotides whose target sequence is the same as the target sequence of the compound of Comparative Example 5 (SNCA-019) described above. Furthermore, the compounds of each of these Examples and Comparative Examples are composed of sense and antisense strands modified as shown in Table 2. For example, the antisense strand region of the compound of Example 4 (SNCA-012.88SPh) has a nucleotide sequence that is completely complementary to the target sequence described above (i.e., the sequence of SEQ ID NO: 3) as the target-corresponding sequence, with two consecutive U (M)s bound to the 3' end as an overhang structure. The sense strand region is a nucleotide sequence that is completely complementary to the target sequence, and has SPh bound to its 5' end as a fatty acid unit via a phosphodiester bond.

[0422] In the sequences, the "2'-O-methylnucleoside" portion was synthesized using the phosphoramidite described in Nucleic Acids Research 17, 3373 (1989). The "DNA" portion was synthesized using the phosphoramidite described in Nucleic Acids Research 11, 4539 (1984). The "3'-O-methylnucleoside" portion may be 3'-O-Methyl Adenosine (n-bz) CED phosphoramidite (ChemGene, catalog number: ANP-2901), 3'-O-Methyl Cytidine (n-bz) CED phosphoramidite (ChemGene, catalog number: ANP-2902), 3'-O-Methyl Guanosine (n-ibu) CED phosphoramidite (ChemGene, catalog number: ANP-2903), or 3'-O-Methyl Uridine CED The "2'-deoxy-2'-fluoronucleoside" moiety was synthesized using the phosphoramidite described in J. Med. Chem. 36, 831 (1993). ENA was synthesized using the phosphoramidite described in WO2000 / 47599. The "2'-O-methoxyethylnucleoside" moiety was synthesized using the phosphoramidite described in Helv. Chim. Acta 78, 486-504 (1995). As the unit to be bound to the 5' end of the antisense strand, synthesis was performed using an amidite unit that is the compound of the Examples described in the literature to synthesize the "VPu" portion, the compound of Reference Example 16 to synthesize the "EPu" portion, or the compound of Reference Example 7 to synthesize the "VPuam" portion. Furthermore, as the unit to be bound to the 5' end of the sense strand, synthesis was performed using 2-cyanoethyl (6-stearamidohexyl) diisopropylphosphoramidite, a compound described in Reference Example 1 of WO2021 / 010301 to synthesize the "S" portion, or the amidite unit of Reference Example 8 to synthesize the "SPh" portion.

[0423] When the amidite of Reference Example 16 was used, prior to the deprotection step using concentrated aqueous ammonia, the product was treated with a solution of trimethylsilane iodide and pyridine in methylene chloride for 40 minutes, and then with a solution of 2-mercaptoethanol in triethylamine and acetonitrile for 45 minutes, thereby deprotecting the ethyl group of the phosphonate moiety.

[0424] Reference Example 1: 2'-azido-2'-deoxy-3',5'-bis-O-(1,1,1-triethylsilyl)uridine

[0425]

[0426] 2'-Azido-2'-deoxyuridine (500 mg, 1.86 mmol) and imidazole (630 mg, 9.29 mmol) were dissolved in dimethylformamide (10 ml), and triethylsilyl chloride (1.25 ml, 7.43 mmol) was added under ice-cooling, followed by stirring at room temperature for 1 hour. The reaction solution was diluted with dichloromethane, washed with saturated aqueous sodium bicarbonate, dried over anhydrous sodium sulfate, filtered, and purified with silica gel (ethyl acetate:hexane = 0-50%) to obtain 560 mg (61%) of the target compound as a solid. １ H-NMR (CDCl ３ ) δ: 8.53 (1H, s), 7.93 (1H, d, J = 8.5Hz), 6.10 (1H, d, J = 4.9Hz), 5. 70 (1H, dd, J = 8.5, 2.1Hz), 4.42 (1H, t, J = 4.9Hz), 4.07 (1H, t, J = 2 .1Hz), 3.95 (1H, dd, J=11.6, 2.1Hz), 3.75 (1H, dd, J=11.6, 1.5Hz) ), 3.64 (1H, t, J=5.2Hz), 1.10-0.90 (18H, m), 0.70-0.60 (12H, m). ESI-MS: 498 (M+H ＋ )

[0427] Reference Example 2: 2'-azido-2'-deoxy-3'-O-(1,1,1-triethylsilyl)uridine

[0428]

[0429] The compound 2'-azido-2'-deoxy-3',5'-bis-O-(1,1,1-triethylsilyl)uridine (1000 mg, 2.01 mmol) obtained in Reference Example 1 was dissolved in dichloromethane (10 mL), and a solution of pyridinium toluenesulfonate (300 mg, 1.21 mmol) in methanol (10 mL) was added at 0°C, followed by stirring at 0°C for 3 hours. Triethylamine (420 μL) was added to the reaction solution, which was then diluted with dichloromethane, washed with saturated aqueous sodium bicarbonate, dried over sodium sulfate, filtered, and the filtrate was concentrated. The product was purified on silica gel (methanol:dichloromethane = 0-15%) to obtain 690 mg (90%) of the target compound as a solid. １ H-NMR (CDCl ３ ) δ: 8.08 (1H, s), 7.61 (1H, d, J = 7.9Hz), 5.78 (2H, dt, J = 15.1, 5.8Hz), 4.53 (1H, dd, J = 5.8, 4.0Hz), 4.10-4.08 (1H, m), 4.02 (1H, t , J=5.8Hz), 3.97 (1H, dt, J=12.0, 2.6Hz), 3.77-3.80 (1H, m), 2.35 (1H, dd, J=6.1, 3.1Hz), 1.08-0.99 (9H, m), 0.75-0.64 (6H, m). ESI-MS: 384 (M+H) ＋

[0430] Reference Example 3: 2'-azido-2'-deoxy-3'-O-(1,1,1-triethylsilyl)-5'-deoxy-5'-oxouridine

[0431]

[0432] The compound 2'-azido-2'-deoxy-3'-O-(1,1,1-triethylsilyl)uridine (900 mg, 2.35 mmol) obtained in Reference Example 2 was dissolved in acetonitrile (100 ml), and 2-iodoxybenzoic acid (3.4 g, 4.70 mmol) was added, followed by stirring at 80°C for 1 hour under a nitrogen atmosphere. The mixture was returned to room temperature, and the solid was filtered. The filtrate was concentrated and purified with silica gel (methanol:dichloromethane = 0-10%) to obtain 700 mg (78%) of the target compound as a solid. １ H-NMR (CDCl ３) δ: 9.75 (1H, s), 8.41 (1H, s), 7.65 (1H, d, J = 7.9Hz), 5.98 (1H, d, J = 6.7Hz), 5.83 (1H, t, J = 4.9Hz), 4.6 5 (1H, t, J = 3.7Hz), 4.54 (1H, d, J = 3.7Hz), 3.80 (1H, t, J = 5.5Hz), 1.05-1.02 (9H, m), 0.76-0.70 (6H, m). ESI-MS: 382 (M+H) ＋

[0433] Reference Example 4: 2'-azido-2'-deoxy-3'-O-(1,1,1-triethylsilyl)-5'-deoxy-5'-2-(bis{[(2,2-dimethoxypropanoyl)oxy]methoxy}phosphoryl)vinyl]uridine

[0434]

[0435] Tetra(pivaloyloxymethyl) methylenebisphosphonate (1 g, 1.57 mmol, Tetrahedron Lett. 1999, 40, 8491-8493) was dissolved in tetrahydrofuran (10 ml), and NaH (80 mg, 1.81 mmol) was added at 0° C., followed by stirring at room temperature for 15 minutes. A tetrahydrofuran (10 ml) solution containing 2'-azido-2'-deoxy-3'-O-(1,1,1-triethylsilyl)-5'-deoxy-5'-oxouridine (300 mg, 0.79 mmol), the compound obtained in Reference Example 3, was added dropwise at 0° C., followed by stirring at room temperature for 2 hours. An aqueous ammonium chloride solution was added, the mixture was extracted with ethyl acetate, dried over sodium sulfate, and filtered. The filtrate was concentrated and purified with silica gel (ethyl acetate:hexane=0-50%) to obtain 350 mg (64%) of a solid target compound as a mixture of E- and Z-isomers. １ H-NMR (DMSO-D ６) δ: 11.47 (1H, d, J = 1.8Hz), 7.70 (1H, t, J = 8.2Hz), 6.77 (1H, ddd, J = 26.7, 13.6, 6.6Hz), 6.20 (0.8H, dd, J = 22. 3, 16.8Hz), 6.07 (0.2H, dd, J = 18.0, 13.6Hz), 5.82 (0.2H, d, J = 4.9Hz), 5.73 (0.8H, d, J = 4.9Hz), 5.65-5.70 (1 H, m), 5.55-5.65 (4H, m), 5.21 (0.2H, dd, J = 10.4, 4.7Hz), 4.57 (0.8H, t, J = 6.1Hz), 4.51-4.47 (1H, m), 4.45 (0 .2H, dd, J=11.6, 6.1Hz), 4.35 (0.8H, t, J=6.1Hz), 1.18-1.14 (18H, m), 0.98-0.92 (9H, m), 0.68-0.59 (6H, m). ESI-MS 686 (M － )

[0436] Reference Example 5: 2'-acetamido-2'-deoxy-3'-O-(1,1,1-triethylsilyl)-5'-deoxy-5'-[2-(bis{[(2,2-dimethoxypropanoyl)oxy]methoxy}phosphoryl)vinyl]uridine

[0437]

[0438] The compound obtained in Reference Example 4 (2.95 g, 4.29 mmol) was dissolved in ethyl acetate (70 mL), and N-methylmorpholine (0.61 mL, 5.58 mmol), acetic anhydride (0.73 mL, 7.73 mmol), and Lindlar's catalyst (2.95 g) were added. Hydrogen gas was added to this mixture using a balloon with stirring at room temperature for 5 hours. After purging with nitrogen, insoluble matter was removed by filtration through Celite, and the resulting solution was concentrated under reduced pressure. Toluene (20 mL) was added to the resulting residue, and the mixture was concentrated under reduced pressure. The mixture was then purified using silica gel chromatography (hexane:ethyl acetate = 90:10 → 50 / 50 → 25 / 75 → 0 / 100) to obtain the target product (2.21 g, 3.14 mmol, 73%) as a colorless glassy solid as a mixture of the E- and Z-isomers. １H-NMR (400MHz, DMSO-d6): 0.54-0.65 (6H, m), 0.89-0.94 (9H, m), 1.12-1.17 (18H, m), 1.85 (3H, s), 4.15-4.7 8 (3H, m), 5.56-6.16 (7H, m), 6.75-6.95 (1H, m), 7.76-7.84 (1H, m), 8.03-8.11 (1H, m), 11.41-11.44 (1H, m). ESI-MS: 704 (M+H) ＋

[0439] (Reference Example 6a) 2'-acetamido-2',5'-dideoxy-5'-[(E)-2-(bis{[(2,2-dimethoxypropanoyl)oxy]methoxy}phosphoryl)vinyl]uridine

[0440]

[0441] (Reference Example 6b) 2'-acetamido-2',5'-dideoxy-5'-[(Z)-2-(bis{[(2,2-dimethoxypropanoyl)oxy]methoxy}phosphoryl)vinyl]uridine

[0442]

[0443] The compound obtained in Reference Example 5 (2.19 g, 3.11 mmol) was dissolved in tetrahydrofuran (10 mL), and water (10 mL) and formic acid (10 mL) were added in an ice bath. The mixture was stirred in an ice bath for 5 minutes and then at room temperature for 45 minutes. After purging with nitrogen, insoluble materials were removed by filtration through Celite, and the resulting solution was concentrated under reduced pressure. The reaction solution was diluted with ethyl acetate (200 mL) and washed with water (three times) and saturated brine. The solvent was concentrated under reduced pressure, and the resulting residue was purified by silica gel chromatography (hexane:ethyl acetate = 50 / 50 → 0 / 100 → methanol:ethyl acetate = 5 / 95 → 10 / 90) to give the (Z) isomer (colorless solid, 0.39 g, 0.66 mmol, 21%) and the colorless solid E isomer (1.19 g, 2.02 mmol, 65%).

[0444] (6a) 1H-NMR (400MHz, DMSO-d6): 1.14-1.16 (18H, m), 1.85 (3H, s), 4.08-4.13 (3 H, m), 4.39-4.44 (1H, m), 4.48-4.55 (1H, m), 5.59-5.68 (5H, m), 5.84 (1H, d, J=7.9Hz), 5.98 (1H, d, J=4.9Hz), 6.08 (1H, ddd, J=22.3, 17.1, 1.5Hz), 6.8 4-6.95 (1H, m), 7.73 (1H, d, J = 8.5Hz), 7.98 (1H, d, J = 8.5Hz), 11.39 (1H, s). ESI-MS: 590 (M+H) ＋

[0445] (6b) １ H-NMR (400MHz, DMSO-d6): 1.15 (18H, d, J = 9.2Hz), 1.85 (3H, s), 4.00 (3 H, td, J = 5.5, 1.8 Hz), 4.66 (1H, td, J = 8.5, 6.1 Hz), 5.19 (1H, d, J = 8 Hz), 5.59-5.67 (5H, m), 5.85-5.95 (3H, m), 6.90 (1H, ddd, J = 54.8, 13, 9.6Hz ), 7.83 (1H, d, J = 8.5Hz), 7.95 (1H, d, J = 8.5Hz), 11.34 (1H, d, J = 1.8Hz). ESI-MAS: 590 (M+H) ＋

[0446] Reference Example 7: 2'-acetamido-2',5'-dideoxy-5'-[(E)-2-(bis{[(2,2-dimethoxypropanoyl)oxy]methoxy}phosphoryl)vinyl]uridine-3'-O-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite

[0447]

[0448] A mixture of the compound obtained in Reference Example 6a (1.14 g, 1.93 mmol), triethylamine (1.1 mL, 7.73 mmol), and anhydrous dichloromethane (30 mL) was ice-cooled, and a solution of 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (>80%, 1.14 g, 3.87 mmol) in anhydrous dichloromethane (5 mL) was added, followed by stirring at room temperature for 75 minutes. The reaction mixture was diluted with toluene (35 mL) and purified by column chromatography using NH silica gel (hexane (1% triethylamine added): ethyl acetate (1% triethylamine added) = 50 / 50 → 0 / 100 → methanol (1% triethylamine added): ethyl acetate (1% triethylamine added) = 15 / 85) to give the target product (805 mg, 1.02 mmol, 53%) as a colorless solid. １ H-NMR (400MHz, CD3CN): 1.16-1.21 (30H, m), 1.87 (3H, d, J = 16.0Hz), 2.69 (2H , td, J=11.6, 5.9Hz), 3.61-3.92 (4H, m), 4.31-4.49 (1H, m), 4.54-5.75 (2H, m) , 5.54-5.67 (5H, m), 5.85 (1H, t, J = 7.0Hz), 6.02-6.20 (1H, m), 6.57 (1H, dd, J = 16.8, 8.2Hz), 6.79-6.98 (1H, m), 7.44 (1H, dd, J = 8.2, 4.6Hz), 8.96 (1H, br). ESI-MS: 790 (M+H) ＋

[0449] Reference Example 8: N-[4-[2-cyanoethoxy-(diisopropylamino)phosphanyl]oxyphenyl]octadecanamide

[0450]

[0451] N-(4-hydroxyphenyl)stearamide (Channelpharm, CP-1140373, 1.00 g, 2.66 mmol) was dissolved in anhydrous chloroform (100 mL), and N,N-diisopropylethylamine (1.86 mL, 10.6 mmol) and 2-cyanoethyldiisopropylchlorophosphoramidite (purity 80%, 1.04 mL, 3.73 mmol) were added under an argon atmosphere, followed by stirring at 30°C for 2 hours. The reaction solution was washed with a saturated aqueous sodium hydrogen carbonate solution and saturated brine, dried over anhydrous sodium sulfate, and the solvent was concentrated under reduced pressure. The resulting residue was purified using silica gel chromatography (NH silica, n-hexane:ethyl acetate=100:0-60:40, containing 1% triethylamine; the impurity-containing fraction was re-purified with n-hexane:dichloromethane=100:0-65:35, containing 1% triethylamine), yielding the target product (0.96 g, 1.67 mmol, 63%) as a white solid. １ H-NMR (400MHz, CDCl ３ ): 0.88 (3H, t, J = 6.7Hz), 1.16 (6H, d, J = 6.7Hz), 1.19-1.42 (34H, m), 1.67-1.76 (2H, m), 2.33 (2H, t, J = 7.6 Hz), 2.67 (2H, t, J = 6.7), 3.65-3.78 (2H, m), 3.86-3.98 (2H, m), 6.96-7.04 (3H, m), 7.41 (2H, d, J = 8.5Hz). LC-MS: 493.3 (M-iPr2N+OH+H) ＋

[0452] Reference Example 9: 5'-O-(4,4'-dimethoxytrityl)-2'-O-methyluridine

[0453]

[0454] 2'-O-methyluridine (5.20 g, 20.1 mmol) was dissolved in pyridine (55 mL), and 4,4'-dimethoxytrityl chloride (8.70 g, 25.7 mmol) was added under ice-cooling, followed by stirring overnight at room temperature. The reaction solvent was distilled off under reduced pressure, and the residue was purified by silica gel chromatography (hexane:ethyl acetate (containing 2% triethylamine) = 67:33 to 0:100) to obtain 14.6 g (including the solvent) of the target product.

[0455] Reference Example 10: 3'-O-tert-butyl(diphenyl)silyl-5'-O-(4,4'-dimethoxytrityl)-2'-O-methyluridine

[0456]

[0457] 5'-O-(4,4'-dimethoxytrityl)-2'-O-methyluridine (14.6 g (including solvent), 20.1 mmol) obtained in Reference Example 9 and imidazole (2.20 g, 32.3 mmol) were dissolved in DMF (150 mL), and tert-butyldiphenylchlorosilane (8.56 g, 31.1 mmol) was added at room temperature, followed by stirring overnight at room temperature. Saturated aqueous sodium bicarbonate was added to the reaction solution, followed by further addition of water, and extraction with methylene chloride was performed. After drying over sodium sulfate, the filtered solution was concentrated and purified by silica gel chromatography (hexane:ethyl acetate (containing 1% triethylamine) = 80:20 to 42:58) to obtain 15.2 g (98%) of the desired product.

[0458] Reference Example 11: N3-benzoyl-3'-O-tert-butyl(diphenyl)silyl-2'-O-methyluridine

[0459]

[0460] 3'-O-tert-butyl(diphenyl)silyl-5'-O-(4,4'-dimethoxytrityl)-2'-O-methyluridine (15.2 g, 19.1 mmol) obtained in Reference Example 10 and diisopropylethylamine (4.45 g, 34.3 mmol) were dissolved in pyridine (100 mL), and benzoyl chloride (4.24 g, 30.2 mmol) was added dropwise at room temperature over 5 minutes, followed by stirring overnight at room temperature. The solvent was distilled off under reduced pressure, and saturated aqueous sodium bicarbonate was added, followed by extraction with methylene chloride. The mixture was dried over sodium sulfate, and the filtered solution was concentrated to obtain the intermediate benzoylated product. Methylene chloride (200 mL), trichloroacetic acid (6 mL), and triethylsilane (6.40 g, 55.0 mmol) were added to the resulting intermediate, followed by stirring at room temperature for 2.5 hours. Water was added to the reaction solution, followed by extraction with methylene chloride. The residue was washed with saturated aqueous sodium bicarbonate, dried over sodium sulfate, and the filtered solution was concentrated and purified by silica gel chromatography (hexane:ethyl acetate=67:33 to 0:100) to obtain 7.45 g (65%) of the desired product. １ H-NMR (400MHz, DMSO-d6): 1.04 (9H, s), 3.09 (3H, s), 3.38 (1H, ddd, J = 2.4, 4.8, 12.1Hz), 3.54 (1H, dd, J=4.2, 4.8Hz), 3.62 (1H, ddd, J=2.4, 4.8, 12.1Hz), 3.98-4.05 (1H, m), 4.37 (1H, dd, J=4.2, 4.8 Hz), 5.28 (1H, dd, J = 4.2, 4.8Hz), 5.90 (1H, d, J = 4.2Hz), 5.93 (1H, d, J = 8.5Hz), 7.40-7.50 (6H, m), 7.57-7.66 (4H, m), 7.67-7.70 (2H, m), 7.77-7.83 (1H, m), 7.93-7.97 (2H, m), 8.13 (1H, d, J=8.5Hz).

[0461] (Reference Example 12) 5-(3-benzoyl-2,4-dioxo-pyrimidin-1-yl)-3-[tert-butyl(diphenyl)silyl]oxy-4-methoxy-tetrahydrofuran-2-carbaldehyde

[0462]

[0463] N3-benzoyl-4'-O-tert-butyl(diphenyl)silyl-2'-O-methyluridine (7.45 g, 12.4 mmol) obtained in Reference Example 11 was dissolved in acetonitrile (120 mL), and 2-iodoxybenzoic acid (9.00 g, 32.12 mmol) was added at room temperature, followed by stirring for 2 hours at 85° C. The solid was collected by filtration, and the solvent was evaporated under reduced pressure to obtain 7.77 g (99%) of the desired product. １ H-NMR (400MHz, DMSO-d6): 1.05 (9H, s), 3.05 (3H, s), 3.78 (1H, dd, J = 4.3, 6.7 Hz), 4.59 (1H, d, J = 3.0Hz), 4.68 (1H, dd, J = 3.0, 4.2Hz), 6.04 (1H, d, J = 8.5Hz) , 6.07 (1H, d, J = 6.7Hz), 7.39-7.51 (6H, m), 7.58-7.66 (4H, m), 7.67-7.72 (2H, m), 7.77-7.84 (1H, m), 7.95-8.00 (2H, m), 8.10 (1H, d, J=8.5Hz), 9.42 (1H, s).

[0464] Reference Example 13: 3-benzoyl-1-[4-tert-butyl(diphenyl)silyl]oxy-5-(2,2-dibromovinyl)-3-methoxy-tetrahydrofuran-2-yl]pyrimidine-2,4-dione

[0465]

[0466] 5-(3-benzoyl-2,4-dioxo-pyrimidin-1-yl)-3-[tert-butyl(diphenyl)silyl]oxy-4-methoxy-tetrahydrofuran-2-carbaldehyde (7.77 g, 12.3 mmol) obtained in Reference Example 12 was dissolved in methylene chloride (123 mL) and added to a methylene chloride (37 mL) solution of carbon tetrabromide (8.25 g, 24.9 mmol) and triphenylphosphine (12.9 g, 49.2 mmol) cooled to 0°C, followed by stirring at room temperature overnight. Saturated aqueous sodium bicarbonate was added to the reaction solution, followed by extraction with methylene chloride. The mixture was dried over sodium sulfate, and the filtered solution was concentrated and purified by silica gel chromatography (hexane:ethyl acetate = 85:15 to 50:50) to obtain 5.00 g (54%) of the desired product. １H-NMR (400MHz, CDCl ３ ): 1.10 (9H, s), 3.17 (1H, dd, J = 0.9, 5.1Hz), 3.30 (3H, s), 3.86 (1H , dd, J=5.1, 7.6 Hz), 4.84 (1H, dd, J=8.8, 7.6 Hz), 5.68 (1H, d, J=0. 9Hz), 5.77 (1H, d, J = 8.5Hz), 6.25 (1H, d, J = 8.8Hz), 7.17 (1H, d, J = 8.5Hz), 7.33-7.53 (9H, m), 7.61-7.72 (5H, m), 7.86-7.91 (2H, m).

[0467] Reference Example 14: 3-benzoyl-1-[4-tert-butyl(diphenyl)silyl]oxy-5-(2-diethoxyphosphorylethynyl)-3-methoxy-tetrahydrofuran-2-yl]pyrimidine-2,4-dione

[0468]

[0469] 3-Benzoyl-1-[4-tert-butyl(diphenyl)silyl]oxy-5-(2,2-dibromovinyl)-3-methoxy-tetrahydrofuran-2-yl]pyrimidine-2,4-dione (5.00 g, 6.63 mmol) obtained in Reference Example 13 was dissolved in DMF (60 mL) and added to a solution of palladium acetate (0.300 g, 1.33 mmol) and 1,1'-diphenylphosphinoferrocene (1.45 g, 2.61 mmol) in DMF (40 mL), propylene oxide (1.16 g, 20.0 mmol) and diethyl phosphite (1.87 g, 13.6 mmol) were added, and the mixture was stirred overnight at 80° C. Water was added to the reaction solution, followed by extraction with ethyl acetate. After drying over sodium sulfate and filtration, the solution was concentrated and purified by silica gel chromatography (hexane:ethyl acetate=67:33 to 30:70) to obtain 1.47 g (30%) of the desired product. １ H-NMR (400MHz, CDCl ３): 1.10 (9H, s), 1.35 (3H, t, J = 6.7Hz), 1.37 (3H, t, J = 6.7Hz), 3.26 (3H, s), 3 .42 (1H, dd, J=3.6, 4.2Hz), 4.08-4.15 (4H, m), 4.20 (1H, dd, J=4.2, 5.4Hz), 4 .. 80 (1H, dd, J = 3.0, 5.4Hz), 5.81 (1H, d, J = 7.9Hz), 6.01 (1H, d, J = 3.6Hz), 7.3 5-7.50 (8H, m), 7.51 (1H, d, J=7.9Hz), 7.64-7.73 (5H, m), 7.88-7.92 (2H, m).

[0470] Reference Example 15: 3-benzoyl-1-[5-(2-diethoxyphosphorylethynyl)-4-hydroxy-3-methoxy-tetrahydrofuran-2-yl]pyrimidine-2,4-dione

[0471]

[0472] 3-Benzoyl-1-[4-tert-butyl(diphenyl)silyl]oxy-5-(2-diethoxyphosphorylethynyl)-3-methoxy-tetrahydrofuran-2-yl]pyrimidine-2,4-dione (1.47 g, 2.01 mmol) obtained in Reference Example 14 was dissolved in tetrahydrofuran (20 mL), and triethylamine-trihydrofluoride (2.0 mL) was added at room temperature, followed by stirring at room temperature for 6 hours. Further triethylamine-trihydrofluoride (1.2 mL) was added at room temperature, followed by stirring at room temperature for 2 hours. Saturated aqueous sodium bicarbonate was added to the reaction solution, followed by extraction with methylene chloride. The solution was dried over sodium sulfate, and the filtered solution was concentrated and purified by silica gel chromatography (hexane:ethyl acetate = 50:50 to 0:100 to ethyl acetate:methanol = 95:5) to obtain 0.575 g (58%) of the desired product. １ H-NMR (400MHz, CDCl ３): 1.40 (6H, t, J = 7.3Hz), 2.88 (1H, d, J = 7.9Hz), 3.59 (3H, s), 4.01 (1H, dd, J=2.4, 4.8Hz), 4.17-4.25 (4H, m), 4.37 (1H, ddd, J=4.8, 6.0, 7.9Hz), 4.72 (1H, dd, J=3.6, 6.0Hz), 5.94 (1H, d, J=8.5Hz), 5.97 (1H, d, J=2.4Hz), 7.49 -7.55 (2H, m), 7.56 (1H, d, J=8.5Hz), 7.65-7.71 (1H, m), 7.93-7.96 (2H, m).

[0473] Reference Example 16: 3-{[5-(3-benzoyl-2,4-dioxo-pyrimidin-1-yl)-2-(2-diethoxyphosphorylethynyl)-4-methoxy-tetrahydrofuran-3-yl]oxy-(diisopropylamino)phosphanyl}oxypropanenitrile

[0474]

[0475] 3-Benzoyl-1-[5-(2-diethoxyphosphorylethynyl)-4-hydroxy-3-methoxy-tetrahydrofuran-2-yl]pyrimidine-2,4-dione (0.575 g, 1.17 mmol) obtained in Reference Example 15 and diisopropylethylamine (0.780 g, 6.03 mmol) were dissolved in methylene chloride (15 mL), and 2-cyanoethyldiisopropylchlorophosphoramidite (0.553 g, 2.34 mmol) was added at 0°C, followed by stirring at room temperature for 5 hours. Saturated aqueous sodium bicarbonate was added to the reaction solution, followed by extraction with methylene chloride. The mixture was dried over sodium sulfate, and the filtered solution was concentrated and purified by silica gel chromatography (hexane:ethyl acetate (containing 2% triethylamine) = 35:65 to 0:100) to obtain 0.555 g (69%) of the desired product. １ H-NMR (400MHz, CDCl ３): 1.20 (12H, d, J = 7.3Hz), 1.36-1.42 (6H, m), 2.62-2.70 (2H, m), 3.52 (3H, s ), 3.58-3.70 (2H, m), 3.78-3.94 (2H, m), 4.19 (1H, dd, J = 3.6, 4.2Hz), 4.14- 4.25 (4H, m), 4.48 (1H, dd, J = 4.2, 8.5Hz), 4.86 (1H, dd, J = 3.6, 4.2Hz), 6.01 (1H, d, J=4.2Hz), 7.47-7.53 (2H, m), 7.63-7.70 (1H, m), 7.94-7.98 (2H, m). ESI-MS: 693 (M+H) ＋ .

[0476] Test Example 1: Evaluation of knockdown activity of SNCA siRNA in H4 cells by reverse transfection (1) and cytotoxicity evaluation The following experiment was performed using the compounds of Examples 1 and 2 and Comparative Examples 1 to 5 as SNCA siRNA. 1 x 10^4 H4 cells were seeded per well in a 96-well plate (Corning) in 100 μL of DMEM medium (Thermo Fisher Scientific) supplemented with 10% FBS (Gibco). Immediately afterwards, two wells were prepared for each type of siRNA sample, and two wells were prepared for a negative control (NT) in which a mixture of four non-targeting siRNAs (Dharmacon) was used instead of SNCA siRNA. To prepare the reagent for transfection of one siRNA sample, 4.8 μL of Opti-MEM (Thermo Fisher Scientific) and 0.2 μL of RNAiMAX (Thermo Fisher Scientific) were mixed, to which 4.5 μL of Opti-MEM and 0.5 μL of siRNA (concentrations of 0.02 μM and 2 μM) were added, and the mixture was incubated at room temperature for 15 minutes. The mixture was then added to a 96-well plate seeded with H4 cells.

[0477] The final siRNA concentrations were 0.1 nM and 10 nM in all samples. To prepare the negative control reagent, 4.8 μL of Opti-MEM (Thermo Fisher Scientific) and 0.2 μL of RNAiMAX (Thermo Fisher Scientific) were mixed, to which 4.5 μL of Opti-MEM and 0.5 μL of a mixture of four non-targeting siRNAs (Dharmacon, concentrations 0.02 μM and 2 μM) were added, followed by incubation at room temperature for 15 minutes.

[0478] One day after transfection, cells were washed once with PBS (Thermo Fisher Scientific) and RNA was extracted. RNA extraction and reverse transcription were performed using the SuperPrep Cell Lysis & RT Kit for qPCR (TOYOBO) according to the kit's protocol. The resulting cDNA was used to measure the amount of endogenous human SNCA (hSNCA) mRNA by quantitative PCR. Human GAPDH (hGAPDH) mRNA was also measured as a housekeeping gene.

[0479] Quantitative PCR was performed using IDT PrimeTime Gene Expression Master Mix (Integrated DNA Technologies) as follows. 5 μL of IDT PrimeTime Gene Expression Master Mix, 0.5 μL of hSNCA primer (Hs00240906_m1 or Hs00240907_m1, Thermo Fisher Scientific), 0.5 μL of hGAPDH primer (Hs99999905_m1, Thermo Fisher Scientific), 2 μL of distilled water, and 2 μL of cDNA were mixed per well of a 384-well PCR plate (Applied Biosystems) (total of 10 μL), and two wells were prepared for each cDNA, and quantitative PCR was performed. The hSNCA mRNA expression level was measured as the average value between two wells and calculated using the ΔΔCt method with the housekeeping gene hGAPDH as the reference. The hSNCA mRNA expression level in the negative control was set to 1.0, and the relative hSNCA mRNA expression level in the samples treated with each siRNA was calculated and expressed as a relative value. The results are shown in Figure 8A below.

[0480] Cytotoxicity was evaluated one day after transfection by measuring absorbance at 560 nm according to the protocol of the LDH-Cytotoxic Test Wako (Fujifilm Wako Pure Chemical Industries, Ltd.). As a positive control, culture supernatant of cells treated with 1% Triton-X100 for 30 minutes at 37°C was used. As a negative control, culture supernatant of cells in which PBS was used instead of Triton X-100 was used. The absorbance of each well was designated A, the absorbance of the positive control P, and the absorbance of the negative control N, and cytotoxicity was calculated according to the following formula. The results are shown in Figure 8B below. Cytotoxicity (%) = (A - N) / (P - N) x 100

[0481] In a test using primer Hs00240906_m1, the compounds of Example 1 (SNCA-023) and Example 2 (SNCA-012) demonstrated stronger inhibition of target SNCA mRNA in H4 cells than the compounds of Comparative Examples 1 to 3 (SNCA-038, SNCA-058, and SNCA-059, respectively). Furthermore, the compounds of Example 1 (SNCA-023) and Example 2 (SNCA-012) demonstrated similar inhibition of target SNCA mRNA compared to the compound of Comparative Example 4 (SNCA-052).

[0482] The compound of Comparative Example 5 (SNCA-019) overlaps with the target region of the probe Hs00240906_m1, so a different probe, Hs00240907_m1, was used. In the test using the primer Hs00240907_m1, the compound of Comparative Example 5 (SNCA-019) showed the same level of inhibition of the target SNCA mRNA as the compound of Comparative Example 4 (SNCA-052).

[0483] The compound of Example 2 (SNCA-012) did not exhibit cytotoxicity in the concentration range of 0.003 nM to 100 nM. These results indicate that the compound of Example 2 (SNCA-012) can strongly suppress target hSNCA mRNA in H4 cells without exhibiting cytotoxicity.

[0484] Test Example 2: Efficacy evaluation test of SNCA siRNA in human SNCA transgenic mice The following experiment was conducted using human SNCA siRNA and human SNCA BAC transgenic mice (Taguchi T, et al. Brain. 143:249-265, 2020). 180 μg / body (or the dose indicated in the figures) of an example compound or a comparative example compound was administered intracerebroventricularly (i.c.v.) once. As a vehicle control group, PBS was administered i.c.v. instead of the drug. Four days after treatment with the Example or Comparative Compound (Day 4, or the number of days indicated in the figures), the mice were euthanized by exsanguination, and the cerebral cortex, midbrain, cerebellum, striatum, brainstem, and / or liver (or the sampling sites indicated in the figures) were sampled to measure the mRNA expression levels of hSNCA, mouse GAPDH (mGapdh), or mouse β-actin (mActb). RNA was extracted using an RNeasy Mini Kit (QIAGEN) according to the kit's protocol. The resulting 250 ng of RNA was reverse transcribed using PrimeScript RT Master Mix (Perfect Real Time) (Takara), and the amount of hSNCA, mGapdh, or mActb mRNA was measured by quantitative PCR using the obtained cDNA.

[0485] Quantitative PCR was performed using IDT PrimeTime Gene Expression Master Mix (Integrated DNA Technologies) as follows. Each well of a 384-well PCR plate (Applied Biosystems) contained 5 μL of IDT PrimeTime Gene Expression Master Mix, 0.5 μL of hSNCA primer (Hs00240906_m1 or Hs00240907_m1, Thermo Fisher Scientific), 0.5 μL of mGapdh primer (Mm99999915_g1, Thermo Fisher Scientific) or 0.5 μL of mActb primer (Mm.PT.39a.22214843.g, Integrated DNA (manufactured by Sigma-Aldrich Technologies), 3 μL of distilled water and 1 μL of cDNA were mixed (total 10 μL), and two wells were prepared for each cDNA, followed by quantitative PCR. hSNCA or mActb mRNA expression levels were measured as the average between two wells and calculated using the ΔΔCt method, with the housekeeping gene mGapdh or mActb as the reference. Relative mRNA levels were calculated and expressed as mean ± standard error (SD) relative to the mRNA level in the vehicle control group, which was set at 1.0. The results for hSNCA mRNA expression levels in the brain are shown in Figures 9 to 13, respectively.

[0486] Human α-synuclein protein expression was measured in the brains of mice for which hSNCA mRNA expression was measured. Protein extraction was performed using a lysis buffer containing 0.5 mL of 1 M Tris-HCl (Nippon Gene Co., Ltd.), 0.3 mL of 5 M NaCl (Thermo Fisher Scientific), 0.02 mL of 0.5 mol / L EDTA solution (Nacalai Tesque), 0.1 mL of Triton X-100 (Thermo Fisher Scientific), one tablet of cComplete protease inhibitor cocktail (Roche), one tablet of PhosSTOP (Roche), and 9.08 mL of distilled water (total 10 mL). The collected brain tissue was homogenized by adding 10 μL of lysis buffer to 1 mg of tissue. The tissue was then subjected to 10-second ultrasonic treatments, repeated 10 times at 30-second intervals, and centrifuged at 20,000 × g for 15 minutes to obtain the supernatant. 60 μg of the collected protein was added to 9.2 μL of 20% sodium dodecyl sulfate solution (Bio-Rad), 15 μL of NuPAGE LDS Sample Buffer (Thermo Fisher Scientific), 6.1 μL of NuPAGE Sample Reducing Agent (Thermo Fisher Scientific), and distilled water to a total volume of 61 μL. Proteins were separated by electrophoresis using a polyacrylamide gel (Thermo Fisher Scientific) and transferred to a PVDF membrane (Invitrogen). Human α-synuclein was detected with a human α-synuclein antibody (LB509, Abcam), and mouse β-actin was detected with a β-actin antibody (C4, Santa Cruz). The protein expression level of human α-synuclein was calculated as a ratio to the expression level of the housekeeping gene β-actin. The relative protein amount was calculated and expressed as a value relative to the protein amount in the vehicle control group, which was set to 1.0 (mean ± standard error). The results of human α-synuclein protein expression levels in the brain are shown in Figure 14.

[0487] 9 shows the results of a test in which the compound of Comparative Example 6 (SNCA-019.88SPh) and the compounds of Example 3 (SNCA-023.88SPh) and Example 4 (SNCA-012.88SPh), which have the base sequences of SNCA-019, SNCA-023, and SNCA-012, were compared in human SNCA BAC transgenic mice, where the chemical modification pattern (xxx.88) of the siRNA was fixed. As a result, the compounds of Example 3 (SNCA-023.88SPh) and Example 4 (SNCA-012.88SPh) strongly suppressed target SNCA mRNA in the mouse cerebral cortex, midbrain, and cerebellum four days after administration, compared to the compound of Comparative Example 6 (SNCA-019.88SPh).

[0488] FIG. 10 shows the results of a test in which the chemical modification pattern (xxx.94) of siRNA was fixed in human SNCA BAC transgenic mice, and the compound of Comparative Example 7 (SNCA-019.94SPh) having the base sequences of SNCA-019, SNCA-023, and SNCA-012 was compared with the compounds of Example 5 (SNCA-023.94SPh) and Example 6 (SNCA-012.94SPh). As a result, compared with the compound of Comparative Example 7 (SNCA-019.94SPh), the compounds of Example 5 (SNCA-023.94SPh) and Example 6 (SNCA-012.94SPh) strongly suppressed target SNCA mRNA in the mouse cerebral cortex, midbrain, cerebellum, and diencephalon / striatum (shown as Striatum in the figure) four days after administration.

[0489] FIG. 11 shows the results of a comparative study in which the compound of Example 4 (SNCA-012.88SPh) or antisense oligonucleotides (compound No. 763391 and compound No. 827599, which are hASO1 and hASO2 described in JCI Insight 2021; 6(5): e135633) that are described in WO2019 / 164562 as being capable of strongly suppressing target SNCA were administered intracerebroventricularly in doses of 30 μg, 100 μg, or 300 μg to human SNCA BAC transgenic mice. As a result, 4 days after administration, the compound of Example 4 (SNCA-012.88SPh) more strongly suppressed target SNCA mRNA in the mouse cerebral cortex, midbrain, cerebellum, and diencephalon / striatum (referred to as "Striatum" in the figure) than the antisense oligonucleotides (compound No. 763391 and compound No. 827599).

[0490] 12 shows the results of a test comparing the durability of the inhibitory activity of target SNCA mRNA in human SNCA BAC transgenic mice after intracerebroventricular administration of 100 μg of the compound of Example 4 (SNCA-012.88SPh) and the antisense oligonucleotide (compound No. 763391) described in WO2019 / 164562. As a result, after 4 days of administration, the compound of Example 4 (SNCA-012.88SPh) more strongly inhibited target SNCA mRNA in the mouse diencephalon / striatum (referred to as "Striatum" in the figure) and brainstem than the antisense oligonucleotide (compound No. 763391), and the inhibition was sustained.

[0491] Figure 13 shows the results of a test comparing the durability of inhibitory activity against the target α-synuclein protein in the same test as in Figure 12. As a result, the compound of Example 4 (SNCA-012.88SPh) inhibited the target SNCA protein in the mouse diencephalon / striatum (shown as Striatum in the figure) and brainstem with a potency equal to or greater than that of the antisense oligonucleotide (Compound No. 763391), and the inhibition was sustained.

[0492] These results suggest that the SNCA siRNA of the present invention may be able to reduce the expression levels of SNCA mRNA and SNCA protein, and provide a therapeutic effect against α-synucleinopathies.

[0493] Test Example 3: Evaluation of knockdown activity of SNCA siRNA in H4 cells by reverse transfection method (2) Using the compounds of Example 4 (SNCA-012.88SPh), Example 7 (SNCA-012.88SPh1), and Example 8 (SNCA-012.88SPh2) as SNCA siRNA, a knockdown activity evaluation test was carried out in the same manner as in Test Example 1. In all samples, the siRNA was serially diluted at a common ratio of 10 to give final concentrations ranging from 100 nM to 0.01 pM. The results are shown in FIG. 14.

[0494] These results indicated that the compound of Example 7 (SNCA-012.88SPh1) strongly inhibited target SNCA mRNA in H4 cells, similar to Example 4 (SNCA-012.88SPh), which showed activity in animal studies. Furthermore, the compound of Example 8 (SNCA-012.88SPh2) inhibited target SNCA mRNA in H4 cells more strongly than Example 4 (SNCA-012.88SPh).

[0495] The siRNA of the present invention can suppress the expression of SNCA mRNA, and is useful for treating or preventing diseases that can be treated or prevented by suppressing the expression of SNCA mRNA.

[0496] SEQ ID NO: 1: Nucleotide sequence of hSNCA mRNA SEQ ID NO: 2: Target sequence of SNCA-023 SEQ ID NO: 3: Target sequence of SNCA-012 SEQ ID NOs: 4 to 28: Nucleotide sequences of the sense strand region or antisense strand region in the compounds of Examples or Comparative Examples

Claims

1. An oligonucleotide or a pharma- ceutically acceptable salt thereof, comprising the following antisense strand region (A) and sense strand region (B), which has a knockdown effect on α-synuclein mRNA; (A) an antisense strand region consisting of a target corresponding sequence substantially complementary to a target sequence consisting of 18 to 21 consecutive bases in the region between nucleotide numbers 676 and 708 of SEQ ID NO: 1, the 5' end of the target corresponding sequence being a nucleoside or phosphonic acid unit, the base of the nucleoside or phosphonic acid unit at the 5' end of the target corresponding sequence being a nucleoside base substantially complementary to the nucleoside at the 3' end of the target sequence, adenine, thymine or uracil, and an overhang structure of 5 bases or less may be added to the 5' end and / or 3' end of the target corresponding sequence; (B) a sense strand region consisting of 18 to 31 bases in length, which contains a nucleotide sequence substantially complementary to the target sequence of the antisense strand region and may further have an overhang structure of 5 bases or less added to the 5' end and / or 3' end of the nucleotide sequence.

2. The oligonucleotide or a pharma- ceutically acceptable salt thereof according to claim 1, wherein the target sequence is a nucleotide sequence consisting of 19 bases from nucleotide numbers 676 to 698 or nucleotide numbers 686 to 708 of SEQ ID NO:

1.

3. The oligonucleotide or a pharma- ceutically acceptable salt thereof according to claim 1, wherein the target sequence is a nucleotide sequence consisting of 19 bases from nucleotide numbers 678 to 696 or nucleotide numbers 688 to 706 of SEQ ID NO:

1.

4. The oligonucleotide or a pharma- ceutically acceptable salt thereof according to any one of claims 1 to 3, wherein the 5'-terminal nucleoside or phosphonic acid base in the target-corresponding sequence of the antisense strand region is adenine or uracil.

5. The oligonucleotide or a pharma- ceutical acceptable salt thereof according to any one of claims 1 to 4, wherein the target corresponding sequence of the antisense strand region is a nucleotide sequence that is completely complementary to a target sequence consisting of 19 bases from nucleotide numbers 678 to 696 or nucleotide numbers 688 to 706 of SEQ ID NO:

1.

6. The oligonucleotide or a pharma- ceutical acceptable salt thereof according to any one of claims 1 to 5, wherein the 5'-end of the target-corresponding sequence of the antisense strand region is a phosphonic acid unit, and the phosphonic acid unit is any one of the following formulas: (In the formula, the end of the dashed line represents a phosphodiester bond, which may be chemically modified, to the 5'-phosphate group of the adjacent nucleotide.) 7. The oligonucleotide or a pharma- ceutically acceptable salt thereof according to any one of claims 1 to 6, wherein at least one of the sugar and / or phosphodiester bonds constituting the oligonucleotide is modified.

8. The oligonucleotide or a pharma- ceutically acceptable salt thereof according to claim 7, wherein the sugar constituting the oligonucleotide is D-ribofuranose and the sugar modification is a modification of the hydroxyl group at the 2'-position of D-ribofuranose.

9. The oligonucleotide or a pharma- ceutically acceptable salt thereof according to claim 8, wherein the sugar modification is 2'-deoxylation, 2'-O-alkylation, 2'-O-alkoxyalkylation, 2'-halogenation, and / or 2'-O,4'-C-alkylenation of D-ribofuranose.

10. The oligonucleotide or a pharma- ceutically acceptable salt thereof according to claim 9, wherein the 2'-O-alkylation is 2'-O-methylation, the 2'-O-alkoxyalkylation is 2'-O-methoxyethylation, the 2'-halogenation is 2'-fluorolation, and the 2'-O,4'-C-alkylenation is 2'-O,4'-C-methylenation and / or 2'-O,4'-C-ethylenation.

11. The oligonucleotide or a pharma- ceutically acceptable salt thereof according to any one of claims 7 to 10, wherein the modification of the phosphodiester bond is phosphorothioate.

12. The oligonucleotide or a pharma- ceutically acceptable salt thereof according to any one of claims 7 to 11, characterized in that the D-ribofuranose in the 3'-terminal nucleoside in the sense strand region and / or antisense strand region is 3'-O-alkylated.

13. The oligonucleotide or a pharma- ceutically acceptable salt thereof according to claim 12, wherein the 3'-O-alkylation is 3'-O-methylation.

14. The oligonucleotide or a pharma- ceutical acceptable salt thereof according to any one of claims 1 to 13, wherein an overhang structure of 3 bases or less is added to the 5'-end and / or 3'-end of the sense strand region and / or the antisense strand region.

15. The oligonucleotide or a pharma- ceutically acceptable salt thereof according to claim 14, wherein the overhang structure is two bases long.

16. The oligonucleotide or a pharma- ceutically acceptable salt thereof according to claim 15, wherein the overhang structure is a nucleoside having two consecutive thymines or a nucleoside having two consecutive uracils.

17. The oligonucleotide or a pharma- ceutically acceptable salt thereof according to claim 16, wherein the overhang structure is two consecutive U (M) added to the 3'-end of the antisense strand region.

18. The oligonucleotide according to claim 1, wherein the sense strand region is an oligonucleotide represented by the following formula (I), the antisense strand region is an oligonucleotide represented by the following formula (II), and the oligonucleotide further has the following characteristics (a) to (g): Sense strand region: 5' S Ｏ５ -S ａ -S １９ -S １８ -S １７ -S １６ -S １５ -S １４ -S １３ -S １２ -S １１ -S １０ -S ９ -S ８ -S ７ -S ６ -S ５ -S ４ -S ３ -S ２ -S １ -S Ｏ３ 3'(I) Antisense strand region: 5' A Ｏ５ -A １ -A ２ -A ３ -A ４ -A ５ -A ６ -A ７ -A ８ -A ９ -A １０ -A １１ -A １２ -A １３ -A １４ -A １５ -A １６ -A １７ -A １８ -A １９ -A ａ -A Ｏ３ 3'(II) (a) S １ is a 3'-modified nucleoside, and S ２ ~S １９ represents one nucleoside, each independently being 2'-OMe RNA, 2'-F RNA or DNA; S ａ represents 0 to 3 nucleosides, each nucleoside independently represents 2'-OMe RNA, 2'-F RNA, or DNA; S Ｏ３ and S Ｏ５ each independently represents 0 to 3 nucleosides, each of which independently represents 2'-OMe RNA, 2'-F RNA, DNA, or a 2'-O,4'-C-bridged modified nucleoside. The bond between each nucleoside represents a phosphodiester bond which may be chemically modified; (b) A １１ , A １２ , A １３ , A １４ and A １５ represents one nucleoside, at least one of which is DNA, RNA, 2'-O,4'-C-bridged modified nucleoside or 2'-MOE RNA, and the rest are 2'-OMe RNA or 2'-F RNA; A １ represents one nucleoside or phosphonic acid unit, A １ is a nucleoside, it represents 2'-OMe RNA, 2'-F RNA or DNA, ２ ~A １０ and A １６ ~A １９ represents one nucleoside, each independently represents 2'-OMe RNA, 2'-F RNA or DNA; A ａ represents 0 to 3 nucleosides, each nucleoside independently represents 2'-OMe RNA, 2'-F RNA, or DNA; A Ｏ３ and A Ｏ５ each independently represents 0 to 3 nucleosides, each of which independently represents 2'-OMe RNA, 2'-F RNA, DNA, or a 2'-O,4'-C-bridged modified nucleoside. The bond between each nucleoside represents a phosphodiester bond which may be chemically modified; (c) A ２ ~A ａ The nucleotide sequence between A and B consists of a nucleotide sequence substantially complementary to the target sequence, １ is a nucleoside or phosphonate unit, the base of the nucleoside or phosphonate unit being a nucleoside base substantially complementary to the corresponding nucleoside of the target sequence, adenine, thymine or uracil; A Ｏ３ and A Ｏ５ (d) S １ ~S ａ and the nucleotide sequence between A １ ~A ａ The nucleotide sequences between are substantially complementary to each other and form a double-stranded structure; (e) S Ｏ５ and A Ｏ３ If both exist, S Ｏ５ and A Ｏ３ are nucleotide sequences that are not complementary to each other; (f) S Ｏ３ and A Ｏ５ If both exist, S Ｏ３ and A Ｏ５ are nucleotide sequences that are not complementary to each other; and (g) the 5'-position of the 5'-terminal nucleoside and / or the 3'-position of the 3'-terminal nucleoside, or the 2'-position of the 3'-terminal nucleoside when the 3'-terminal nucleoside is a 3'-modified nucleoside, in the sense strand region and / or the antisense strand region may be chemically modified.

19. In formula (II), S １ The oligonucleotide or a pharma- ceutically acceptable salt thereof according to claim 18, wherein is 3'-OMe RNA.

20. The oligonucleotide or a pharma- ceutically acceptable salt thereof according to claim 18 or 19, wherein the 2'-O,4'-C-bridged modified nucleoside is LNA or ENA.

21. In formula (II), A １１ , A １２ , A １３ , A １４ and A １５ The oligonucleotide or its pharma- ceutical acceptable salt according to any one of claims 18 to 20, wherein two or more of the above may be the same or different, and are DNA, RNA, 2'-O,4'-C-bridged modified nucleoside, or 2'-MOE RNA.

22. In formula (II), A １４ The oligonucleotide or a pharma- ceutically acceptable salt thereof according to claim 21, wherein is RNA or a 2'-O,4'-C-bridged modified nucleoside.

23. In formula (II), A １３ is a 2'-O,4'-C-bridged modified nucleoside or a 2'-OMe RNA; １４ The oligonucleotide or a pharma- ceutically acceptable salt thereof according to claim 22, wherein is RNA.

24. In formula (II), A １１ -A １２ -A １３ -A １４ -A １５ is any of the following; A １１ (2'-OMe RNA)-A １２ (2'-OMe RNA)-A １３ (ENA)-A １４ (RNA)-A １５ (2'-OMe RNA), A １１ (2'-F RNA)-A １２ (2'-OMe RNA)-A １３ (ENA)-A １４ (RNA)-A １５ (2'-OMe RNA), A １１ (2'-OMe RNA)-A １２ (2'-F RNA)-A １３ (ENA)-A １４ (RNA)-A １５ (2'-OMe RNA), A １１ (2'-OMe RNA)-A １２ (2'-OMe RNA)-A １３ (LNA)-A １４ (RNA)-A １５ (2'-OMe RNA), A １１ (2'-F RNA)-A １２ (2'-OMe RNA)-A １３ (LNA)-A １４ (RNA)-A １５ (2'-OMe RNA), A １１ (2'-OMe RNA)-A １２ (2'-F RNA)-A １３ (LNA)-A １４ (RNA)-A １５ (2'-OMe RNA), A １１ (2'-F RNA)-A １２ (2'-OMe RNA)-A １３ (2'-OMe RNA)-A １４ (RNA)-A １５ (2'-OMe RNA), or, A １１ (2'-OMe RNA)-A １２ (2'-F RNA)-A １３ (2'-OMe RNA)-A １４ (RNA)-A １５ 24. The oligonucleotide according to claim 23, which is: (2'-OMe RNA), or a pharma- ceutically acceptable salt thereof.

25. In formula (II), A １２ is DNA, and A １４ The oligonucleotide or a pharma- ceutically acceptable salt thereof according to claim 22, wherein is a 2'-O,4'-C-bridged modified nucleoside.

26. In formula (II), A １１ -A １２ -A １３ -A １４ -A １５ is any of the following: A １１ (2'-F RNA)-A １２ (DNA)-A １３ (2'-OMe RNA)-A １４ (ENA)-A １５ (2'-OMe RNA), A １１ (2'-OMe RNA)-A １２ (DNA)-A １３ (2'-OMe RNA)-A １４ (ENA)-A １５ (2'-OMe RNA), A １１ (2'-F RNA)-A １２ (DNA)-A １３ (2'-OMe RNA)-A １４ (LNA)-A １５ (2'-OMe RNA), or １１ (2'-OMe RNA)-A １２ (DNA)-A １３ (2'-OMe RNA)-A １４ (LNA)-A １５ 26. The oligonucleotide according to claim 25, which is: (2'-OMe RNA), or a pharma- ceutically acceptable salt thereof.

27. In formula (II), A ２ , A ６ and A １６ The oligonucleotide or a pharma- ceutically acceptable salt thereof according to any one of claims 18 to 26, wherein is 2'-F RNA.

28. In the formula (II), further, A ８ , A ９ and A １０ one or two nucleosides selected from the group consisting of: ３ ~A ５ , A ７ , A １７ ~A １９ and A ａ The oligonucleotide or a pharma- ceutically acceptable salt thereof according to claim 27, wherein the nucleoside is 2'-OMe RNA.

29. In formula (II), A ２ , A ６ , A ８ , A １０ and A １６ is 2'-F RNA, and A ３ ~A ５ , A ７ , A ９ , A １７ ~A １９ and A ａ The oligonucleotide or a pharma- ceutically acceptable salt thereof according to claim 28, wherein the nucleoside is 2'-OMe RNA.

30. In the formula (II), A １ The oligonucleotide or a pharma- ceutically acceptable salt thereof according to claim 29, wherein is 2'-OMe RNA.

31. In the formula (II), A １ The oligonucleotide according to claim 29, or a pharma- ceutically acceptable salt thereof, wherein: (In the formula, the end of the dashed line represents a phosphodiester bond, which may be chemically modified, to the 5'-phosphate group of the adjacent nucleotide.) 32. In formula (I), S １１ , S １２ , S １３ and S １５ The oligonucleotide or a pharma- ceutically acceptable salt thereof according to any one of claims 18 to 31, wherein is 2'-F RNA.

33. In formula (I), S ２ ~S １０ , S １４ , S １６ ~S １９ and S ａ The oligonucleotide or a pharma- ceutically acceptable salt thereof according to claim 32, wherein is 2'-OMe RNA.

34. An oligonucleotide or a pharma- ceutically acceptable salt thereof according to any one of claims 18 to 33, characterized in that, when RNA is employed in formula (II), the bond between the RNA and the adjacent nucleoside on its 3' side is a phosphorothioate bond.

35. The oligonucleotide or a pharma- ceutically acceptable salt thereof according to any one of claims 18 to 34, characterized in that in 2 to 5 nucleotides from the 5' and 3' ends of the oligonucleotide, each internucleoside bond is a phosphorothioate bond.

36. S Ｏ３ , S Ｏ５ , A Ｏ５ and A Ｏ３ The oligonucleotide or a pharma- ceutically acceptable salt thereof according to any one of claims 18 to 35, wherein the number of nucleosides is 0 to 2.

37. S Ｏ３ The oligonucleotide or a pharma- ceutically acceptable salt thereof according to claim 36, wherein the number of nucleosides is 0.

38. S Ｏ３ , S Ｏ５ and A Ｏ５ The number of nucleosides in Ｏ３ The oligonucleotide or a pharma- ceutically acceptable salt thereof according to claim 37, wherein the number of nucleosides is 2.

39. The oligonucleotide or a pharma- ceutical acceptable salt thereof according to any one of claims 1 to 38, characterized in that the antisense strand region and the sense strand region each form a double-stranded oligonucleotide as an independent oligonucleotide.

40. The oligonucleotide or a pharma- ceutically acceptable salt thereof according to any one of claims 1 to 38, characterized in that the nucleoside at the 5'-terminus of the antisense strand region and the nucleoside at the 3'-terminus of the sense strand region are linked by a linker structure.

41. The linker structure is represented by the following formula: The oligonucleotide according to claim 40 or a pharma- ceutically acceptable salt thereof, having a structure represented by the following formula: [wherein the end of the dashed line represents that the oxygen atom bonded to the phenyl group is bonded to the 5'-phosphate group of a nucleotide other than the phosphonate unit at the 5'-end of the adjacent antisense strand region by a phosphodiester bond or a phosphorothioate bond, and the methylene group at the other end represents that the 3'-phosphate group (the 2'-phosphate group in the case of a nucleotide modified at the 3'-position) of the 3'-end of the adjacent sense strand region by a phosphodiester bond or a phosphorothioate bond.] 42. The oligonucleotide or a pharma- ceutically acceptable salt thereof according to any one of claims 1 to 41, characterized in that the 5'-position of the 5'-terminal nucleoside of the oligonucleotide and / or the 3'-position of the 3'-terminal nucleoside, or the 2'-position thereof if the 3'-terminal nucleoside is a 3'-modified nucleoside, is chemically modified with a fatty acid unit.

43. The oligonucleotide or a pharma- ceutically acceptable salt thereof according to claim 42, wherein the fatty acid unit is any unit represented by the following formula: [In the formula, the end of the dashed line represents a phosphodiester bond with the 5'-terminal phosphate group and / or the 3'-terminal phosphate group (the 2'-position phosphate group in a 3'-modified nucleotide) of an adjacent nucleotide.] 44. The oligonucleotide or a pharma- ceutically acceptable salt thereof according to claim 43, wherein the fatty acid unit is a unit represented by the following formula: [In the formula, the end of the dashed line represents a phosphodiester bond with the 5'-terminal phosphate group and / or the 3'-terminal phosphate group (the 2'-position phosphate group in a nucleotide modified at the 3'-position) of an adjacent nucleotide.] 45. The oligonucleotide according to claim 1 or a pharma- ceutically acceptable salt thereof, wherein the sense strand region excluding the overhang structure and the antisense strand region excluding the overhang structure are any of the following formulas (I-1) to (I-6), the sense strand region and the antisense strand region each form a double-stranded oligonucleotide as an independent oligonucleotide, the sense strand region and the antisense strand region each independently may contain an overhang structure, and the nucleotide at the 3' end and / or the 5' end of the sense strand region and / or the antisense strand region may be chemically modified. (Formula) (I-1) Sense strand region 5' U(M)^C(M)^U(M)G(M)C(F)U(M)G(F)A(F)C(F)A(M)G(M)A(M)U(M)G(M)U(M)U(M)C(M)^C(M)^A(3M) 3 Antisense strand region 5' VPu^G(F)^G(M)A(F)A(M)C(F)A(M)U(M)C(F)U(M)G(M)T(D)C(M)A(E)G(M)C(F)A(M)G(M)^A(M) 3' (I-2) Sense strand region 5' G(M)^A(M)^U(M)G(M)U(F)U(M)C(F)C(F)A(F)U(M)C(M)C(M)U(M)G(M)U(M)A(M)C(M)^A(M)^A(3M) 3' Antisense strand region 5' VPu^U(F)^G(M)U(F)A(M)C(F)A(M)G(M)G(F)A(M)U(M)G(D)G(M)A(E)A(M)C(F)A(M)U(M)^C(M) 3' (I-3) Sense strand region 5' U(M)^C(M)^U(M)G(M)C(F)U(M)G(F)A(F)C(F)A(M)G(M)A(M)U(M)G(M)U(M)U(M)C(M)^C(M)^A(3M) 3' Antisense strand region 5' VPu^G(F)^G(M)A(M)A(M)C(F)A(M)U(M)C(F)U(M)G(F)U(M)C(M)A(R)^G(M)C(F)A(M)G(M)^A(M) 3' (I-4) Sense strand region 5' G(M)^A(M)^U(M)G(M)U(F)U(M)C(F)C(F)A(F)U(M)C(M)C(M)U(M)G(M)U(M)A(M)C(M)^A(M)^A(3M) 3'Antisense strand region 5' VPu^U(F)^G(M)U(M)A(M)C(F)A(M)G(M)G(F)A(M)U(F)G(M)G(M)A(R)^A(M)C(F)A(M)U(M)^C(M) 3' (I-5) Sense strand region 5' G(M)^A(M)^U(M)G(M)U(F)U(M)C(F)C(F)A(F)U(M)C(M)C(M)U(M)G(M)U(M)A(M)C(M)^A(M)^A(3M) 3' Antisense strand region 5' EPu^U(F)^G(M)U(F)A(M)C(F)A(M)G(M)G(F)A(M)U(M)G(D)G(M)A(E)A(M)C(F)A(M)U(M)^C(M) 3' (I-6) Sense strand region 5' G(M)^A(M)^U(M)G(M)U(F)U(M)C(F)C(F)A(F)U(M)C(M)C(M)U(M)G(M)U(M)A(M)C(M)^A(M)^A(3M) 3' Antisense strand region 5' VPuam^U(F)^G(M)U(F)A(M)C(F)A(M)G(M)G(F)A(M)U(M)G(D)G(M)A(E)A(M)C(F)A(M)U(M)^C(M) 3' [wherein, for the bases of the nucleic acid, A represents adenine, U represents uracil, G represents guanine, and C represents cytosine (however, when C is ENA, it represents 2'-O,4'-C-ethylene-bridged-5-methylcytidine), for the nucleic acid, (R) represents RNA, (D) represents DNA, (M) represents 2'-OMe RNA, (3M) represents 3'-OMe RNA, (F) represents 2'-F RNA, and (E) represents ENA, and for the phosphonic acid unit, VPu, EPu, and VPuam are represented by the following formulae, respectively. (In the formula, the end of the dashed line indicates that the 5'-phosphate group of the 5'-terminal nucleotide of the adjacent sense strand region is bonded by a phosphorothioate bond (-P(=S)(OH)-). In addition, in the formula, "^" indicates that the bond between nucleosides is a phosphorothioate bond (-P(=S)(OH)-), and unless otherwise specified, indicates that the bond between nucleosides is a phosphodiester bond (-P(=O)(OH)-). However, even if not otherwise specified, two internucleoside bonds in the three nucleosides from the 5'-terminus and 3'-terminus of the sense strand region, and two internucleoside bonds in the three nucleosides from the 5'-terminus and three internucleoside bonds in the four nucleosides from the 3'-terminus of the antisense strand region are phosphorothioate bonds. ] 46. ​​The oligonucleotide or a pharma- ceutically acceptable salt thereof according to claim 45, wherein an overhang structure of 3 bases or less is added to the 5'-end and / or 3'-end of the sense strand region and / or the antisense strand region.

47. The oligonucleotide or a pharma- ceutically acceptable salt thereof according to claim 46, wherein the overhang structure is two bases long.

48. The oligonucleotide or a pharma- ceutically acceptable salt thereof according to claim 47, wherein the overhang structure is a nucleoside containing two consecutive thymines or a nucleoside containing two consecutive uracils.

49. The oligonucleotide or a pharma- ceutically acceptable salt thereof according to claim 48, wherein the overhang structure is two consecutive U (M) added to the 3' end of the antisense strand region.

50. The oligonucleotide or a pharma- ceutical acceptable salt thereof according to any one of claims 45 to 49, characterized in that the 5'-end of the sense strand region is chemically modified with a fatty acid unit represented by any unit represented by the following formulas: (In the formula, the end of the dashed line represents a phosphodiester bond, which may be chemically modified, to the 5'-phosphate group of the adjacent nucleotide.) 51. The oligonucleotide or a pharma- ceutical acceptable salt thereof according to claim 1, wherein the sense strand region and the antisense strand region are represented by any one of the following formulas (I-7) to (I-12), and the sense strand region and the antisense strand region each serve as independent oligonucleotides to form a double-stranded oligonucleotide. (Formula) (I-7) Sense strand region 5' SPhU(M)^C(M)^U(M)G(M)C(F)U(M)G(F)A(F)C(F)A(M)G(M)A(M)U(M)G(M)U(M)U(M)C(M)^C(M)^A(3M) 3 Antisense strand region 5' VPu^G(F)^G(M)A(F)A(M)C(F)A(M)U(M)C(F)U(M)G(M)T(D)C(M)A(E)G(M)C(F)A(M)G(M)^A(M)^U(M)^U(M) 3' (I-8) Sense strand region 5' SPhG(M)^A(M)^U(M)G(M)U(F)U(M)C(F)C(F)A(F)U(M)C(M)C(M)U(M)G(M)U(M)A(M)C(M)^A(M)^A(3M) 3' Antisense strand region 5' VPu^U(F)^G(M)U(F)A(M)C(F)A(M)G(M)G(F)A(M)U(M)G(D)G(M)A(E)A(M)C(F)A(M)U(M)^C(M)^U(M)^U(M) 3' (I-9) Sense strand region 5' SPhU(M)^C(M)^U(M)G(M)C(F)U(M)G(F)A(F)C(F)A(M)G(M)A(M)U(M)G(M)U(M)U(M)C(M)^C(M)^A(3M) 3' Antisense strand region 5' VPu^G(F)^G(M)A(M)A(M)C(F)A(M)U(M)C(F)U(M)G(F)U(M)C(M)A(R)^G(M)C(F)A(M)G(M)^A(M)^U(M)^U(M) 3' (I-10) Sense strand region 5' SPhG(M)^A(M)^U(M)G(M)U(F)U(M)C(F)C(F)A(F)U(M)C(M)C(M)U(M)G(M)U(M)A(M)C(M)^A(M)^A(3M) 3' Antisense strand region 5' VPu^U(F)^G(M)U(M)A(M)C(F)A(M)G(M)G(F)A(M)U(F)G(M)G(M)A(R)^A(M)C(F)A(M)U(M)^C(M)^U(M)^U(M) 3' (I-11)Sense strand region 5' SPhG(M)^A(M)^U(M)G(M)U(F)U(M)C(F)C(F)A(F)U(M)C(M)C(M)U(M)G(M)U(M)A(M)C(M)^A(M)^A(3M) 3' Antisense strand region 5' EPu^G(F)^G(M)A(F)A(M)C(F)A(M)U(M)C(F)U(M)G(M)T(D)C(M)A(E)G(M)C(F)A(M)G(M)^A(M)^U(M)^U(M) 3' (I-12) Sense strand region 5' SPhG(M)^A(M)^U(M)G(M)U(F)U(M)C(F)C(F)A(F)U(M)C(M)C(M)U(M)G(M)U(M)A(M)C(M)^A(M)^A(3M) 3' Antisense strand region 5' VPuam^G(F)^G(M)A(F)A(M)C(F)A(M)U(M)C(F)U(M)G(M)T(D)C(M)A(E)G(M)C(F)A(M)G(M)^A(M)^U(M)^U(M)) 3' [Wherein, SPh is the following formula (wherein the end of the dashed line indicates a phosphodiester bond with the 5'-phosphate group of the 5'-terminal nucleotide of the adjacent sense strand region), with respect to the nucleic acid base, A represents adenine, U represents uracil, G represents guanine, and C represents cytosine (however, when C is ENA, it represents 2'-O,4'-C-ethylene-bridged-5-methylcytidine), with respect to the nucleic acid, (R) represents RNA, (D) represents DNA, (M) represents 2'-OMe RNA, (3M) represents 3'-OMe RNA, (F) represents 2'-F RNA, and (E) represents ENA, and with respect to the phosphonic acid unit, VPu, EPu, and VPuam are represented by the following formulae, respectively: (In the formula, the end of the dashed line indicates a phosphorothioate bond (-P(=S)(OH)-) with the 5'-phosphate group of the 5'-terminal nucleotide of the adjacent sense strand region.) In addition, in the formula, "^" indicates that the bond between nucleosides is a phosphorothioate bond (-P(=S)(OH)-), and indicates that the bond between nucleosides is a phosphodiester bond (-P(=O)(OH)-) unless otherwise specified. The 3' position of the 3'-terminal nucleotide of the sense strand region and the 3' position of the 3'-terminal nucleotide of the antisense strand region are both hydroxyl groups.) 52. A pharmaceutical composition comprising as an active ingredient the oligonucleotide or a pharma- ceutically acceptable salt thereof according to any one of claims 1 to 51.

53. The pharmaceutical composition according to claim 52, for treating or preventing a disease that can be treated or prevented by inhibiting the expression of α-synuclein.

54. The pharmaceutical composition according to claim 53 for the prevention or treatment of an alpha-synucleinopathy.

55. The pharmaceutical composition of claim 54, wherein the alpha-synucleinopathy is Parkinson's disease, Parkinson's dementia, dementia with Lewy bodies, multiple system atrophy, or pure autonomic failure.

56. A method for treating or preventing an α-synucleinopathy, comprising administering to a subject an effective amount of the oligonucleotide according to any one of claims 1 to 51 or a pharma- ceutically acceptable salt thereof.

57. The method of claim 56, wherein the alpha-synucleinopathy is Parkinson's disease, Parkinson's dementia, dementia with Lewy bodies, multiple system atrophy, or pure autonomic failure.

58. An oligonucleotide or a pharma- ceutically acceptable salt thereof according to any one of claims 1 to 51 for use in the treatment or prevention of an α-synucleinopathic disease.

59. Use of an oligonucleotide according to any one of claims 1 to 51 or a pharma- ceutically acceptable salt thereof in the manufacture of a pharmaceutical composition according to any one of claims 52 to 55.

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