Sirna for suppressing expression of phospholamban

Abstract

The present invention provides, for example, a new siRNA that has a gene expression suppression effect and / or an RNA interference effect on an mRNA encoding PLN.　Provided is: an oligonucleotide having a knockdown effect on a PLN mRNA, the oligonucleotide comprising an antisense strand region including a target corresponding sequence complementary to a target sequence in an mRNA encoding PLN, and a sense strand region including a nucleotide sequence complementary to the target corresponding sequence of the antisense strand region; or a pharmaceutically acceptable salt of the oligonucleotide.

Description

siRNA that inhibits the expression of phospholamban

[0001] The present invention relates to siRNAs having RNA interference activity and / or gene expression-inhibiting activity against mRNA encoding phospholamban (PLN), uses of the siRNAs, methods for inhibiting PLN gene expression using the siRNAs, and pharmaceuticals containing the siRNAs.

[0002] siRNA against the mRNA encoding the PLN protein suppresses the expression of PLN mRNA, thereby inhibiting Ca, which plays a central role in cardiac contraction and expansion. ２＋ In a failing heart, myocardial Ca ２＋ Since abnormalities in the regulation of PLN are known, it is expected that activation of SERCA2A can suppress the onset and progression of heart failure. Therefore, studies are being conducted to target the suppression of PLN production and to use siRNA to suppress PLN mRNA expression.

[0003] In cardiac myocytes with heart failure, contractile and diastolic forces are weakened, and Ca ２＋ Regulation i.e. Ca ２＋ It has been revealed that Ca handling plays an important role in contractile and diastolic responses. ２＋ Handling is controlled by various proteins within the cell. When the cell membrane is excited, L-type Ca ２＋ The channel opens and extracellular Ca ２＋ When Ca enters the cell, it is released from the sarcoplasmic reticulum (SR) into the cytoplasm via the ryanodine receptor. ２＋ is released. ２＋ When the concentration increases, contraction is induced, and when relaxation occurs, cytoplasmic Ca ２＋ Most of this Ca is re-incorporated into the SR. ２＋ The uptake of SERCA2A, an ATPase localized in the SR membrane, is important. It is known that the expression level of SERCA2A is closely related to contractile and diastolic functions (Non-Patent Document 1), and it has been reported that SERCA2A expression is reduced in the myocardium of heart failure patients and heart failure pathological model mice (Non-Patent Document 2).

[0004] SERCA2A activity is negatively regulated by PLN, an endogenous inhibitor of SERCA2A. Under physiological conditions, PLN is phosphorylated by PKA, which is activated by β-adrenergic stimulation. Phosphorylation of PLN significantly alters the protein-protein interaction with SERCA2A, reducing its inhibitory effect on SERCA2A. In other words, β-adrenergic stimulation activates SERCA2A, enhancing contractility and diastolic function. It is believed that in cardiac myocytes with heart failure, a decrease in cAMP levels reduces PKA activity and PLN phosphorylation levels, resulting in the suppression of SERCA2A (Non-Patent Document 3). It has been revealed that heart failure attenuates contractility and diastolic function through two mechanisms: a decrease in SERCA2A expression and a decrease in PLN phosphorylation levels.

[0005] Since PLN-deficient mice exhibit cardiac function equivalent to that of β-adrenergic stimulation under unstimulated conditions, it has been revealed that PLN is responsible for most of the cardiac enhancement effect during β-adrenergic stimulation (Non-Patent Document 4). Meanwhile, it is known that PLN-overexpressing mice exhibit symptoms of heart failure, and that PLN gene mutations cause dilated cardiomyopathy (Non-Patent Document 5). Based on these findings, it has been reported that PLN deficiency or inhibition improves symptoms in various animal models of heart failure. For example, PLN deficiency or inhibition has been reported to improve cardiac function and suppress arrhythmias in myocardial infarction models, dilated cardiomyopathy models, and arrhythmia induction models (Non-Patent Documents 6, 7, and 8). Therefore, activation of SERCA2A through PLN inhibition or reduced expression is expected as a treatment for heart failure.

[0006] 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 (e.g., Non-Patent Document 9).

[0007] 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.

[0008] 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 10).

[0009] 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 1, 2, and 3.

[0010] 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 11).

[0011] 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 12, Non-Patent Document 13, Non-Patent Document 14, Non-Patent Document 15, 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 16).

[0012] 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 17).

[0013] 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 3'-terminal overhanging region of one or both of the sense and antisense strands of siRNA, RNAi activity is reduced (e.g., Non-Patent Document 18).

[0014] 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 19).

[0015] 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 4, 5, 6, and 7). 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, 8, and 9).

[0016] A group of siRNA base sequences has been reported as siRNA for human PLN, consisting of 19 base pairs formed by sense and antisense strands, but these have only been extracted by in silico analysis, and the actual strength of RNAi activity has not been clarified (Patent Document 10). Furthermore, although it is necessary to deliver siRNA to the heart for the treatment of heart failure, no specific method for this has been described.

[0017] US Patent No. 9,399,775 US Patent No. 9,796,974 US Patent No. 10,612,024 International Publication No. 2012 / 058210 Pamphlet International Publication No. 2013 / 074974 Pamphlet International Publication No. 2016 / 028649 Pamphlet International Publication No. 2018 / 185241 Pamphlet International Publication No. 2004 / 045543 Pamphlet

[0018] Circulation Research. 1998;83:1205-1214Circ Res. 1994;75:434-442. J Biol Chem. 1991 Sep 15;266(26):17486-93 Circ Res. 1996 May; 78(5):839-47 Sci Rep. 2020 Jun 17;10(1):9819J Clin Invest. 2004 Mar; 113(5):727-36Cell. 1999 Oct 29;99(3):313-22J Physiol. 2016 Jun 1; 594(11):3005-30 Nature, 2001, Vol. 411, pp. 494-498 Antisense Nucleic Acid Drug Development, 2000, Vol. 10, pp. 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, p. 7967-7975 Nucleic Acids Research, 2003, Vol. 31, pp. 2705-2716 Journal of Medicinal Chemistry, 2005, Vol. 48, pp. 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 PLN siRNA that can prevent, ameliorate, and / or treat heart failure, as well as for PLN siRNA that has low toxicity to cells, is stable in blood, and has excellent drug delivery properties to the heart.

[0020] An object of the present invention is to provide a novel siRNA that exhibits RNA interference activity and / or gene expression suppression activity (these activities are also referred to as knockdown activity) against mRNA encoding PLN in vivo. Another object of the present invention is to provide a novel PLN siRNA with improved cytotoxicity, stability, and / or drug deliverability. 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 suppressing PLN mRNA expression in vivo.

[0021] As a result of intensive research, the present inventors have found that PLN siRNA, which targets a base sequence at a specific position in PLN mRNA, exhibits RNA interference and / or gene expression suppression in vivo. In particular, the present inventors have found that siRNA containing oligonucleotides that appropriately combine DNA, RNA, 2'-O-methyl RNA, 2'-F RNA, LNA, ENA, and 3'-O-methyl RNA exhibits RNA interference and / or gene expression suppression and is highly active in vivo. Furthermore, the present inventors have found that adding a fatty acid unit for transport derived from stearic acid or the like to PLN siRNA improves drug delivery to the heart and enables PLN mRNA suppression in the heart, thereby completing the present invention.

[0022] That is, the present invention provides the following: [1] An oligonucleotide or a pharmaceutically acceptable salt thereof, which has a knockdown effect on phospholamban 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 273 and 295 of SEQ ID NO: 1, wherein the 5'-end of the target corresponding sequence is 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 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 nucleotide sequence consisting of 19 bases from nucleotide numbers 275 to 293 of SEQ ID NO: 1. [3] The oligonucleotide or a pharmaceutically acceptable salt thereof according to [1] or [2], 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. [4] The oligonucleotide or pharmaceutically acceptable salt thereof according to any one of [1] to [3], wherein the target-corresponding sequence of the antisense strand region has a nucleotide sequence that is completely complementary to 18 bases from nucleotide numbers 275 to 292 of SEQ ID NO: 1, with respect to a target sequence consisting of 19 bases from nucleotide numbers 275 to 293 of SEQ ID NO: 1, and the 5'-end of the target-corresponding sequence is a nucleoside having uracil or a phosphonate unit having uracil.[5] The oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of [1] to [4], 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] (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.) [6] The oligonucleotide according to any one of [1] to [5], wherein at least one of the sugars and / or phosphodiester bonds constituting the oligonucleotide is modified, or a pharmaceutically acceptable salt thereof. [7] The oligonucleotide according to [6], wherein the sugar constituting the oligonucleotide is D-ribofuranose, and the sugar modification is modification of the hydroxyl group at position 2' of D-ribofuranose, or a pharmaceutically acceptable salt thereof. [8] The oligonucleotide according to [7], 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. [9] The oligonucleotide or pharmaceutically acceptable salt thereof according to [8], 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.

[10] The oligonucleotide or pharmaceutically acceptable salt thereof according to any one of [6] to [9], wherein the phosphodiester bond modification is phosphorothioate.

[11] The oligonucleotide or pharmaceutically acceptable salt thereof according to any one of [6] to

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

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

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

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

[12] , 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.

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

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

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

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

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

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

[17] 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 may be chemically modified.

[18] In formula (II), S １

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

[17] or

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

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

[17] or

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

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

[17] to

[19] , 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

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

[20] , 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

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

[21] , 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.

[24] In formula (II), A １２ is DNA, and A １４

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

[21] , 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.

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

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

[17] to

[25] , 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

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

[26] , 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

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

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

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

[28] , wherein A is 2'-OMe RNA. １

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

[28] , wherein:

[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.)

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

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

[17] to

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

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

[17] to

[32] , 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.

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

[17] to

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

[35] S Ｏ３ , S Ｏ５ , A Ｏ５ and A Ｏ３

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

[17] to

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

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

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

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

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

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

[37] , 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.

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

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

[0025] [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.]

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

[40] , 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.

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

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

[0026] [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.]

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

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

[0027] [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.]

[44] 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-8) and (I-17), 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' G(M)^C(M)^U(M)A(M)C(F)A(M)G(F)A(F)A(F)U(M)C(M)U(M)A(M)U(M)U(M)U(M)A(M)^U(M)^A(3M) 3' Antisense strand region 5' U(M)^A(F)^U(M)A(M)A(M)A(F)U(M)A(M)G(F)A(M)U(F)U(M)C(E)U(R)^G(M)U(F)A(M)G(M)^C(M) 3' (I-2) Sense strand region 5' G(M)^C(M)^U(M)A(M)C(F)A(M)G(F)A(F)A(F)U(M)C(M)U(M)A(M)U(M)U(M)U(M)A(M)^U(M)^A(3M) 3' Antisense strand region 5' U(M)^A(F)^U(M)A(M)A(M)A(F)U(M)A(M)G(F)A(M)U(M)U(F)C(E)U(R)^G(M)U(F)A(M)G(M)^C(M) 3' (I-3) Sense strand region 5' G(M)^C(M)^U(M)A(M)C(F)A(M)G(F)A(F)A(F)U(M)C(M)U(M)A(M)U(M)U(M)U(M)A(M)^U(M)^A(3M) 3' Antisense strand region 5' VPu^A(F)^U(M)A(M)A(M)A(F)U(M)A(M)G(F)A(M)U(M)U(F)C(E)U(R)^G(M)U(F)A(M)G(M)^C(M) 3' (I-4)Sense strand region: 5’ G(M)^C(M)^U(M)A(M)C(F)A(M)G(F)A(F)A(F)U(M)C(M)U(M)A(M)U(M)U(M)U(M)A(M)^U(M)^A(3M) 3’ Antisense strand region: 5’ EPu^A(F)^U(M)A(M)A(M)A(F)U(M)A(M)G(F)A(M)U(M)U(F)C(E)U(R)^G(M)U(F)A(M)G(M)^C(M) 3’ (I-5) Sense strand region: 5’ G(M)^C(M)^U(M)A(M)C(F)A(M)G(F)A(F)A(F)U(M)C(M)U(M)A(M)U(M)U(M)U(M)A(M)^U(M)^A(3M) 3’ Antisense strand region: 5’ VPuam^A(F)^U(M)A(M)A(M)A(F)U(M)A(M)G(F)A(M)U(M)U(F)C(E)U(R)^G(M)U(F)A(M)G(M)^C(M) 3’ (I-6) Sense strand region: 5’ G(M)^C(M)^U(M)A(M)C(F)A(M)G(F)A(F)A(F)U(M)C(M)U(M)A(M)U(M)U(M)U(M)A(M)^U(M)^A(3M) 3’ Antisense strand region: 5’ VPu^A(F)^U(M)A(M)A(M)A(F)U(M)A(M)G(F)A(M)U(M)U(F)C(E)U(R)^G(M)U(F)A(M)G(M)^C(M) 3’ (I-7) Sense strand region: 5’ G(M)^C(M)^U(M)A(M)C(F)A(M)G(F)A(F)A(F)U(M)C(M)U(M)A(M)U(M)U(M)U(M)A(M)^U(M)^A(3M) 3’ Antisense strand region: 5’ EPu^A(F)^U(M)A(M)A(M)A(F)U(M)A(M)G(F)A(M)U(M)U(F)C(E)U(R)^G(M)U(F)A(M)G(M)^C(M) 3’ (I-8) Sense strand region: 5’ G(M)^C(M)^U(M)A(M)C(F)A(M)G(F)A(F)A(F)U(M)C(M)U(M)A(M)U(M)U(M)U(M)A(M)^U(M)^A(3M) 3’ Antisense strand region: 5’ VPuam^A(F)^U(M)A(M)A(M)A(F)U(M)A(M)G(F)A(M)U(M)U(F)C(E)U(R)^G(M)U(F)A(M)G(M)^C(M) 3’ (I-17)Sense strand region 5' G(M)^C(M)^U(M)A(M)C(F)A(M)G(F)A(F)A(F)U(M)C(M)U(M)A(M)U(M)U(M)U(M)A(M)^U(M)^A(3M) 3' Antisense strand region 5' EPuam^A(F)^U(M)A(M)A(M)A(F)U(M)A(M)G(F)A(M)U(M)U(F)C(E)U(R)^G(M)U(F)A(M)G(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, (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, VPuam, and EPuam represent the following formulae, respectively.

[0028] (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.

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

[44] , 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 antisense strand region.

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

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

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

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

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

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

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

[44] to

[48] , 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:

[0029] (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.)

[50] The oligonucleotide or a pharmaceutically acceptable salt thereof according to [1], wherein the sense strand region and the antisense strand region are any of the following formulae (I-9) to (I-16) and (I-18), and the sense strand region and the antisense strand region each serve as independent oligonucleotides to form a double-stranded oligonucleotide. (Formula) (I-9) Sense strand region 5' G(M)^C(M)^U(M)A(M)C(F)A(M)G(F)A(F)A(F)U(M)C(M)U(M)A(M)U(M)U(M)U(M)A(M)^U(M)^A(3M) 3' Antisense strand region 5' U(M)^A(F)^U(M)A(M)A(M)A(F)U(M)A(M)G(F)A(M)U(F)U(M)C(E)U(R)^G(M)U(F)A(M)G(M)^C(M)^U(M)^U(M) 3' (I-10) Sense strand region 5' G(M)^C(M)^U(M)A(M)C(F)A(M)G(F)A(F)A(F)U(M)C(M)U(M)A(M)U(M)U(M)U(M)A(M)^U(M)^A(3M) 3' Antisense strand region 5' U(M)^A(F)^U(M)A(M)A(M)A(F)U(M)A(M)G(F)A(M)U(M)U(F)C(E)U(R)^G(M)U(F)A(M)G(M)^C(M)^U(M)^U(M) 3' (I-11) Sense strand region 5' SG(M)^C(M)^U(M)A(M)C(F)A(M)G(F)A(F)A(F)U(M)C(M)U(M)A(M)U(M)U(M)U(M)A(M)^U(M)^A(3M) 3' Antisense strand region 5' VPu^A(F)^U(M)A(M)A(M)A(F)U(M)A(M)G(F)A(M)U(M)U(F)C(E)U(R)^G(M)U(F)A(M)G(M)^C(M)^U(M)^U(M) 3' (I-12) Sense strand region 5' SG(M)^C(M)^U(M)A(M)C(F)A(M)G(F)A(F)A(F)U(M)C(M)U(M)A(M)U(M)U(M)U(M)A(M)^U(M)^A(3M) 3'Antisense strand region: 5’ EPu^A(F)^U(M)A(M)A(M)A(F)U(M)A(M)G(F)A(M)U(M)U(F)C(E)U(R)^G(M)U(F)A(M)G(M)^C(M)^U(M)^U(M) 3’ (I-13) Sense strand region: 5’ SG(M)^C(M)^U(M)A(M)C(F)A(M)G(F)A(F)A(F)U(M)C(M)U(M)A(M)U(M)U(M)U(M)A(M)^U(M)^A(3M) 3’ Antisense strand region: 5’ VPuam^A(F)^U(M)A(M)A(M)A(F)U(M)A(M)G(F)A(M)U(M)U(F)C(E)U(R)^G(M)U(F)A(M)G(M)^C(M)^U(M)^U(M) 3’ (I-14) Sense strand region: 5’ SPhG(M)^C(M)^U(M)A(M)C(F)A(M)G(F)A(F)A(F)U(M)C(M)U(M)A(M)U(M)U(M)U(M)A(M)^U(M)^A(3M) 3’ Antisense strand region: 5’ VPu^A(F)^U(M)A(M)A(M)A(F)U(M)A(M)G(F)A(M)U(M)U(F)C(E)U(R)^G(M)U(F)A(M)G(M)^C(M)^U(M)^U(M) 3’ (I-15) Sense strand region: 5’ SPhG(M)^C(M)^U(M)A(M)C(F)A(M)G(F)A(F)A(F)U(M)C(M)U(M)A(M)U(M)U(M)U(M)A(M)^U(M)^A(3M) 3’ Antisense strand region: 5’ EPu^A(F)^U(M)A(M)A(M)A(F)U(M)A(M)G(F)A(M)U(M)U(F)C(E)U(R)^G(M)U(F)A(M)G(M)^C(M)^U(M)^U(M) 3’ (I-16) Sense strand region: 5’ SPhG(M)^C(M)^U(M)A(M)C(F)A(M)G(F)A(F)A(F)U(M)C(M)U(M)A(M)U(M)U(M)U(M)A(M)^U(M)^A(3M) 3’ Antisense strand region: 5’ VPuam^A(F)^U(M)A(M)A(M)A(F)U(M)A(M)G(F)A(M)U(M)U(F)C(E)U(R)^G(M)U(F)A(M)G(M)^C(M)^U(M)^U(M) 3’ (I-18)Sense strand region 5' SG(M)^C(M)^U(M)A(M)C(F)A(M)G(F)A(F)A(F)U(M)C(M)U(M)A(M)U(M)U(M)U(M)A(M)^U(M)^A(3M) 3' Antisense strand region 5' EPuam^A(F)^U(M)A(M)A(M)A(F)U(M)A(M)G(F)A(M)U(M)U(F)C(E)U(R)^G(M)U(F)A(M)G(M)^C(M)^U(M)^U(M) 3' [wherein S and SPh are the following formulae, respectively]

[0030] (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, (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, VPuam, and EPuam as phosphonate units are represented by the following formulae, respectively:

[0031] (wherein 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)-). 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.]

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

[50] or a pharmaceutically acceptable salt thereof.

[52] The pharmaceutical composition according to

[51] , for treating or preventing a disease that can be treated or prevented by suppressing the expression of phospholamban.

[53] The pharmaceutical composition according to

[52] , for preventing or treating heart failure.

[54] A method for treating or preventing heart failure, comprising administering to a subject an effective amount of the oligonucleotide according to any one of [1] to

[50] or a pharmaceutically acceptable salt thereof.

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

[50] or a pharmaceutically acceptable salt thereof for use in the treatment or prevention of heart failure.

[56] Use of the oligonucleotide according to any one of [1] to

[50] or a pharmaceutically acceptable salt thereof in the manufacture of the pharmaceutical composition according to any one of

[51] to

[53] .

[0032] The siRNA of the present invention can suppress the expression of PLN mRNA. Furthermore, the siRNA of the present invention can suppress the expression of PLN mRNA in the heart in vivo, thereby preventing, ameliorating, and / or treating heart failure.

[0033]

[0033] Figure 1 shows the modification patterns of double-stranded siRNA. In the figure, black circles represent 2'-O-methyl RNA, white circles represent 2'-deoxy-2'-fluoro RNA, black triangles represent DNA, white triangles represent 3'-O-methyl RNA, white diamonds represent ENA, black diamonds represent RNA, white circles with vertical bars represent LNA, white circles with horizontal bars represent 2'-O-methoxyethyl RNA, the lines connecting marks represent phosphodiester bonds, and when an "s" is written on the line connecting marks, it represents a phosphorothioate bond. In the figure, the upper 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 lower 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 ９ is 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 phospholamban (hPLN). The underlined sequence (sequences 275 to 293) indicates the target sequence of hPLN-023. This figure is a continuation of Figure 7-1. Figure 8A shows the cytotoxicity of PLN siRNA in HEK293A cells. Figure 8B shows the knockdown activity of PLN siRNA against PLN mRNA in HEK293A cells. Figure 8C shows the knockdown activity of PLN siRNA against PLN mRNA in human iPS cell-derived cardiomyocytes. Figure 9A shows the knockdown activity of PLN siRNA against PLN mRNA in monkeys. Figure 9B shows the knockdown activity of PLN siRNA against PLN protein in monkeys. 9C shows the amount of PLN siRNA in the hearts of monkeys administered PLN siRNA s.c.; 9D shows the knockdown activity of PLN siRNA against PLN mRNA in HEK293A cells; 9E shows the knockdown activity of PLN siRNA against PLN mRNA in HEK293A cells; 9F shows the knockdown activity of PLN siRNA against PLN mRNA in HEK293A cells; 9G shows the knockdown activity of PLN siRNA against PLN mRNA in HEK293A cells;

[0034] <1. Explanation of Terms> As used herein, the term "target gene" refers to mRNA for a gene encoding phospholamban (PLN), and may be immature mRNA before undergoing RNA processing (also referred to as pre-mRNA or pre-mRNA), 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.

[0035] "PLN" is a membrane protein present in the sarcoplasmic reticulum, and is composed of a C-terminal transmembrane domain and an N-terminal cytoplasmic domain. PLN in the non-phosphorylated cytoplasmic domain acts as a SERCA2A inhibitor, and when it is phosphorylated, the inhibition is released and Ca2+ is released into the sarcoplasmic reticulum via SERCA2A. ２＋ Therefore, PLN promotes Ca uptake in cardiac myocytes. ２＋ It is known to play an important role in handling and the associated contractile and diastolic responses.

[0036] "Heart failure" refers to a clinical syndrome associated with some kind of cardiac dysfunction, such as dyspnea, fatigue, edema, and decreased exercise tolerance. Therefore, improving cardiac function, i.e., contractility and relaxation, is considered to be extremely important in treating heart failure. Heart failure includes heart failure with reduced left ventricular ejection fraction (EF) (HFrEF), heart failure with preserved EF (HFpEF), ischemic heart disease, cardiomyopathy (hypertrophic, restrictive, and dilated), valvular disease, hypertension, and heart failure associated with tachycardia or bradyarrhythmia. Ischemic heart disease includes, for example, coronary artery disease, angina pectoris, myocardial infarction, and the like, caused by hypertension, diabetes, obesity, and the like.

[0037] As used herein, nucleic acid or nucleic acid molecule is a general term for nucleosides, nucleotides, oligonucleotides, and polynucleotides.

[0038] "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.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 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).

[0045] (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.)

[0046] 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).

[0047] (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.)

[0048] 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.

[0049] 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).

[0050]

[0051] (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.)

[0052] 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.).

[0053] 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.

[0054] 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).

[0055] (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.)

[0056] 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).

[0057] (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.)

[0058] 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).

[0059] (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.)

[0060] 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 individually 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.

[0061] 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).

[0062] (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.)

[0063] 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 abbreviated 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 conveniently written as C(3m). It can also be used interchangeably with 3'-O-methoxyethylcytidine), 3'-O-methoxyethyl uridine as U(3m), and 3'-O-methoxyethyl-5-methyluridine as T(3m). Additionally, 3'-O-methoxyethyl nucleosides that do not specify a base are sometimes abbreviated as N(3m).

[0064] 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).

[0065] 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)).

[0066] 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)).

[0067] 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).

[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] 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).

[0070] (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.)

[0071] 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.

[0072]

[0073] (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.)

[0074] 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).

[0075]

[0076] (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.)

[0077] As used herein, the term "phosphonate unit" refers to a compound in which a compound having any of the phosphonates represented by the following formulae is bound to the 4'-position of the 5'-terminal nucleoside of the antisense strand in the oligonucleotide of the present invention (facing upward relative to the five-membered ring of the sugar moiety, and being β-position in relation to the base). 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.

[0078]

[0079] (In the formula, R represents a nucleoside and indicates that it is bonded to the 4' carbon atom of the nucleoside.)

[0080] 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.

[0081] 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.

[0082] (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.)

[0083] 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.

[0084] 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).

[0085] 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.

[0086] 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.

[0087] 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."

[0088] 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."

[0089] 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.

[0090] 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.

[0091] 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.

[0092] 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.

[0093] 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.

[0094] 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.

[0095] 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, a gene encoding phospholamban, 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.

[0096] 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.

[0097] 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.

[0098] 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.

[0099] 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.

[0100] 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.

[0101] 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;

[0102] 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.

[0103] 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.

[0104] The RNAi-oligonucleotides of the present invention can suppress the expression of PLN 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 PLN mRNA, the RNAi-oligonucleotides of the present invention can suppress Ca, which plays a central role in cardiac contraction and expansion. ２＋The RNAi-oligonucleotides of the present invention can activate ATPase (SERCA2A). Although 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 PLN mRNA expression and / or activating SERCA2A. Examples of such diseases include heart failure.

[0105] The present invention provides pharmaceutical use inventions, such as a pharmaceutical composition for treating or preventing heart failure containing the RNAi-oligonucleotide of the present invention as an active ingredient, a method for treating or preventing heart failure comprising administering an effective amount of the oligonucleotide, the oligonucleotide for use in treating or preventing heart failure, and use of the RNAi-oligonucleotide of the present invention in the manufacture of a pharmaceutical composition for treating or preventing heart failure. Heart failure that can be treated or prevented by the RNAi-oligonucleotide of the present invention includes, for example, heart failure with reduced left ventricular ejection fraction (EF) (HFrEF) and heart failure with preserved EF (HFpEF). Heart failure refers to a clinical syndrome associated with some type of cardiac dysfunction, such as dyspnea, fatigue, edema, and reduced exercise tolerance, and therefore includes heart failure associated with ischemic heart disease, cardiomyopathy (hypertrophic, restrictive, or dilated), valvular disease, hypertension, and tachycardiac and bradyarrhythmia. Ischemic heart disease includes, for example, coronary artery disease, angina pectoris, myocardial infarction, and the like caused by high blood pressure, diabetes, obesity, and the like.

[0106] 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 heart failure, 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 combined with other drugs that have a different mechanism of action against heart failure symptoms to provide a greater therapeutic effect. For example, drugs such as angiotensin converting enzyme (ACE) inhibitors, angiotensin II receptor blockers (ARBs), diuretics, beta-blockers, sodium-glucose cotransporter 2 (SGLT2) inhibitors, angiotensin receptor neprilysin inhibitors (ARNIs), hyperpolarization-activated cyclic nucleotide-gated (HCN) channel blockers, and calcium channel blockers are sometimes prescribed for heart failure, but these drugs have a different mechanism of action from the RNAi-oligonucleotide of the present invention. Therefore, the combination of these drugs with the RNAi-oligonucleotides of the present invention may provide greater therapeutic effects against heart failure than either drug alone.

[0107] When the RNAi-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 other heart failure therapeutic agents. Examples of other heart failure therapeutic agents include ACE inhibitors, ARBs, diuretics, β-blockers, SGLT2 inhibitors, ARNIs, HCN channel blockers, calcium channel blockers, and the like. Examples of ACE inhibitors include captopril, enalapril, lisinopril, temocapril, and the like. Examples of ARBs include losartan, cilexetil, valsartan, telmisartan, olmesartan, irbesartan, azilsartan, and the like. Examples of diuretics include trichlormethiazide, indapamide, furosemide, spironolactone, eplerenone, esaxerenone, etc. Examples of beta-blockers include atenolol, aceptol, propranolol, carteolol, nipradilol, etc. Examples of SGLT2 inhibitors include dapagliflozin, canagliflozin, empagliflozin, etc. Examples of ARNIs include sacubitril valsartan. Examples of HCN channel blockers include ivabradine. Examples of calcium antagonists include nifedipine, amlodipine, diltiazem, etc.

[0108] <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 -S１３ -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 ~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 １５At 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 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; (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.

[0109] <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 ~S １９ 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.

[0110] 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)). Ｏ３The 2'-position is preferably a hydroxyl group.

[0111] S3M - S of the complementary region in the sense strand region of the oligonucleotide １ The 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.

[0112] 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.

[0113] 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 between 0 and 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.

[0114] 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

[0115] 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

[0116] 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.

[0117] 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.]

[0118] <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:

[0119] A １ may 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.

[0120] 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.

[0121] In one example of the present invention, the complementary region A 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.

[0122] 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)

[0123] In one example of the present invention, the complementary region A 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.

[0124] 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)

[0125] In one example of the present invention, the complementary region A 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.

[0126] 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.

[0127] 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.

[0128] 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.

[0129] 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.

[0130] 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.

[0131] 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.

[0132]

[0133]

[0134] [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.]

[0135] 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.

[0136] 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.

[0137] <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.

[0138] 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.

[0139] 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.

[0140] 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.

[0141] 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.

[0142] 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.

[0143]

[0144] (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.)

[0145] 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.

[0146] 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.

[0147] 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)).

[0148] Amidite reagents corresponding to various nucleosides may be purchased commercially or may be synthesized according to known methods.

[0149] 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.

[0150] 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).

[0151] 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).

[0152] Commercially available phosphoramidite reagents can be used for 2'-O-allylguanosine, 2'-O-allyl adenosine, 2'-O-allyl cytidine, and 2'-O-allyl uridine.

[0153] 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.

[0154] 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).

[0155] 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).

[0156] Commercially available phosphoramidite reagents can be used for 2'-O-propargylguanosine, 2'-O-propargyladenosine, 2'-O-propargylcytidine, and 2'-O-propargyluridine.

[0157] 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.

[0158] For the 3'-modified nucleoside 3'-F RNA, the corresponding phosphoramidite reagents can be synthesized according to WO2017027645 for 3'-deoxy-3'-fluoroguanosine, WO2018198076 for 3'-deoxy-3'-fluoroadenosine, and US2011 / 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.

[0159] 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.

[0160] 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.

[0161] 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.

[0162] 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.

[0163] 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).

[0164] 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).

[0165] 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.

[0166] 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).

[0167] 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.

[0168] 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.

[0169] 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.

[0170] 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.

[0171] 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).

[0172] 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.

[0173] 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).

[0174] 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.

[0175] 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.

[0176]

[0177] (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.)

[0178] 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.

[0179]

[0180] (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.)

[0181] 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.

[0182] 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 atoms 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.

[0183] 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.

[0184] Vpu can be synthesized using the method described in J. Med. Chem. 2018, 61, 3, 734-744.

[0185] Vpuam, Vpuam2, Vpuam3, and Vpuam4 can be synthesized using the following steps A-1 to A-10.

[0186]

[0187] (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.

[0188] 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).

[0189] 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.

[0190] 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.

[0191] 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.

[0192] 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.

[0193] 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.

[0194] 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.

[0195] 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.

[0196] The reaction temperature varies depending on the reagents, raw materials, solvent, etc. used, but is usually -10 to 100°C, preferably 0 to 50°C.

[0197] 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.

[0198] 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.

[0199] 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.

[0200] 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.

[0201] 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.

[0202] 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.

[0203] 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.

[0204] 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.

[0205] 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.

[0206] 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.

[0207] 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.

[0208] 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.

[0209] 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.

[0210] 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.

[0211] 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).

[0212] 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.

[0213] 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.

[0214] 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.

[0215] 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.

[0216] 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.

[0217] 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.

[0218] 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.

[0219] 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).

[0220] 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.

[0221] 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.

[0222] 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.

[0223] 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.

[0224] 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.

[0225] 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.

[0226] 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.

[0227] 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.

[0228] 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).

[0229] 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.

[0230] 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.

[0231] The reaction temperature is not particularly limited, but is usually 0 to 80°C, preferably room temperature.

[0232] 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.

[0233] 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.

[0234] Steps A-8 and A-10 are steps in which compound (8) or compound (9) produced by the above-described 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.

[0235] 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, or chromatography, etc.

[0236] EPu can be synthesized using the following steps B-1 to B-10.

[0237]

[0238] (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.

[0239] 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.

[0240] 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).

[0241] 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.

[0242] 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.

[0243] The reaction temperature is usually -20 to 80°C, preferably -10 to 50°C, depending on the reagents, raw materials, solvent, etc. used.

[0244] 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.

[0245] 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.

[0246] 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).

[0247] 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.

[0248] 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.

[0249] The reaction temperature is usually -20 to 150°C, preferably -10 to 50°C, depending on the reagents, raw materials, solvent, etc. used.

[0250] 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.

[0251] 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.

[0252] Step B-3 is a step of preparing compound (17) by reacting compound (16) with a protecting reagent in an inert solvent.

[0253] 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).

[0254] 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.

[0255] 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.

[0256] The reaction temperature is usually -20 to 150°C, preferably -10 to 80°C, depending on the reagents, raw materials, solvent, etc. used.

[0257] 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.

[0258] 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.

[0259] 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.

[0260] 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.

[0261] 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.

[0262] 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.

[0263] 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.

[0264] 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.

[0265] 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.

[0266] 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.

[0267] 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.

[0268] 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.

[0269] 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.

[0270] 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.

[0271] 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.

[0272] 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.

[0273] 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.

[0274] 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.

[0275] 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.

[0276] 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.

[0277] 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.

[0278] 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.

[0279] 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.

[0280] 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.

[0281] 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.

[0282] 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).

[0283] 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.

[0284] 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.

[0285] 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.

[0286] 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.

[0287] 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).

[0288] The reaction temperature is not particularly limited, but is usually from -20 to 100°C, preferably from 10 to 40°C.

[0289] 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.

[0290] EPuam can be synthesized using the following steps C-1 to C-13.

[0291]

[0292] (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].)

[0293] 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.

[0294] 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.

[0295] 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.

[0296] 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.

[0297] 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.

[0298] 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.

[0299] 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.

[0300] 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.

[0301] 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.

[0302] 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.

[0303] The reaction temperature is usually -20 to 80°C, preferably -10 to 50°C, depending on the reagents, raw materials, solvent, etc. used.

[0304] 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.

[0305] 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.

[0306] 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.

[0307] 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.

[0308] 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.

[0309] 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.

[0310] 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.

[0311] 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).

[0312] 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.

[0313] 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.

[0314] The reaction temperature is usually -20 to 150°C, preferably -10 to 50°C, depending on the reagents, raw materials, solvent, etc. used.

[0315] 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.

[0316] 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.

[0317] 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.

[0318] 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.

[0319] The acetylating agent to be used includes, for example, acetyl chloride, acetic anhydride, ethyl acetate, etc. Acetic anhydride is preferred.

[0320] 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.

[0321] 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.

[0322] 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.

[0323] Step C-6 is a step of preparing compound (31) by reacting compound (30) with a protecting reagent in an inert solvent.

[0324] 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).

[0325] 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.

[0326] 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.

[0327] The reaction temperature is usually -20 to 150°C, preferably -10 to 80°C, depending on the reagents, raw materials, solvent, etc. used.

[0328] 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.

[0329] 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.

[0330] 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.

[0331] 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.

[0332] The reaction temperature is usually -20 to 80°C, preferably -10 to 50°C, depending on the reagents, raw materials, solvent, etc. used.

[0333] 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.

[0334] 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.

[0335] 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.

[0336] 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.

[0337] 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.

[0338] 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.

[0339] 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.

[0340] 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.

[0341] 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.

[0342] 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.

[0343] 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.

[0344] 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.

[0345] 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).

[0346] 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.

[0347] 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.

[0348] 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.

[0349] 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.

[0350] 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.

[0351] 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).

[0352] 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.

[0353] 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.

[0354] 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.

[0355] 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.

[0356] 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.

[0357] 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.

[0358] 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.

[0359] 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.

[0360] 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).

[0361] 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.

[0362] 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.

[0363] The reaction temperature is not particularly limited, but is usually 0 to 80°C, preferably room temperature.

[0364] 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.

[0365] 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.

[0366] 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).

[0367] The reaction temperature is not particularly limited, but is usually from -20 to 100°C, preferably from 10 to 40°C.

[0368] 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.

[0369] 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)).

[0370] 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).

[0371] 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.

[0372] The oligonucleotide of the present invention or a pharmaceutically acceptable salt thereof can suppress the expression of PLN mRNA. Furthermore, the oligonucleotide of the present invention or a pharmaceutically acceptable salt thereof can suppress the expression of PLN mRNA, thereby inhibiting Ca, which plays a central role in cardiac contraction and expansion. ２＋It can activate ATPase (SERCA2A). The mechanism of action is not particularly limited, but 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 PLN mRNA expression and / or activating SERCA2A. In a preferred embodiment, when administered to a patient with heart failure, it can suppress PLN mRNA expression, and is expected to have an effect of improving the symptoms of heart failure. 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.

[0373] 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.

[0374] 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.

[0375] 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.

[0376] 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.

[0377] 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).

[0378] 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.

[0379] 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.

[0380] In addition to the above, a colloidal dispersion system can be used to deliver the oligonucleotide of the present invention to a patient. Colloidal dispersion systems are expected to enhance the stability of compounds in vivo and to efficiently transport 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-).

[0381] 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.

[0382] 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).

[0383] Representative phospholipids include phosphatidylcholine, dipalmitoylphosphatidylcholine, and distearoylphosphatidylcholine.

[0384] Targeting of colloidal dispersion systems, including liposomes, can be either passive or active.

[0385] Passive targeting is achieved by taking advantage of the natural tendency of liposomes to distribute to reticuloendothelial cells of organs containing sinusoidal capillaries.

[0386] 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.

[0387] 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.

[0388] 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.

[0389] Agents that enhance the intracellular stability and / or targeting of the contents can also be added to the colloidal dispersion system.

[0390] 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.

[0391] Pharmaceutical compositions and formulations for topical administration include transdermal patches, ointments, lotions, creams, gels, lozenges, suppositories, sprays, liquids and powders.

[0392] The oligonucleotide of the present invention can suppress the expression of PLN mRNA. Furthermore, the oligonucleotide of the present invention has low toxicity to cells and can be used as a highly safe pharmaceutical. Furthermore, by suppressing the expression of PLN mRNA, the oligonucleotide of the present invention can suppress the Ca 2+ receptor, which plays a central role in cardiac contraction and expansion. ２＋ The oligonucleotides of the present invention can activate ATPase (SERCA2A). Although 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 PLN mRNA expression and / or activating SERCA2A. Such diseases include, for example, heart failure.

[0393] The present invention provides pharmaceutical use inventions, such as pharmaceutical compositions containing the oligonucleotide of the present invention as an active ingredient for use in treating or preventing heart failure, methods for treating or preventing heart failure comprising administering an effective amount of the oligonucleotide, the oligonucleotide for use in treating or preventing heart failure, and use of the oligonucleotide of the present invention in the manufacture of pharmaceutical compositions for treating or preventing heart failure. Examples of heart failure that can be treated or prevented by the oligonucleotide of the present invention include heart failure with reduced left ventricular ejection fraction (EF) (HFrEF) and heart failure with preserved EF (HFpEF). Heart failure refers to clinical syndromes associated with some form of cardiac dysfunction, such as dyspnea, fatigue, edema, and reduced exercise tolerance, and includes heart failure associated with ischemic heart disease, cardiomyopathy (hypertrophic, restrictive, or dilated), valvular disease, hypertension, and tachycardiac and bradyarrhythmia. Ischemic heart disease includes, for example, coronary artery disease, angina pectoris, and myocardial infarction caused by hypertension, diabetes, obesity, etc.

[0394] The present invention provides a method for treating or preventing heart failure, comprising using the oligonucleotide of the present invention in combination with one or more other drugs. The 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 oligonucleotide of the present invention can be used in combination with other drugs that have a different mechanism of action against heart failure symptoms to provide a greater therapeutic effect. For example, drugs such as angiotensin converting enzyme (ACE) inhibitors, angiotensin II receptor blockers (ARBs), diuretics, beta-blockers, sodium-glucose cotransporter 2 (SGLT2) inhibitors, angiotensin receptor neprilysin inhibitors (ARNIs), hyperpolarization-activated cyclic nucleotide-gated (HCN) channel blockers, and calcium channel blockers are sometimes prescribed for heart failure, but these drugs have different mechanisms of action from PLN siRNA. Therefore, the combination of these drugs with PLN siRNA can provide a greater therapeutic effect against heart failure than a single drug.

[0395] 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 other heart failure therapeutic agents. Examples of other heart failure therapeutic agents include ACE inhibitors, ARBs, diuretics, β-blockers, SGLT2 inhibitors, ARNIs, HCN channel blockers, calcium channel blockers, and the like. Examples of ACE inhibitors include captopril, enalapril, lisinopril, temocapril, and the like. Examples of ARBs include losartan, cilexetil, valsartan, telmisartan, olmesartan, irbesartan, azilsartan, and the like. Examples of diuretics include trichlormethiazide, indapamide, furosemide, spironolactone, eplerenone, esaxerenone, etc. Examples of beta-blockers include atenolol, aceptol, propranolol, carteolol, nipradilol, etc. Examples of SGLT2 inhibitors include dapagliflozin, canagliflozin, empagliflozin, etc. Examples of ARNIs include sacubitril valsartan. Examples of HCN channel blockers include ivabradine. Examples of calcium antagonists include nifedipine, amlodipine, diltiazem, etc.

[0396] The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.

[0397] (Examples 1 to 9) Each Example shown in Table 2 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 are shown in Table 1. 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 shown 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 a double-stranded oligonucleotide was confirmed using native gel or HPLC, resulting in an Example compound having a double-stranded structure. The compounds of each Example are shown in Table 2.

[0398]

[0399] In the sequences in the table, N(R) represents RNA, 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). In the table, VPu, EPu, VPuam, EPuam, S, and SPh 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, EPuam, S, or SPh is bonded to the 5'-terminal of each oligonucleotide, VPu, EPu, VPuam, EPuam, S, or SPh is bonded to the adjacent nucleotide in the bonding mode shown in the table, respectively. Furthermore, VPu, EPu, VPuam, EPuam, S, or SPh has no bonding site other than the bond to the adjacent oligonucleotide portion.

[0400] Furthermore, two consecutive U (M) are bound as an overhang structure to the 3' end of the antisense strand region in Table 1. The "molecular weight" in the table indicates the actual measured value by negative ion ESI mass spectrometry.

[0401]

[0402] (In the formula, the tip of the dashed line represents bonding to the adjacent nucleotide.)

[0403]

[0404] The target sequence for the compounds of Examples 1 to 8 and 10 is a sequence consisting of 19 bases from the 275th to 293rd bases in the base sequence of human PLN mRNA shown in SEQ ID NO: 1 (disclosed as accession number NM_002667.5 in NCBI Genbank), i.e., 5' GCUACAGAAUCUAUUUAUC 3' (SEQ ID NO: 2). The antisense strand region has a nucleotide sequence substantially complementary to the target sequence, and the sense strand region has a nucleotide sequence completely complementary to the antisense strand region. For example, in the compound of Example 2 (hPLN-023.93), the target-corresponding sequence of the antisense strand region is 5' U(M)^A(F)^U(M)A(M)A(M)A(F)U(M)A(M)G(F)A(M)U(M)U(F)C(E)U(R)^G(M)U(F)A(M)G(M)^C(M) 3' (SEQ ID NO: 11), and the nucleotide sequence from 2 to 19 counting from the 5' end of the antisense strand region is a nucleotide sequence that is completely complementary to the target sequence. The antisense strand region of the compound of Example 2 further comprises U(M)^U(M) linked to the 3' end of the target-corresponding sequence via a phosphorothioate bond as an overhang structure. The sense strand region of the compound of Example 2 is a region that forms a double-stranded structure with the target-corresponding sequence (i.e., a complementary region), and its nucleotide sequence is the nucleotide sequence shown in SEQ ID NO: 3. The nucleotide sequence of the sense strand region is a nucleotide sequence that is completely complementary to the antisense strand region in the complementary region. The compound of Example 2 has a 93-type modification pattern shown in Figure 6.

[0405] The target sequence in the compound of Example 9 (hPLN-023.93CG) is a sequence consisting of 19 bases from the 275th to the 293rd in the base sequence of human PLN mRNA shown in SEQ ID NO: 1 (disclosed as accession number NM_002667.5 in NCBI Genbank). This target sequence corresponds to SEQ ID NO: 379146 described in WO 2004 / 045543. The antisense strand region in the compound of Example 9 has a sequence that is completely complementary to the target sequence as a target-corresponding sequence, and further has U(M)^U(M) bound to its 3' end as an overhang structure via a phosphorothioate bond. The sense strand region of the compound of Example 9 is a region that forms a double-stranded structure with the target-corresponding sequence (i.e., a complementary region), and its nucleotide sequence is the nucleotide sequence shown in SEQ ID NO: 9. The nucleotide sequence of the sense strand region is a nucleotide sequence that is completely complementary to the antisense strand region in the complementary region. The compound of Example 9 was modified with the 93-type pattern shown in FIG.

[0406] 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 was a compound of an example described in the literature for synthesizing the "VPu" portion, a compound of Reference Example 16 for synthesizing the "EPu" portion, a compound of Reference Example 7 for synthesizing the "VPuam" portion, or a compound of Reference Example 27 for synthesizing the "EPuam" portion.Furthermore, as the unit 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.

[0407] When the amidite of Reference Example 16 or Reference Example 27 was used, prior to the deprotection step using concentrated aqueous ammonia, the ethyl group of the phosphonate moiety was deprotected by treating the compound 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.

[0408] Reference Example 1: 2'-azido-2'-deoxy-3',5'-bis-O-(1,1,1-triethylsilyl)uridine

[0409]

[0410] 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 ＋ )

[0411] Reference Example 2: 2'-azido-2'-deoxy-3'-O-(1,1,1-triethylsilyl)uridine

[0412]

[0413] 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) ＋

[0414] Reference Example 3: 2'-azido-2'-deoxy-3'-O-(1,1,1-triethylsilyl)-5'-deoxy-5'-oxouridine

[0415]

[0416] 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) ＋

[0417] 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

[0418]

[0419] 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 － )

[0420] 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

[0421]

[0422] 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. １ H-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) ＋

[0423] (Reference Example 6a) 2'-acetamido-2',5'-dideoxy-5'-[(E)-2-(bis{[(2,2-dimethoxypropanoyl)oxy]methoxy}phosphoryl)vinyl]uridine

[0424]

[0425] (Reference Example 6b) 2'-acetamido-2',5'-dideoxy-5'-[(Z)-2-(bis{[(2,2-dimethoxypropanoyl)oxy]methoxy}phosphoryl)vinyl]uridine

[0426]

[0427] 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%).

[0428] (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) ＋

[0429] (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) ＋

[0430] 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

[0431]

[0432] 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) ＋

[0433] Reference Example 8: N-[4-[2-cyanoethoxy-(diisopropylamino)phosphanyl]oxyphenyl]octadecanamide

[0434]

[0435] 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) ＋

[0436] Reference Example 9: 5'-O-(4,4'-dimethoxytrityl)-2'-O-methyluridine

[0437]

[0438] 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.

[0439] Reference Example 10: 3'-O-tert-butyl(diphenyl)silyl-5'-O-(4,4'-dimethoxytrityl)-2'-O-methyluridine

[0440]

[0441] 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.

[0442] Reference Example 11: N3-benzoyl-3'-O-tert-butyl(diphenyl)silyl-2'-O-methyluridine

[0443]

[0444] 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).

[0445] (Reference Example 12) 5-(3-benzoyl-2,4-dioxo-pyrimidin-1-yl)-3-[tert-butyl(diphenyl)silyl]oxy-4-methoxy-tetrahydrofuran-2-carbaldehyde

[0446]

[0447] N3-benzoyl-3'-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).

[0448] 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

[0449]

[0450] 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).

[0451] Reference Example 14: 3-benzoyl-1-[4-tert-butyl(diphenyl)silyl]oxy-5-(2-diethoxyphosphorylethynyl)-3-methoxy-tetrahydrofuran-2-yl]pyrimidine-2,4-dione

[0452]

[0453] 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).

[0454] Reference Example 15: 3-benzoyl-1-[5-(2-diethoxyphosphorylethynyl)-4-hydroxy-3-methoxy-tetrahydrofuran-2-yl]pyrimidine-2,4-dione

[0455]

[0456] 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).

[0457] 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

[0458]

[0459] 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) ＋ .

[0460] Reference Example 17: 2'-trifluoroacetamido uridine

[0461]

[0462] Commercially available 2'-aminouridine (10.1 g, 41.5 mmol) was dissolved in methanol (100 mL), and triethylamine (11.5 mL, 83.0 mmol) and ethyl trifluoroacetate (11.8 g, 83.0 mmol) were added in that order, followed by stirring at room temperature overnight. The solvent was removed by distillation under reduced pressure, and acetonitrile (50 mL) was added and the mixture was concentrated under reduced pressure three times to obtain 17.5 g (99%) of the target product. 1H-NMR (400MHz, CD3OD): 3.75 (1H, dd, J = 3.1, 11.6Hz), 3.81 (1H, dd, J = 2.4, 11.6Hz), 4.09 (1H, dt, J = 3.1, 2.4Hz), 4.31 (dd, J = 2 .4, 6.1Hz), 4.61 (dd, J = 6.1, 7.9Hz), 5.73 (d, J = 7.9Hz), 61.4 (1H, d, J = 7.9Hz), 8.01 (1H, d, J = 7.9Hz).

[0463] Reference Example 18: 5'-O-(4,4'-dimethoxytrityl)-2'-trifluoroacetamido uridine

[0464]

[0465] 2'-Trifluoroacetamidouridine (17.5 g, 41.5 mmol) obtained in Reference Example 17 was dissolved in pyridine (100 mL) and cooled to 0°C. 4,4'-Dimethoxytrityl chloride (17.1 g, 50.5 mmol) was added over 15 minutes, and the mixture was stirred at room temperature for 3 hours. The solvent was distilled off under reduced pressure, and toluene (50 mL) was added and concentrated under reduced pressure. This operation was repeated three times, after which water was added and the mixture was extracted with ethyl acetate. The mixture was dried over magnesium sulfate, and the filtered solution was concentrated. The solid was washed with isopropyl alcohol and filtered to obtain 23.1 g (87%) of the target product. 1H-NMR (400 MHz, CD3OD): 3.40 (1H, dd, J = 2.4, 11.0Hz), 3.48 (1H, dd, J = 3.1, 11.0Hz), 3.78 (6H, s), 4 .16 (1H, dt, J=3.1, 2.4Hz), 4.45 (1H, dd, J=2.4, 5.5Hz), 4.84-4.60 (1H, m) , 5.31 (1H, d, J = 7.9Hz), 6.17 (1H, d, J = 7.3Hz), 6.89 (4H, d, J = 8.6Hz), 7.22 -7.26 (1H, m), 7.30-7.34 (6H, m), 7.42-7.45 (2H, m), 7.84 (1H, d, J=7.9Hz).

[0466] Reference Example 19: 5'-O-(4,4'-dimethoxytrityl)-2'-aminouridine

[0467]

[0468] 5'-O-(4,4'-dimethoxytrityl)-2'-trifluoroacetamidouridine (23.1 g, 36.0 mmol) obtained in Reference Example 18 was dissolved in 1,4-dioxane (100 mL), and 28% aqueous ammonia (200 mL) was added over 10 minutes, followed by stirring at room temperature overnight. After adding water, the mixture was extracted with ethyl acetate and dried over magnesium sulfate. The filtered solution was concentrated to obtain 20.9 g (99%) of the target product. 1H-NMR (400 MHz, CDCl3): 3.41 (1H, dd, J = 3.1, 11.0Hz), 3.47 (1H, dd, J = 2.4, 11.0Hz), 3.61 (1H, t, J = 6.1Hz), 3.73 (6H, s), 4.20 (1H, dd, J = 3.1, 6.1Hz), 4.23 (1H, dt, J = 2.4, 3.1 Hz), 5.42 (1H, d, J = 7.9Hz), 5.89 (1H, d, J = 6.1Hz), 6.83-6.85 (4H, m), 7.14 -7.18 (1H, m), 7.22-7.31 (6H, m), 7.36-7.45 (2H, m), 7.84 (1H, d, J=7.9Hz).

[0469] Reference Example 20: 3'-O-tert-butyl(dimethyl)silyl-5'-O-(4,4'-dimethoxytrityl)-2'-aminouridine

[0470]

[0471] 5'-O-(4,4'-dimethoxytrityl)-2'-aminouridine (20.9 g, 36.0 mmol) obtained in Reference Example 19 and imidazole (9.80 g, 144 mmol) were dissolved in pyridine (200 mL), and tert-butyl(dimethyl)silyl chloride (9.80 g, 65.0 mmol) was added, followed by stirring at room temperature for 2.5 hours. After adding water, the mixture was extracted with ethyl acetate and dried over magnesium sulfate. The filtered solution was concentrated and purified by silica gel chromatography (hexane:ethyl acetate = 90:10 to 25:75) to obtain 22.6 g (95%) of the target product. 1H-NMR (400 MHz, CDCl3): 0.04 (3H, s), 0.88 (6H, s), 3.32 (1H, dd, J = 3.1, 11.0Hz), 3.49 (1H, dd, J = 5.5, 6 .1Hz), 3.51 (1H, dd, J=2.4, 11.0Hz), 3.80 (6H, s), 4.09 (1H, dt, J=3.1, 2.4Hz) , 4.24 (1H, dd, J = 3.1, 5.5Hz), 5.39 (1H, d, J = 7.9Hz), 5.88 (1H, d, J = 6.1Hz), 6. 83-6.86 (4H, m), 7.23-7.33 (7H, m), 7.36-7.39 (2H, m), 7.85 (1H, d, J=7.9Hz).

[0472] Reference Example 21: 2'-acetamido-3'-O-tert-butyl(dimethyl)silyl-5'-O-(4,4'-dimethoxytrityl)uridine (11.7 g, 15.7 mmol)

[0473]

[0474] 3'-O-tert-butyl(dimethyl)silyl-5'-O-(4,4'-dimethoxytrityl)-2'-aminouridine (22.6 g, 34.3 mmol) obtained in Reference Example 20 was dissolved in pyridine (200 mL), acetic anhydride (16.2 mL, 171 mmol) was added, and the mixture was stirred at room temperature for 1.5 hours. The solvent was distilled off under reduced pressure, toluene (50 mL) was added, and the mixture was concentrated under reduced pressure. This operation was repeated three times, after which water was added and the mixture was extracted with ethyl acetate. The organic layer was washed with 0.25 M hydrochloric acid and then saturated aqueous sodium bicarbonate, and then dried over magnesium sulfate. The solution after filtration was concentrated and purified by silica gel chromatography (hexane:ethyl acetate = 70:30 to 30:70) to obtain 21.8 g (91%) of the target product. 1H-NMR (400 MHz, CDCl3): 0.05 (3H, s), 0.90 (9H, s), 2.01 (3H, s), 3.34 (1H, dd, J = 3.1, 11.0Hz), 3.41 (1H, dd, J = 3.1, 11.0H z), 3.80 (6H, s), 4.07 (1H, dt, J = 1.2, 3.1Hz), 4.32 (1H, dd, J = 1.8, 6.1Hz), 4.75 (1H, dd, J = 6.1, 8. 6Hz), 5.50 (1H, dd, J = 1.8, 8.6Hz), 5.97 (1H, d, J = 8.6Hz), 6.04 (1H, d, J = 8.6Hz), 6.83-6.87 (4H, m ), 7.22-7.24 (2H, m), 7.29-7.33 (5H, m), 7.41-7.43 (2H, m), 7.67 (1H, d, J=8.6Hz), 8.29 (1H, br).

[0475] Reference Example 22: 2'-acetamido-N3-benzoyl-3'-O-tert-butyl(dimethyl)silyluridine

[0476]

[0477] 2'-Acetamido-3'-O-tert-butyl(dimethyl)silyl-5'-O-(4,4'-dimethoxytrityl)uridine (11.7 g, 15.7 mmol) obtained in Reference Example 21 and diisopropylethylamine (5.0 mL, 28.2 mmol) were dissolved in pyridine (80 mL), and benzoyl chloride (3.32 g, 23.5 mmol) was added dropwise at room temperature over 5 minutes, followed by stirring at room temperature overnight. 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 (250 mL) and trifluoroacetic acid (9.0 mL, 156.7 mmol) were added to the obtained intermediate, followed by stirring at room temperature for 40 minutes. Saturated aqueous sodium bicarbonate was added to the reaction solution, followed by extraction with methylene chloride. The mixture was dried over magnesium sulfate, and the filtered solution was concentrated and purified by silica gel chromatography (hexane:ethyl acetate = 67:33 to 0:100) to obtain 7.00 g (83%) of the target compound. 1H-NMR (400 MHz, CDCl3): 0.11 (6H, s), 0.92 (9H, s), 1.94 (3H, s), 2.26 (1H, brs), 3.79 (1H, ddd, J = 2.4, 6.1, 11.5 Hz), 3.93 (1H, ddd, J = 2.4, 3.6, 11.5 Hz), 4.06 (1H, dt, J = 1.8, 2.4 Hz), 4.44 (1H, dd, J = 1.8, 6.1Hz), 4.61 (1H, ddd, J = 7.3, 6.1, 9.1Hz), 5.84 (1H, d, J = 9.1Hz), 5.88 (1H, d, J = 8.5Hz), 6.01 (1H, d, J=7.3Hz), 7.48-7.52 (2H, m), 7.62-7.66 (1H, m), 7.72 (1H, d, J=8.5Hz), 7.99-8.01 (2H, m).

[0478] (Reference Example 23) 4'-acetamido-5'-(3-benzoyl-2,4-dioxo-pyrimidin-1-yl)-3'-[tert-butyl(dimethyl)silyl]oxy-tetrahydrofuran-2'-carbaldehyde

[0479]

[0480] 2'-Acetamido-N3-benzoyl-3'-O-tert-butyl(dimethyl)silyluridine (7.00 g, 13.9 mmol) obtained in Reference Example 22 was dissolved in acetonitrile (150 mL), and 2-iodoxybenzoic acid (10.00 g, 34.75 mmol) was added at room temperature, followed by stirring for 2.5 hours at 85° C. The solid was collected by filtration, and the solvent was evaporated under reduced pressure to obtain 6.11 g (88%) of the desired product. 1H-NMR (400MHz, CDCl3): 0.14 (3H, s), 0.17 (3H, s), 0.92 (9H, s), 1.95 (3H, s), 4.3 5 (1H, ddd, J = 5.4, 6.1, 6.7Hz), 4.50 (1H, d, J = 1.8Hz), 4.63 (1H, dd, J = 1.8, 5.4Hz) , 5.95 (1H, d, J = 8.5Hz), 5.97 (1H, d, J = 6.1Hz), 6.06 (1H, d, J = 6.7Hz), 7.49-7.53 ( 2H, m), 7.64-7.68 (1H, m), 7.84 (1H, d, J=8.5Hz), 7.96-8.00 (2H, m), 9.72 (1H, s).

[0481] Reference Example 24: 3-benzoyl-1-[3-acetamido-4-tert-butyl(diphenyl)silyl]oxy-5-(2,2-dibromovinyl)-tetrahydrofuran-2-yl]pyrimidine-2,4-dione

[0482]

[0483] 4'-Acetamido-5'-(3-benzoyl-2,4-dioxo-pyrimidin-1-yl)-3'-[tert-butyl(dimethyl)silyl]oxy-tetrahydrofuran-2'-carbaldehyde (8.48 g, 24.4 mmol) obtained in Reference Example 23 was dissolved in methylene chloride (20 mL) and added to a methylene chloride (20 mL) solution of carbon tetrabromide (8.25 g, 24.9 mmol) and triphenylphosphine (13.1 g, 48.7 mmol) cooled to 0°C. The mixture was stirred overnight at room temperature. 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. A 1:1 mixture of hexane and ethyl acetate (20 mL) was added, and the resulting white solid was filtered off and purified by silica gel chromatography (hexane and ethyl acetate = 70:30 to 0:100) to obtain 2.52 g (31%) of the desired product. 1H-NMR (400MHz, CDCl3): 0.09 (3H, s), 0.13 (3H, s), 0.92 (9H, s), 1.98 (3H, s), 4 .41 (1H, m), 4.43 (1H, dd, J = 3.6, 6.1Hz), 4.58 (1H, dd, J = 3.6, 9.1Hz), 5.68 (1H, d , J=5.4Hz), 5.89 (1H, d, J=8.5Hz), 6.19 (1H, d, J=4.8Hz), 6.53 (1H, d, J=9.1Hz), 7.38 (1H, d, J=8.5Hz), 7.50-7.54 (2H, m), 7.64-7.68 (1H, m), 7.96-7.99 (2H, m).

[0484] Reference Example 25: 3-benzoyl-1-[3-acetamido-4-tert-butyl(dimethyl)silyl]oxy-5-(2-diethoxyphosphorylethynyl)-tetrahydrofuran-2-yl]pyrimidine-2,4-dione

[0485]

[0486] 3-Benzoyl-1-[4-tert-butyl(dimethyl)silyl]oxy-5-(2,2-dibromovinyl)-3-methoxy-tetrahydrofuran-2-yl]pyrimidine-2,4-dione (2.52 g, 3.83 mmol) obtained in Reference Example 24 was dissolved in DMF (20 mL) and added to a solution of palladium acetate (0.18 g, 0.77 mmol) and 1,1'-diphenylphosphinoferrocene (0.87 g, 1.53 mmol) in DMF (30 mL), propylene oxide (1.16 g, 20.0 mmol) and diethyl phosphite (1.00 mL, 7.67 mmol) were added, and the mixture was stirred overnight at 80° C. Water and saturated brine were added to the reaction solution, followed by extraction with ethyl acetate. The mixture was dried over sodium sulfate, filtered, and the resulting solution was concentrated and purified by silica gel chromatography (hexane:ethyl acetate = 35:65 to 0:100 to ethyl acetate:methanol = 93:7) to obtain 1.31 g (54%) of the target compound. 1H-NMR (400 MHz, CDCl3): 0.14 (3H, s), 0.16 (3H, s), 0.92 (9H, s), 1.35-1.42 (6H, m), 1.92 (3H, s), 4.17-4.25 (4H, m), 4.51 (1H, dd, J = 1.2, 4.8 Hz), 4.66 (1H, ddd, J = 4.8, 7.9, 8.5 Hz). , 4.70 (1H, dd, J = 1.2, 3.6Hz), 5.87 (1H, d, J = 8.5Hz), 5.96 (1H, d, J = 7.9Hz), 6.08 (1H, d, J =7.9Hz), 7.50-7.54 (2H, m), 7.58 (1H, d, J = 7.9Hz), 7.63-7.69 (1H, m), 7.97-7.99 (2H, m).

[0487] Reference Example 26: 3-benzoyl-1-[3-acetamido-5-(2-diethoxyphosphorylethynyl)-4-hydroxy-tetrahydrofuran-2-yl]pyrimidine-2,4-dione

[0488]

[0489] 3-Benzoyl-1-[3-acetamido-4-tert-butyl(dimethyl)silyl]oxy-5-(2-diethoxyphosphorylethynyl)-tetrahydrofuran-2-yl]pyrimidine-2,4-dione (1.31 g, 2.07 mmol) obtained in Reference Example 25 was dissolved in tetrahydrofuran (20 mL), and triethylamine-trihydrofluoride (4.0 mL) was added at room temperature. The mixture was stirred overnight at room temperature. 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 (ethyl acetate:methanol = 100:0 to 85:15) to obtain 0.32 g (30%) of the desired product. 1H-NMR (400MHz, CD3OD): 1.35 (3H, t, J = 6.7Hz), 1.36 (3H, t, J = 6.7Hz), 1.9 3 (3H, s), 4.18 (2H, q, J = 6.7Hz), 4.20 (2H, q, J = 6.7Hz), 4.48 (1H, m), 4.90 ( 1H, dd, J=1.2, 3.6Hz), 5.97 (1H, d, J=8.5Hz), 6.04 (1H, d, J=7.7Hz), 7.55- 5.59 (2H, m), 7.71-7.75 (1H, m), 7.78 (1H, d, J=8.5Hz), 7.96-7.99 (2H, m).

[0490] (Reference Example 27) 3-{[4-acetamido-5-(3-benzoyl-2,4-dioxo-pyrimidin-1-yl)-2-(2-diethoxyphosphorylethynyl)-tetrahydrofuran-3-yl]oxy-(diisopropylamino)phosphanyl}oxypropanenitrile

[0491]

[0492] 3-Benzoyl-1-[3-acetamido-5-(2-diethoxyphosphorylethynyl)-4-hydroxy-tetrahydrofuran-2-yl]pyrimidine-2,4-dione (0.32 g, 0.62 mmol) obtained in Reference Example 26 and triethylamine (0.45 mL, 3.08 mmol) were dissolved in methylene chloride (4.5 mL), and 2-cyanoethyldiisopropylchlorophosphoramidite (0.30 mL, 0.92 mmol) was added at 0°C, followed by stirring at room temperature for 1.5 hours. Methylene chloride and saturated aqueous sodium bicarbonate were 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 basic silica gel chromatography (ethyl acetate (containing 2% triethylamine):acetone = 100:0 to 60:40) to obtain 0.25 g (56%) of the desired product. 1H-NMR (400MHz, CDCl3): 1.18-1.29 (18H, m), 1.90-1.95 (3H, m), 2.74-2.78 (2H, m), 3.76-3.97 (4H, m), 4.15-4.25 (4H, m), 4.52-5.1 0 (3H, m), 5.97 (1H, d, J = 8.6Hz), 6.07-6.35 (2H, m), 7.49-7.53 (2H, m), 7.63 (1H, d, J = 8.6Hz), 7.65-7.86 (1H, m), 7.93-8.03 (2H, m).

[0493] Test Example 1: Evaluation of knockdown activity and cytotoxicity of PLN siRNA in HEK293A cells by reverse transfection (1) The following experiment was carried out using the compounds of Example 1 (hPLN-023.92) and Example 2 (hPLN-023.93) as PLN siRNA. HEK293A cells were transfected with genes to establish a stable human PLN expression line. 1 x 10 cells per well were placed in 90 μL of DMEM medium (Wako) supplemented with 10% FBS (Sigma). ４HEK293A cells were seeded. To prepare the reagent for transfection of one siRNA sample, 4.7 μL of Opti-MEM (Life Technology) and 0.3 μL of RNAiMAX (Thermo Fisher Scientific) were mixed, to which 3 μL of Opti-MEM and 2 μL of siRNA (50x the final concentration) were added, followed by incubation at room temperature for 5 minutes. As a negative control, distilled water was added instead of siRNA. The RNAiMAX / siRNA mixture was added to a 96-well plate previously seeded with HEK293 cells. The final siRNA concentration was 0.1 nM, 1 nM, or 10 nM.

[0494] Three days after transfection, the culture supernatant was collected and cytotoxicity was measured using the Cytotoxicity LDH Assay Kit-WST (Dojindo Laboratories) kit according to the kit's protocol. The measured absorbance at 490 nm is shown in Figure 8A. After collecting the culture supernatant, the cells were washed once with PBS (Wako) 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 obtained cDNA was used to measure the mRNA level of human PLN by quantitative PCR. The mRNA level of human GAPDH was measured as a housekeeping gene.

[0495] Quantitative PCR was performed using IDT PrimeTime Gene Expression Master Mix (Integrated DNA Technologies) as follows: The obtained cDNA was diluted two-fold with distilled water. 5 μL of IDT PrimeTime Gene Expression Master Mix, 0.5 μL of hPLN probe (Hs.PT.58.23189767, Integrated DNA Technologies) or 0.5 μL of hGAPDH probe (Hs.PT.39a.22214836, Integrated DNA Technologies), 2.5 μL of distilled water, and 2 μL of cDNA were mixed (total 10 μL) per well of a 384-well PCR plate (Applied Biosystems). Two wells were prepared for each cDNA, and quantitative PCR was performed. hPLN and hGAPDH mRNA expression levels were measured as the average between the two wells. The hPLN mRNA expression level was normalized to that of the housekeeping gene hGAPDH. The hPLN mRNA expression level in the negative control was set to 1.0, and the relative hPLN mRNA expression level in the samples treated with each siRNA was calculated and expressed as a relative value. The results are shown in Figure 8B.

[0496] These results demonstrate that the compounds of Example 1 (hPLN-023.92) and Example 2 (hPLN-023.93) can strongly suppress target PLN mRNA in HEK293A cells stably expressing human PLN without exhibiting cytotoxicity.

[0497] Test Example 2: Evaluation of knockdown activity of PLN siRNA in human iPS cell-derived cardiomyocytes by forward transfection. Human iPS cell-derived cardiomyocytes (manufactured by Myoridge, Inc.) were cultured according to the manual, and 6 days after seeding, siRNA transfection was performed. To prepare the reagent for transfection of one siRNA sample, 4.7 μL of Opti-MEM (manufactured by Life Technology) and 0.3 μL of RNAiMAX (manufactured by Thermo Fisher Scientific) were mixed, to which 3 μL of Opti-MEM and 2 μL of siRNA (concentration 50x the final concentration) were added, followed by incubation at room temperature for 5 minutes. As a negative control, distilled water was added instead of siRNA. Transfection was performed by mixing 10 μL of this mixture with 90 μL of maintenance medium (manufactured by Myoridge) and replacing the medium. The final concentration of siRNA was 0.1 nM, 1 nM, or 10 nM. Three days after transfection, RNA extraction was performed in the same manner as for HEK293A cells.

[0498] Quantitative PCR was performed using IDT PrimeTime Gene Expression Master Mix (Integrated DNA Technologies) as follows: The obtained cDNA was diluted two-fold with distilled water. Per well of a 384-well PCR plate (Applied Biosystems), 5 μL of IDT PrimeTime Gene Expression Master Mix, 0.5 μL of hPLN probe (Hs.PT.58.23189767, Integrated DNA Technologies) or 0.5 μL of 18S probe (4352930E, Applied Biosystems), 2.5 μL of distilled water, and 2 μL of cDNA were mixed (total 10 μL), and two wells were prepared for each cDNA, and quantitative PCR was performed. hPLN and 18S mRNA expression levels were measured as the average between two wells. The hPLN mRNA expression level was normalized to that of the housekeeping gene 18S. The hPLN mRNA expression level in the negative control was set to 1.0, and the relative hPLN mRNA expression level in the samples treated with each siRNA was calculated and expressed as a relative value. The results are shown in Figure 8C.

[0499] These results indicate that the compounds of Example 1 (hPLN-023.92) and Example 2 (hPLN-023.93) have inhibitory activity against the target PLN mRNA. In particular, the compound of Example 2 (hPLN-023.93) has stronger inhibitory activity against the target PLN mRNA.

[0500] Test Example 3: Test to evaluate the PLN knockdown activity of the compound of Example 3 in cynomolgus monkeys The compound of Example 3 (hPLN-023.93SV) was dissolved in phosphate-buffered saline (PBS, manufactured by Cell Science Institute) and the following experiment was performed. Three male cynomolgus monkeys (manufactured by Shin Nippon Scientific Co., Ltd.) were subcutaneously administered (s.c.) once with 30 mg / kg (30 mpk) of the compound of Example 3. One animal in the vehicle control group was administered s.c. with PBS instead of the drug. The vehicle control group and one animal in the drug-administered group were euthanized by exsanguination 28 days after administration, and the hearts were collected to measure the mRNA and protein expression levels of monkey PLN and monkey GAPDH. One animal in the drug administration group was similarly sampled 7 days after administration, and the other animal was sampled 56 days after administration, and the mRNA expression level and protein expression level in the heart of each animal were measured.

[0501] RNA was extracted using Trizol Reagent (Invitrogen) and RNeasy Mini Kit (QIAGEN) according to the kit's protocol. The mRNA levels of monkey PLN and monkey GAPDH were measured by quantitative PCR using cDNA obtained by reverse transcription of RNA using Rever Tra Ace qPCR RT Master Mix (Toyobo).

[0502] 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 monkey PLN (cPLN) probe (Mf04255299_s1, Thermo Fisher Scientific), 2.5 μL of distilled water, and 2 μL of cDNA were mixed per well of a 384-well PCR plate (Applied Biosystems) (total 10 μL), and two wells were prepared for each cDNA, and quantitative PCR was performed. A monkey GAPDH (cGAPDH) probe (Mf04392546_g1, Thermo Fisher Scientific) was used as the housekeeping gene. The cDNAs were mixed at the ratios described above, and two wells were prepared for each cDNA. Quantitative PCR was performed. The mRNA levels of cPLN and cGAPDH were measured as the average value between the two wells. The mRNA expression level of cPLN was normalized by the mRNA expression level of the housekeeping gene, cGAPDH. The relative cPLN mRNA expression level in the compound-treated group of Example 3 was calculated and expressed as a mean ± standard error (SD) value relative to the mRNA expression level in the vehicle control group, which was set to 1.0. The results are shown in Figure 9A.

[0503] Proteins were extracted using the following procedure, and protein expression levels were quantified by Western blotting. RIPA Buffer (Sigma-Aldrich) was mixed with 10% SDS Solution (Nacalai Teque, final concentration 1%) and protease inhibitor (Roche) to prepare a lysis buffer. Heart tissue pieces were added with lysis buffer and disrupted using a Tissue Lyzer (QIAGEN). After incubation at 4°C for 10 minutes, they were sonicated using a probe sonicator. The pieces were centrifuged at 13,000 rpm for 10 minutes, and the supernatant was collected as a protein sample. The protein concentration was measured using a Pierce BCA Protein Assay Kit (Thermofischer Sciences) and adjusted to 2 mg / mL. The protein was then adjusted to 1 mg / mL with 2x Laemmli Sample Buffer (BIO-RAD) containing 20% ​​mercaptoethanol (Sigma-Aldrich). After preparation, the protein was denatured by incubation at 98°C for 10 minutes.

[0504] The denatured protein sample was applied at 10 μL / well to Perfect NT gel 5-20%, 16-wells (DRC) and separated by SDS-PAGE. The protein was transferred to a PVDF membrane (BIO-RAD) and blocked in 5% skim milk (Wako) / TBST solution (Wako, Thermofischer Sciences). The membrane was immersed in a primary antibody solution diluted with Can Get Signal Immunoreaction Enhancer Solution (TOYOBO) and incubated overnight at 4°C. The primary antibodies used were anti-phospholamban antibody [EPR21897] (manufactured by Abcam) and GAPDH antibody (6C5) (manufactured by Santa Cruz). After washing with TBST, the sections were immersed in a secondary antibody solution diluted with Can Get Signal Immunoreaction Enhancer Solution and incubated at room temperature for 1 hour. The secondary antibodies used were Anti-Mouse IgG, HRP-Linked F(ab')2 Fragment Sheep (GE Healthcare) and Anti-Rabbit IgG, HRP-Linked F(ab')2 Fragment Donkey (GE Healthcare). After washing with TBST, detection was performed with ECL Select Western Blotting Detection Reagent (Amersham) and images were taken with an Amersham Imager 600 (GE Healthcare). The band sizes of PLN and GAPDH were quantified using Amersham Imager 600 analysis software, and PLN protein expression levels were normalized with the expression level of GAPDH, a housekeeping gene. The relative cPLN protein expression levels in the compound-treated group of Example 3 were calculated and expressed as a relative value when the cPLN protein expression level in the vehicle control group was set to 1.0. The results are shown in Figure 9B.

[0505] The siRNA concentration in monkey hearts was measured as follows: Buffer RLT Plus Lysis buffer (QIAGEN) was added in an amount equal to 0 times the monkey heart weight, and the mixture was homogenized using a Shake Master auto (Bio Medical Sciences). The homogenized solution was then diluted 5-fold with Buffer RLT Lysis buffer and centrifuged at 4°C and 15,000 rpm (CF15RX, rotor: T15A33, Hitachi Koki) for 2 minutes. On a 96-well plate (Applied Biosystems), 5 μL of the centrifuged supernatant was mixed with 5 μL of a complementary probe (Gene Design) for the antisense strand of the test substance and 40 μL of HEPES Buffer (GE Healthcare), and the mixture was incubated at 95 ° C for 5 minutes and 42 ° C for 60 minutes using a thermal cycler (Applied Biosystems). Hybridization of the test substance and the complementary probe was then carried out. Then, 10 μL of S1 Nuclease (18 U / μ L, Takara Bio Inc.) was added to each well and incubated at 37 ° C for 60 minutes. After the reaction, the mixture was centrifuged at 4 ° C, 3000 rpm (CF15RX, rotor: T5S32, Hitachi Koki) for 10 minutes. Five microliters of the supernatant was mixed with 5 μL of Rexxip ADA (Gyros Protein Technologies) in equal volume to form the measurement sample, and quantitative measurements were performed using a fully automated immunoassay platform, Gyrolab xp workstation (Gyros Protein Technologies). A calibration curve was generated using Gyrolab Evaluator Software, regressed with a four-parameter logistic model (weighted response). The results are shown in Figure 9C.

[0506] These results demonstrate that the sc administration of the compound of Example 3 (hPLN-023.93SV) having a fatty acid unit added thereto to monkeys delivered PLN siRNA to the heart and suppressed the expression levels of PLN mRNA and protein.

[0507] Test Example 4: Evaluation of knockdown activity and cytotoxicity of PLN siRNA in HEK293A cells by reverse transfection (2) Using the compounds of Example 2 (hPLN-023.93), Example 3 (hPLN-023.93SV), Example 4 (hPLN-023.93SV1), Example 5 (hPLN-023.93SV2), Example 6 (hPLN-023.93SPh), Example 7 (hPLN-023.93SPh1), and Example 8 (hPLN-023.93SPh2) as PLN siRNAs, a comparison was made in the same test as in Test Example 1. The results are shown in FIG.

[0508] These results indicate that the compounds of Example 3 (hPLN-023.93SV), Example 4 (hPLN-023.93SV1), Example 5 (hPLN-023.93SV2), Example 6 (hPLN-023.93SPh), Example 7 (hPLN-023.93SPh1), and Example 8 (hPLN-023.93SPh2) have potent target PLN mRNA inhibitory activity comparable to or greater than that of Example 2 (hPLN-023.93).

[0509] Test Example 5: Comparative test of PLN siRNAs of Example 2 and Example 9 Using the compounds of Example 2 (hPLN-023.93) and Example 9 (hPLN-023.93CG) as PLN siRNAs, knockdown activity was evaluated in the same manner as in Test Example 1. These compounds differ by one base in the nucleotide sequence at the 3' end of the sense strand region and the 5' end of the antisense strand region. The results are shown in Figure 11.

[0510] This result indicates that the compound of Example 2 (hPLN-023.93) has stronger target PLN mRNA suppression activity than the compound of Example 9 (hPLN-023.93CG).

[0511] Test Example 6: Evaluation of knockdown activity and cytotoxicity of PLN siRNA in HEK293A cells by reverse transfection (3) Using the compounds of Example 2 (hPLN-023.93), Example 3 (hPLN-023.93SV), Example 4 (hPLN-023.93SV1), Example 5 (hPLN-023.93SV2), and Example 10 (hPLN-023.93SV3) as PLN siRNAs, a comparison was made in the same test as in Test Example 1. However, the treatment time was 48 hours. The results are shown in Figure 12.

[0512] These results indicate that the compounds of Example 3 (hPLN-023.93SV), Example 4 (hPLN-023.93SV1), Example 5 (hPLN-023.93SV2), and Example 10 (hPLN-023.93SV3) have potent target PLN mRNA inhibitory activity comparable to or greater than that of Example 2 (hPLN-023.93).

[0513] The siRNA of the present invention can suppress the expression of PLN mRNA, which plays a central role in heart failure, and is useful for treating or preventing diseases that can be treated or prevented by suppressing the expression of PLN mRNA.

[0514] SEQ ID NO: 1: Nucleotide sequence of hPLN mRNA SEQ ID NO: 2: Target sequence of hPLN-023 SEQ ID NOs: 3 to 10: Nucleotide sequences of the sense strand region or antisense strand region of the compounds of the examples SEQ ID NO: 11: Target-corresponding sequence of the compound of Example 2 (hPLN-023.93) SEQ ID NO: 12: Nucleotide sequence of the antisense strand region of Example 10 (hPLN-023.93SV3)

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 phospholamban 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 273 and 295 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; and (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 275 to 293 of SEQ ID NO:

1.

3. The oligonucleotide or a pharma- ceutically acceptable salt thereof according to claim 1 or 2, wherein the 5'-terminal nucleoside or phosphonic acid base in the target-corresponding sequence of the antisense strand region is adenine or uracil.

4. The oligonucleotide or a pharma- ceutical acceptable salt thereof according to any one of claims 1 to 3, wherein the target corresponding sequence of the antisense strand region has a nucleotide sequence that is completely complementary to the 18 bases from nucleotide numbers 275 to 292 of SEQ ID NO: 1 with respect to the 19 bases from nucleotide numbers 275 to 293 of SEQ ID NO: 1, and the 5' end of the target corresponding sequence is a nucleoside having uracil or a phosphonic acid unit having uracil.

5. The oligonucleotide or a pharma- ceutical acceptable salt thereof according to any one of claims 1 to 4, 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.) 6. The oligonucleotide or a pharma- ceutically acceptable salt thereof according to any one of claims 1 to 5, wherein at least one of the sugar and / or phosphodiester bonds constituting the oligonucleotide is modified.

7. The oligonucleotide or a pharma- ceutically acceptable salt thereof according to claim 6, 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.

8. The oligonucleotide or a pharma- ceutically acceptable salt thereof according to claim 7, 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.

9. The oligonucleotide or a pharma- ceutically acceptable salt thereof according to claim 8, 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.

10. The oligonucleotide or a pharma- ceutically acceptable salt thereof according to any one of claims 6 to 9, wherein the modification of the phosphodiester bond is phosphorothioate.

11. The oligonucleotide or a pharma- ceutically acceptable salt thereof according to any one of claims 6 to 10, characterized in that D-ribofuranose in the 3'-terminal nucleoside of the sense strand region and / or antisense strand region is 3'-O-alkylated.

12. The oligonucleotide or a pharma- ceutically acceptable salt thereof according to claim 11, wherein the 3'-O-alkylation is 3'-O-methylation.

13. The oligonucleotide or a pharma- ceutical acceptable salt thereof according to any one of claims 1 to 12, 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.

14. The oligonucleotide or a pharma- ceutically acceptable salt thereof according to claim 13, wherein the overhang structure is two bases long.

15. The oligonucleotide or a pharma- ceutically acceptable salt thereof according to claim 14, wherein the overhang structure is a nucleoside having two consecutive thymines or a nucleoside having two consecutive uracils.

16. The oligonucleotide or a pharma- ceutically acceptable salt thereof according to claim 15, wherein the overhang structure is two consecutive U (M) added to the 3'-end of the antisense strand region.

17. 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.

18. In formula (II), S １ The oligonucleotide or a pharma- ceutically acceptable salt thereof according to claim 17, wherein is 3'-OMe RNA.

19. The oligonucleotide or a pharma- ceutically acceptable salt thereof according to claim 17 or 18, wherein the 2'-O,4'-C-bridged modified nucleoside is LNA or ENA.

20. 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 17 to 19, 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.

21. In formula (II), A １４ The oligonucleotide or a pharma- ceutically acceptable salt thereof according to claim 20, wherein is RNA or a 2'-O,4'-C-bridged modified nucleoside.

22. 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 21, wherein said oligonucleotide is RNA.

23. 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 23. The oligonucleotide according to claim 22, which is: (2'-OMe RNA), or a pharma- ceutically acceptable salt thereof.

24. In formula (II), A １２ is DNA, and A １４ The oligonucleotide or a pharma- ceutically acceptable salt thereof according to claim 21, wherein is a 2'-O,4'-C-bridged modified nucleoside.

25. 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 25. The oligonucleotide according to claim 24, which is: (2'-OMe RNA), or a pharma- ceutically acceptable salt thereof.

26. 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 17 to 25, wherein is 2'-F RNA.

27. In the formula (II), 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 26, wherein the nucleoside is 2'-OMe RNA.

28. 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 27, wherein the nucleoside is 2'-OMe RNA.

29. In the formula (II), A １ The oligonucleotide or a pharma- ceutically acceptable salt thereof according to claim 28, wherein is 2'-OMe RNA.

30. In the formula (II), A １ The oligonucleotide according to claim 28, 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.) 31. 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 17 to 30, wherein is 2'-F RNA.

32. 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 31, wherein is 2'-OMe RNA.

33. An oligonucleotide or a pharma- ceutically acceptable salt thereof according to any one of claims 17 to 32, 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.

34. The oligonucleotide or a pharma- ceutically acceptable salt thereof according to any one of claims 17 to 33, characterized in that in 2 to 5 nucleotides from the 5' and 3' ends of the oligonucleotide, each internucleoside bond is a phosphorothioate bond.

35. S Ｏ３ , S Ｏ５ , A Ｏ５ and A Ｏ３ The oligonucleotide or a pharma- ceutically acceptable salt thereof according to any one of claims 17 to 34, wherein the number of nucleosides is 0 to 2.

36. S Ｏ３ The oligonucleotide or a pharma- ceutically acceptable salt thereof according to claim 35, wherein the number of nucleosides is 0.

37. 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 36, wherein the number of nucleosides is 2.

38. The oligonucleotide or a pharma- ceutically acceptable salt thereof according to any one of claims 1 to 37, characterized in that the antisense strand region and the sense strand region each form a double-stranded oligonucleotide as an independent oligonucleotide.

39. The oligonucleotide or a pharma- ceutically acceptable salt thereof according to any one of claims 1 to 37, 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.

40. The linker structure is represented by the following formula: The oligonucleotide according to claim 39 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.] 41. The oligonucleotide or a pharma- ceutically acceptable salt thereof according to any one of claims 1 to 40, 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.

42. The oligonucleotide or a pharma- ceutically acceptable salt thereof according to claim 41, 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.] 43. The oligonucleotide or a pharma- ceutically acceptable salt thereof according to claim 42, 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 3'-modified nucleotide) of an adjacent nucleotide.] 44. 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-8) and (I-17), 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 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' G(M)^C(M)^U(M)A(M)C(F)A(M)G(F)A(F)A(F)U(M)C(M)U(M)A(M)U(M)U(M)U(M)A(M)^U(M)^A(3M) 3' Antisense strand region 5' U(M)^A(F)^U(M)A(M)A(M)A(F)U(M)A(M)G(F)A(M)U(F)U(M)C(E)U(R)^G(M)U(F)A(M)G(M)^C(M) 3' (I-2) Sense strand region 5' G(M)^C(M)^U(M)A(M)C(F)A(M)G(F)A(F)A(F)U(M)C(M)U(M)A(M)U(M)U(M)U(M)A(M)^U(M)^A(3M) 3' Antisense strand region 5' U(M)^A(F)^U(M)A(M)A(M)A(F)U(M)A(M)G(F)A(M)U(M)U(F)C(E)U(R)^G(M)U(F)A(M)G(M)^C(M) 3' (I-3) Sense strand region 5' G(M)^C(M)^U(M)A(M)C(F)A(M)G(F)A(F)A(F)U(M)C(M)U(M)A(M)U(M)U(M)U(M)A(M)^U(M)^A(3M) 3' Antisense strand region 5' VPu^A(F)^U(M)A(M)A(M)A(F)U(M)A(M)G(F)A(M)U(M)U(F)C(E)U(R)^G(M)U(F)A(M)G(M)^C(M) 3' (I-4) Sense strand region 5' G(M)^C(M)^U(M)A(M)C(F)A(M)G(F)A(F)A(F)U(M)C(M)U(M)A(M)U(M)U(M)U(M)A(M)^U(M)^A(3M) 3'Antisense strand region 5’ EPu^A(F)^U(M)A(M)A(M)A(F)U(M)A(M)G(F)A(M)U(M)U(F)C(E)U(R)^G(M)U(F)A(M)G(M)^C(M) 3’ (I-5) Sense strand region 5’ G(M)^C(M)^U(M)A(M)C(F)A(M)G(F)A(F)A(F)U(M)C(M)U(M)A(M)U(M)U(M)U(M)A(M)^U(M)^A(3M) 3’ Antisense strand region 5’ VPuam^A(F)^U(M)A(M)A(M)A(F)U(M)A(M)G(F)A(M)U(M)U(F)C(E)U(R)^G(M)U(F)A(M)G(M)^C(M) 3’ (I-6) Sense strand region 5’ G(M)^C(M)^U(M)A(M)C(F)A(M)G(F)A(F)A(F)U(M)C(M)U(M)A(M)U(M)U(M)U(M)A(M)^U(M)^A(3M) 3’ Antisense strand region 5’ VPu^A(F)^U(M)A(M)A(M)A(F)U(M)A(M)G(F)A(M)U(M)U(F)C(E)U(R)^G(M)U(F)A(M)G(M)^C(M) 3’ (I-7) Sense strand region 5’ G(M)^C(M)^U(M)A(M)C(F)A(M)G(F)A(F)A(F)U(M)C(M)U(M)A(M)U(M)U(M)U(M)A(M)^U(M)^A(3M) 3’ Antisense strand region 5’ EPu^A(F)^U(M)A(M)A(M)A(F)U(M)A(M)G(F)A(M)U(M)U(F)C(E)U(R)^G(M)U(F)A(M)G(M)^C(M) 3’ (I-8) Sense strand region 5’ G(M)^C(M)^U(M)A(M)C(F)A(M)G(F)A(F)A(F)U(M)C(M)U(M)A(M)U(M)U(M)U(M)A(M)^U(M)^A(3M) 3’ Antisense strand region 5’ VPuam^A(F)^U(M)A(M)A(M)A(F)U(M)A(M)G(F)A(M)U(M)U(F)C(E)U(R)^G(M)U(F)A(M)G(M)^C(M) 3’ (I-17) Sense strand region 5’ G(M)^C(M)^U(M)A(M)C(F)A(M)G(F)A(F)A(F)U(M)C(M)U(M)A(M)U(M)U(M)U(M)A(M)^U(M)^A(3M) 3’Antisense strand region 5' EPuam^A(F)^U(M)A(M)A(M)A(F)U(M)A(M)G(F)A(M)U(M)U(F)C(E)U(R)^G(M)U(F)A(M)G(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, (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, VPuam, and EPuam represent 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. ] 45. The oligonucleotide or a pharma- ceutically acceptable salt thereof according to claim 44, 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.

46. ​​The oligonucleotide or a pharma- ceutically acceptable salt thereof according to claim 45, wherein the overhang structure is two bases long.

47. The oligonucleotide or a pharma- ceutically acceptable salt thereof according to claim 46, wherein the overhang structure is a nucleoside containing two consecutive thymines or a nucleoside containing two consecutive uracils.

48. The oligonucleotide or a pharma- ceutically acceptable salt thereof according to claim 47, wherein the overhang structure is two consecutive U (M) added to the 3' end of the antisense strand region.

49. The oligonucleotide or a pharma- ceutical acceptable salt thereof according to any one of claims 44 to 48, 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.) 50. The oligonucleotide or a pharma- ceutical acceptable salt thereof according to claim 1, wherein the sense strand region and the antisense strand region are any of the following formulas (I-9) to (I-16) and (I-18), and the sense strand region and the antisense strand region each serve as independent oligonucleotides to form a double-stranded oligonucleotide. (Formula) (I-9) Sense strand region 5' G(M)^C(M)^U(M)A(M)C(F)A(M)G(F)A(F)A(F)U(M)C(M)U(M)A(M)U(M)U(M)U(M)A(M)^U(M)^A(3M) 3' Antisense strand region 5' U(M)^A(F)^U(M)A(M)A(M)A(F)U(M)A(M)G(F)A(M)U(F)U(M)C(E)U(R)^G(M)U(F)A(M)G(M)^C(M)^U(M)^U(M) 3' (I-10) Sense strand region 5' G(M)^C(M)^U(M)A(M)C(F)A(M)G(F)A(F)A(F)U(M)C(M)U(M)A(M)U(M)U(M)U(M)A(M)^U(M)^A(3M) 3' Antisense strand region 5' U(M)^A(F)^U(M)A(M)A(M)A(F)U(M)A(M)G(F)A(M)U(M)U(F)C(E)U(R)^G(M)U(F)A(M)G(M)^C(M)^U(M)^U(M) 3' (I-11) Sense strand region 5' SG(M)^C(M)^U(M)A(M)C(F)A(M)G(F)A(F)A(F)U(M)C(M)U(M)A(M)U(M)U(M)U(M)A(M)^U(M)^A(3M) 3' Antisense strand region 5' VPu^A(F)^U(M)A(M)A(M)A(F)U(M)A(M)G(F)A(M)U(M)U(F)C(E)U(R)^G(M)U(F)A(M)G(M)^C(M)^U(M)^U(M) 3' (I-12) Sense strand region 5' SG(M)^C(M)^U(M)A(M)C(F)A(M)G(F)A(F)A(F)U(M)C(M)U(M)A(M)U(M)U(M)U(M)A(M)^U(M)^A(3M) 3' Antisense strand region 5' EPu^A(F)^U(M)A(M)A(M)A(F)U(M)A(M)G(F)A(M)U(M)U(F)C(E)U(R)^G(M)U(F)A(M)G(M)^C(M)^U(M)^U(M) 3' (I-13)Sense strand region: 5’ SG(M)^C(M)^U(M)A(M)C(F)A(M)G(F)A(F)A(F)U(M)C(M)U(M)A(M)U(M)U(M)U(M)A(M)^U(M)^A(3M) 3’ Antisense strand region: 5’ VPuam^A(F)^U(M)A(M)A(M)A(F)U(M)A(M)G(F)A(M)U(M)U(F)C(E)U(R)^G(M)U(F)A(M)G(M)^C(M)^U(M)^U(M) 3’ (I-14) Sense strand region: 5’ SPhG(M)^C(M)^U(M)A(M)C(F)A(M)G(F)A(F)A(F)U(M)C(M)U(M)A(M)U(M)U(M)U(M)A(M)^U(M)^A(3M) 3’ Antisense strand region: 5’ VPu^A(F)^U(M)A(M)A(M)A(F)U(M)A(M)G(F)A(M)U(M)U(F)C(E)U(R)^G(M)U(F)A(M)G(M)^C(M)^U(M)^U(M) 3’ (I-15) Sense strand region: 5’ SPhG(M)^C(M)^U(M)A(M)C(F)A(M)G(F)A(F)A(F)U(M)C(M)U(M)A(M)U(M)U(M)U(M)A(M)^U(M)^A(3M) 3’ Antisense strand region: 5’ EPu^A(F)^U(M)A(M)A(M)A(F)U(M)A(M)G(F)A(M)U(M)U(F)C(E)U(R)^G(M)U(F)A(M)G(M)^C(M)^U(M)^U(M) 3’ (I-16) Sense strand region: 5’ SPhG(M)^C(M)^U(M)A(M)C(F)A(M)G(F)A(F)A(F)U(M)C(M)U(M)A(M)U(M)U(M)U(M)A(M)^U(M)^A(3M) 3’ Antisense strand region: 5’ VPuam^A(F)^U(M)A(M)A(M)A(F)U(M)A(M)G(F)A(M)U(M)U(F)C(E)U(R)^G(M)U(F)A(M)G(M)^C(M)^U(M)^U(M) 3’ (I-18) Sense strand region: 5’ SG(M)^C(M)^U(M)A(M)C(F)A(M)G(F)A(F)A(F)U(M)C(M)U(M)A(M)U(M)U(M)U(M)A(M)^U(M)^A(3M) 3’Antisense strand region 5' EPuam^A(F)^U(M)A(M)A(M)A(F)U(M)A(M)G(F)A(M)U(M)U(F)C(E)U(R)^G(M)U(F)A(M)G(M)^C(M)^U(M)^U(M) 3' [wherein S and SPh are represented by the following formulas, respectively. (wherein the dashed line indicates that it is bound to the 5'-phosphate group of the 5'-terminal nucleotide of the adjacent sense strand region by a phosphodiester bond), with respect to the nucleic acid bases, A indicates adenine, U indicates uracil, G indicates guanine, and C indicates cytosine (however, when C is ENA, it indicates 2'-O,4'-C-ethylene-bridged-5-methylcytidine), with respect to the nucleic acid, (R) indicates RNA, (M) indicates 2'-OMe RNA, (3M) indicates 3'-OMe RNA, (F) indicates 2'-F RNA, and (E) indicates ENA, and with respect to the phosphonic acid units, VPu, EPu, VPuam, and EPuam 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.) 51. 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 50.

52. A pharmaceutical composition according to claim 51 for treating or preventing a disease which can be treated or prevented by inhibiting the expression of phospholamban.

53. The pharmaceutical composition according to claim 52 for the prevention or treatment of heart failure.

54. A method for treating or preventing heart failure, comprising administering to a subject an effective amount of the oligonucleotide according to any one of claims 1 to 50 or a pharma- ceutically acceptable salt thereof.

55. An oligonucleotide or a pharma- ceutically acceptable salt thereof according to any one of claims 1 to 50 for use in the treatment or prevention of heart failure.

56. Use of an oligonucleotide according to any one of claims 1 to 50 or a pharma- ceutically acceptable salt thereof in the manufacture of a pharmaceutical composition according to any one of claims 51 to 53.

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