Drugs for the treatment of glycogenopathies type Ia

Abstract

The present invention establishes a molecular therapy for glycogenopathies type Ia. The present invention is an oligonucleotide, a pharmacologically acceptable salt or solvate thereof, wherein the oligonucleotide is an oligonucleotide having 15 to 30 bases and containing a nucleotide sequence complementary to the cDNA of the G6PC gene having the c.648G>T mutation, and containing a sequence complementary to a region of any of the 82nd to 92nd sites from the 5' end of exon 5 of the G6PC gene having the c.648G>T mutation. The medicine of the present invention (e.g., a drug for treating glycogenopathies type Ia) contains the oligonucleotide, a pharmaceutically acceptable salt or solvate thereof.

Description

[0001] Information about divisional applications

[0002] This case is a divisional application. The parent case of the divisional application is an invention patent application with application date of March 5, 2019, application number 201980006746.4, and invention name “Glycogenosis Type Ia Therapeutic Drug”.

[0003] [Related applications]

[0004] This application is based on Japanese Patent Application No. 2018-043524 (filing date: March 9, 2018), Japanese Patent Application No. 2018-128015 (filing date: July 5, 2018), and PCT Patent Application No. PCT / JP2019 / 008713 (filing date: March 5, 2019). This application includes all the contents of the basic applications by reference. Technical Field

[0005] The present invention relates to a drug for treating glycogenosis type Ia, and more particularly, to an oligonucleotide and a medicine containing the same that repairs the G6PC gene having the c.648G>T mutation in glycogenosis type Ia patients at the mRNA level and enables the expression of normal G6PC protein. Background Art

[0006] Glycogenosis type Ia is an autosomal recessive metabolic disorder with glucose-6-phosphate dephosphorylase (G6PC) as the causative gene, and the main symptoms are hypoglycemia, hepatomegaly, and nephromegaly. Survival prognosis is greatly improved by controlling blood sugar using dietary therapy, but even with good control, more than half of them present with hepatic adenoma and albuminuria (Non-patent Document 1). Several prevalent mutations in patients with glycogenosis type Ia have been reported, and the G6PC c.648G>T mutation that causes splicing abnormalities in East Asia is known as a prevalent mutation (Non-patent Document 2).

[0007] As a method for delivering oligonucleotides to hepatocytes of the liver, the industry has reported a complex with a nucleic acid medicine (antisense or siRNA, etc.) to which N-acetyl-D-galactosamine (GalNAc) or the like is covalently bonded as a ligand that can be bonded to the asialoglycoprotein receptor (ASGPR) (Non-patent Document 3 and Patent Documents 1 to 9). One to three GalNAcs are bonded to one oligonucleotide. In addition, as an example of two GalNAcs bonded, Non-patent Document 4 describes it.

[0008] Prior art literature

[0009] Non-patent literature

[0010] Non-patent document 1: Eur J Pediatr. 2002, Vol. 161, Suppl 1: S20-34

[0011] Non-patent document 2: Hum Mutat. 2008, Vol. 29, p. 921-930.

[0012] Non-patent document 3: Methods in Enzymology, 1999, Vol. 313, pp. 297-321

[0013] Non-patent document 4: Bioorganic & Medicinal Chemistry Letters 26 (2016) 3690-3693

[0014] Patent Literature

[0015] Patent Document 1: International Publication No. 2009 / 073809

[0016] Patent Document 2: International Publication No. 2014 / 076196

[0017] Patent Document 3: International Publication No. 2014 / 179620

[0018] Patent Document 4: International Publication No. 2015 / 006740

[0019] Patent Document 5: International Publication No. 2015 / 105083

[0020] Patent Document 6: International Publication No. 2016 / 055601

[0021] Patent Document 7: International Publication No. 2017 / 023817

[0022] Patent Document 8: International Publication No. 2017 / 084987

[0023] Patent Document 9: International Publication No. 2017 / 131236 Summary of the invention

[0024] [Problems to be solved by the invention]

[0025] The object of the present invention is to establish a molecular therapy for glycogenopathies type Ia.

[0026] [Technical means to solve the problem]

[0027] The present inventors have established the following treatment method ( Figure 1), which repairs abnormal splicing of mRNA and induces the production of normal G6PC protein by administering antisense oligonucleotides (ASO) to patients with glycogen disease type Ia with the G6PC c.648G>T mutation. Abnormal splicing of mRNA occurs in the G6PC c.648G>T mutation, and 91 bases of exon 5 are missing from the junction of intron 4 and exon 5, causing inactivation of the enzyme due to frame shift. When the abnormal splicing of mRNA is corrected, it is expected that the translated amino acid sequence is the same as the normal type (CUG(Leu)→CUU(Leu)). Therefore, in patients with the G6PC c.648G>T mutation, the treatment method of repairing abnormal splicing of mRNA can produce active normal G6PC protein, and the risk of hypoglycemia, organomegaly, and hepatic adenoma in patients with glycogen disease type Ia can be reduced. This research is the world's first research to develop a molecular therapy for glycogenotype Ia.

[0028] The gist of the present invention is as follows.

[0029] (1) An oligonucleotide, or a pharmacologically acceptable salt or solvate thereof, wherein the oligonucleotide comprises a nucleotide sequence complementary to a cDNA of a G6PC gene having a c.648G>T mutation and has a base number of 15 to 30, and comprises a sequence complementary to a region of any of the 82nd to 92nd sites from the 5' end of exon 5 of the G6PC gene having a c.648G>T mutation.

[0030] (2) An oligonucleotide as described in (1), or a pharmacologically acceptable salt or solvate thereof, wherein the oligonucleotide is an oligonucleotide having 15 to 21 bases and contains a sequence complementary to a region including any of the 86th to 92nd sites from the 5' end of exon 5 of a G6PC gene having a c.648G>T mutation.

[0031] (3) The oligonucleotide according to (1), or a pharmacologically acceptable salt or solvate thereof, wherein the oligonucleotide is an oligonucleotide having 15 to 21 bases and contains a sequence complementary to a region including the 92nd site from the 5' end of exon 5 of the G6PC gene having the c.648G>T mutation.

[0032] (4) The oligonucleotide according to (1), or a pharmacologically acceptable salt or solvate thereof, wherein the oligonucleotide is an oligonucleotide having 15 to 18 bases and contains a sequence complementary to a region including the 92nd site from the 5' end of exon 5 of the G6PC gene having the c.648G>T mutation.

[0033] (5) The oligonucleotide according to (1), or a pharmacologically acceptable salt or solvate thereof, wherein the oligonucleotide is an 18-base oligonucleotide comprising a sequence complementary to a region including the 92nd site from the 5' end of exon 5 of the G6PC gene having the c.648G>T mutation.

[0034] (6) The oligonucleotide according to (1), or a pharmacologically acceptable salt or solvate thereof, wherein the oligonucleotide is a 17-base oligonucleotide comprising a sequence complementary to a region including the 92nd site from the 5' end of exon 5 of the G6PC gene having the c.648G>T mutation.

[0035] (7) The oligonucleotide according to (1), or a pharmacologically acceptable salt or solvate thereof, wherein the oligonucleotide is a 16-base oligonucleotide comprising a sequence complementary to a region including the 92nd site from the 5' end of exon 5 of the G6PC gene having the c.648G>T mutation.

[0036] (8) The oligonucleotide according to (1), or a pharmacologically acceptable salt or solvate thereof, wherein the oligonucleotide is a 15-base oligonucleotide containing a sequence complementary to a region including the 92nd site from the 5' end of exon 5 of the G6PC gene having the c.648G>T mutation.

[0037] (9) An oligonucleotide as described in (1), or a pharmacologically acceptable salt or solvate thereof, wherein the oligonucleotide comprises a sequence of at least 15 consecutive nucleotides of any of the sequences Nos. 1 to 32, 40 to 42, and 44 to 48 (wherein t in the sequence may be u and u may be t).

[0038] (10) The oligonucleotide according to any one of (1) to (9), or a pharmacologically acceptable salt or solvate thereof, wherein the oligonucleotide further has an oligonucleotide cleavable in vivo added to the 5' end and / or the 3' end.

[0039] (11) The oligonucleotide according to any one of (1) to (10), or a pharmacologically acceptable salt or solvate thereof, wherein at least one of the sugar and / or phosphodiester bonds constituting the oligonucleotide is modified.

[0040] (12) The oligonucleotide according to any one of (1) to (10), or a pharmacologically acceptable salt or solvate thereof, wherein the sugar constituting the oligonucleotide is D-ribofuranose, and the modification of the sugar is modification of the hydroxyl group at the 2'-position of D-ribofuranose.

[0041] (13) The oligonucleotide according to any one of (1) to (10), or a pharmacologically acceptable salt or solvate thereof, wherein the sugar constituting the oligonucleotide is D-ribofuranose, and the modification of the sugar is 2'-O-alkylation and / or 2'-, 4'-cross-linking of the D-ribofuranose.

[0042] (14) The oligonucleotide according to any one of (1) to (10), or a pharmacologically acceptable salt or solvate thereof, wherein the sugar constituting the oligonucleotide is D-ribofuranose, and the modification of the sugar is 2'-O-alkylation and / or 2'-O,4'-C-alkylation of D-ribofuranose.

[0043] (15) The oligonucleotide according to any one of (1) to (14), or a pharmacologically acceptable salt or solvate thereof, wherein the modification of the phosphodiester bond is phosphorothioate.

[0044] (16) The oligonucleotide according to any one of (1) to (15), or a pharmacologically acceptable salt or solvate thereof, wherein the oligonucleotide has a GalNAc unit bound to the 5' terminus and / or the 3' terminus.

[0045] (17) The oligonucleotide according to any one of (1) to (15), or a pharmacologically acceptable salt or solvate thereof, wherein the oligonucleotide has a GalNAc unit bound to the 5′ end.

[0046] (18) The oligonucleotide according to (16) or (17), or a pharmacologically acceptable salt or solvate thereof, wherein the GalNAc unit is of the formula

[0047] [Chemistry 1]

[0048]

[0049] [In the formula, Ra represents

[0050] [Chemistry 2]

[0051]

[0052] The group represented by Rb is expressed as

[0053] [Chemistry 3]

[0054]

[0055] The group or hydrogen atom represented by XX

[0056] [Chemistry 4]

[0057]

[0058] The group represented by, G represents 5-acetamido-2-hydroxymethyl-3,4-dihydroxytetrahydropyran-6-yl (GalNAc), Z represents an oxygen atom or a sulfur atom, L 1 and L 2 One of them represents a methylene group (CH 2 ), the other represents not separated by atoms, p, q, r, s, t and u independently represent 0 or 1, n and n' independently represent integers of 1 to 15, m and m' independently represent integers of 0 to 5, when Rb is not a hydrogen atom, v represents 1, and when Rb is a hydrogen atom, v represents 1 to 7; wherein, when n is 1, m is an integer of 0 to 5, when n is an integer of 2 to 15, m is 0, when n' is 1, m' is an integer of 1 to 5, and when n' is an integer of 2 to 15, m' is 0; a hydroxyl group, an XX group, or an OG group may be bonded to a bond farther from the phosphorus atom].

[0059] (19) The oligonucleotide according to (16) or (17), or a pharmacologically acceptable salt or solvate thereof, wherein the GalNAc unit is of the formula

[0060] [Chemistry 5]

[0061]

[0062] [Where G, Z, L 1 , L 2 , n and m have the same meanings as described above].

[0063] (20) The oligonucleotide according to (16) or (17), or a pharmacologically acceptable salt or solvate thereof, wherein the GalNAc unit is of the formula

[0064] [Chemistry 6]

[0065]

[0066] [Where G, Z, L 1 , L 2 , q, n and m have the same meanings as above, Ra' represents the formula

[0067] [Chemistry 7]

[0068]

[0069] The group represented by] The group represented by.

[0070] (21) The oligonucleotide according to (16) or (17), or a pharmacologically acceptable salt or solvate thereof, wherein the GalNAc unit is of the formula

[0071] [Chemistry 8]

[0072]

[0073] [Where G, Z, L 1 , L 2 , s, n, m and v have the same meanings as above, and Rb' is expressed as

[0074] [Chemistry 9]

[0075]

[0076] (wherein n' and m' have the same meanings as described above) or a group represented by a hydrogen atom].

[0077] (22) The oligonucleotide according to (16) or (17), or a pharmacologically acceptable salt or solvate thereof, wherein the GalNAc unit is or is of the formula

[0078] [Chemistry 10]

[0079]

[0080] [Where G, Z, L 1 , L 2 , n and m have the same meanings as described above].

[0081] (23) The oligonucleotide according to (16) or (17), or a pharmacologically acceptable salt or solvate thereof, wherein the GalNAc unit is of the formula

[0082] [Chemistry 11]

[0083]

[0084] [Where G, Z, L 1 , L 2 , n, m and Ra' have the same meanings as described above].

[0085] (24) The oligonucleotide according to (1), or a pharmacologically acceptable salt or solvate thereof, wherein the oligonucleotide is of the formula

[0086] [Chemistry 12]

[0087] RO-Xg-Xf-Xe-Xd-Xc-Xb-Xa-T

[0088] [wherein, R represents a hydrogen atom, an XX group, or a G group, T represents an oligonucleotide having no hydroxyl group at the 5' end, and Xg represents a group selected from X 1 ~X 6 and X 9 ~X 17The GalNAc unit in the group consisting of, or RO-Xg represents a group consisting of X 7 , X 8 , X 18 , X 19 , X 20 , X 21 and X 22 In the GalNAc unit of the group consisting of: Xa, Xb, Xc, Xd, Xe and Xf are independently selected from X 1 ~X 6 and X 9 ~X 17 or a GalNAc unit or a single bond in the group consisting of their optical isomers],

[0089] [Chemistry 13]

[0090]

[0091] [Chemistry 14]

[0092]

[0093] [Chemistry 15]

[0094]

[0095] [Chemistry 16]

[0096]

[0097] [Chemistry 17]

[0098]

[0099] [Chemistry 18]

[0100]

[0101] [Chemistry 19]

[0102]

[0103] [Chemistry 20]

[0104]

[0105] [Chemistry 21]

[0106]

[0107] [Chemistry 22]

[0108]

[0109] [Chemistry 23]

[0110]

[0111] [Chemistry 24]

[0112]

[0113] [Chemistry 25]

[0114]

[0115] [Chemistry 26]

[0116]

[0117] [Chemistry 27]

[0118]

[0119] [Chemistry 28]

[0120]

[0121] [Chemistry 29]

[0122]

[0123] [Chemistry 30]

[0124]

[0125] [Chemistry 31]

[0126]

[0127] [Chemistry 32]

[0128]

[0129] [Chemistry 33]

[0130]

[0131] [Chemistry 34]

[0132]

[0133] (25) A medicine comprising the oligonucleotide according to any one of (1) to (24), or a pharmacologically acceptable salt or solvate thereof.

[0134] (26) A therapeutic agent for glycogenopathies type Ia, comprising the oligonucleotide according to any one of (1) to (24), or a pharmaceutically acceptable salt or solvate thereof.

[0135] (27) A method for treating glycogenopathies type Ia, comprising administering to a subject a pharmaceutically effective amount of the oligonucleotide according to any one of (1) to (24), or a pharmaceutically acceptable salt or solvate thereof.

[0136] (28) The oligonucleotide according to any one of (1) to (24), or a pharmaceutically acceptable salt or solvate thereof, which is used for a method for treating glycogenopathies type Ia.

[0137] (29) A formulation for oral or parenteral administration, comprising the oligonucleotide according to any one of (1) to (24), or a pharmacologically acceptable salt or solvate thereof.

[0138] (30) The oligonucleotide according to any one of (1) to (24), or a pharmacologically acceptable salt or solvate thereof, for use as a medicine.

[0139] [Effects of the Invention]

[0140] According to the present invention, in patients with glycogen disease type Ia, the G6PC gene with the c.648G>T mutation in patients with glycogen disease type Ia can be repaired at the mRNA level, resulting in the expression of G6PC protein, achieving the normalization of hypoglycemia and normalization of liver hypertrophy in patients with glycogen disease type Ia, and inhibiting the progression to liver cancer.

[0141] This specification includes the contents described in the specifications and / or drawings of the Japanese patent applications, Japanese Patent Application No. 2018-43524, and Japanese Patent Application No. 2018-128015, which are the basis of the priority of this case. BRIEF DESCRIPTION OF THE DRAWINGS

[0142] Figure 1 (A) is a diagram illustrating the process of generating G6PC protein from G6PC gene. (B) is a diagram showing the splicing pattern of mRNA of G6PC gene having c.648G>T mutation in patients with glycogenotype Ia.

[0143] Figure 2 A schematic diagram illustrating the principle of treatment using the glycogen disease type Ia therapeutic agent ASO of the present invention is shown.

[0144] Figure 3 This is a diagram showing the sequence of exon 5 of the mRNA of the G6PC gene having the c.648G>T mutation in which ASOs (21e_001 to 21e_012) can bind.

[0145] Figure 4 This is a diagram showing the sequence of ASOs (21m_001 to 21m_012) that can bind to exon 5 of the mRNA of the G6PC gene having the c.648G>T mutation.

[0146] Figure 5 The following graphs show the effect of using ASO to correct abnormal splicing of G6PC mRNA measured by qRT-PCR (quantitative real time polymerase chain reaction). (A) is the case of using ASO (21e_001 to 21e_012). (B) is the case of using ASO (21m_001 to 21m_012). RQ: Relative Quantification

[0147] Figure 6 The figure shows the effect of using ASO to repair abnormal splicing of G6PC mRNA and produce normal human G6PC specific peptide measured by LC-MS / MS (Liquid chromatography-mass spectrometry / Mass spectrometry). (A) is the case of using ASO (21e_001 to 21e_012). (B) is the case of using ASO (21m_001 to 21m_012).

[0148] Figure 7 The figures show that abnormal splicing of G6PC mRNA was repaired using ASOs to show G6PC enzyme activity. (A) is the case where ASOs (21e_001 to 21e_012) were used. (B) is the case where ASOs (21m_001 to 21m_012) were used.

[0149] Figure 8 This is a diagram showing the sequence in which ASOs (21e_001 to 21e_006 and 21e_013 to 21e_022) can bind to exon 5 of the mRNA of the G6PC gene having the c.648G>T mutation.

[0150] Fig. 9 This is a graph showing the effect of correcting abnormal splicing of G6PC mRNA measured by qRT-PCR using ASO (21e_001 to 21e_006 and 21e_013 to 21e_022). RQ: Relative Quantification

[0151] Fig.10 This figure shows the effect of repairing abnormal splicing of G6PC mRNA using ASO (21e_001 to 21e_006 and 21e_013 to 21e_022) and producing normal human G6PC-specific peptides measured by LC-MS / MS.

[0152] Fig.11 This is a diagram showing the sequence of ASOs (18e_001 to 18e_017) that can bind to exon 5 of the mRNA of the G6PC gene having the c.648G>T mutation.

[0153] Fig.12 This is a diagram showing the sequence of ASOs (18m_001 to 18m_017) that can bind to exon 5 of the mRNA of the G6PC gene having the c.648G>T mutation.

[0154] Fig.13 (A) is a graph showing the effect of using ASO (18e_001 to 18e_017) to correct abnormal splicing of G6PC mRNA measured by qRT-PCR. (B) is a graph showing the effect of using ASO (18m_001 to 18m_017) to correct abnormal splicing of G6PC mRNA measured by qRT-PCR. RQ: Relative Quantification

[0155] Fig.14 (A) is a graph showing the effect of repairing abnormal splicing of G6PC mRNA using ASO (18e_001 to 18e_017) on the production of normal human G6PC-specific peptides as measured by LC-MS / MS. (B) is a graph showing the effect of repairing abnormal splicing of G6PC mRNA using ASO (18m_001 to 18m_017) on the production of normal human G6PC-specific peptides as measured by LC-MS / MS.

[0156] Fig.15 This is a diagram showing the sequence of exon 5 of the mRNA of the G6PC gene having the c.648G>T mutation in which ASOs (18e_018 to 18e_031) can bind.

[0157] Fig.16 (A) is a graph showing the effect of using ASO (18e_018 to 18e_031) to correct abnormal splicing of G6PC mRNA measured by qRT-PCR. (B) is a graph showing the effect of using ASO (18e_018 to 18e_031) to repair abnormal splicing of G6PC mRNA and produce normal human G6PC-specific peptides measured by LC-MS / MS. RQ: Relative Quantification

[0158] Fig.17It is a diagram showing the sequence of ASOs (21e_002, 18e_005, 21m_002, 18e_022, 18m_005, 15e_001, 15ed_001, 18e_008, 18e_025, 18m_008, 15e_002, and 15ed_002) that can bind to exon 5 of the mRNA of the G6PC gene having the c.648G>T mutation.

[0159] Fig.18 This is a graph showing the effect of correcting abnormal splicing of G6PC mRNA measured by qRT-PCR using ASO (21e_002, 18e_005, 21m_002, 18m_005, 18e_022, 15e_001, 15ed_001, 18e_008, 18e_025, 18m_008, 15e_002, and 15ed_002). RQ: Relative Quantification; Actn: β-actin

[0160] Fig.19 This is a graph showing the effect of the compounds of Examples 91 to 95 on correcting abnormal splicing of G6PC mRNA in the liver measured by qRT-PCR when administered to isotype knock-in mice. RQ: Relative Quantification; Actn: β-actin; mpk: mg / kg

[0161] Fig. 20 This is a graph showing the effect of correcting abnormal splicing of G6PC mRNA in the liver measured by qRT-PCR when the compounds of Example 91 and Examples 96 to 103 were administered to isotype knock-in mice. RQ: Relative Quantification; Actn: β-actin; mpk: mg / kg

[0162] Fig.21 This is a graph showing the effect of correcting abnormal splicing of G6PC mRNA in the liver measured by qRT-PCR when the compounds of Example 83, Examples 87 to 91, and Examples 104 to 107 were administered to isotype knock-in mice. RQ: Relative Quantification; Actn: β-actin; mpk: mg / kg

[0163] Fig. 22This is a graph showing the effect of correcting abnormal splicing of G6PC mRNA in the liver measured by qRT-PCR when the compounds of Example 104 and Examples 108 to 115 were administered to isotype knock-in mice. RQ: Relative Quantification; Actn: β-actin; mpk: mg / kg

[0164] Fig.23 This is a graph showing the effect of correcting abnormal splicing of G6PC mRNA in the liver measured by qRT-PCR when the compounds of Examples 105, 113, and 131 to 137 were administered to isotype knock-in mice. RQ: Relative Quantification; Actn: β-actin; mpk: mg / kg

[0165] Fig.24 This is a diagram showing the fragment analysis of the abnormal splicing repair sequence in the cultured cells obtained using the compound of Example 1.

[0166] Fig.25 This is a graph showing the effect of correcting abnormal splicing of G6PC mRNA in the liver measured by qRT-PCR when the compounds of Examples 133 and 143 to 149 were administered to isotypic knock-in mice. RQ: Relative Quantification; mpk: mg / kg

[0167] Fig.26 This is a graph showing the effect of correcting abnormal splicing of G6PC mRNA in the liver measured by qRT-PCR when the compounds of Examples 149 and 152 to 160 were administered to isotype knock-in mice. RQ: Relative Quantification; mpk: mg / kg DETAILED DESCRIPTION

[0168] Hereinafter, embodiments of the present invention will be described in further detail.

[0169] The present invention provides an oligonucleotide that repairs the G6PC gene with c.648G>T mutation in patients with glycogen disease type Ia at the mRNA level and allows the expression of normal G6PC protein, and a pharmacologically acceptable salt or solvate thereof. The oligonucleotide of the present invention is an oligonucleotide having 15 to 30 bases and containing a nucleotide sequence complementary to the cDNA of the G6PC gene with c.648G>T mutation, and contains a sequence complementary to a region of any of the 82nd to 92nd sites from the 5' end of exon 5 of the G6PC gene with c.648G>T mutation.

[0170] In the present invention, patients with glycogenopathic type Ia who have c.648G>T mutation can be used as subjects. Not only patients who are homozygous for c.648G>T mutation, but also patients who are compound heterozygous for c.648G>T mutation and other mutations can be used as subjects.

[0171] The c.648G>T mutation of the G6PC gene is a mutation from G to T at nucleotide number 728 of the Homo sapiens glucose-6-phosphatase catalytic subunit (G6PC), transcript variant 1, mRNA (NCBI-GenBank accession number NM_000151.3). It is located at the 86th position from the 5' end of exon 5. The oligonucleotide of the present invention may contain a sequence complementary to a region including any of the 86th to 92nd positions from the 5' end of exon 5 of the G6PC gene having the c.648G>T mutation, and preferably contains a sequence complementary to a region including the 92nd position.

[0172] As an oligonucleotide containing a nucleotide sequence complementary to the cDNA of the G6PC gene with the c.648G>T mutation and having a base number of 15 to 30, and containing a sequence complementary to a region of any part of No. 82 to No. 92 from the 5' end of exon 5 of the G6PC gene with the c.648G>T mutation, an oligonucleotide containing all or part of any sequence of sequence numbers 1 to 32, 40 to 42, 44 to 48 (wherein t or T in the sequence may also be u or U, and u or U may also be t or T) can be exemplified. In the present invention, the so-called "part of the sequence" is usually more than 80% of the entire sequence, preferably 85%, further preferably 90%, and most preferably 94%. The number of bases of the oligonucleotide of the present invention is more appropriately 15 to 30, preferably 15 to 21, and more preferably 15 to 18.

[0173] The nucleotides constituting the oligonucleotide (antisense oligonucleotide) of the present invention may be any of natural DNA, natural RNA, DNA / RNA chimeras, and modified forms thereof, and preferably at least one of them is a modified nucleotide.

[0174] As modified nucleotides in the present invention, examples include sugar-modified nucleotides (e.g., nucleotides in which D-ribofuranose is 2'-O-alkylated, nucleotides in which D-ribofuranose is 2'-O,4'-C-alkylated, and nucleotides in which D-ribofuranose is 2'-,4'-crosslinked), nucleotides in which phosphodiester bonds are modified (e.g., thioesterified), nucleotides in which bases are modified, and nucleotides in which these are combined. Nucleotides in which at least one D-ribofuranose constituting the antisense oligonucleotide is 2'-O-alkylated or 2'-O,4'-C-alkylated have a high binding force to RNA and high resistance to nucleases, and therefore, a therapeutic effect higher than that of natural nucleotides (i.e., oligoDNA, oligoRNA) can be expected. In addition, nucleotides in which at least one phosphodiester bond constituting the oligonucleotide is thioesterified also have a high resistance to nucleases, and therefore, a therapeutic effect higher than that of natural nucleotides (i.e., oligoDNA, oligoRNA) can be expected. Since an oligonucleotide containing both a modified sugar and a modified phosphate as described above has higher resistance to nucleases, a higher therapeutic effect can be expected.

[0175] Regarding the oligonucleotide (antisense oligonucleotide) of the present invention, examples of sugar modifications include: 2'-O-alkylation of D-ribofuranose (e.g., 2'-O-methylation, 2'-O-aminoethylation, 2'-O-propylation, 2'-O-allylation, 2'-O-methoxyethylation, 2'-O-butylation, 2'-O-pentylation, 2'-O-propargylation, etc.), 2'-O,4-D-ribofuranose, '-C-alkylene (for example, 2'-O,4'-C-ethylidene, 2'-O,4'-C-methylidene, 2'-O,4'-C-propylidene, 2'-O,4'-C-tetramethylidene, 2'-O,4'-C-pentamethylene, etc.), 2'-deoxy-2'-C,4'-C-methyleneoxymethylidene of D-ribofuranose, S-cEt (2',4'-constrained ethyl), AmNA (Amide-bridged nucleic acid), 3'-deoxy-3'-amino-2'-deoxy-D-ribofuranose, 3'-deoxy-3'-amino-2'-deoxy-2'-fluoro-D-ribofuranose, etc.

[0176] Regarding the oligonucleotide (antisense oligonucleotide) of the present invention, examples of 2'-, 4'-cross-linking modifications of sugars include: 2'-O, 4'-C-alkylation of D-ribofuranose (for example, 2'-O, 4'-C-ethylidene, 2'-O, 4'-C-methylidene, 2'-O, 4'-C-propylidene, 2'-O, 4'-C-tetramethylidene, 2'-O, 4'-C-pentamethylene, etc.), 2'-deoxy-2'-C, 4'-C-methyleneoxymethylidene of D-ribofuranose, S-cEt (2', 4'-restricted ethyl), AmNA, etc.

[0177] Regarding the oligonucleotide (antisense oligonucleotide) of the present invention, examples of modifications of the phosphodiester bond include a phosphorothioate bond, a methylphosphonate bond, a methylphosphonothioate bond, a phosphorodithioate bond, a phosphoroamidate bond, and the like.

[0178] In the present invention, examples of base modifications include: 5-methylation, 5-fluorination, 5-bromination, 5-iodination, and N4-methylation of cytosine, 5-demethylation (uracil), 5-fluorination, 5-bromination, and 5-iodination of thymine, N6-methylation and 8-bromination of adenine, and N2-methylation and 8-bromination of guanine.

[0179] The oligonucleotide (antisense oligonucleotide) of the present invention can be synthesized using a commercially available synthesizer (e.g., Model 392 obtained by the phosphoramidite method of PerkinElmer) according to the method described in the literature (Nucleic Acids Research, 12, 4539 (1984)). As for the phosphoramidite reagent that can be used at this time, natural nucleosides and 2'-O-methyl nucleosides (i.e., 2'-O-methylguanosine, 2'-O-methyladenosine, 2'-O-methylcytidine, 2'-O-methyluridine) can use commercially available reagents. 2'-O-alkylguanosine, adenosine, cytidine, and uridine in which the number of carbon atoms in the alkyl group is 2 to 6 are described below.

[0180] 2'-O-aminoethylguanosine, adenosine, cytidine, and uridine can be synthesized according to the literature (Blommers et al. Biochemistry (1998), 37, 17714-17725).

[0181] 2'-O-propylguanosine, adenosine, cytidine, and uridine can be synthesized according to the literature (Lesnik, EA et al. Biochemistry (1993), 32, 7832-7838).

[0182] Commercially available reagents can be used for 2'-O-allylguanosine, adenosine, cytidine, and uridine.

[0183] 2'-O-methoxyethylguanosine, adenosine, cytidine, and uridine can be synthesized according to patent (US Pat. No. 6,261,840) or literature (Martin, P. Helv. Chim. Acta. (1995) 78, 486-504).

[0184] 2'-O-butylguanosine, adenosine, cytidine, and uridine can be synthesized according to the literature (Lesnik, EA et al. Biochemistry (1993), 32, 7832-7838).

[0185] 2'-O-pentylguanosine, adenosine, cytidine, and uridine can be synthesized according to the literature (Lesnik, EA et al. Biochemistry (1993), 32, 7832-7838).

[0186] Commercially available reagents can be used for 2'-O-propargylguanosine, adenosine, cytidine, and uridine.

[0187] 2'-O,4'-C-methyleneguanosine, adenosine, cytidine, 5-methylcytidine and thymidine can be produced according to the method described in WO99 / 14226, and 2'-O,4'-C-alkyleneguanosine, adenosine, cytidine, 5-methylcytidine and thymidine having 2 to 5 carbon atoms in the alkylene group can be produced according to the method described in WO00 / 47599.

[0188] The nucleoside of D-ribofuranose methylened with 2'-deoxy-2'-C,4'-C-methyleneoxy can be synthesized according to the literature (Wang, G. et al. Tetrahedron (1999), 55, 7707-7724).

[0189] S-cEt (constrained ethyl) can be synthesized according to the literature (Seth, PP et al. J. Org. Chem (2010), 75, 1569-1581).

[0190] AmNA can be synthesized according to the literature (Yahara, A. et al. ChemBioChem (2012), 13, 2513-2516.) or WO2014 / 109384.

[0191] In the present invention, the nucleic acid base sequence can record adenine as (A) or (a), guanine as (G) or (g), cytosine as (C) or (c), thymine as (T) or (t), and uracil as (U) or (u). 5-methylcytosine can be used instead of cytosine. Uracil (U) or (u) and thymine (T) or (t) are compatible in nucleic acid bases. Any of uracil (U) or (u) and thymine (T) or (t) can be used to form base pairs with adenine (A) or (a) of a complementary chain.

[0192] After coupling with the phosphoramidite reagent, sulfur, tetraethylthiuram disulfide (TETD, Applied Biosystems), Beaucage reagent (Glen Research), or xanthane hydride are reacted to synthesize antisense oligonucleotides having thiophosphate bonds (Tetrahedron Letters, 32, 3005 (1991), J. Am. Chem. Soc. 112, 1253 (1990), PCT / WO98 / 54198).

[0193] As the controlled pore glass (CPG) used in the synthesizer, a commercially available controlled pore glass to which 2'-O-methyl nucleoside is bonded can be used. In addition, according to the literature (Oligonucleotide Synthesis, Edited by MJ Gait, Oxford University Press, 1984), nucleosides produced according to the method described in WO99 / 14226 for 2'-O,4'-C-methyleneguanosine, adenosine, 5-methylcytidine and thymidine, and nucleosides produced according to the method described in WO00 / 47599 for 2'-O,4'-C-alkyleneguanosine, adenosine, 5-methylcytidine and thymidine having 2 to 5 carbon atoms in the alkylene group can be bonded to CPG. By using modified CPG (described in Example 12b of Japanese Patent Laid-Open No. 7-87982), an oligonucleotide to which a 2-hydroxyethyl phosphate group is bonded at the 3' end can be synthesized. In addition, by using 3'-amino-modified group C3 CPG, 3'-amino-modified group C7 CPG, glycerol CPG (Glen Research), 3'-spacer C3 SynBase CPG 1000, and 3'-spacer C9 SynBase CPG 1000 (link technologies), oligonucleotides having a hydroxyalkylphosphate group or an aminoalkylphosphate group bound to the 3' terminus can be synthesized.

[0194] The oligonucleotide (antisense oligonucleotide) of the present invention may be bonded with GalNAc via a linker and a phosphate moiety.

[0195] The so-called GalNAc unit in the present invention is a phosphate group bonded to a linking group bonded to GalNAc, and may further have one bond. The phosphate group of the GalNAc unit may be bonded to the 5' end and / or 3' end of the oligonucleotide, and is preferably bonded to the 5' end of the oligonucleotide. When the GalNAc unit has a bond, the bond may be bonded to a hydroxyl group, GalNAc, a linking group bonded to GalNAc, a phosphate group of other GalNAc units, or a phosphate group at the 3' end of the oligonucleotide. The number of GalNAc bonded to one GalNAc unit is preferably 1 to 7, more preferably 1 to 5, particularly preferably 1 to 3, and the optimal number is 2.

[0196] In the present invention, one GalNAc unit may be bonded to an oligonucleotide, and multiple GalNAc units may be bonded to an oligonucleotide in succession. The number of GalNAc units bonded to one oligonucleotide is preferably 1 to 7, more preferably 1 to 5, particularly preferably 1 to 3, and the best number is 1.

[0197] In the present invention, the oligonucleotide (antisense oligonucleotide) bonded to the GalNAc unit may have a base sequence different from the antisense oligonucleotide, including a phosphodiester bond, and an oligonucleotide sequence that can be cut in vivo. As the chain length of the oligonucleotide that can be cut, it is suitable to be 1 to 6 nucleotides, and further suitable to be 1 to 3 nucleotides. As long as the oligonucleotide that can be cut is cut, it is not particularly limited, and all natural oligodeoxynucleotides containing DNA and all natural oligonucleotides containing RNA can be listed. As the base sequence, as long as it is a sequence that can be cut, it is not particularly limited, and 5'-TCATCA-3', 5'-CATCA-3', 5'-ATCA-3', 5'-TCA-3', 5'-CA-3', 5'-A-3', etc. can be listed.

[0198] As an example of the GalNAc unit of the present invention, for example, the general formula

[0199] [Chemistry 35]

[0200]

[0201] [In the formula, Ra represents

[0202] [Chemistry 36]

[0203]

[0204] The group represented by Rb is expressed as

[0205] [Chemistry 37]

[0206]

[0207] The group or hydrogen atom represented by XX

[0208] [Chemistry 38]

[0209]

[0210] The group represented by, G represents 5-acetamido-2-hydroxymethyl-3,4-dihydroxytetrahydropyran-6-yl (GalNAc), Z represents an oxygen atom or a sulfur atom, L 1 and L 2 One of them represents a methylene group (CH 2 ), the other represents no atom, p, q, r, s, t and u are independently 0 or 1, n and n' are independently integers of 1 to 15, m and m' are independently integers of 0 to 5, when Rb is not a hydrogen atom, v is 1, when Rb is a hydrogen atom, v is 1 to 7; wherein, when n is 1, m is an integer of 0 to 5, when n is an integer of 2 to 15, m is 0, when n' is 1, m' is an integer of 1 to 5, when n' is an integer of 2 to 15, m' is 0; a hydroxyl group, an XX group, or an OG group may be bonded to a bond far from the phosphorus atom], preferably a group represented by the formula

[0211] [Chemistry 39]

[0212]

[0213] [Where G, Z, L 1 , L 2 , n and m have the same meanings as described above], a group represented by the formula

[0214] [Chemistry 40]

[0215]

[0216] [Where G, Z, L 1 , L 2 , q, n and m have the same meanings as above, Ra' represents the formula

[0217] [Chemistry 41]

[0218]

[0219] The group represented by] The group represented by

[0220] [Chemistry 42]

[0221]

[0222] [Where G, Z, L 1 , L 2 , s, n, m and v have the same meanings as above, and Rb' is expressed as

[0223] [Chemistry 43]

[0224]

[0225] (wherein n' and m' have the same meanings as above)] a group represented by or a hydrogen atom], a group represented by

[0226] [Chemistry 44]

[0227]

[0228] [Where G, Z, L 1 , L 2 , n and m have the same meanings as above], or a group represented by the formula

[0229] [Chemistry 45]

[0230]

[0231] [Where G, Z, L 1 , L 2 , n, m and Ra' have the same meanings as described above].

[0232] In the present invention, the oligonucleotide to which the GalNAc unit is bonded is, for example, of the formula

[0233] [Chemistry 46]

[0234] RO-Xg-Xf-Xe-Xd-Xc-Xb-Xa-T

[0235] An oligonucleotide represented by [wherein R represents a hydrogen atom, an XX group, or a G group, T represents an oligonucleotide having no hydroxyl group at the 5' end, Xg represents a GalNAc unit having a bonding bond, and Xa, Xb, Xc, Xd, Xe, and Xf independently represent a GalNAc unit having a bonding bond or a single bond].

[0236] Examples of the "GalNAc unit having a bonding bond" in Xa, Xb, Xc, Xd, Xe, Xf and Xg include the following groups.

[0237] [Chemistry 47]

[0238]

[0239] [Chemistry 48]

[0240]

[0241] [Chemistry 49]

[0242]

[0243] [Chemistry 50]

[0244]

[0245] [Chemistry 51]

[0246]

[0247] [Chemistry 52]

[0248]

[0249] [Chemistry 53]

[0250]

[0251] [Chemistry 54]

[0252]

[0253] [Chemistry 55]

[0254]

[0255] [Chemistry 56]

[0256]

[0257] [Chemistry 57]

[0258]

[0259] [Chemistry 58]

[0260]

[0261] [Chemistry 59]

[0262]

[0263] [Chemistry 60]

[0264]

[0265] [Chemistry 61]

[0266]

[0267] Examples of the "GalNAc unit having a bonding bond" Xg and RO-Xg- in which R includes an XX group or a G group include the following groups.

[0268] [Chemistry 62]

[0269]

[0270] [Chemistry 63]

[0271]

[0272] [Chemistry 64]

[0273]

[0274] [Chemistry 65]

[0275]

[0276] [Chemistry 66]

[0277]

[0278] [Chemistry 67]

[0279]

[0280] [Chemistry 68]

[0281]

[0282] The oligonucleotide to which the GalNAc unit of the present invention is bonded can be synthesized by using an amide containing the GalNAc unit in the same manner as an amide reagent for nucleosides, using a commercially available synthesizer (e.g., Model 392 obtained by the phosphoramidite method of PerkinElmer) etc., according to the method described in the literature (Nucleic Acids Research, 12, 4539 (1984)). When bonded to the 5' end of the oligonucleotide, it can be synthesized by coupling the amide containing the GalNAc unit after the chain extension of the oligonucleotide portion is terminated. In addition, when bonded to the 3' end of the oligonucleotide, modified CPG (described in Example 12b of Japanese Patent Laid-Open No. 7-87982), 3'-amino-modified group C3 CPG, 3'-amino-modified group C7 CPG, glycerol CPG, (Glen Research), 3'-spacer C3 SynBase CPG 1000, 3'-spacer C9 SynBase CPG 1000 (link technologies), etc. can be used to couple the amide containing the GalNAc unit, and then extend the chain of the oligonucleotide portion to synthesize an oligonucleotide having a hydroxyalkyl phosphate group or an aminoalkyl phosphate group at the 3' end and further bonded to the GalNAc unit.

[0283] Synthesis of amides containing GalNAc units

[0284] The amide containing the GalNAc unit of the present invention can be produced according to the method A to the method I described below. In the method A to the method I, after each reaction is completed, the target compound of each reaction is collected from the reaction mixture according to a conventional method. For example, it is obtained by the following method: the reaction mixture is appropriately neutralized, and when there is an insoluble matter, it is removed by filtration, and then an immiscible organic solvent such as water and ethyl acetate is added to separate the organic layer containing the target, and after washing with water, it is dried with anhydrous sodium sulfate, etc., and the solvent is distilled off. The obtained compound can be separated and refined by conventional methods, such as silica gel column chromatography, as needed. Or it can be refined by reprecipitation and recrystallization.

[0285] A method

[0286] Method A is a method for preparing compound (8). If a compound having a stereochemical configuration opposite to that of compound (2) is used, then -P(R 3 )R 4 The stereo configuration of the secondary hydroxyl group also becomes opposite.

[0287] [Chemistry 69]

[0288]

[0289] Where R 1 is a common protecting group for hydroxyl groups, R 2 is 4,4'-dimethoxytrityl. 3 )R 4 Indicates: [R 3 and R 4 The same or different groups represent amino groups substituted with hydroxyl, protected hydroxyl, mercapto, protected mercapto, amino, alkoxy having 1 to 4 carbon atoms, alkylthio having 1 to 4 carbon atoms, cyanoalkoxy having 1 to 5 carbon atoms, or alkyl having 1 to 4 carbon atoms]. Wherein X is a carbon atom or an oxygen atom. Wherein n is an integer of 1 to 15, and m is an integer of 0 to 5. When n is 1, m is an integer of 0 to 5, and when n is an integer of 2 to 15, m is 0.

[0290] Step A-1 is a step for producing compound (3), wherein compound (1) and compound (2) are reacted in an inert solvent in the presence of an acid to form an ether bond when the epoxide ring is opened.

[0291] Examples of the inert solvent used in the reaction include hydrocarbons, aromatic hydrocarbons, halogenated hydrocarbons, ethers, acetonitrile, and the like, and dichloromethane is preferred.

[0292] Examples of the acid used in the reaction include organic acids and Lewis acids. Examples of the organic acid include trifluoromethanesulfonic acid, and examples of the Lewis acid include boron trifluoride-diethyl ether complex. Trifluoromethanesulfonic acid is preferred.

[0293] The reaction temperature varies depending on the compound (1) and the compound (2), the acid, the inert solvent and the like, but is usually -20°C to reflux temperature, preferably 30°C to 45°C.

[0294] The reaction time varies depending on the compound (1) and the compound (2), the acid, the inert solvent, the reaction temperature and the like, but is usually 15 minutes to 72 hours, preferably 2 hours to 24 hours.

[0295] Step A-2 is a step for producing compound (5), wherein compound (3) and compound (4) are reacted in an inert solvent in the presence of an acid to form a glycosidic bond.

[0296] Examples of the inert solvent used in the reaction include hydrocarbons, aromatic hydrocarbons, halogenated hydrocarbons, ethers, acetonitrile, and the like, and dichloromethane is preferred.

[0297] Examples of the acid used in the reaction include organic acids, Lewis acids, etc. Examples of the organic acid include trifluoromethanesulfonic acid, and examples of the Lewis acid include boron trifluoride-diethyl ether complex, preferably boron trifluoride-diethyl ether complex.

[0298] The reaction temperature varies depending on the compound (3), the acid, the inert solvent and the like, but is usually -20°C to reflux temperature, preferably 30°C to 45°C.

[0299] The reaction time varies depending on the compound (3), the acid, the inert solvent, the reaction temperature and the like, but is generally 15 minutes to 72 hours, preferably 2 hours to 24 hours.

[0300] Step A-3 is a step for producing compound (6) by removing R 1 The removal of the protecting group varies depending on its type and can generally be carried out according to methods known in the art of organic synthetic chemistry, for example, according to conventional methods such as those described in TH Greene, PGWuts, Protective Groups in organic Synthesis. Third Edition, 1999, John Wiley & Sons, Inc., etc.

[0301] Step A-4 is a step for preparing compound (7), comprising reacting R 2 Introduced into the primary hydroxyl group of compound (6).

[0302] Examples of the inert solvent used in the reaction include hydrocarbons, aromatic hydrocarbons, halogenated hydrocarbons, ethers, acetonitrile, and the like, and dichloromethane or pyridine is preferred.

[0303] Examples of the base used in the reaction include triethylamine, N,N-diisopropylethylamine, pyridine, and 4-dimethylaminopyridine, and N,N-diisopropylethylamine or pyridine is preferred.

[0304] The reaction temperature varies depending on the base, inert solvent, etc., but is usually -20°C to reflux temperature, preferably 10°C to 30°C.

[0305] The reaction time varies depending on the base, the inert solvent, the reaction temperature and the like, but is usually 15 minutes to 24 hours, preferably 1 hour to 4 hours.

[0306] Step A-5 is a step for preparing compound (8), wherein -P(R 3 )R 4 Introduced into the secondary hydroxyl group of compound (7).

[0307] Examples of the inert solvent used in the reaction include hydrocarbons, aromatic hydrocarbons, halogenated hydrocarbons, ethers, acetonitrile, and the like, and dichloromethane is preferred.

[0308] Examples of the base used in the reaction include triethylamine, N,N-diisopropylethylamine, pyridine, and 4-dimethylaminopyridine, and N,N-diisopropylethylamine is preferred.

[0309] The reaction temperature varies depending on the base, inert solvent, etc., but is usually -20°C to reflux temperature, preferably 10°C to 30°C.

[0310] The reaction time varies depending on the base, the inert solvent, the reaction temperature and the like, but is usually 15 minutes to 24 hours, preferably 1 hour to 4 hours.

[0311] Method B

[0312] Method B is a method for preparing compound (14) or branched compound (19). If a compound having a stereochemical configuration opposite to that of compound (2) is used, -P(R 3 )R 4 The stereo configuration of the secondary hydroxyl group also becomes opposite.

[0313] [Chemistry 70]

[0314]

[0315] Where R 1 , R 2 , R 3 , R 4 , X, n and m represent the same meanings as above, R 5 and R 6 It is a common protecting group for amino group.

[0316] Step B-1 is a step for producing compound (10), wherein compound (9) and compound (2) are reacted in an inert solvent in the presence of an acid to form an ether bond during the ring opening of the epoxide. The process can be carried out in the same manner as step A-1 described above.

[0317] Step B-2 is a step for preparing compound (11) by removing R 5 The removal of the protecting group varies depending on its type and can generally be carried out according to methods known in the art of organic synthetic chemistry, for example, according to conventional methods such as those described in TH Greene, PGWuts, Protective Groups in organic Synthesis. Third Edition, 1999, John Wiley & Sons, Inc., etc.

[0318] Step B-3 is a step for producing compound (13), wherein compound (11) and compound (12) are subjected to amide condensation in an inert solvent in the presence of a condensing agent and a base.

[0319] The inert solvent used in the reaction can be appropriately selected according to the type of the condensing agent used, and examples thereof include water, alcohols, and aprotic polar solvents, and dichloromethane or N,N-dimethylformamide is preferred.

[0320] Examples of the base used in the reaction include triethylamine, N,N-diisopropylethylamine, pyridine, and 4-dimethylaminopyridine, and N,N-diisopropylethylamine is preferred.

[0321] Examples of the condensing agent used in the reaction include WSC (1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide) and DIC (N,N'-diisopropylcarbodiimide) as carbodiimide-based condensing agents, CDI (N,N'-carbonyldiimidazole) as imidazole-based condensing agents, and DMT-MM (4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride) as triazine-based condensing agents. The condensation agent may be HATU (O-(7-azabenzotriazole-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate), TSTU (O-(N-succinimidyl)-N,N,N',N'-tetramethyluronium tetrafluoroborate), or PyBOP (1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate), etc., preferably WSC or HATU. In addition, additives such as HOAt (1-hydroxy-7-azabenzotriazole), HOBt (1-hydroxybenzotriazole), and Oxyma (ethyl (hydroxyimino) cyanoacetate) may be added as needed.

[0322] The reaction temperature varies depending on the condensing agent, base, solvent and the like, but is usually -20°C to reflux temperature, preferably 10°C to 30°C.

[0323] The reaction time varies depending on the condensing agent, base, solvent, reaction temperature and the like, but is usually 15 minutes to 72 hours, preferably 1 hour to 24 hours.

[0324] The conversion of compound (13) to compound (14) can be carried out in the same manner as in the above-mentioned method A.

[0325] Step B-4 is to remove R as a protecting group. 1 The step can be performed in the same manner as step A-3.

[0326] Step B-5 is to convert R 2 The step of introducing the primary hydroxyl group can be performed in the same manner as step A-4.

[0327] Step B-6 is to convert -P(R 3 )R 4 The step of introducing the secondary hydroxyl group can be performed in the same manner as step A-4.

[0328] Step B-7 is a step for producing compound (16), wherein compound (11) and compound (15) are subjected to amide condensation in an inert solvent in the presence of a condensing agent and a base. This can be carried out in the same manner as step B-3 described above.

[0329] Step B-8 is a step for preparing compound (17) by removing R 6 The removal of the protecting group varies depending on its type and can generally be carried out according to methods known in the art of organic synthetic chemistry, for example, according to conventional methods such as those described in TH Greene, PGWuts, Protective Groups in organic Synthesis. Third Edition, 1999, John Wiley & Sons, Inc., etc.

[0330] Step B-9 is a step for producing compound (18), wherein compound (12) and compound (17) are subjected to amide condensation in an inert solvent in the presence of a condensing agent and a base. This can be carried out in the same manner as step B-3 described above.

[0331] The conversion of compound (18) to compound (19) can be carried out in the same manner as in the above-mentioned method A.

[0332] Step B-10 is to remove R as a protecting group. 1 The step can be performed in the same manner as step A-3.

[0333] Step B-11 is to convert R 2 The step of introducing the primary hydroxyl group can be performed in the same manner as step A-4.

[0334] Step B-12 is to convert -P(R 3 )R 4 The step of introducing the secondary hydroxyl group can be performed in the same manner as step A-5.

[0335] C Method

[0336] Method C is a method for synthesizing compound (26) or branched compound (30). If a compound having a secondary hydroxyl group of compound (23) with a stereo configuration opposite to that of compound (23) is used, -P(R 3 )R 4 The stereo configuration of the secondary hydroxyl group also becomes opposite.

[0337] [Chemistry 71]

[0338]

[0339] Where R 2 , R 3 , R 4 , X, n and m represent the same meanings as above, R 8 is a common protecting group for hydroxyl groups, R 7 and R 9 The same as or different from each other are common protecting groups for amino groups.

[0340] Step C-1 is a step for producing compound (21), wherein compound (20) and compound (4) are reacted in an inert solvent in the presence of an acid to form a glycosidic bond. This can be carried out in the same manner as step A-2 described above.

[0341] Step C-2 is a step for preparing compound (22) by removing R 7 The removal of the protecting group varies depending on its type and can generally be carried out according to methods known in the art of organic synthetic chemistry, for example, according to conventional methods such as those described in TH Greene, PGWuts, Protective Groups in organic Synthesis. Third Edition, 1999, John Wiley & Sons, Inc., etc.

[0342] Step C-3 is a step for producing compound (24), wherein compound (23) is converted into an active ester in an inert solvent in the presence of an activating agent and a base, and then reacted with compound (22) to form a carbamate.

[0343] Examples of the inert solvent used in the reaction include hydrocarbons, aromatic hydrocarbons, halogenated hydrocarbons, and ethers, and dichloromethane is preferred.

[0344] The activating agent used in the reaction is not particularly limited as long as it forms an activated ester, and examples thereof include bis(pentafluorophenyl) carbonate, bis(4-nitrophenyl) carbonate, 4-nitrophenyl chloroformate, etc. 4-nitrophenyl chloroformate is preferred.

[0345] Examples of the base used in the reaction include triethylamine, N,N-diisopropylethylamine, pyridine, and 4-dimethylaminopyridine. It is preferred to use pyridine when preparing an active ester and then add N,N-diisopropylethylamine when reacting with an amine.

[0346] The reaction temperature varies depending on the activating agent, base, inert solvent and the like, but is usually -20°C to reflux temperature, preferably 10°C to 30°C.

[0347] The reaction time varies depending on the activating agent, the base, the inert solvent, the reaction temperature and the like, but is generally 15 minutes to 24 hours, preferably 1 hour to 4 hours.

[0348] Step C-4 is a step for preparing compound (25) by removing R 8 The removal of the protecting group varies depending on its type and can generally be carried out according to methods known in the art of organic synthetic chemistry, for example, according to conventional methods such as those described in TH Greene, PGWuts, Protective Groups in organic Synthesis. Third Edition, 1999, John Wiley & Sons, Inc., etc.

[0349] The conversion of compound (25) to compound (26) can be carried out in the same manner as in the above-mentioned method A.

[0350] Step C-5 is to convert R 2 The step of introducing the primary hydroxyl group can be performed in the same manner as step A-4.

[0351] Step C-6 is to convert -P(R 3 )R 4 The step of introducing the secondary hydroxyl group can be performed in the same manner as step A-5.

[0352] Step C-7 is a step for producing compound (28), wherein compound (22) and compound (27) are subjected to amide condensation in an inert solvent in the presence of a condensing agent and a base. This can be carried out in the same manner as step B-3 of method B described above.

[0353] The conversion of compound (28) to compound (29) can be carried out in the same manner as described above.

[0354] Step C-8 is to remove R which is the protecting group of the amino group of compound (28). 9 The step of can be carried out in the same manner as step C-2.

[0355] Step C-9 is a step of performing urethanization, and can be performed in the same manner as step C-3.

[0356] Step C-10 is to remove R as a protecting group. 8 The step of can be carried out in the same manner as step C-4.

[0357] The conversion of compound (29) to compound (30) can be carried out in the same manner as in the above-mentioned method A.

[0358] Step C-11 is to convert R 2 The step of introducing the primary hydroxyl group can be performed in the same manner as step A-4.

[0359] Step C-12 is to convert -P(R 3 )R 4 The step of introducing the secondary hydroxyl group can be performed in the same manner as step A-5.

[0360] D method

[0361] Method D is a method for preparing compound (35). The order of the secondary amide condensation can be changed, or the steps can be appropriately selected according to the state of the protecting group of each compound. If a compound having a stereo configuration opposite to that of the secondary hydroxyl group of compound (23) is used, then -P(R 3 )R 4 The stereo configuration of the secondary hydroxyl group also becomes opposite.

[0362] [Chemistry 72]

[0363]

[0364] Where R 2 , R 3 , R 4 , R 8 , X, n and m represent the same meanings as above, R 10 It is a common protecting group for amino group.

[0365] Step D-1 is a step for producing compound (32), wherein compound (31) and compound (12) are subjected to amide condensation in an inert solvent in the presence of a condensing agent and a base. This can be carried out in the same manner as step B-3 described above.

[0366] Step D-2 is a step for preparing compound (33) by removing R 10 The removal of the protecting group varies depending on its type and can generally be carried out according to methods known in the art of organic synthetic chemistry, for example, according to conventional methods such as those described in TH Greene, PGWuts, Protective Groups in organic Synthesis. Third Edition, 1999, John Wiley & Sons, Inc., etc.

[0367] Step D-3 is a step for producing compound (34), wherein compound (23) is converted to an active ester in an inert solvent in the presence of an activating agent and a base, and then reacted with compound (33) to form a carbamate. This can be carried out in the same manner as step C-3 described above.

[0368] The conversion of compound (34) to compound (35) can be carried out in the same manner as in the above-mentioned method C.

[0369] Step D-4 is to remove R as a protecting group. 8 The step of can be carried out in the same manner as step C-4.

[0370] Step D-5 is to convert R 2 The step of introducing the primary hydroxyl group can be carried out in the same manner as step C-5.

[0371] Step D-6 is to convert -P(R 3 )R 4 The step of introducing the secondary hydroxyl group can be carried out in the same manner as step C-6.

[0372] Step D-7 is a step for producing compound (36), wherein compound (23) is converted to an active ester in an inert solvent in the presence of an activating agent and a base, and then reacted with compound (31) to form a carbamate. This can be carried out in the same manner as step C-3 described above.

[0373] Step D-8 is to remove only R which is the protecting group of the amino group of compound (36). 10 To prepare compound (37), or to simultaneously remove R as a protective group for the hydroxyl group 8 With R as a protecting group for the amino group 10 The removal of the protecting group varies depending on the type of the protecting group, and can generally be carried out according to a method known in the art of organic synthetic chemistry, for example, according to a conventional method such as that described in TH Greene, PGWuts, Protective Groups in organic Synthesis. Third Edition, 1999, John Wiley & Sons, Inc., etc.

[0374] Step D-9 is a step for producing compound (34), wherein compound (37) and compound (12) are subjected to amide condensation in an inert solvent in the presence of a condensing agent and a base. This can be carried out in the same manner as step B-3 described above.

[0375] Step D-10 is a step for preparing compound (39), wherein compound (38) and compound (12) are subjected to amide condensation in an inert solvent in the presence of a condensing agent and a base. This can be carried out in the same manner as step B-3 described above.

[0376] The conversion of compound (39) to compound (35) can be carried out through steps D-5 and D-6.

[0377] E-method

[0378] Method E is a method for preparing a branched compound (44) using the compound (31) or the intermediate (38) synthesized in method D. The order of the secondary amide condensation can be changed, or the steps can be appropriately selected according to the state of the protecting groups of each compound. If a compound having a stereo configuration opposite to that of the secondary hydroxyl group of compound (23) is used, -P(R 3 )R 4 The stereo configuration of the secondary hydroxyl group also becomes opposite.

[0379] [Chemistry 73]

[0380]

[0381] Where R 2 , R 3 , R 4 , R 6 , R 8 , R 10 , X, n and m have the same meanings as described above.

[0382] Step E-1 is a step for producing compound (40), wherein compound (31) and compound (15) are subjected to amide condensation in an inert solvent in the presence of a condensing agent and a base. This can be carried out in the same manner as step B-3 described above.

[0383] Step E-2 is to remove only R which is the protecting group of the amino group of compound (40). 6 The removal of the protecting group varies depending on the type of the protecting group, and can generally be carried out according to a method known in the art of organic synthetic chemistry, for example, according to a conventional method such as that described in TH Greene, PGWuts, Protective Groups in organic Synthesis. Third Edition, 1999, John Wiley & Sons, Inc., etc.

[0384] Step E-3 is a step for producing compound (42), wherein compound (41) and compound (12) are subjected to amide condensation in an inert solvent in the presence of a condensing agent and a base. This can be carried out in the same manner as step B-3 described above.

[0385] The conversion of compound (42) to compound (43) can be carried out in the same manner as in the above-mentioned method D.

[0386] Step E-4 is to remove R as the protecting group of the amino group of compound (42). 10 The step can be carried out in the same manner as step D-2 described above.

[0387] Step E-5 is a step of performing urethanization and can be performed in the same manner as in the above-mentioned step D-3.

[0388] The conversion of compound (43) to compound (44) can be carried out in the same manner as in the above-mentioned method C.

[0389] Step E-6 is to remove R which is the protecting group of the hydroxyl group of compound (43). 8 The step of can be carried out in the same manner as step C-4.

[0390] Step E-7 is to convert R 2 The step of introducing the primary hydroxyl group can be carried out in the same manner as step C-5.

[0391] Step E-8 is to convert -P(R 3 )R 4 The step of introducing the secondary hydroxyl group can be carried out in the same manner as step C-6.

[0392] Step E-9 is a step for preparing compound (45), wherein compound (38) and compound (15) are subjected to amide condensation in an inert solvent in the presence of a condensing agent and a base. This can be carried out in the same manner as step B-3 described above.

[0393] The conversion of compound (45) to compound (46) can be carried out in the same manner as in steps E-2 and E-3 described above.

[0394] Step E-10 is to remove R as the protecting group of the amino group of compound (45). 6 The removal of the protecting group varies depending on its type and can generally be carried out according to methods known in the art of organic synthetic chemistry, for example, according to conventional methods such as those described in TH Greene, PGWuts, Protective Groups in organic Synthesis. Third Edition, 1999, John Wiley & Sons, Inc., etc.

[0395] Step E-11 is a step of introducing compound (15) by amide condensation and can be carried out in the same manner as the above-mentioned step B-3.

[0396] The conversion of compound (46) to compound (44) can be carried out through steps E-7 and E-8.

[0397] F Method

[0398] Method F is a method for producing a branched compound (49) using the intermediate (3) synthesized by method A. When a compound (3) having a secondary hydroxyl group with an opposite stereoconfiguration is used, the stereoconfiguration of the secondary hydroxyl group bonded with GalNAc in compound (49) also becomes opposite.

[0399] [Chemistry 74]

[0400]

[0401] Where R 1 , R 3 , R 4 , X, n and m have the same meanings as described above.

[0402] Step F-1 is a step for producing compound (47), wherein compound (3) and compound (4) are reacted in an inert solvent in the presence of an acid to form two glycosidic bonds. This can be carried out in the same manner as step A-2 described above.

[0403] Step F-2 is a step for preparing compound (48) by removing R 1 The process can be carried out in the same manner as step A-3 described above.

[0404] Step F-3 is a step for preparing compound (49) by introducing -P(R 3 )R 4 The process can be carried out in the same manner as step A-5 described above.

[0405] G Method

[0406] Method G is a method for producing a branched compound (55) using the intermediate (22) used in Method C. When a compound (52) or (56) having a secondary hydroxyl group with a reversed stereoconfiguration is used, the stereoconfiguration of the secondary hydroxyl group bonded with GalNAc in compound (55) also becomes reversed.

[0407] [Chemistry 75]

[0408]

[0409] Where R 3 , R4 , X, n and m represent the same meanings as above, r represents 0 or 1, R 11 is a common protecting group for amino group, R 12 and R 13 It is a common protecting group for hydroxyl group.

[0410] Step G-1 is to carry out amide condensation of compound (22) and compound (50) in an inert solvent in the presence of a condensing agent and a base. This can be carried out in the same manner as Step B-3 described above.

[0411] Step G-2 is to remove R as a protecting group. 11 The removal of the protecting group varies depending on its type and can generally be carried out according to methods known in the art of organic synthetic chemistry, for example, according to conventional methods such as those described in TH Greene, PGWuts, Protective Groups in organic Synthesis. Third Edition, 1999, John Wiley & Sons, Inc., etc.

[0412] Step G-3 is a step of performing carbamate conversion, which can be performed in the same manner as the above-mentioned step C-3, wherein when r=0, compound (22) is carbamate converted, and when r=1, compound (52) is carbamate converted.

[0413] Step G-4 is to remove R as a protecting group 12 The removal of the protecting group varies depending on its type and can generally be carried out according to methods known in the art of organic synthetic chemistry, for example, according to conventional methods such as those described in TH Greene, PGWuts, Protective Groups in organic Synthesis. Third Edition, 1999, John Wiley & Sons, Inc., etc.

[0414] Step G-5 is a step for preparing compound (55) by introducing -P(R 3 )R 4 The process can be carried out in the same manner as step A-5 described above.

[0415] Step G-6 is a step of performing carbamate conversion. It can be performed in the same manner as the above-mentioned step C-3. When r=0, compound (22) is carbamate converted, and when r=1, compound (52) is carbamate converted.

[0416] Step G-7 is to remove R as a protecting group. 13The removal of the protecting group varies depending on its type and can generally be carried out according to methods known in the art of organic synthetic chemistry, for example, according to conventional methods such as those described in TH Greene, PGWuts, Protective Groups in organic Synthesis. Third Edition, 1999, John Wiley & Sons, Inc., etc.

[0417] Step G-8 is a step of performing carbamate conversion and can be performed in the same manner as in the above-mentioned step C-3.

[0418] H Method

[0419] Method H is a method for preparing a branched compound (61) using the intermediate (22) used in Method C or the intermediate (51) used in Method G. The stereoconfiguration of the compound (52) or the compound (56) is not limited.

[0420] [Chemistry 76]

[0421]

[0422] Where R 3 , R 4 ,X,n,m,r,R 12 and R 13 Means the same as described above.

[0423] Step H-1 is a step of performing carbamate conversion, which can be performed in the same manner as Step C-3 described above, wherein when r=0, compound (22) is carbamate converted, and when r=1, compound (51) is carbamate converted.

[0424] Step H-2 is to remove R as a protecting group. 13 The removal of the protecting group varies depending on its type and can generally be carried out according to methods known in the art of organic synthetic chemistry, for example, according to conventional methods such as those described in TH Greene, PGWuts, Protective Groups in organic Synthesis. Third Edition, 1999, John Wiley & Sons, Inc., etc.

[0425] Step H-3 is a step for preparing compound (61), wherein -P(R 3 )R 4 The process can be carried out in the same manner as step A-5 described above.

[0426] Step H-4 is a step of performing carbamate conversion, which can be performed in the same manner as the above-mentioned step C-3, wherein when r=0, compound (22) is carbamate converted, and when r=1, compound (52) is carbamate converted.

[0427] Step H-5 is to remove R as a protecting group. 12 The removal of the protecting group varies depending on its type and can generally be carried out according to methods known in the art of organic synthetic chemistry, for example, according to conventional methods such as those described in TH Greene, PGWuts, Protective Groups in organic Synthesis. Third Edition, 1999, John Wiley & Sons, Inc., etc.

[0428] Step H-6 is a step of performing urethanization and can be performed in the same manner as the above-mentioned step C-3.

[0429] I method

[0430] Method I is a method for preparing branched compounds (73) and (74). For example, by using compound (70), the protecting group of the 6-hydroxyl group of the GalNAc moiety can be appropriately selected. 14 When R is acetyl, compound (22) synthesized by method C is used as compound (70). 14 In the case of 4,4'-dimethoxytrityl group, for example, a compound which can be synthesized according to the Reference Example described below is used as compound (70).

[0431] [Chemistry 77]

[0432]

[0433] Where R 12 and R 13 is a common protective group for hydroxyl, preferably benzyl. 14 is a common protective group for hydroxyl, preferably acetyl or 4,4'-dimethoxytrityl. 15 is a common protecting group for the carbonyl group, preferably a benzyl group. 3 )R 4 Indicates: [R 3 and R 4The same or different groups represent amino groups substituted with hydroxyl, protected hydroxyl, mercapto, protected mercapto, amino, alkoxy having 1 to 4 carbon atoms, alkylthio having 1 to 4 carbon atoms, cyanoalkoxy having 1 to 5 carbon atoms, or alkyl having 1 to 4 carbon atoms]. Wherein X is carbon or oxygen. Wherein n is an integer of 1 to 15, and m is an integer of 0 to 5. When n is 1, m is an integer of 0 to 5, and when n is an integer of 2 to 15, m is 0. Wherein r is independently 0 or 1. Wherein v is 1 to 7.

[0434] Step I-1 is a step of performing urethanization and can be performed in the same manner as Step C-3 described above.

[0435] Step I-2 is to remove R as a protecting group 12 or R 13 With R 15 The removal of the protecting group varies depending on its type and can generally be carried out according to methods known in the art of organic synthetic chemistry, for example, according to conventional methods such as those described in TH Greene, PGWuts, Protective Groups in organic Synthesis. Third Edition, 1999, John Wiley & Sons, Inc., etc.

[0436] Step I-3 is to carry out amide condensation with compound (22) in an inert solvent in the presence of a condensing agent and a base. It can be carried out in the same manner as Step B-3 described above.

[0437] Step I-4 is a step for producing compound (73) or compound (74) by introducing -P(R 3 )R 4 The process can be carried out in the same manner as step A-5 described above.

[0438] The oligonucleotide (antisense oligonucleotide) of the present invention can be used in the form of medicine, especially for the treatment of glycogenopathies type Ia. The treatment includes both prevention and post-treatment.

[0439] The oligonucleotide (antisense oligonucleotide) of the present invention can also be used in the form of a pharmacologically acceptable salt. The so-called "pharmacologically acceptable salt" refers to a salt of an oligonucleotide (antisense oligonucleotide), and examples of such salts include: alkali metal salts such as sodium salts, potassium salts, and lithium salts; alkaline earth metal salts such as calcium salts and magnesium salts; metal salts such as aluminum salts, iron salts, zinc salts, copper salts, nickel salts, and cobalt salts; inorganic salts such as ammonium salts; such as tert-octylamine salts, dibenzylamine salts, morpholine salts, glucosamine salts, phenylglycine alkyl ester salts, ethylenediamine salts, N-methyl reduced glucosamine salts, guanidine salts, diethylamine salts, triethylamine salts, dicyclohexylamine salts, N,N'-dibenzylethylenediamine salts, chloroprocaine salts, procaine salts, diethanolamine salts, N-benzyl-phenethylamine salts, piperazine salts, tetradecene ... Amine salts such as organic salts of methylammonium salts and tris(hydroxymethyl)aminomethane salts; hydrohalide salts such as hydrofluoride, hydrochloride, hydrobromide, and hydroiodide; inorganic acid salts such as nitrate, perchlorate, sulfate, and phosphate; lower alkane sulfonates such as methanesulfonate, trifluoromethanesulfonate, and ethanesulfonate; aryl sulfonates such as benzenesulfonate and p-toluenesulfonate; organic acid salts such as acetate, malate, fumarate, succinate, citrate, tartrate, oxalate, and maleate; amino acid salts such as glycine, lysine, arginine, ornithine, glutamate, and aspartate, preferably alkali metal salts, more preferably sodium salts. These salts can be manufactured by known methods.

[0440] Furthermore, an oligonucleotide (antisense oligonucleotide) and a pharmacologically acceptable salt thereof may exist in the form of a solvate (eg, a hydrate), and such a solvate may also be used.

[0441] When the oligonucleotide (antisense oligonucleotide) of the present invention, its pharmacologically acceptable salt or solvate is used for the treatment of glycogen disease Ia, it can be administered orally by tablets, capsules, granules, powders or syrups, or non-orally by injections, suppositories, patches or external preparations, as a mixture thereof or with suitable pharmacologically acceptable excipients, diluents, etc.

[0442] These preparations are prepared using excipients (e.g., organic excipients such as lactose, saccharide, glucose, mannitol, sorbitol, starch derivatives such as corn starch, potato starch, α-starch, dextrin, cellulose derivatives such as crystalline cellulose, gum arabic, dextran, pullulan, inorganic excipients such as light anhydrous silicic acid, synthetic aluminum silicate, calcium silicate, magnesium aluminum silicate, phosphates such as calcium hydrogen phosphate, carbonates such as calcium carbonate, sulfates such as calcium sulfate, etc., etc. ), lubricants (e.g., stearic acid; metal stearates such as calcium stearate and magnesium stearate; talc; colloidal silicon dioxide; waxes such as beeswax and spermaceti; 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 silicic acid hydrate; the starch derivatives, etc.), binders (e.g., hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl pyrrolidone, polyethylene glycol, and the excipients disintegrants (e.g., cellulose derivatives such as low-substituted hydroxypropyl cellulose, carboxymethyl cellulose, carboxymethyl cellulose calcium, and internally cross-linked sodium carboxymethyl cellulose; chemically modified starch-celluloses such as carboxymethyl starch, sodium carboxymethyl starch, and cross-linked polyvinyl pyrrolidone); emulsifiers (e.g., colloidal clays such as bentonite and colloidal magnesium aluminum silicate; metal hydroxides such as magnesium hydroxide and aluminum hydroxide; anionic surfactants such as sodium lauryl sulfate and calcium stearate; benzalkonium chloride The invention relates to a preparation method of the present invention which comprises the following additives: cationic surfactants such as polyoxyethylene alkyl ethers, polyoxyethylene sorbitan fatty acid esters, nonionic surfactants such as sucrose fatty acid esters, stabilizers (such as parabens such as methyl paraben and propyl paraben, alcohols such as chlorobutanol, benzyl alcohol, and phenylethyl alcohol, benzalkonium chloride, phenols such as phenol and cresol, thimerosal, dehydroacetic acid, sorbic acid, etc.), flavoring and deodorizing agents (for example, commonly used sweeteners, sour flavors, and spices, etc.), diluents, etc., and the like, and the preparation method is known in the art.

[0443] The therapeutic drug of the present invention may contain 0.1 to 250 μmoles / mL of oligonucleotide (antisense oligonucleotide), preferably 1 to 50 μmoles / mL of oligonucleotide (antisense oligonucleotide), a pharmacologically acceptable salt or solvate thereof, 0.02 to 10% w / v of a carbohydrate or polyol, and 0.01 to 0.4% w / v of a pharmacologically acceptable surfactant.

[0444] As the carbohydrate, monosaccharides or disaccharides are particularly preferred. Examples of these carbohydrates and polyols include glucose, galactose, mannose, lactose, maltose, mannitol, and sorbitol. These may be used alone or in combination.

[0445] In addition, preferred examples of surfactants in the present invention include polyoxyethylene sorbitan mono-tri-esters, alkylphenyl polyoxyethylene, sodium taurocholate, sodium cholate, and polyol esters. Among them, polyoxyethylene sorbitan mono-tri-esters are particularly preferred, and as esters, oleate, laurate, stearate, and palmitate are particularly preferred. These may be used alone or in combination.

[0446] Furthermore, the therapeutic agent of the present invention may preferably contain 0.03 to 0.09 M of a pharmacologically acceptable neutral salt, such as sodium chloride, potassium chloride and / or calcium chloride.

[0447] In addition, the therapeutic agent of the present invention may further preferably contain a pharmacologically acceptable buffer at 0.002 to 0.05 M. Preferred examples of buffers include sodium citrate, sodium glycine, sodium phosphate, and tris(hydroxymethyl)aminomethane. These buffers may be used alone or in combination.

[0448] Furthermore, the therapeutic agent can be supplied in a solution state. However, since there are situations where a certain period of time must be preserved, etc., in order to stabilize the oligonucleotide (antisense oligonucleotide) and prevent the reduction of the therapeutic effect, it is usually preferred to freeze-dry in advance. In this case, it is reconstituted (reconstruction) with a dissolving solution (distilled water for injection, etc.) when used, that is, it is made into a liquid state for administration. Therefore, the therapeutic agent of the present invention also includes a therapeutic agent in a freeze-dried state used for reconstructing with a dissolving solution in a manner that each component becomes a specific concentration range. In order to promote the solubility of the freeze-dried material, it can be further made to contain amino acids such as albumin and glycine.

[0449] The oligonucleotide (antisense oligonucleotide) of the present invention can be encapsulated using a lipid described in International Publication No. 2015 / 005253 and administered in the form of nucleic acid lipid nanoparticles or liposomes described in International Publication No. 2015 / 005253.

[0450] When the oligonucleotide (antisense oligonucleotide) of the present invention, or a pharmacologically acceptable salt or solvate thereof is administered to humans, for example, a daily dosage of about 0.01 to 100 mg / kg (body weight), preferably 0.1 to 20 mg / kg (body weight) for adults, can be administered subcutaneously, intravenously, or intravenously once or in divided doses. The dosage or frequency of administration can be appropriately changed depending on the type of disease, symptoms, age, administration method, etc.

[0451] The administration of the oligonucleotide (antisense oligonucleotide) of the present invention, its pharmacologically acceptable salt or solvate to patients with glycogen disease type Ia can be carried out in the following manner, for example. That is, the oligonucleotide (antisense oligonucleotide), its pharmacologically acceptable salt or solvate is manufactured by a method known to the industry, and sterilized by a conventional method, for example, a 125mg / mL injection solution is prepared. In a manner where the dosage of the oligonucleotide (antisense oligonucleotide) is, for example, 10mg per 1kg of body weight, the solution is drip-administered into the patient's vein, for example, in the form of an infusion. Administration is carried out, for example, at intervals of 1 week, and thereafter, the therapeutic effect is confirmed by blood sugar level, blood lactate level, hepatomegaly / glycogen level obtained by CT (Computed Tomography, computer tomography technology), and the like, and the treatment is appropriately repeated.

[0452] Example

[0453] The present invention will be specifically described below by way of examples. It should be noted that these examples are intended to illustrate the present invention and are not intended to limit the scope of the present invention.

[0454] (Examples 1 to 11)

[0455] HO-A e2s -G m1s -A m1s -T e2s -A m1s -A m1s -A e2s -A m1s -U m1s -C e2s -C m1s -G m1s -A e2s -U m1s -G m1s -G e2s -C m1s -G m1s -A e2 - s A m1s -G e2t -H(21e_002)(Sequence number 1)

[0456] The synthesis was performed using a nucleic acid automatic synthesizer ("ABI 394 DNA / RNA Synthesizer" manufactured by Applied Biosystems) by the phosphoramidite method (Nucleic Acids Research, 12, 4539 (1984)). As reagents, activator solution-3 (0.25 mol / L 5-benzylthio-1H-tetrazole / acetonitrile solution, manufactured by Wako Pure Chemical Industries, Ltd., product number 013-20011), CAP A for AKTA (1-methylimidazole / acetonitrile solution, manufactured by Sigma-Aldrich, product number L040050), Cap B1 for AKTA (acetic anhydride / acetonitrile solution, manufactured by Sigma-Aldrich, product number L050050), Cap B2 for AKTA (pyridine / acetonitrile solution, manufactured by Sigma-Aldrich, product number L050150), and DCA Deblock (dichloroacetic acid / toluene solution, manufactured by Sigma-Aldrich, product number L023050) were used. As a sulfurizing agent for forming a phosphorothioate bond, acetonitrile (dehydrated, Kanto Chemical, product number 01837-05) and pyridine (dehydrated, Kanto Chemical, product number 11339-05) 1:1 (v / v) solution of phenylacetyl disulfide (Carbosynth, product number FP07495) were used to make 0.2 M. As an amide reagent, the phosphoramidite of 2'-O-Me nucleoside (adenosine form product number ANP-5751, cytidine form product number ANP-5752, guanosine form product number ANP-5753, uridine form product number ANP-5754) was produced by ChemGenes. The non-natural phosphoramidites were Example 14 (5'-O-dimethoxytrityl-2'-O,4'-C-ethylidene-6-N-benzoyladenosine-3'-O-(2-cyanoethyl N,N-diisopropyl) phosphoramidite) and Example 27 (5'-O-dimethoxytrityl-2'-O,4'-C-ethylidene-2-N-isobutyrylguanosine-3'-O-(2-cyanoethyl N,N-diisopropyl) phosphoramidite) of Japanese Patent Laid-Open No. 2000-297097. N-diisopropyl) phosphoramidite), Example 22 (5'-O-dimethoxytrityl-2'-O,4'-C-ethylidene-4-N-benzoyl-5-methylcytidine-3'-O-(2-cyanoethyl N,N-diisopropyl) phosphoramidite), Example 9 (5'-O-dimethoxytrityl-2'-O,4'-C-ethylidene-5-methyluridine-3'-O-(2-cyanoethyl N,N-diisopropyl) phosphoramidite).Glen Unysupport 0.1 μmol (manufactured by Glen Research) was used as a solid phase support to synthesize the expressed compound. The procedure used a 0.2 μmol scale attached to a nucleic acid automatic synthesizer, wherein the time required for amide condensation was set to 600 seconds and the time required for sulfurization was set to 150 seconds.

[0457] The protected oligonucleotide-related substance having the target sequence was treated with 300 μL of concentrated aqueous ammonia to cut the oligomer from the support and remove the protecting groups of the cyanoethyl group on the phosphorus atom and the nucleic acid base. Purification was performed using ClarityQSP (manufactured by Phenomenex) according to the attached instructions.

[0458] The base sequence of this compound is a sequence complementary to the 92nd to 112th sequence from the 5' end of exon 5 of the c.648G>T variant G6PC gene in which the G at nucleotide number 728 is mutated to T in the catalytic subunit (G6PC) of Homo sapiens glucose-6-phosphatase, transcript variant 1, mRNA (NCBI-GenBank accession number NM_000151.3).

[0459] The compounds of Examples 2 to 11 were also synthesized in the same manner as in Example 1. The data of Example 1 and Examples 2 to 11 are shown in Table 1.

[0460] Table 1

[0461]

[0462] In the sequence in the table, capital letters represent 2'-O,4'-C-ethylene cross-linked nucleic acid, and lowercase letters represent 2'-OMe-RNA. C in 2'-O,4'-C-ethylene cross-linked nucleic acid is 5-methylcytosine. Start and end represent the numbering from the 5' end of exon 5 of the c.648G>T variant G6PC gene in which the G at nucleotide number 728 of the homo sapiens glucose-6-phosphatase catalytic subunit (G6PC), transcript variant 1, mRNA (NCBI-GenBank accession number NM_000151.3) is mutated to T. The compounds were identified by negative ion ESI (electrospray ionization) mass analysis, and the measured values ​​are shown in the table.

[0463] (Examples 12 to 22)

[0464] HO-A m1s -G m1s -A m1s -U m1s -A m1s -A m1s -A m1s -Am1s -U m1s -C m1s -C m1s -G m1s -A m1s -U m1s -G m1s -G m1s -C m1s -G m1s -A m1s -AG m1t -H(21m_002)(Sequence number 1)

[0465] Using a nucleic acid automatic synthesizer (MerMade 192X manufactured by BioAutomation), synthesis and purification were carried out in the same manner as in Example 1 to obtain the target compound.

[0466] The base sequence of this compound is a sequence complementary to the 92nd to 112th sequence from the 5' end of exon 5 of the c.648G>T variant G6PC gene in which the G at nucleotide number 728 is mutated to T in the catalytic subunit (G6PC) of Homo sapiens glucose-6-phosphatase, transcript variant 1, mRNA (NCBI-GenBank accession number NM_000151.3).

[0467] The compounds of Examples 13 to 22 were also synthesized in the same manner as in Example 12. The data of Example 12 and Examples 13 to 22 are shown in Table 2.

[0468] Table 2

[0469]

[0470] The lowercase letters in the sequence in the table represent 2'-OMe-RNA. The start and end represent the numbering from the 5' end of exon 5 of the G6PC gene, transcript variant 1, mRNA (NCBI-GenBank accession number NM_000151.3), in which the G at nucleotide number 728 is mutated to T, c.648G>T variant. The compounds were identified by negative ion ESI mass analysis, and the measured values ​​are shown in the table.

[0471] (Examples 23 to 50)

[0472] HO-G e2s -A m1s -U m1s -A e2s -A m1s -A m1s -A e2s -U m1s -C m1s -Ce2s -G m1s -A m1s -T e2s -G m1s -G m1s -C e2s -G m1s -A m1s -A e2s -G m1s -C e2t -H(21e_015)(Sequence number 12)

[0473] The target compound was obtained by synthesis and purification in the same manner as in Example 12.

[0474] The base sequence of this compound is a sequence complementary to the 91st to 111th sequence from the 5' end of exon 5 of the c.648G>T variant G6PC gene in which the G at nucleotide number 728 is mutated to T in the catalytic subunit (G6PC) of Homo sapiens glucose-6-phosphatase, transcript variant 1, mRNA (NCBI-GenBank accession number NM_000151.3).

[0475] The compounds of Examples 24 to 50 were also synthesized in the same manner as in Example 23. The data of Example 23 and Examples 24 to 50 are shown in Table 3.

[0476] Table 3

[0477]

[0478] In the sequence in the table, capital letters represent 2'-O,4'-C-ethylene cross-linked nucleic acid, and lowercase letters represent 2'-OMe-RNA. C in 2'-O,4'-C-ethylene cross-linked nucleic acid is 5-methylcytosine. Start and end represent the numbering from the 5' end of exon 5 of the c.648G>T variant G6PC gene in which the G at nucleotide number 728 of the catalytic subunit of glucose-6-phosphatase (G6PC), transcript variant 1, mRNA (NCBI-GenBank accession number NM_000151.3) is mutated to T. The compounds were identified by negative ion ESI mass analysis, and the measured values ​​are shown in the table.

[0479] (Examples 51 to 69)

[0480] HO-U m1s -A e2s -A m1s -A m1s -A e2s -U m1s -C m1s -C e2s -G m1s -Am1s -T e2s -G m1s -G m1s -C e2s -G m1s -A m1s -A e2s -G m1t -H(18e_005)(Sequence number 20)

[0481] The synthesis was performed in the same manner as in Example 12. The protected oligonucleotide-related substance having the target sequence was treated with 450 μL of concentrated aqueous ammonia to cut the oligomer from the support, and the protecting group cyanoethyl on the phosphorus atom and the protecting group on the nucleic acid base were removed. The mixed solution of the oligomer was mixed with 300 μL of Clarity QSP DNA Loading Buffer (manufactured by Phenomenex) and added to a Clarity SPE 96-well plate (manufactured by Phenomenex). After adding 1 mL of a Clarity QSP DNA Loading Buffer: water = 1:1 solution, 3 mL of water, 3 mL of a 3% dichloroacetic acid (DCA) aqueous solution, and 6 mL of water in sequence, the components extracted by a 20 mM Tris aqueous solution: acetonitrile = 9:1 solution were collected. After the solvent was distilled off, the target compound was obtained.

[0482] The base sequence of this compound is a sequence complementary to the 91st to 109th sequence from the 5' end of exon 5 of the c.648G>T variant G6PC gene in which the G at nucleotide number 728 is mutated to T in the catalytic subunit (G6PC) of Homo sapiens glucose-6-phosphatase, transcript variant 1, mRNA (NCBI-GenBank accession number NM_000151.3).

[0483] The compounds of Examples 52 to 69 were also synthesized in the same manner as in Example 51. The data of Example 51 and Examples 52 to 69 are shown in Table 4.

[0484] Table 4

[0485]

[0486] In the sequence in the table, capital letters represent 2'-O,4'-C-ethylene cross-linked nucleic acid, and lowercase letters represent 2'-OMe-RNA. C in 2'-O,4'-C-ethylene cross-linked nucleic acid is 5-methylcytosine. Start and end represent the numbering from the 5' end of exon 5 of the c.648G>T variant G6PC gene in which the G at nucleotide number 728 of the catalytic subunit of glucose-6-phosphatase (G6PC), transcript variant 1, mRNA (NCBI-GenBank accession number NM_000151.3) is mutated to T. The compounds were identified by negative ion ESI mass analysis, and the measured values ​​are shown in the table.

[0487] (Examples 70 to 82)

[0488] HO-U m1s -A m1s -A m1s -A m1s -A m1s -U m1s -C m1s -C m1s -G m1s -A m1s -U m1s -G m1s -G m1s -C m1s -G m1s -A m1s -A m1s -G m1t -H(18m_005)(Serial No. 20)

[0489] Using a nucleic acid automatic synthesizer (MerMade 192X manufactured by BioAutomation), synthesis and purification were carried out in the same manner as in Example 12 to obtain the target compound.

[0490] The base sequence of this compound is a sequence complementary to the 92nd to 109th sequence from the 5' end of exon 5 of the c.648G>T variant G6PC gene in which the G at nucleotide number 728 is mutated to T in the catalytic subunit (G6PC) of Homo sapiens glucose-6-phosphatase, transcript variant 1, mRNA (NCBI-GenBank accession number NM_000151.3).

[0491] The compounds of Examples 71 to 82 were also synthesized in the same manner as in Example 70. The data of Example 70 and Examples 71 to 82 are shown in Table 5.

[0492] Table 5

[0493]

[0494] The lowercase letters in the sequence in the table represent 2'-OMe-RNA. The start and end represent the numbering from the 5' end of exon 5 of the G6PC gene, transcript variant 1, mRNA (NCBI-GenBank accession number NM_000151.3), in which the G at nucleotide number 728 is mutated to T, c.648G>T variant. The compounds were identified by negative ion ESI mass analysis, and the measured values ​​are shown in the table.

[0495] (Reference Example 1)

[0496] HO-A e2s -G m1s -A m1s -T e2s -A m1s -A m1s -A e2s -A m1s -U m1s -C e2s -C m1s -G m1s -A e2s -U m1s -G m1s -G e2s -C m1s -G m1s -A e2 - s A m1s -G e2t -H(21e_001)(Sequence number 33)

[0497] The target compound was obtained by synthesis and purification in the same manner as in Example 1.

[0498] The base sequence of the compound is a sequence complementary to the 95th to 115th sequence from the 5' end of exon 5 of the c.648G>T variant G6PC gene in which the G at nucleotide number 728 is mutated to T in the mRNA of glucose-6-phosphatase catalytic subunit (G6PC) of Homo sapiens, transcript variant 1 (NCBI-GenBank accession number NM_000151.3). The compound was identified by negative ion ESI mass spectrometry (measured value: 7520.85).

[0499] (Reference Examples 2-3)

[0500] HO-G e2s -C m1s -A m1s -G e2s -A m1s -U m1s -A e2s -A m1s -Am1s -A e2s -U m1s -C m1s -C e2s -G m1s -A m1s -T e2s -G m1s -G m1s -C e2s -G m1s -A e2t -H(21e_013)(Sequence number 34)

[0501] The target compound was obtained by synthesis and purification in the same manner as in Example 12.

[0502] The base sequence of this compound is a sequence complementary to the 94th to 114th sequence from the 5' end of exon 5 of the c.648G>T variant G6PC gene in which the G at nucleotide number 728 is mutated to T in the catalytic subunit (G6PC) of Homo sapiens glucose-6-phosphatase, transcript variant 1, mRNA (NCBI-GenBank accession number NM_000151.3).

[0503] The compound of Reference Example 3 was also synthesized in the same manner as in Reference Example 2. The data of Reference Example 2 and Reference Example 3 are shown in Table 6.

[0504] Table 6

[0505]

[0506] In the sequence in the table, capital letters represent 2'-O,4'-C-ethylene cross-linked nucleic acid, and lowercase letters represent 2'-OMe-RNA. C in 2'-O,4'-C-ethylene cross-linked nucleic acid is 5-methylcytosine. Start and end represent the numbering from the 5' end of exon 5 of the c.648G>T variant G6PC gene in which the G at nucleotide number 728 of the catalytic subunit of glucose-6-phosphatase (G6PC), transcript variant 1, mRNA (NCBI-GenBank accession number NM_000151.3) is mutated to T. The compounds were identified by negative ion ESI mass analysis, and the measured values ​​are shown in the table.

[0507] (Reference Examples 4 to 15)

[0508] HO-C m1s -A e2s -G m1s -A m1s -T e2s -A m1s -A m1s -A e2s -A m1s -Um1s -C e2s -C m1s -G m1s -A e2s -U m1s -G m1s -G e2s -C m1t -H(18e_001)(Sequence number 36)

[0509] The target compound was obtained by synthesis and purification in the same manner as in Example 51.

[0510] The base sequence of this compound is a sequence complementary to the 96th to 113th sequence from the 5' end of exon 5 of the c.648G>T variant G6PC gene in which the G at nucleotide number 728 is mutated to T in the catalytic subunit (G6PC) of Homo sapiens glucose-6-phosphatase, transcript variant 1, mRNA (NCBI-GenBank accession number NM_000151.3).

[0511] The compounds of Reference Examples 5 to 15 were also synthesized in the same manner as in Reference Example 4. The data of Reference Example 4 and Reference Examples 5 to 15 are shown in Table 7.

[0512] Table 7

[0513]

[0514] In the sequence in the table, capital letters represent 2'-O,4'-C-ethylene cross-linked nucleic acid, and lowercase letters represent 2'-OMe-RNA. C in 2'-O,4'-C-ethylene cross-linked nucleic acid is 5-methylcytosine. Start and end represent the numbering from the 5' end of exon 5 of the G6PC gene of the c.648G>T variant in which the G at nucleotide number 728 of the homo sapiens glucose-6-phosphatase catalytic subunit (G6PC), transcript variant 1, mRNA (NCBI-GenBank accession number NM_000151.3) is mutated to T. The compounds were identified by negative ion ESI mass analysis, and the measured values ​​are shown in the table.

[0515] (Reference Examples 16 to 20)

[0516] HO-C m1s -A m1s -G m1s -A m1s -U m1s -A m1s -A m1s -A m1s -A m1s -U m1s -C m1s -C m1s -G m1s-A m1s -U m1s -G m1s -G m1s -C m1t -H(18m_001)(Sequence number 36)

[0517] The target compound was obtained by synthesis and purification in the same manner as in Example 70.

[0518] The base sequence of this compound is a sequence complementary to the 96th to 113th sequence from the 5' end of exon 5 of the c.648G>T variant G6PC gene in which the G at nucleotide number 728 is mutated to T in the catalytic subunit (G6PC) of Homo sapiens glucose-6-phosphatase, transcript variant 1, mRNA (NCBI-GenBank accession number NM_000151.3).

[0519] The compounds of Reference Examples 17 to 20 were also synthesized in the same manner as in Reference Example 16. The data of Reference Example 16 and Reference Examples 17 to 20 are shown in Table 8.

[0520] Table 8

[0521]

[0522] The lowercase letters in the sequence in the table represent 2'-OMe-RNA. The start and end represent the numbering from the 5' end of exon 5 of the G6PC gene, transcript variant 1, mRNA (NCBI-GenBank accession number NM_000151.3), in which the G at nucleotide number 728 is mutated to T, c.648G>T variant. The compounds were identified by negative ion ESI mass analysis, and the measured values ​​are shown in the table.

[0523] (Examples 83 to 86)

[0524] HO-A m1s -A e2s -U m1s -C m1s -C e2s -G m1s -A m1s -T e2s -G m1s -G m1s -C e2s -G m1s -A m1s -A e2s -G m1t -H(15e_001)(Sequence number 40)

[0525] Using AKTA Oligopilot, using Primer Support 5G Unylinker 350, 100 μmol (manufactured by GE Healthcare) as a solid phase support, the synthesis was carried out in the same manner as in Example 1. The protected oligonucleotide-related substance having the target sequence was treated with 25 mL of concentrated ammonia water, thereby cutting off the oligomer from the support, and removing the protecting group cyanoethyl on the phosphorus atom and the protecting group on the nucleic acid base. The mixed solution of the oligomer was deprotected and purified using phenomenex Clarity QSP 5g, and the target compound was obtained after the solvent was distilled off.

[0526] The base sequence of this compound is a sequence complementary to the 92nd to 106th sequence from the 5' end of exon 5 of the c.648G>T variant G6PC gene in which the G at nucleotide number 728 is mutated to T in the catalytic subunit (G6PC) of Homo sapiens glucose-6-phosphatase, transcript variant 1, mRNA (NCBI-GenBank accession number NM_000151.3).

[0527] The compounds of Examples 84 to 86 were also synthesized in the same manner as in Example 83. The data of Example 83 and Examples 84 to 86 are shown in Table 9.

[0528] Table 9

[0529]

[0530] In the sequence in the table, capital letters represent 2'-O,4'-C-ethylene cross-linked nucleic acid, lowercase letters represent 2'-OMe-RNA, and underlined lowercase letters represent DNA. The C of 2'-O,4'-C-ethylene cross-linked nucleic acid is 5-methylcytosine. The start and end represent the numbering from the 5' end of exon 5 of the c.648G>T variant G6PC gene in which the G at nucleotide number 728 of the catalytic subunit of glucose-6-phosphatase (G6PC), transcript variant 1, mRNA (NCBI-GenBank accession number NM_000151.3) is mutated to T. The compounds were identified by negative ion ESI mass spectrometry, and the measured values ​​are shown in the table.

[0531] (Examples 87 to 90)

[0532] HO-A m1s -A e2s -U m1s -C e2s -C e2s -G m1s -A e2s -T e2s -G m1s -G m1s -Ce2s -G m1s -A m1s -A e2s -G m1t -H(15e_001.1)(Sequence number 40)

[0533] The synthesis was carried out in the same manner as in Example 1, except that Primer Support 5G Unylinker 350, 10 μmol (manufactured by GE Healthcare) was used as a solid support and the 10 μmol program was used. Purification was carried out in the same manner as in Example 83 to obtain the target compound.

[0534] The base sequence of this compound is a sequence complementary to the 92nd to 106th sequence from the 5' end of exon 5 of the c.648G>T variant G6PC gene in which the G at nucleotide number 728 is mutated to T in the catalytic subunit (G6PC) of Homo sapiens glucose-6-phosphatase, transcript variant 1, mRNA (NCBI-GenBank accession number NM_000151.3).

[0535] The compounds of Examples 88 to 90 were also synthesized in the same manner as in Example 87. The data of Example 87 and Examples 88 to 90 are shown in Table 10.

[0536] Table 10

[0537]

[0538] In the sequence in the table, capital letters represent 2'-O,4'-C-ethylene cross-linked nucleic acid, and lowercase letters represent 2'-OMe-RNA. C in 2'-O,4'-C-ethylene cross-linked nucleic acid is 5-methylcytosine. Start and end represent the numbering from the 5' end of exon 5 of the c.648G>T variant G6PC gene in which the G at nucleotide number 728 of the catalytic subunit of glucose-6-phosphatase (G6PC), transcript variant 1, mRNA (NCBI-GenBank accession number NM_000151.3) is mutated to T. The compounds were identified by negative ion ESI mass analysis, and the measured values ​​are shown in the table.

[0539] (Example 91)

[0540] HO-XXXA p -A m1s -A e2s -U m1s -C m1s -C e2s -G m1s -A m1s -T e2s -G m1s -Gm1s -C e2s -G m1s -A m1s -A e2s -G m1t -H (Sequence number 42)

[0541] The synthesis was performed in the same manner as in Example 1. However, Primer Support 5G Unylinker 350, 10 μmol (manufactured by GE Healthcare) was used as a solid support, and a 10 μmol program was used. The X portion was condensed three times using the GalNAc phosphoramidite unit 1 described in the literature (Bioorg. Med. Chem. (2016) 24, 26-32). Purification was performed in the same manner as in Example 83 to obtain the target compound.

[0542] The base sequence of the compound is a sequence complementary to the 92nd to 107th sequence from the 5' end of exon 5 of the c.648G>T variant G6PC gene in which the G at nucleotide number 728 is mutated to T in the mRNA of glucose-6-phosphatase catalytic subunit (G6PC) of Homo sapiens, transcript variant 1 (NCBI-GenBank accession number NM_000151.3). The compound was identified by negative ion ESI mass spectrometry (measured value: 7131.24).

[0543] (Example 92)

[0544] HO-XXXA m1s -A e2s -U m1s -C m1s -C e2s -G m1s -A m1s -T e2s -G m1s -G m1s -C e2s -G m1s -A m1s -A e2s -G m1t -H (Sequence number 40)

[0545] The synthesis was carried out in the same manner as in Example 91.

[0546] The base sequence of the compound is a sequence complementary to the 92nd to 106th sequence from the 5' end of exon 5 of the c.648G>T variant G6PC gene in which the G at nucleotide number 728 is mutated to T in the mRNA of glucose-6-phosphatase catalytic subunit (G6PC) of Homo sapiens, transcript variant 1 (NCBI-GenBank accession number NM_000151.3). The compound was identified by negative ion ESI mass spectrometry (measured value: 6818.18).

[0547] (Example 93)

[0548] HO-XXXT p -C p -A p -A m1s -A e2s -U m1s -C m1s -C e2s -G m1s -A m1s -T e2s -G m1s -G m1s -C e2s -G m1s -A m1s -A e2s -G m1t -H (Sequence number 43)

[0549] The synthesis was carried out in the same manner as in Example 91.

[0550] The base sequence of the compound has a sequence complementary to the 92nd to 107th sequence from the 5' end of exon 5 of the c.648G>T variant G6PC gene in which the G at nucleotide number 728 is mutated to T in the mRNA of glucose-6-phosphatase catalytic subunit (G6PC) of Homo sapiens, transcript variant 1 (NCBI-GenBank accession number NM_000151.3). The compound was identified by negative ion ESI mass spectrometry (measured value: 7738.35).

[0551] (Example 94)

[0552] HO-XXXA p -A m1s -A e2s -U m1s -C e2s -C m1s -G e2s -A m1s -T e2s -G m1s -G e2s -C m1s -Ge2s -A m1s -A e2s -G m1t -H (Sequence number 42)

[0553] The synthesis was carried out in the same manner as in Example 91.

[0554] The base sequence of the compound has a sequence complementary to the 92nd to 107th sequence from the 5' end of exon 5 of the c.648G>T variant G6PC gene in which the G at nucleotide number 728 is mutated to T in the mRNA of glucose-6-phosphatase catalytic subunit (G6PC) of Homo sapiens, transcript variant 1 (NCBI-GenBank accession number NM_000151.3). The compound was identified by negative ion ESI mass spectrometry (measured value: 7141.22).

[0555] (Example 95)

[0556] HO-XXXA p -A e2s -A m1s -T e2s -C m1s -C e2s -G m1s -A e2s -U m1s -G e2s -G m1s -C e2s -G m1s -A e2s -A m1s -G e2t -H (Sequence number 42)

[0557] The synthesis was carried out in the same manner as in Example 91.

[0558] The base sequence of the compound has a sequence complementary to the 92nd to 107th sequence from the 5' end of exon 5 of the c.648G>T variant G6PC gene in which the G at nucleotide number 728 is mutated to T in the catalytic subunit of glucose-6-phosphatase (G6PC) of Homo sapiens, transcript variant 1, mRNA (NCBI-GenBank accession number NM_000151.3). The compound was identified by negative ion ESI mass spectrometry (measured value: 7167.24).

[0559] (Reference Example 21)

[0560] (21A)

[0561] Synthesis of [(2R,3R,4R,5R,6R)-5-acetamide-3,4-diacetoxy-6-[5-(benzyloxycarbonylamino)pentyloxy]tetrahydropyran-2-yl]methyl acetate (Compound 21A)

[0562] [Chemistry 78]

[0563]

[0564] Trifluoromethanesulfonic acid (450 μL, 5.23 mmol) was added to a suspension of N-benzyloxycarbonyl-1-hydroxypentyl-5-amine (J. Am. Chem. Soc., 2006, 128, 4058-4073.) (8.05 g, 33.9 mmol) and acetic acid [(2R, 3R, 4R, 5R)-5-acetamide-3, 4, 6-triacetoxy-tetrahydropyran-2-yl] methyl ester (WO2011053614) (12.0 g, 30.8 mmol) in dichloromethane (200 mL), and stirred overnight at 45° C. After the reaction was completed, the solvent was distilled off under reduced pressure and concentrated, and added to a mixed solution of ethyl acetate / saturated sodium bicarbonate aqueous solution. The organic layer was washed with saturated sodium bicarbonate aqueous solution and saturated saline / phosphate buffer (pH 7.0), dried with anhydrous sodium sulfate, filtered, and the solvent was distilled off under reduced pressure to obtain a crude product. It was purified by silica gel column chromatography (hexane:ethyl acetate = 100:0-0:100, v / v) to obtain an amorphous target product 21A (17.0 g, yield 94%).

[0565] 1 H-NMR (CDCl 3 )δ:7.37-7.32(5H,m),5.69(1H,d,J=9.1Hz),5.35(1H,d,J=2.4Hz),5.29(1H,d,J=11.5Hz),5.10(2H,s),4.94(1H,br),4.68(1H,d,J=7.9Hz),4. 20-4.09(2H,m),3.97-3.86(3H,m),3.49-3.44(1H,m),3.20-3.17(2H,m) ,2.14(3H,s),2.05(3H,s),2.01(3H,s),1.93(3H,s),1.66-1.34(6H,m).

[0566] C 27 H 38 N 2 O 11 :[M+H] + Calculated value 567, measured value 567.

[0567] (21B)

[0568] Synthesis of [(2R,3R,4R,5R,6R)-5-acetamide-3,4-diacetoxy-6-(5-aminopentyloxy)tetrahydropyran-2-yl]methyl acetate (Compound 21B)

[0569] [Chemistry 79]

[0570]

[0571] The compound 21A (17.0 g, 30.0 mmol) synthesized in step (21A) was dissolved in ethanol (200 ml). 10% palladium / carbon (wet basis) (2 g) was added and stirred vigorously at room temperature under a hydrogen atmosphere. After the reaction was completed, the mixture was filtered and the obtained liquid was removed by vacuum distillation to obtain a crude product of the target compound 21B (12.64 g, yield 97%). No further purification was performed and the product was used in the next reaction.

[0572] 1 H-NMR (CDCl 3 )δ:5.78(1H,d,J=8.5Hz),5.36-5.32(2H,m),4.72(1H,d,J=8.5Hz),4.21-4.09(2H,m),3.96-3.88(3H,m),3.5 2-3.47(1H,m),2.70(2H,t,J=6.7Hz),2.15(3H,s),2.05(3H,s),2.01(3H,s),1.96(3H,s),1.75-1.36(6H,m).

[0573] C 19 H 32 N 2 O 9 :[M+H] + Calculated value 433, measured value 433.

[0574] (21C)

[0575] Synthesis of [(2R,3R,4R,5R,6R)-5-acetamide-3,4-diacetoxy-6-[5-[[(4S)-2,2-dimethyl-1,3-dioxolan-4-yl]methoxycarbonylamino]pentyloxy]tetrahydropyran-2-yl]methyl acetate (Compound 21C)

[0576] [Chemistry 80]

[0577]

[0578] (R)-(-)-2,2-dimethyl-1,3-dioxolane-4-methanol (1.68 g, 12.8 mmol) and triethylamine (3.22 mL, 23.1 mmol) were dissolved in dichloromethane (100 mL). 4-Nitrophenyl chloroformate (2.56 g, 12.7 mmol) was added and stirred at room temperature for 1 hour. Then, triethylamine (5 mL, 36 mmol) and a dichloromethane solution (20 mL) of compound 21B (5.0 g, 11.56 mmol) synthesized in step (21B) were added and stirred at 45°C for 1 hour. After the reaction was completed, the solvent was distilled off under reduced pressure to obtain a crude product. It was purified by silica gel column chromatography (hexane: ethyl acetate = 100: 0-0: 100, v / v) to obtain an amorphous target product 21C (3.46 g, yield 51%).

[0579] 1 H-NMR (CDCl 3 )δ:5.75(1H,d,J=8.5Hz),5.36(1H,d,J=3.0Hz),5.30(1H,dd,J=11.2,3.3Hz ),4.97(1H,br),4.71(1H,d,J=8.5Hz),4.33-4.29(1H,m),4.23-3.88(8H,m), 3.74-3.72(1H,m),3.49-3.46(1H,m),3.19-3.14(2H,m),2.15(3H,s),2.05(3 H,s),2.01(3H,s),1.96(3H,s),1.63-1.53(6H,m),1.44(3H,s),1.37(3H,s).

[0580] C 26 H 42 N 2 O 13 :[M+H] + Calculated value 591, measured value 591.

[0581] (21D)

[0582] Synthesis of [(2R,3R,4R,5R,6R)-5-acetamide-3,4-diacetoxy-6-[5-[[(2S)-2,3-dihydroxypropoxy]carbonylamino]pentyloxy]tetrahydropyran-2-yl]methyl acetate (Compound 21D)

[0583] [Chemistry 81]

[0584]

[0585] Pure water (2 mL) and trifluoroacetic acid (6 mL) were added to the compound 21C (4.38 g, 7.42 mmol) synthesized in step (21C), and the mixture was stirred at room temperature for 1.5 hours. After the reaction was completed, the solvent was distilled off under reduced pressure. Toluene was then azeotroped to remove water and a crude product was obtained. It was purified by silica gel column chromatography (hexane: ethyl acetate = 100: 0-0: 100, v / v) to obtain an amorphous target product 21D (2.47 g, yield 61%).

[0586] 1 H-NMR (CDCl 3 )δ:5.75(1H,d,J=7.3Hz),5.35(1H,d,J=3.0Hz),5.22(1H,dd,J=11.5,3.0Hz),5.13(1H,br s),4.61(1H,d,J=8.5Hz),4.23-3.87(7H,m),3.67-3.60(3H,m),3.49-3.46(1H,m),3.2 2-3.16(2H,m),2.16(3H,s),2.05(3H,s),2.01(3H,s),1.97(3H,s),1.61-1.50(6H,m).

[0587] C 23 H 38 N 2 O 13 :[M+H] + Calculated value 551, measured value 551.

[0588] (21E)

[0589] Synthesis of [(2R,3R,4R,5R,6R)-5-acetamide-3,4-diacetoxy-6-[5-[[(2S)-3-[bis(4-methoxyphenyl)-phenyl-methoxy]-2-hydroxy-propoxy]carbonylamino]pentyloxy]tetrahydropyran-2-yl]methyl acetate (Compound 21E)

[0590] [Chemistry 82]

[0591]

[0592] The compound 21D (2.47 g, 4.52 mmol) synthesized in step (21D) was dissolved in pyridine (15 mL). 4,4'-dimethoxytriphenylchloromethane (1.82 g, 5.38 mmol) was added and stirred at room temperature. After the reaction was completed, the solvent was distilled off under reduced pressure to obtain a crude product. It was purified by silica gel column chromatography (hexane: ethyl acetate = 100: 0-0: 100, v / v, then ethyl acetate: methanol = 95: 5, v / v) to obtain an amorphous target product 21E (1.84 g, yield 48%).

[0593] 1 H-NMR (CDCl 3 )δ:7.43-7.42(2H,m),7.32-7.29(6H,m),7.24-7.19(1H,m),6.84-6.81(4H,m),5.74(1H,d,J =8.5Hz),5.35(1H,d,J=3.0Hz),5.26(1H,dd,J=11.2,3.3Hz),4.94(1H,br),4.65(1H,d,J=8.5 Hz),4.22-4.09(4H,m),3.97-3.91(4H,m),3.79(6H,s),3.47-3.43(1H,m),3.18-3.15(4H,m), 3.00(1H,d,J=4.8Hz),2.14(3H,s),2.04(3H,s),1.99(3H,s),1.95(3H,s),1.65-1.34(6H,m).

[0594] C 44 H 56 N 2 O 15 :[M+Na] + Calculated value 875, measured value 875.

[0595] (21F)

[0596] Synthesis of [(2R,3R,4R,5R,6R)-5-acetamide-3,4-diacetoxy-6-[5-[[(2S)-3-[bis(4-methoxyphenyl)-phenyl-methoxy]-2-[2-cyanoethoxy-(diisopropylamino)phosphino]oxypropoxy]carbonylamino]pentyloxy]tetrahydropyran-2-yl]methyl acetate (Compound 21F)

[0597] [Chemistry 83]

[0598]

[0599] The compound 21E (1.84 g, 2.16 mmol) synthesized in step (21E) was added with an appropriate amount of pyridine and azeotroped under reduced pressure, and then dissolved in dichloromethane (21 mL). N,N-diisopropylethylamine (2.25 mL, 12.9 mmol) and 2-cyanoethyldiisopropylchlorophosphoramidite (0.96 mL, 4.31 mmol) were added and stirred at room temperature for 1.5 hours. After the reaction was completed, the solvent was distilled off under reduced pressure. A crude product was obtained by adding an appropriate amount of toluene and azeotroping. It was purified by silica gel column chromatography (hexane: ethyl acetate = 100: 0-0: 100, v / v, containing 1% triethylamine) to obtain the amorphous target 21F (1.87 g, yield 82%).

[0600] 1 H-NMR (CDCl 3 )δ:7.46-7.40(2H,m),7.35-7.17(7H,m),6.82-6.80(4H,m),5.73-5.63(1H,m),5. 37-5.35(1H,m),5.30-5.26(1H,m),4.97-4.94(0.5H,m),4.72-4.68(1H,m),4.42- 4.40(0.5H,m),4.22-4.10(4H,m),3.99-3.43(14H,m),3.18-3.12(4H,m),2.67-2. 62(1H,m),2.46-2.44(1H,m),2.14(3H,s),2.01-1.96(9H,m),1.65-0.97(18H,m).

[0601] (Example 96)

[0602] HO-X 1 -X 1 -X 1 -A m1s -A e2s -U m1s -C m1s -C e2s -G m1s -A m1s -T e2s -G m1s -G m1s -C e2s -G m1s -A m1s -A e2s -G m1t -H (Sequence number 40)

[0603] Replace the part of X with X 1 , and synthesized in the same manner as in Example 91.1 The compound 21F synthesized in Reference Example 21 was used for the condensation three times.

[0604] The base sequence of the compound is a sequence complementary to the 92nd to 106th sequence from the 5' end of exon 5 of the c.648G>T variant G6PC gene in which the G at nucleotide number 728 is mutated to T in the mRNA of glucose-6-phosphatase catalytic subunit (G6PC) of Homo sapiens, transcript variant 1 (NCBI-GenBank accession number NM_000151.3). The compound was identified by negative ion ESI mass spectrometry (measured value: 6830.15).

[0605] (Reference Example 22)

[0606] (22A)

[0607] Synthesis of [(2R,3R,4R,5R,6R)-5-acetamide-3,4-diacetoxy-6-[5-[[(4R)-2,2-dimethyl-1,3-dioxolan-4-yl]methoxycarbonylamino]pentyloxy]tetrahydropyran-2-yl]methyl acetate (Compound 22A)

[0608] [Chemistry 84]

[0609]

[0610] (S)-(+)-2,2-dimethyl-1,3-dioxolane-4-methanol (1.68 g, 2.54 mmol) and triethylamine (3.22 mL, 23.1 mmol) were dissolved in dichloromethane (100 mL). 4-Nitrophenyl chloroformate (2.56 g, 12.72 mmol) was added and stirred at room temperature for 1 hour. Then, triethylamine (5 mL, 36 mmol) and compound 21B (5.2 g, 13 mmol) synthesized in step (21B) were added and stirred at 45°C for 1 hour. After the reaction was completed, the solvent was distilled off under reduced pressure to obtain a crude product. It was purified by silica gel column chromatography (hexane: ethyl acetate = 100: 0-0: 100, v / v) to obtain an amorphous target product 22A (3.82 g, yield 54%).

[0611] 1 H-NMR (CDCl 3)δ:5.71(1H,d,J=8.5Hz),5.36(1H,d,J=3.0Hz),5.31(1H,dd,J=11.2,3.0Hz),4. 97(1H,br),4.71(1H,d,J=8.5Hz),4.33-4.30(1H,m),4.22-3.99(5H,m),3.95-3.8 9(3H,m),3.74-3.71(1H,m),3.50-3.46(1H,m),3.19-3.14(2H,m),2.15(3H,s),2. 05(3H,s),2.01(3H,s),1.96(3H,s),1.69-1.47(6H,m),1.44(3H,s),1.37(3H,s).

[0612] C 26 H 42 N 2 O 13 :[M+H] + Calculated value 591, measured value 591.

[0613] (22B)

[0614] Synthesis of [(2R,3R,4R,5R,6R)-5-acetamide-3,4-diacetoxy-6-[5-[[(2R)-2,3-dihydroxypropoxy]carbonylamino]pentyloxy]tetrahydropyran-2-yl]methyl acetate (Compound 22B)

[0615] [Chemistry 85]

[0616]

[0617] Pure water (2 mL) and trifluoroacetic acid (6 mL) were added to the compound 22A (3.82 g, 6.47 mmol) synthesized in step (22A), and the mixture was stirred at room temperature for 1.5 hours. After the reaction was completed, the solvent was distilled off under reduced pressure. Toluene was then azeotroped to remove water and a crude product was obtained. It was purified by silica gel column chromatography (hexane: ethyl acetate = 100: 0-0: 100, v / v) to obtain an amorphous target product 22B (2.49 g, yield 70%).

[0618] 1 H-NMR (CDCl 3)δ:5.98(1H,d,J=8.5Hz),5.37-5.20(3H,m),4.63(1H,d,J=8.5Hz),4.21-3.88(7H,m),3.69-3.57(3H,m),3 .51-3.44(1H,m),3.21-3.14(2H,m),2.16(3H,s),2.05(3H,s),2.01(3H,s),1.97(3H,s),1.66-1.34(6H,m).

[0619] C 23 H 38 N 2 O 13 :[M+H] + Calculated value 551, measured value 551.

[0620] (22C)

[0621] Synthesis of [(2R,3R,4R,5R,6R)-5-acetamide-3,4-diacetoxy-6-[5-[[(2R)-3-[bis(4-methoxyphenyl)-phenyl-methoxy]-2-hydroxy-propoxy]carbonylamino]pentyloxy]tetrahydropyran-2-yl]methyl acetate (Compound 22C)

[0622] [Chemistry 86]

[0623]

[0624] The compound 22B (2.49 g, 4.52 mmol) synthesized in step (22B) was dissolved in pyridine (15 mL). 4,4'-dimethoxytriphenylchloromethane (1.84 g, 5.43 mmol) was added and stirred at room temperature. After the reaction was completed, the solvent was distilled off under reduced pressure to obtain a crude product. It was purified by silica gel column chromatography (hexane: ethyl acetate = 100: 0-0: 100, v / v, then ethyl acetate: methanol = 95: 5, v / v) to obtain an amorphous target product 22C (2.1 g, yield 54%).

[0625] 1 H-NMR (CDCl 3)δ:7.42(2H,d,J=7.9Hz),7.32-7.16(6H,m),6.83(4H,d,J=9.1Hz),5.74(1H,d,J=8.5Hz ),5.35(1H,d,J=3.0Hz),5.27(1H,dd,J=11.2,3.0Hz),4.93(1H,br),4.66(1H,d,J=8.5Hz ),4.25-4.11(4H,m),4.01-3.89(4H,m),3.79(6H,s),3.50-3.43(1H,m),3.21-3.13(4H, m),2.98(1H,br),2.13(3H,s),2.04(3H,s),1.99(3H,s),1.94(3H,s),1.65-1.34(6H,m).

[0626] C 44 H 56 N 2 O 15 :[M+Na] + Calculated value 875, measured value 875.

[0627] (22D)

[0628] Synthesis of [(2R,3R,4R,5R,6R)-5-acetamide-3,4-diacetoxy-6-[5-[[(2R)-3-[bis(4-methoxyphenyl)-phenyl-methoxy]-2-[2-cyanoethoxy-(diisopropylamino)phosphino]oxypropoxy]carbonylamino]pentyloxy]tetrahydropyran-2-yl]methyl acetate (Compound 22D)

[0629] [Chemistry 87]

[0630]

[0631] Compound 22C (2.10 g, 2.50 mmol) synthesized in step (22C) was added with an appropriate amount of pyridine and dissolved in dichloromethane (20 mL) after azeotroping under reduced pressure. N,N-diisopropylethylamine (2.6 mL, 15 mmol) and 2-cyanoethyldiisopropylchlorophosphoramidite (1.11 ml, 4.9 mmol) were added and stirred at room temperature for 1.5 hours. After the reaction was completed, the solvent was distilled off under reduced pressure. A crude product was obtained by adding an appropriate amount of toluene and azeotroping. It was purified by silica gel column chromatography (hexane: ethyl acetate = 100: 0-0: 100, v / v, containing 1% triethylamine) to obtain the amorphous target 22D (1.63 g, yield 63%).

[0632] 1 H-NMR (CDCl 3)δ:7.46-7.40(2H,m),7.35-7.17(7H,m),6.85-6.78(4H,m),5.70-5.57(1H,m),5. 38-5.34(1H,m),5.32-5.24(1H,m),4.96-4.91(0.5H,m),4.73-4.65(1H,m),4.44- 4.38(0.5H,m),4.23-4.08(4H,m),4.00-3.41(14H,m),3.27-3.03(4H,m),2.68-2. 60(1H,m),2.49-2.42(1H,m),2.14(3H,s),2.07-1.91(9H,m),1.65-0.97(18H,m).

[0633] (Example 97)

[0634] HO-X 2 -X 2 -X 2 -A m1s -A e2s -U m1s -C m1s -C e2s -G m1s -A m1s -T e2s -G m1s -G m1s -C e2s -G m1s -A m1s -A e2s -G m1t -H (Sequence number 40)

[0635] Replace the part of X with X 2 , and synthesized in the same manner as in Example 91. 2 The compound 22D synthesized in Reference Example 22 was used for the condensation three times.

[0636] The base sequence of the compound is complementary to the sequence from 92 to 106 from the 5' end of exon 5 of the c.648G>T variant G6PC gene in which the G at nucleotide number 728 is mutated to T in the mRNA of glucose-6-phosphatase catalytic subunit (G6PC) of Homo sapiens, transcript variant 1 (NCBI-GenBank accession number NM_000151.3). The compound was identified by negative ion ESI mass spectrometry (measured value: 6830.14).

[0637] (Reference Example 23)

[0638] (23A)

[0639] Synthesis of [(2R,3R,4R,5R,6R)-5-acetamide-3,4-diacetoxy-6-[8-(benzyloxycarbonylamino)octyloxy]tetrahydropyran-2-yl]methyl acetate (Compound 23A)

[0640] [Chemistry 88]

[0641]

[0642] Trifluoromethanesulfonic acid (310 μL, 3.6 mmol) was added to a suspension of N-(8-hydroxyoctyl)carbamic acid benzyl ester (J. Med. Chem, 1993, 36, 3721-3726.) (6.5 g, 23 mmol) and acetic acid [(2R, 3R, 4R, 5R)-5-acetamide-3,4,6-trioxy-tetrahydropyran-2-yl] methyl ester (WO2011053614) (8.2 g, 21 mmol) in dichloromethane (150 mL), and the mixture was stirred overnight at 45°C. After the reaction was completed, half of the solvent was distilled off under reduced pressure and concentrated, and the mixture was added to a mixed solution of ethyl acetate / saturated sodium bicarbonate aqueous solution. The organic layer was washed with saturated sodium bicarbonate aqueous solution and saturated saline / phosphate buffer (pH 7.0), dried with anhydrous sodium sulfate, filtered, and the solvent was distilled off under reduced pressure to obtain a crude product. This was purified by silica gel column chromatography (hexane:ethyl acetate=100:0-0:100, v / v) to obtain an amorphous target compound 23A (12.68 g, yield 99%).

[0643] 1 H-NMR (CDCl 3 )δ:7.39-7.29(5H,m),5.65(1H,d,J=8.5Hz),5.35(1H,d,J=3.0Hz),5.29( 1H,dd,J=11.5,3.0Hz),5.10(2H,s),4.83(1H,br),4.68(1H,d,J=7.9Hz), 4.20-4.09(2H,m),3.99-3.85(3H,m),3.50-3.42(1H,m),3.22-3.15(2H,m ),2.14(3H,s),2.05(3H,s),2.00(3H,s),1.95(3H,s),1.62-1.27(12H,m).

[0644] C 30 H 44 N 2 O 11 :[M+H] + Calculated value 609, measured value 609.

[0645] (23B)

[0646] Synthesis of [(2R,3R,4R,5R,6R)-5-acetamido-3,4-diacetoxy-6-(8-aminooctyloxy)tetrahydropyran-2-yl]methyl acetate (Compound 23B)

[0647] [Chemistry 89]

[0648]

[0649] The compound 23A (12.68 g, 20.83 mmol) synthesized in step (23A) was dissolved in ethanol (200 mL). 10% palladium / carbon (wet basis) (2 g) was added and stirred vigorously at room temperature under a hydrogen atmosphere. After the reaction was completed, the mixture was filtered and the obtained liquid was removed by vacuum distillation to obtain a crude product of the target compound 23B (10.1 g). No further purification was performed and the mixture was used in the next reaction.

[0650] C 22 H 38 N 2 O 9 :[M+H] + Calculated value 475, measured value 475.

[0651] (23C)

[0652] Synthesis of [(2R,3R,4R,5R,6R)-5-acetamide-3,4-diacetoxy-6-[8-[[(4S)-2,2-dimethyl-1,3-dioxolan-4-yl]methoxycarbonylamino]octyloxy]tetrahydropyran-2-yl]methyl acetate (Compound 23C)

[0653] [Chemistry 90]

[0654]

[0655] (R)-(-)-2,2-dimethyl-1,3-dioxolane-4-methanol (1.6 g, 12 mmol) and triethylamine (3.1 mL, 22 mmol) were dissolved in dichloroethane (30 mL). 4-Nitrophenyl chloroformate (2.2 g, 12 mmol) was added and stirred at room temperature for 1.5 hours. Subsequently, triethylamine (4.5 mL, 32 mmol) and compound 23B (5.1 g, 11 mmol) synthesized in step (23B) were added and stirred at 45°C for 4 hours. After the reaction was completed, the solvent was distilled off under reduced pressure to obtain a crude product. It was purified by silica gel column chromatography (hexane: ethyl acetate = 100: 0-0: 100, v / v) to obtain the amorphous target product 23C (2.45 g, yield 36%).

[0656] 1 H-NMR (CDCl 3 )δ:5.64(1H,d,J=7.9Hz),5.36(1H,d,J=3.0Hz),5.29(1H,dd,J=11.2,3.0Hz),4.85(1H,br),4.69(1H,d,J=7.9Hz),4.35-3.86(9H,m),3.76-3.70 (1H,m),3.50-3.43(1H,m),3.20-3.12(2H,m),2.14(3H,s),2.05(3H,s), 2.01(3H,s),1.96(3H,s),1.65-1.28(12H,m),1.44(3H,s),1.37(3H,s).

[0657] C 29 H 48 N 2 O 13 :[M+H] + Calculated value 633, measured value 633.

[0658] (23D)

[0659] Synthesis of [(2R,3R,4R,5R,6R)-5-acetamide-3,4-diacetoxy-6-[8-[[(2S)-2,3-dihydroxypropoxy]carbonylamino]octyloxy]tetrahydropyran-2-yl]methyl acetate (Compound 23D)

[0660] [Chemistry 91]

[0661]

[0662] Pure water (2 mL) and trifluoroacetic acid (6 mL) were added to the compound 23C (2.45 g, 3.87 mmol) synthesized in step (23C), and the mixture was stirred at room temperature for 1.5 hours. After the reaction was completed, the solvent was distilled off under reduced pressure. Toluene was then azeotroped to remove water and a crude product was obtained. It was purified by silica gel column chromatography (hexane: ethyl acetate = 100: 0-0: 100, v / v) to obtain an amorphous target product 23D (1.18 g, yield 51%).

[0663] 1 H-NMR (CDCl 3)δ:5.87(1H,d,J=7.9Hz),5.35(1H,d,J=3.0Hz),5.27(1H,dd,J=11.2,3.0Hz),5.07(1H,br),4.66(1H,d,J=8.5Hz),4.27-3.86(7H,m ),3.70-3.57(2H,m),3.50-3.43(2H,m),3.24-3.13(2H,m),2.15(3H,s),2.05(3H,s),2.01(3H,s),1.96(3H,s),1.67-1.24(12H,m).

[0664] C 26 H 44 N 2 O 13 :[M+H] + Calculated value 593, measured value 593.

[0665] (23E)

[0666] Synthesis of [(2R,3R,4R,5R,6R)-5-acetamide-3,4-diacetoxy-6-[8-[[(2S)-3-[bis(4-methoxyphenyl)-phenyl-methoxy]-2-hydroxy-propoxy]carbonylamino]octyloxy]tetrahydropyran-2-yl]methyl acetate (Compound 23E)

[0667] [Chemistry 92]

[0668]

[0669] The compound 23D (1.18 g, 1.99 mmol) synthesized in step (23D) was dissolved in pyridine (20 mL). 4,4'-dimethoxytriphenylchloromethane (0.81 g, 2.39 mmol) was added and stirred at room temperature. After the reaction was completed, the solvent was distilled off under reduced pressure. After adding an appropriate amount of toluene for azeotropy, a crude product was obtained. It was purified by silica gel column chromatography (hexane: ethyl acetate = 100: 0-0: 100, v / v, then ethyl acetate: methanol = 95: 5, v / v) to obtain an amorphous target 23E (0.6 g, yield 34%).

[0670] 1 H-NMR (CDCl 3)δ:7.44-7.40(2H,m),7.33-7.19(7H,m),6.85-6.81(4H,m),5.61(1H,d,J=8.5Hz), 5.35(1H,d,J=3.0Hz), 5.29(1H,dd,J=11.2,3.3Hz), 4.79(1H,br), 4.69(1H,d,J=8. 5Hz),4.24-4.09(4H,m),4.00-3.86(4H,m),3.79(6H,s),3.50-3.43(1H,m),3.21-3 .12(4H,m),2.14(3H,s),2.04(3H,s),2.00(3H,s),1.95(3H,s),1.63-1.26(12H,m).

[0671] C 47 H 62 N 2 O 15 :[M+Na] + Calculated value 917, measured value 917.

[0672] (23F)

[0673] Synthesis of [(2R,3R,4R,5R,6R)-5-acetamide-3,4-diacetoxy-6-[8-[[(2S)-3-[bis(4-methoxyphenyl)-phenyl-methoxy]-2-[2-cyanoethoxy-(diisopropylamino)phosphino]oxypropoxy]carbonylamino]octyloxy]tetrahydropyran-2-yl]methyl acetate (Compound 23F)

[0674] [Chemistry 93]

[0675]

[0676] The compound 23E (0.60 g, 0.67 mmol) synthesized in step (23E) was added with an appropriate amount of pyridine and dissolved in dichloroethane (5 mL) after azeotroping under reduced pressure. N, N-diisopropylethylamine (0.70 ml, 4.0 mmol) and 2-cyanoethyldiisopropylchlorophosphoramidite (0.30 mL, 1.3 mmol) were added and stirred at room temperature for 1.5 hours. After the reaction was completed, the solvent was distilled off under reduced pressure. A crude product was obtained by adding an appropriate amount of toluene and azeotroping. It was purified by silica gel column chromatography (hexane: ethyl acetate = 100: 0-0: 100, v / v, containing 1% triethylamine) to obtain the amorphous target 23F (0.51 g, yield 69%).

[0677] 1 H-NMR (CDCl 3)δ:7.46-7.40(2H,m),7.34-7.17(7H,m),6.84-6.78(4H,m),5.56-5.48(1H,m),5.37-5 .34(1H,m),5.32-5.28(1H,m),4.87-4.80(0.5H,m),4.73-4.68(1H,m),4.43-4.37(0.5 H,m),4.23-4.08(4H,m),3.97-3.43(14H,m),3.26-3.06(4H,m),2.66-2.61(1H,m),2.4 7-2.41(1H,m),2.14(3H,s),2.06-1.93(9H,m),1.64-1.23(12H,m),1.21-0.98(12H,m).

[0678] (Example 98)

[0679] HO-X 3 -X 3 -X 3 -A m1s -A e2s -U m1s -C m1s -C e2s -G m1s -A m1s -T e2s -G m1s -G m1s -C e2s -G m1s -A m1s -A e2s -G m1t -H (Sequence number 40)

[0680] Replace the part of X with X 3 , and synthesized in the same manner as in Example 91. 3 The compound 23F synthesized in Reference Example 23 was used for the condensation three times.

[0681] The base sequence of the compound is a sequence complementary to the 92nd to 106th sequence from the 5' end of exon 5 of the c.648G>T variant G6PC gene in which the G at nucleotide number 728 is mutated to T in the mRNA of glucose-6-phosphatase catalytic subunit (G6PC) of Homo sapiens, transcript variant 1 (NCBI-GenBank accession number NM_000151.3). The compound was identified by negative ion ESI mass spectrometry (measured value: 6957.27).

[0682] (Reference Example 24)

[0683] (24A)

[0684] Synthesis of 7-[(2S)-3-benzyloxy-2-hydroxypropoxy]heptan-1-ol (Compound 24A)

[0685] [Chemistry 94]

[0686]

[0687] Benzyl (S)-(+)-glycidyl ether (5.0 g, 30 mmol) was dissolved in dichloromethane (150 mL). 1,7-heptanediol (6.0 g, 45 mmol) and boron trifluoride-diethyl ether complex (0.76 mL, 6.1 mmol) were added and stirred at room temperature for 2 days. The reaction solution was added to a mixed solution of ethyl acetate / saturated sodium bicarbonate aqueous solution and extracted with ethyl acetate. The organic layer was washed with saturated saline / saturated sodium bicarbonate aqueous solution, dried with anhydrous sodium sulfate, filtered, and the solvent was distilled off under reduced pressure to obtain a crude product. It was purified by silica gel column chromatography (hexane: ethyl acetate = 50:50, v / v) to obtain the target product 24A (3.3 g, yield 37%) as a colorless oil.

[0688] 1 H-NMR (CDCl 3 )δ:7.39-7.28(5H,m),4.57(2H,s),4.02-3.96(1H,m),3.67-3.61(2H,m),3. 59-3.43(6H,m),2.48(1H,d,J=4.2Hz),1.61-1.52(4H,m),1.37-1.30(6H,m).

[0689] C 17 H 28 O 4 :[M+Na] + Calculated value 319, measured value 319.

[0690] (24B)

[0691] Synthesis of [(2R,3R,4R,5R,6R)-5-acetamide-3,4-diacetoxy-6-[7-[(2S)-3-benzyloxy-2-hydroxy-propoxy]heptyloxy]tetrahydropyran-2-yl]methyl acetate (Compound 24B-1)

[0692] [Chemistry 95]

[0693]

[0694] Synthesis of [(2R,3R,4R,5R,6R)-5-acetamide-6-[7-[(2S)-2-[(2R,3R,4R,5R,6R)-3-acetamide-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxy-3-benzyloxy-propoxy]heptyloxy]-3,4-diacetoxytetrahydropyran-2-yl]methyl acetate (Compound 24B-2)

[0695] [Chemistry 96]

[0696]

[0697] Trifluoromethanesulfonic acid (0.15 mL, 1.7 mmol) was added to a suspension of compound 15A (3.3 g, 11 mmol) synthesized in step (24A) and acetic acid [(2R, 3R, 4R, 5R)-5-acetamide-3, 4, 6-triacetoxy-tetrahydropyran-2-yl] methyl ester (WO2011053614) (4.0 g, 10 mmol) of a known compound in the literature in dichloromethane (200 mL), and the mixture was stirred at 45°C for 3 days. After the reaction was completed, half of the solvent was distilled off under reduced pressure and concentrated, and the mixture was added to a mixed solution of ethyl acetate / saturated sodium bicarbonate aqueous solution and extracted with ethyl acetate. The organic layer was washed with saturated sodium bicarbonate aqueous solution and saturated saline / phosphate buffer (pH 7.0), dried with anhydrous sodium sulfate, filtered, and the solvent was distilled off under reduced pressure to obtain a crude product. It was purified by silica gel column chromatography (hexane:ethyl acetate=100:0-0:100, v / v) to obtain amorphous target products 24B-1 (4.5 g, yield 70%) and 24B-2 (1.9 g, yield 20%).

[0698] (Compound 24B-1)

[0699] 1 H-NMR (CDCl 3 )δ:7.39-7.28(5H,m),5.51(1H,d,J=8.5Hz),5.37-5.29(2H,m),4.71(1H,d,J=8.5Hz),4.56(2H,s),4.20-4.09(2H,m),4.03-3.85(4H, m),3.58-3.41(7H,m),2.58(1H,d,J=4.2Hz),2.14(3H,s),2.05(3H,s),2.00(3H,s),1.95(3H,s),1.63-1.52(4H,m),1.36-1.29(6H,m).

[0700] C 31 H 47 NO12 :[M+H] + Calculated value 626, measured value 626.

[0701] (Compound 24B-2)

[0702] 1 H-NMR (CDCl 3 )δ:7.41-7.28(5H,m),5.65(1H,d,J=8.5Hz),5.48(1H,d,J=8.5Hz),5.42-5.23(4H,m),4.84(1H,d,J=8.5Hz),4.72 (1H,d,J=8.5Hz),4.59-4.49(2H,m),4.20-3.38(17H,m),2.19-1.92(24H,m),1.61-1.47(4H,m),1.38-1.26(6H,m).

[0703] C 45 H 66 N 2 O 20 :[M+H] + Calculated value 955, measured value 955.

[0704] (24C)

[0705] Synthesis of [(2R,3R,4R,5R,6R)-5-acetamide-3,4-diacetoxy-6-[7-[(2R)-2,3-dihydroxypropoxy]heptyloxy]tetrahydropyran-2-yl]methyl acetate (Compound 24C)

[0706] [Chemistry 97]

[0707]

[0708] The compound 24B-1 (4.5 g, 7.2 mmol) synthesized in step (24B) was dissolved in ethanol (100 mL). 10% palladium / carbon (wet basis) (2.0 g) was added and stirred vigorously at room temperature for 5 hours under a hydrogen environment. After the reaction was completed, the mixture was filtered and the obtained liquid was distilled off under reduced pressure. After adding an appropriate amount of toluene for azeotropy, an appropriate amount of pyridine was added for azeotropy to obtain a crude product of the target product 24C. No further purification was performed and it was used for the next reaction.

[0709] 1 H-NMR (CDCl 3)δ:5.55(1H,d,J=9.1Hz),5.37-5.27(2H,m),4.69(1H,d,J=8.5Hz),4.21-4.09(2H,m),3.99-3.42(11H,m),2. 69(1H,d,J=5.4Hz),2.15(3H,s),2.05(3H,s),2.01(3H,s),1.96(3H,s),1.65-1.52(4H,m),1.40-1.29(6H,m).

[0710] C 24 H 41 NO 12 :[M+H] + Calculated value 536, measured value 536.

[0711] (24D)

[0712] Synthesis of [(2R,3R,4R,5R,6R)-5-acetamide-3,4-diacetoxy-6-[7-[(2S)-3-[bis(4-methoxyphenyl)-phenyl-methoxy]-2-hydroxy-propoxy]heptyloxy]tetrahydropyran-2-yl]methyl acetate (Compound 24D)

[0713] [Chemistry 98]

[0714]

[0715] The crude product of compound 24C synthesized in step (24C) was dissolved in pyridine (30 mL). 4,4'-dimethoxytriphenylchloromethane (2.7 g, 7.9 mmol) was added and stirred at room temperature for 13 hours. After the reaction was completed, the solvent was distilled off under reduced pressure. After adding toluene for azeotropy, a crude product was obtained. It was purified by silica gel column chromatography (hexane: ethyl acetate = 50:50-0:100, v / v, containing 1% triethylamine) to obtain an amorphous target 24D (3.0 g, yield 2 step 49%).

[0716] 1 H-NMR (CDCl 3)δ:7.45-7.41(2H,m),7.34-7.20(7H,m),6.85-6.80(4H,m),5.50(1H,d,J=8.5Hz ),5.37-5.29(2H,m),4.71(1H,d,J=8.5Hz),4.20-4.09(2H,m),3.98-3.85(4H,m), 3.79(6H,s),3.55-3.40(5H,m),3.22-3.13(2H,m),2.51(1H,d,J=4.2Hz),2.14(3H ,s),2.05(3H,s),2.00(3H,s),1.93(3H,s),1.60-1.52(4H,m),1.35-1.27(6H,m).

[0717] C 45 H 59 NO 14 :[M+Na] + Calculated value 860, measured value 860.

[0718] (24E)

[0719] Synthesis of [(2R,3R,4R,5R,6R)-5-acetamide-3,4-diacetoxy-6-[7-[(2S)-3-[bis(4-methoxyphenyl)-phenyl-methoxy]-2-[2-cyanoethyl-(diisopropylamino)phosphino]oxypropoxy]heptyloxy]tetrahydropyran-2-yl]methyl acetate (Compound 24E)

[0720] [Chemistry 99]

[0721]

[0722] The compound 24D (3.0 g, 3.6 mmol) synthesized in step (24D) was dissolved in dichloromethane (50 mL) after azeotroping with an appropriate amount of pyridine. N,N-diisopropylethylamine (2.5 mL, 14 mmol) and 2-cyanoethyldiisopropylchlorophosphoramidite (0.88 mL, 3.9 mmol) were added and stirred at room temperature for 1.5 hours. After the reaction was completed, the solvent was distilled off under reduced pressure to obtain a crude product. It was purified by silica gel column chromatography (hexane: ethyl acetate = 50:50-0:100, v / v, containing 1% triethylamine) to obtain an amorphous target 24E (3.0 g, yield 82%).

[0723] 1 H-NMR (CDCl 3)δ:7.48-7.41(2H,m),7.36-7.15(7H,m),6.85-6.77(4H,m),5.59-5.41 (1H,m),5.38-5.26(2H,m),4.75-4.68(1H,m),4.20-4.07(2H,m),3.97- 3.06(21H,m),2.67-2.40(2H,m),2.14(3H,s),2.04(3H,s),2.00(3H,s) ,1.92(3H,s),1.64-1.46(4H,m),1.37-1.22(6H,m),1.22-0.97(12H,m).

[0724] (Example 99)

[0725] HO-X 4 -X 4 -X 4 -A m1s -A e2s -U m1s -C m1s -C e2s -G m1s -A m1s -T e2s -G m1s -G m1s -C e2s -G m1s -A m1s -A e2s -G m1t -H (Sequence number 40)

[0726] Replace the part of X with X 4 , and synthesized in the same manner as in Example 91. 4 The compound 24E synthesized in Reference Example 24 was used for the condensation three times.

[0727] The base sequence of this compound is complementary to the sequence from 92 to 106 from the 5' end of exon 5 of the c.648G>T variant G6PC gene in which the G at nucleotide number 728 is mutated to T in the catalytic subunit of glucose-6-phosphatase (G6PC) of Homo sapiens, transcript variant 1, mRNA (NCBI-GenBank accession number NM_000151.3). The compound was identified by negative ion ESI mass spectrometry (measured value: 6785.22)

[0728] (Reference Example 25)

[0729] (25A)

[0730] Synthesis of 7-[(2R)-3-benzyloxy-2-hydroxypropoxy]heptan-1-ol (Compound 25A)

[0731] [Chemical 100]

[0732]

[0733] Benzyl (R)-(-)-glycidyl ether (5.0 g, 30 mmol) was dissolved in dichloromethane (150 mL). 1,7-heptanediol (6.0 g, 45 mmol) and boron trifluoride-diethyl ether complex (0.76 mL, 6.1 mmol) were added and stirred at room temperature for 2 days. The reaction solution was added to a mixed solution of ethyl acetate / saturated sodium bicarbonate aqueous solution and extracted with ethyl acetate. The organic layer was washed with saturated saline / saturated sodium bicarbonate aqueous solution, dried with anhydrous sodium sulfate, filtered, and the solvent was distilled off under reduced pressure to obtain a crude product. It was purified by silica gel column chromatography (hexane: ethyl acetate = 50:50, v / v) to obtain the target product 25A (4.0 g, yield 44%) as a colorless oil.

[0734] 1 H-NMR (CDCl 3 )δ:7.39-7.27(5H,m),4.56(2H,s),4.04-3.94(1H,m),3.67-3.59(2H,m),3. 58-3.40(6H,m),2.51(1H,d,J=4.2Hz),1.63-1.49(4H,m),1.41-1.30(6H,m).

[0735] C 17 H 28 O 4 :[M+Na] + Calculated value 319, measured value 319.

[0736] (25B)

[0737] Synthesis of [(2R,3R,4R,5R,6R)-5-acetamide-3,4-diacetoxy-6-[7-[(2R)-3-benzyloxy-2-hydroxy-propoxy]heptyloxy]tetrahydropyran-2-yl]methyl acetate (Compound 25B-1)

[0738] [Chemistry 101]

[0739]

[0740] Synthesis of [(2R,3R,4R,5R,6R)-5-acetamide-6-[7-[(2R)-2-[(2R,3R,4R,5R,6R)-3-acetamide-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxy-3-benzyloxy-propoxy]heptyloxy]-3,4-diacetoxytetrahydropyran-2-yl]methyl acetate (Compound 25B-2)

[0741] [Chemistry 102]

[0742]

[0743] To a suspension of compound 25A (4.0 g, 14 mmol) synthesized in step (25A) and acetic acid [(2R, 3R, 4R, 5R)-5-acetamide-3, 4, 6-triacetoxy-tetrahydropyran-2-yl] methyl ester (WO2011053614) (4.8 g, 12 mmol) of a known compound in the literature in dichloromethane (200 mL), trifluoromethanesulfonic acid (0.18 mL, 2.1 mmol) was added, and the mixture was stirred at 45°C for 3 days. After the reaction was completed, half of the solvent was distilled off under reduced pressure and concentrated, and the mixture was added to a mixed solution of ethyl acetate / saturated sodium bicarbonate aqueous solution and extracted with ethyl acetate. The organic layer was washed with saturated sodium bicarbonate aqueous solution and saturated saline / phosphate buffer (pH 7.0), dried with anhydrous sodium sulfate, filtered, and the solvent was distilled off under reduced pressure to obtain a crude product. It was purified by silica gel column chromatography (hexane:ethyl acetate=100:0-0:100, v / v) to obtain amorphous target products 25B-1 (4.8 g, yield 62%) and 25B-2 (1.9 g, yield 16%).

[0744] (Compound 25B-1)

[0745] 1 H-NMR (CDCl 3 )δ:7.39-7.27(5H,m),5.50(1H,d,J=9.1Hz),5.37-5.28(2H,m),4.70(1H,d,J=8.5Hz),4.57(2H,s),4.20-4.08(2H,m),4.03-3.85(4H, m),3.58-3.42(7H,m),2.58(1H,d,J=4.2Hz),2.14(3H,s),2.05(3H,s),2.00(3H,s),1.95(3H,s),1.64-1.50(4H,m),1.38-1.29(6H,m).

[0746] C 31 H 47 NO12 :[M+H] + Calculated value 626, measured value 626.

[0747] (Compound 25B-2)

[0748] 1 H-NMR (CDCl 3 )δ:7.39-7.28(5H,m),5.85-5.21(6H,m),4.90-4.73(2H,m),4.53(2H,s),4. 22-3.38(17H,m),2.20-1.91(24H,m),1.63-1.51(4H,m),1.41-1.30(6H,m).

[0749] C 45 H 66 N 2 O 20 :[M+H] + Calculated value 955, measured value 955.

[0750] (25C)

[0751] Synthesis of [(2R,3R,4R,5R,6R)-5-acetamide-3,4-diacetoxy-6-[7-[(2S)-2,3-dihydroxypropoxy]heptyloxy]tetrahydropyran-2-yl]methyl acetate (Compound 25C)

[0752] [Chemistry 103]

[0753]

[0754] The compound 25B-1 (4.8 g, 7.7 mmol) synthesized in step (25B) was dissolved in ethanol (100 mL). 10% palladium / carbon (wet basis) (2.0 g) was added and stirred vigorously at room temperature for 5 hours under a hydrogen environment. After the reaction was completed, the mixture was filtered and the obtained liquid was distilled off under reduced pressure. After adding an appropriate amount of toluene for azeotropy, an appropriate amount of pyridine was added for azeotropy to obtain a crude product of the target compound 25C. No further purification was performed and the mixture was used for the next reaction.

[0755] 1 H-NMR (CDCl 3)δ:5.62(1H,d,J=8.5Hz),5.38-5.26(2H,m),4.69(1H,d,J=8.5Hz),4.21-4.09(2H,m),4.00-3.43(11H,m),2.74(1H,br s),2.15(3H,s),2.05(3H,s),2.01(3H,s),1.96(3H,s),1.68-1.50(4H,m),1.41-1.29(6H,m).

[0756] C 24 H 41 NO 12 :[M+H] + Calculated value 536, measured value 536.

[0757] (25D)

[0758] Synthesis of [(2R,3R,4R,5R,6R)-5-acetamide-3,4-diacetoxy-6-[7-[(2R)-3-[bis(4-methoxyphenyl)-phenyl-methoxy]-2-hydroxy-propoxy]heptyloxy]tetrahydropyran-2-yl]methyl acetate (Compound 25D)

[0759] [Chemistry 104]

[0760]

[0761] The crude product of compound 25C synthesized in step (25C) was dissolved in pyridine (30 mL). 4,4'-dimethoxytriphenylchloromethane (2.7 g, 8.0 mmol) was added and stirred at room temperature for 1.5 hours. After the reaction was completed, the solvent was distilled off under reduced pressure. After adding toluene for azeotropy, a crude product was obtained. It was purified by silica gel column chromatography (hexane: ethyl acetate = 50:50-0:100, v / v, containing 1% triethylamine) to obtain an amorphous target 25D (4.5 g, yield 2 step 70%).

[0762] 1 H-NMR (CDCl 3)δ:7.46-7.40(2H,m),7.34-7.18(7H,m),6.86-6.79(4H,m),5.50(1H,d,J=8.5Hz ),5.37-5.28(2H,m),4.70(1H,d,J=8.5Hz),4.20-4.08(2H,m),3.98-3.84(4H,m), 3.79(6H,s),3.55-3.39(5H,m),3.21-3.14(2H,m),2.51(1H,d,J=4.2Hz),2.14(3H ,s),2.04(3H,s),2.00(3H,s),1.94(3H,s),1.65-1.47(4H,m),1.36-1.27(6H,m).

[0763] C 45 H 59 NO 14 :[M+Na] + Calculated value 860, measured value 860.

[0764] (25E)

[0765] Synthesis of [(2R,3R,4R,5R,6R)-5-acetamide-3,4-diacetoxy-6-[7-[(2R)-3-[bis(4-methoxyphenyl)-phenyl-methoxy]-2-[2-cyanoethyl-(diisopropylamino)phosphino]oxypropoxy]heptyloxy]tetrahydropyran-2-yl]methyl acetate (Compound 25E)

[0766] [Chemistry 105]

[0767]

[0768] The compound 25D (4.5 g, 5.4 mmol) synthesized in step (25D) was dissolved in dichloromethane (50 mL) after azeotroping with an appropriate amount of pyridine. N,N-diisopropylethylamine (3.7 mL, 21 mmol) and 2-cyanoethyldiisopropylchlorophosphoramidite (1.3 ml, 5.9 mmol) were added and stirred at room temperature for 1 hour. After the reaction was completed, the solvent was distilled off under reduced pressure. A crude product was obtained by adding an appropriate amount of toluene for azeotroping. It was purified by silica gel column chromatography (hexane: ethyl acetate = 50:50-0:100, v / v, containing 1% triethylamine) to obtain the amorphous target 25E (4.5 g, yield 81%).

[0769] 1 H-NMR (CDCl 3)δ:7.48-7.40(2H,m),7.36-7.16(7H,m),6.84-6.77(4H,m),5.58-5.42 (1H,m),5.37-5.27(2H,m),4.74-4.68(1H,m),4.20-4.07(2H,m),3.97- 3.06(21H,m),2.67-2.39(2H,m),2.14(3H,s),2.05(3H,s),2.00(3H,s) ,1.92(3H,s),1.63-1.45(4H,m),1.36-1.23(6H,m),1.21-0.98(12H,m).

[0770] (Example 100)

[0771] HO-X 5 -X 5 -X 5 -A m1s -A e2s -U m1s -C m1s -C e2s -G m1s -A m1s -T e2s -G m1s -G m1s -C e2s -G m1s -A m1s -A e2s -G m1t -H (Sequence number 40)

[0772] Replace the part of X with X 5 , and synthesized in the same manner as in Example 91. 5 The compound 25E synthesized in Reference Example 16 was used for the condensation three times.

[0773] The base sequence of the compound is a sequence complementary to the 92nd to 106th sequence from the 5' end of exon 5 of the c.648G>T variant G6PC gene in which the G at nucleotide number 728 is mutated to T in the mRNA of glucose-6-phosphatase catalytic subunit (G6PC) of Homo sapiens, transcript variant 1 (NCBI-GenBank accession number NM_000151.3). The compound was identified by negative ion ESI mass spectrometry (measured value: 6785.21).

[0774] (Reference Example 26)

[0775] (26A)

[0776] Synthesis of 13-[(2S)-3-benzyloxy-2-hydroxypropoxy]tridecane-1-ol (Compound 26A)

[0777] [Chemistry 106]

[0778]

[0779] Benzyl (S)-(+)-glycidyl ether (4.0 g, 24 mmol) was dissolved in dichloromethane (150 mL). Tridecane-1,13-diol (7.6 g, 35 mmol) and boron trifluoride-diethyl ether complex (0.61 mL, 4.9 mmol) were added and stirred at room temperature for 2 days. The reaction solution was added to a mixed solution of ethyl acetate / saturated sodium bicarbonate aqueous solution and extracted with ethyl acetate. The organic layer was washed with saturated saline / saturated sodium bicarbonate aqueous solution, dried with anhydrous sodium sulfate, filtered, and the solvent was distilled off under reduced pressure to obtain a crude product. It was purified by silica gel column chromatography (hexane: ethyl acetate = 50:50, v / v) to obtain the target product 26A (3.8 g, yield 41%) as a colorless oil.

[0780] 1 H-NMR (CDCl 3 )δ:7.38-7.27(5H,m),4.56(2H,s),4.02-3.94(1H,m),3.69-3.39(8H,m),2.49(1H,d,J=4.2Hz),1.61-1.50(4H,m),1.39-1.21(18H,m).

[0781] C 23 H 40 O 4 :[M+H] + Calculated value 381, measured value 381.

[0782] (26B)

[0783] Synthesis of [(2R,3R,4R,5R,6R)-5-acetamide-3,4-diacetoxy-6-[13-[(2S)-3-benzyloxy-2-hydroxy-propoxy]tridecyloxy]tetrahydropyran-2-yl]methyl acetate (Compound 26B)

[0784] [Chemistry 107]

[0785]

[0786] To a suspension of compound 26A (3.8 g, 9.9 mmol) synthesized in step (26A) and acetic acid [(2R, 3R, 4R, 5R)-5-acetamide-3, 4, 6-triacetoxy-tetrahydropyran-2-yl] methyl ester (WO2011053614) (3.5 g, 9.0 mmol) of a known compound in the literature in dichloromethane (60 mL), trifluoromethanesulfonic acid (0.13 mL, 1.5 mmol) was added, and the mixture was stirred at 45°C for 17 hours. After the reaction was completed, half of the solvent was distilled off under reduced pressure and concentrated, and the mixture was added to a mixed solution of ethyl acetate / saturated sodium bicarbonate aqueous solution and extracted with ethyl acetate. The organic layer was washed with saturated sodium bicarbonate aqueous solution and saturated saline / phosphate buffer (pH 7.0), dried with anhydrous sodium sulfate, filtered, and the solvent was distilled off under reduced pressure to obtain a crude product. This was purified by silica gel column chromatography (hexane:ethyl acetate=100:0-0:100, v / v) to obtain an amorphous target compound 26B (3.7 g, yield 58%).

[0787] 1 H-NMR (CDCl 3 )δ:7.38-7.27(5H,m),5.39-5.30(3H,m),4.72(1H,d,J=8.5Hz),4.57(2H,s),4.20-4.09(2H,m),4.02-3.85(4H,m),3.58-3. 40(7H,m),2.48(1H,d,J=4.2Hz),2.14(3H,s),2.05(3H,s),2.01(3H,s),1.96(3H,s),1.63-1.51(4H,m),1.37-1.21(18H,m).

[0788] C 37 H 59 NO 12 :[M+H] + Calculated value 710, measured value 710.

[0789] (26C)

[0790] Synthesis of [(2R,3R,4R,5R,6R)-5-acetamide-3,4-diacetoxy-6-[13-[(2R)-2,3-dihydroxypropoxy]tridecyloxy]tetrahydropyran-2-yl]methyl acetate (Compound 26C)

[0791] [Chemistry 108]

[0792]

[0793] The compound 26B (3.7 g, 5.2 mmol) synthesized in step (26B) was dissolved in ethanol (100 mL). 10% palladium / carbon (wet basis) (2.0 g) was added and stirred vigorously at room temperature for 5 hours under a hydrogen environment. After the reaction was completed, the mixture was filtered and the obtained liquid was distilled off under reduced pressure to obtain a crude product. It was purified by silica gel column chromatography (ethyl acetate: methanol = 100: 0-90: 10, v / v) to obtain the target product 26C (1.4 g, yield 43%) as a colorless oil.

[0794] 1 H-NMR (CDCl 3 )δ:5.42-5.29(3H,m),4.57(1H,d,J=8.5Hz),4.21-4.09(2H,m),3.95-3.44(11H,m),2.60(1H,d,J= 4.8Hz),2.14(3H,s),2.05(3H,s),2.01(3H,s),1.96(3H,s),1.64-1.51(4H,m),1.37-1.22(18H,m).

[0795] C 30 H 53 NO 12 :[M+Na] + Calculated value 642, measured value 642.

[0796] (26D)

[0797] Synthesis of [(2R,3R,4R,5R,6R)-5-acetamide-3,4-diacetoxy-6-[13-[(2S)-3-[bis(4-methoxyphenyl)-phenylmethoxy]-2-hydroxy-propoxy]tridecyloxy]tetrahydropyran-2-yl]methyl acetate (Compound 26D)

[0798] [Chemistry 109]

[0799]

[0800] The compound 26C (1.4 g, 2.3 mmol) synthesized in step (26C) was dissolved in pyridine (10 mL). 4,4'-dimethoxytriphenylchloromethane (0.84 g, 2.5 mmol) was added and stirred at room temperature for 15 hours. After the reaction was completed, the solvent was distilled off under reduced pressure. After adding toluene for azeotropy, a crude product was obtained. It was purified by silica gel column chromatography (hexane: ethyl acetate = 50:50-20:80, v / v, containing 1% triethylamine) to obtain an amorphous target 26D (1.9 g, yield 91%).

[0801] 1 H-NMR (CDCl 3 )δ:7.45-7.41(2H,m),7.34-7.18(7H,m),6.85-6.80(4H,m),5.40-5.30(3H, m),4.72(1H,d,J=8.5Hz),4.20-4.09(2H,m),3.97-3.84(4H,m),3.79(6H,s) ,3.56-3.40(5H,m),3.22-3.13(2H,m),2.43(1H,d,J=4.8Hz),2.14(3H,s),2 .05(3H,s),2.01(3H,s),1.95(3H,s),1.63-1.50(4H,m),1.35-1.21(18H,m).

[0802] C 51 H 71 NO 14 :[M+Na] + Calculated value 944, measured value 944.

[0803] (26E)

[0804] Synthesis of [(2R,3R,4R,5R,6R)-5-acetamide-3,4-diacetoxy-6-[13-[(2S)-3-[bis(4-methoxyphenyl)-phenylmethoxy]-2-[2-cyanoethyl-(diisopropylamino)phosphino]oxypropoxy]tridecyloxy]tetrahydropyran-2-yl]methyl acetate (Compound 26E)

[0805] [Chemistry 110]

[0806]

[0807] The compound 26D (1.9 g, 2.1 mmol) synthesized in step (26D) was dissolved in dichloromethane (50 mL) after azeotroping with an appropriate amount of pyridine. N,N-diisopropylethylamine (1.4 mL, 8.0 mmol) and 2-cyanoethyldiisopropylchlorophosphoramidite (0.49 mL, 2.2 mmol) were added and stirred at room temperature for 1.5 hours. After the reaction was completed, the solvent was distilled off under reduced pressure to obtain a crude product. It was purified by silica gel column chromatography (hexane: ethyl acetate = 100: 0-50: 50, v / v, containing 1% triethylamine) to obtain an amorphous target 26E (1.7 g, yield 75%).

[0808] 1 H-NMR (CDCl 3)δ:7.48-7.41(2H,m),7.36-7.16(7H,m),6.84-6.78(4H,m),5.41-5.30(3H,m),4.72(1H,d,J=8.5Hz),4.20-4.09(2H,m),3.95-3.07(21 H,m),2.66-2.40(2H,m),2.14(3H,s),2.05(3H,s),2.00(3H,s),1.95(3H,s),1.63-1.45(4H,m),1.35-1.21(18H,m),1.21-0.99(12H,m).

[0809] (Example 101)

[0810] HO-X 6 -X 6 -X 6 -A m1s -A e2s -U m1s -C m1s -C e2s -G m1s -A m1s -T e2s -G m1s -G m1s -C e2s -G m1s -A m1s -A e2s -G m1t -H (Sequence number 40)

[0811] Replace the part of X with X 6 , and synthesized in the same manner as in Example 91. 6 The compound 26E synthesized in Reference Example 26 was used for the condensation three times.

[0812] The base sequence of the compound is complementary to the sequence from 92 to 106 from the 5' end of exon 5 of the c.648G>T variant G6PC gene in which the G at nucleotide number 728 is mutated to T in the catalytic subunit of glucose-6-phosphatase (G6PC) of Homo sapiens, transcript variant 1, mRNA (NCBI-GenBank accession number NM_000151.3). The compound was identified by negative ion ESI mass spectrometry (measured value: 7037.48).

[0813] (Reference Example 27)

[0814] (27A)

[0815] Synthesis of [(2R,3R,4R,5R,6R)-5-acetamide-6-[7-[(2R)-2-[(2R,3R,4R,5R,6R)-3-acetamide-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxy-3-hydroxy-propoxy]heptyloxy]-3,4-diacetoxytetrahydropyran-2-yl]methyl acetate (Compound 27A)

[0816] [Chemistry 111]

[0817]

[0818] The compound 24B-2 (1.9 g, 2.0 mmol) synthesized in step (24B) was dissolved in ethanol (60 mL). 10% palladium / carbon (wet basis) (2.0 g) was added and stirred vigorously at room temperature for 8 hours under a hydrogen environment. After the reaction was completed, the obtained liquid was filtered and removed by vacuum distillation. After adding an appropriate amount of toluene for azeotropy, an appropriate amount of pyridine was added for azeotropy to obtain a crude product (1.3 g) of the target compound 27A. No further purification was performed and it was used for the next reaction.

[0819] 1 H-NMR (CDCl 3 )δ:5.84(1H,d,J=8.5Hz),5.69(1H,d,J=8.5Hz),5.41-5.14(4H,m),4.85(1H,d,J=8.5Hz),4.70(1H,d,J= 8.5Hz),4.22-3.40(17H,m),2.86-2.81(1H,m),2.20-1.93(24H,m),1.65-1.49(4H,m),1.39-1.23(6H,m).

[0820] C 38 H 60 N 2 O 20 :[M+H] + Calculated value 865, measured value 865.

[0821] (27B)

[0822] Synthesis of [(2R,3R,4R,5R,6R)-5-acetamide-6-[7-[(2S)-2-[(2R,3R,4R,5R,6R)-3-acetamide-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxy-3-[2-cyanoethoxy-(diisopropylamino)phosphino]oxypropoxy]heptyloxy]-3,4-diacetoxytetrahydropyran-2-yl]methyl acetate (Compound 27B)

[0823] [Chemistry 112]

[0824]

[0825] The crude product (1.3 g) of compound 27A synthesized in step (27A) was dissolved in dichloromethane (50 mL). Diisopropylethylamine (1.0 mL, 5.8 mmol) and 2-cyanoethyldiisopropylchlorophosphoramidite (0.36 mL, 1.6 mmol) were added, and the mixture was stirred at room temperature for 1 hour. Furthermore, 2-cyanoethyldiisopropylchlorophosphoramidite (0.36 mL, 1.6 mmol) was added, and the mixture was stirred at room temperature for 1 hour. After the reaction was completed, the solvent was distilled off under reduced pressure. A crude product was obtained by adding an appropriate amount of toluene for azeotropy. It was purified by silica gel column chromatography (ethyl acetate:acetonitrile = 100:0-90:10, v / v) to obtain the amorphous target product 27B (0.56 g, yield 26%).

[0826] 1 H-NMR (CDCl 3 )δ:6.04-5.69(2H,m),5.39-5.06(4H,m),4.86-4.69(2H,m),4.25-3.40(21H,m) ,2.80-2.67(2H,m),2.21-1.92(24H,m),1.66-1.48(4H,m),1.38-1.15(18H,m).

[0827] (Example 102)

[0828] HO-X 7 -X 4 -A m1s -A e2s -U m1s -C m1s -C e2s -G m1s -A m1s -T e2s -G m1s -G m1s -C e2s -G m1s -A m1s -A e2s -G m1t -H (Sequence number 40)

[0829] Replace the part of X with X 4 With X 7 , and synthesized in the same manner as in Example 91. 4 The compound 24E synthesized in Reference Example 15 was used for one condensation. 7The compound 27B synthesized in Reference Example 27 was used for the first condensation.

[0830] The base sequence of the compound is complementary to the sequence from 92 to 106 from the 5' end of exon 5 of the c.648G>T variant G6PC gene in which the G at nucleotide number 728 is mutated to T in the mRNA of glucose-6-phosphatase catalytic subunit (G6PC) of Homo sapiens, transcript variant 1 (NCBI-GenBank accession number NM_000151.3). The compound was identified by negative ion ESI mass spectrometry (measured value: 6517.17).

[0831] (Reference Example 28)

[0832] (28A)

[0833] Synthesis of [(2R,3R,4R,5R,6R)-5-acetamide-6-[7-[(2S)-2-[(2R,3R,4R,5R,6R)-3-acetamide-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxy-3-hydroxy-propoxy]heptyloxy]-3,4-diacetoxytetrahydropyran-2-yl]methyl acetate (Compound 28A)

[0834] [Chemistry 113]

[0835]

[0836] The compound 25B-2 (1.9 g, 2.0 mmol) synthesized in step (25B) was dissolved in ethanol (60 mL). 10% palladium / carbon (wet basis) (2.0 g) was added and stirred vigorously at room temperature for 5 hours under a hydrogen atmosphere. After the reaction was completed, the obtained liquid was filtered and removed by vacuum distillation. After adding an appropriate amount of toluene for azeotropy, an appropriate amount of pyridine was added for azeotropy to obtain a crude product (1.8 g) of the target compound 28A. No further purification was performed and it was used for the next reaction.

[0837] 1 H-NMR (CDCl 3 )δ:5.93(1H,d,J=8.5Hz),5.71(1H,d,J=8.5Hz),5.42-5.22(4H,m),4.82-4.73(2H,m),4.23 -3.35(17H,m),2.98-2.92(1H,m),2.21-1.92(24H,m),1.66-1.48(4H,m),1.42-1.23(6H,m).

[0838] C 38 H 60 N 2O 20 :[M+H] + Calculated value 865, measured value 865.

[0839] (28B)

[0840] Synthesis of [(2R,3R,4R,5R,6R)-5-acetamide-6-[7-[(2R)-2-[(2R,3R,4R,5R,6R)-3-acetamide-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxy-3-[2-cyanoethoxy-(diisopropylamino)phosphino]oxypropoxy]heptyloxy]-3,4-diacetoxytetrahydropyran-2-yl]methyl acetate (Compound 28B)

[0841] [Chemistry 114]

[0842]

[0843] The crude product (1.8 g) of compound 28A synthesized in step (28A) was dissolved in dichloromethane (50 mL). Diisopropylethylamine (1.5 mL, 8.4 mmol) and 2-cyanoethyldiisopropylchlorophosphoramidite (0.51 mL, 2.3 mmol) were added, and the mixture was stirred at room temperature for 5.5 hours. Furthermore, 2-cyanoethyldiisopropylchlorophosphoramidite (0.51 mL, 2.3 mmol) was added, and the mixture was stirred at room temperature for 1 hour. After the reaction was completed, the solvent was distilled off under reduced pressure. A crude product was obtained by adding an appropriate amount of toluene for azeotropy. It was purified by silica gel column chromatography (ethyl acetate:acetonitrile = 100:0-90:10, v / v) to obtain the amorphous target product 28B (1.5 g, yield 2 step 71%).

[0844] 1 H-NMR (CDCl 3 )δ:5.91-5.67(2H,m),5.41-5.18(4H,m),4.91-4.72(2H,m),4.26-3.35(21H,m) ,2.79-2.63(2H,m),2.20-1.92(24H,m),1.67-1.50(4H,m),1.40-1.13(18H,m).

[0845] (Example 103)

[0846] HO-X 8 -X 5 -A m1s -A e2s -U m1s -C m1s -C e2s -G m1s -Am1s -T e2s -G m1s -G m1s -C e2s -G m1s -A m1s -A e2s -G m1t -H (Sequence number 40)

[0847] Replace the part of X with X 5 With X 8 , and synthesized in the same manner as in Example 91. 5 The compound 25E synthesized in Reference Example 25 was used for one condensation. 8 The compound 28B synthesized in Reference Example 28 was used for the first condensation.

[0848] The base sequence of the compound is a sequence complementary to the 92nd to 106th sequence from the 5' end of exon 5 of the c.648G>T variant G6PC gene in which the G at nucleotide number 728 is mutated to T in the mRNA of glucose-6-phosphatase catalytic subunit (G6PC) of Homo sapiens, transcript variant 1 (NCBI-GenBank accession number NM_000151.3). The compound was identified by negative ion ESI mass spectrometry (measured value: 6517.10).

[0849] (Reference Example 29)

[0850] (29A)

[0851] Synthesis of (2R)-1-benzyloxy-3-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]propan-2-ol (Compound 29A)

[0852] [Chemistry 115]

[0853]

[0854] Dissolve triethylene glycol (6.9 g, 46 mmol) and benzyl (S)-(+)-glycidyl ether (5.0 g, 30 mmol) in dichloromethane (75 mL), add boron trifluoride-diethyl ether complex (0.77 mL, 6.1 mmol), and stir at room temperature for 16 hours. After the reaction is completed, add saturated sodium bicarbonate aqueous solution and extract with dichloromethane. After washing the organic layer with saturated saline / saturated sodium bicarbonate aqueous solution, dry and filter with anhydrous sodium sulfate, and remove the solvent by vacuum distillation to obtain a crude product. Separate and purify by reverse phase HPLC (high performance liquid chromatography) (GL Science, InertsilODS-3) with 0.1% trifluoroacetic acid aqueous solution and 0.1% trifluoroacetic acid acetonitrile solution as eluent, collect the components containing the target, and remove the solvent by vacuum distillation. The target compound was dissolved in dichloromethane, and the organic layer was washed with saturated brine and saturated brine / saturated sodium bicarbonate aqueous solution, dried over anhydrous sodium sulfate, filtered, and the solvent was distilled off under reduced pressure to obtain the target compound 29A (5.6 g, yield 58%) as an oil.

[0855] 1 H-NMR (CDCl 3 )δ:7.37-7.25(5H,m),4.55(2H,s),4.05-3.97(1H,m),3.65-3.56(16H,m).

[0856] C 16 H 26 O 6 :[M+H] + Calculated value 315, measured value 315.

[0857] (29B)

[0858] Synthesis of [(2R,3R,4R,5R,6R)-5-acetamide-3,4-diacetoxy-6-[2-[2-[2-[(2R)-3-benzyloxy-2-hydroxy-propoxy]ethoxy]ethoxy]ethoxy]tetrahydropyran-2-yl]methyl acetate (Compound 29B)

[0859] [Chemistry 116]

[0860]

[0861] Trifluoromethanesulfonic acid (0.24 mL, 2.8 mmol) was added to a suspension of compound 29A (5.6 g, 18 mmol) synthesized in step (29A) and acetic acid [(2R, 3R, 4R, 5R)-5-acetamide-3, 4, 6-triacetoxy-tetrahydropyran-2-yl] methyl ester (WO2011053614) (6.3 g, 16 mmol) of a known compound in the literature in dichloromethane (100 mL), and the mixture was stirred at 45° C. for 16 hours. After the reaction was completed, a saturated aqueous sodium bicarbonate solution was added, and extraction was performed with dichloromethane. The organic layer was washed with saturated brine / saturated aqueous sodium bicarbonate solution, dried with anhydrous sodium sulfate, filtered, and the solvent was distilled off under reduced pressure to obtain a crude product. The target product was separated and purified by reverse phase HPLC (GL Science, Inertsil ODS-3) using 0.1% trifluoroacetic acid aqueous solution and 0.1% trifluoroacetic acid acetonitrile solution as eluents, and the fractions containing the target product were collected and the solvent was distilled off under reduced pressure. The target product was dissolved in ethyl acetate, and the organic layer was washed with saturated saline and saturated saline / saturated sodium bicarbonate aqueous solution, and then dried with anhydrous sodium sulfate, filtered, and the solvent was distilled off under reduced pressure to obtain an amorphous target product 29B (4.9 g, yield 47%).

[0862] 1 H-NMR (CDCl 3 )δ:7.38-7.27(5H,m),6.69(1H,d,J=9.1Hz),5.28(1H,d,J=3.0Hz),5.02(1H,dd,J=11.2,3.0Hz),4.77 (1H,d,J=8.5Hz),4.56(2H,s),4.29-3.47(21H,m),2.15(3H,s),2.04(3H,s),1.97(3H,s),1.96(3H,s).

[0863] C 30 H 45 NO 14 :[M+H] + Calculated value 644, measured value 644.

[0864] (29C)

[0865] Synthesis of [(2R,3R,4R,5R,6R)-5-acetamide-3,4-diacetoxy-6-[2-[2-[2-[(2R)-2,3-dihydroxypropoxy]ethoxy]ethoxy]ethoxy]tetrahydropyran-2-yl]methyl acetate (Compound 29C)

[0866] [Chemistry 117]

[0867]

[0868] The compound 29B (4.9 g, 7.6 mmol) synthesized in step (29B) was dissolved in ethanol (100 mL). 10% palladium / carbon (wet basis) (4.0 g) was added and stirred vigorously at room temperature for 8 hours under a hydrogen atmosphere. After the reaction was completed, the mixture was filtered and the obtained liquid was distilled off under reduced pressure. An appropriate amount of toluene was added for azeotropic distillation to obtain the target oil 29C (4.0 g, yield 95%).

[0869] 1 H-NMR (CDCl 3 )δ:6.83-6.76(1H,m),5.32(1H,d,J=3.0Hz),5.11(1H,dd,J=11.2,3.0Hz),4.79(1H,d,J=9.1H z),4.27-4.08(3H,m),3.97-3.53(18H,m),2.16(3H,s),2.06(3H,s),2.00(3H,s),1.97(3H,s).

[0870] C 23 H 39 NO 14 :[M+H] + Calculated value 554, measured value 554.

[0871] (29D)

[0872] Synthesis of [(2R,3R,4R,5R,6R)-5-acetamide-3,4-diacetoxy-6-[2-[2-[2-[(2S)-3-[bis(4-methoxyphenyl)-phenylmethoxy]-2-hydroxy-propoxy]ethoxy]ethoxy]ethoxy]tetrahydropyran-2-yl]methyl acetate (Compound 29D)

[0873] [Chemistry 118]

[0874]

[0875] Add an appropriate amount of pyridine to the compound 29C (4.0 g, 7.2 mmol) synthesized in step (29C) and perform azeotropy under reduced pressure. Dissolve in pyridine (30 mL), add 4,4'-dimethoxytriphenylmethane (2.7 g, 8.0 mmol), and stir at room temperature for 3 hours. After the reaction is completed, the solvent is distilled off under reduced pressure. Add an appropriate amount of toluene and perform azeotropy to obtain a crude product. Purify it by silica gel column chromatography (ethyl acetate: ethyl acetate / methanol (5 / 1) = 100:0-50:50, v / v, containing 1% triethylamine) to obtain an amorphous target 29D (3.5 g, yield 57%).

[0876] 1 H-NMR (CDCl 3 )δ:7.44-7.40(2H,m),7.33-7.18(7H,m),6.85-6.80(4H,m),6.59(1H,d ,J=9.7Hz),5.29(1H,d,J=3.0Hz),5.00(1H,dd,J=11.2,3.0Hz),4.77(1H ,d,J=8.5Hz),4.27-4.07(3H,m),4.00-3.82(4H,m),3.79(6H,s),3.71- 3.49(12H,m),3.20-3.10(2H,m),2.14(3H,s),2.01(3H,s),1.93(6H,s).

[0877] C 44 H 57 NO 16 :[M+Na] + Calculated value 878, measured value 878.

[0878] (29E)

[0879] Synthesis of [(2R,3R,4R,5R,6R)-5-acetamide-3,4-diacetoxy-6-[2-[2-[2-[(2S)-3-[bis(4-methoxyphenyl)-phenylmethoxy]-2-[2-cyanoethoxy-(diisopropylamino)phosphino]oxypropoxy]ethoxy]ethoxy]ethoxy]tetrahydropyran-2-yl]methyl acetate (Compound 29E)

[0880] [Chemistry 119]

[0881]

[0882] Compound 29D (3.5 g, 4.1 mmol) synthesized in step (29D) was dissolved in dichloromethane (40 mL) after azeotroping with an appropriate amount of pyridine. N,N-diisopropylethylamine (2.8 mL, 16 mmol) and 2-cyanoethyldiisopropylchlorophosphoramidite (1.1 mL, 4.9 mmol) were added and stirred at room temperature for 3 hours. After the reaction was completed, the solvent was distilled off under reduced pressure. A crude product was obtained by adding an appropriate amount of toluene for azeotroping. It was purified by silica gel column chromatography (hexane: ethyl acetate = 50:50-0:100, v / v, containing 1% triethylamine) to obtain the amorphous target 29E (2.2 g, yield 51%).

[0883] 1 H-NMR (CDCl 3)δ:7.47-7.40(2H,m),7.35-7.16(7H,m),6.85-6.77(4H,m),6.40(1H, d,J=9.1Hz),5.33-5.28(1H,m),5.03-4.93(1H,m),4.81-4.74(1H,m),4 .28-4.08(3H,m),3.96-3.46(26H,m),3.27-3.04(2H,m),2.69-2.41(2 H,m),2.15(3H,s),2.04(3H,s),1.97-1.90(6H,m),1.30-0.98(12H,m).

[0884] (Example 104)

[0885] HO-X 9 -X 9 -X 9 -A m1s -A e2s -U m1s -C m1s -C e2s -G m1s -A m1s -T e2s -G m1s -G m1s -C e2s -G m1s -A m1s -A e2s -G m1t -H (Sequence number 40)

[0886] Replace the part of X with X 9 , and synthesized in the same manner as in Example 91. 9 The compound 29E synthesized in Reference Example 29 was used for the condensation three times.

[0887] The base sequence of the compound is a sequence complementary to the 92nd to 106th sequence from the 5' end of exon 5 of the c.648G>T variant G6PC gene in which the G at nucleotide number 728 is mutated to T in the mRNA of glucose-6-phosphatase catalytic subunit (G6PC) of Homo sapiens, transcript variant 1 (NCBI-GenBank accession number NM_000151.3). The compound was identified by negative ion ESI mass spectrometry (measured value: 6839.16).

[0888] (Reference Example 30)

[0889] (30A)

[0890] (2R)-1-Benzyloxy-3-[2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethoxy]propan-2-ol (Synthesis of Compound 30A)

[0891] [Chemistry 120]

[0892]

[0893] Tetraethylene glycol (8.87 g, 45.7 mmol) and benzyl (S)-(+)-glycidyl ether (4.66 mL, 30.5 mmol) were dissolved in dichloromethane (75 mL), and boron trifluoride-diethyl ether complex (0.76 mL, 6.1 mmol) was added and stirred at room temperature overnight. After the reaction was completed, a saturated aqueous sodium bicarbonate solution was added. After washing the organic layer with a saturated aqueous sodium bicarbonate solution and a saturated saline solution, it was dried and filtered with anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure to obtain a crude product. The product was separated and purified by reverse phase HPLC (GL Science, Inertsil ODS-3) using a 0.1% trifluoroacetic acid aqueous solution and a 0.1% trifluoroacetic acid acetonitrile solution as eluents, and the components containing the target product were collected and the solvent was distilled off under reduced pressure. The target product was dissolved in dichloromethane, and the organic layer was washed with saturated aqueous sodium bicarbonate solution and saturated brine, dried over anhydrous sodium sulfate, and filtered. The solvent was distilled off under reduced pressure to obtain the target product 30A (5.3 g, yield 49%) as an oil.

[0894] 1 H-NMR (CDCl 3 )δ:7.37-7.23(5H,m),4.56(2H,s),4.01(1H,br),3.75-3.41(20H,m).

[0895] C 18 H 30 O 7 :[M+H] + Calculated value 359, measured value 359.

[0896] (30B)

[0897] Acetate [(2R,3R,4R,5R,6R)-5-acetamide-3,4-diacetoxy-6-[2-[2-[2-[(2R)-3-benzyloxy-2-hydroxy-propoxy]ethoxy]ethoxy]ethoxy]ethoxy]tetrahydropyran-2-yl]methyl ester (Synthesis of Compound 30B)

[0898] [Chemistry 121]

[0899]

[0900] Trifluoromethanesulfonic acid (0.2 mL, 2.3 mmol) was added to a suspension of the compound synthesized in step 30A (5.3 g, 15 mmol) and the known compound in the literature [(2R, 3R, 4R, 5R)-5-acetamide-3,4,6-triacetoxy-tetrahydropyran-2-yl] methyl acetate (WO2011053614) (5.2 g, 13 mmol) in dichloromethane (100 mL), and the mixture was stirred at 45°C for 22 hours. After the reaction was completed, the solvent was distilled off under reduced pressure and concentrated, and ethyl acetate was added. The organic layer was washed with saturated sodium bicarbonate aqueous solution and saturated saline, dried with anhydrous sodium sulfate, filtered, and the solvent was distilled off under reduced pressure to obtain a crude product. The product was separated and purified by reverse phase HPLC (GLScience, Inertsil ODS-3) using 0.1% trifluoroacetic acid aqueous solution and 0.1% trifluoroacetic acid acetonitrile solution as eluents, and the fractions containing the target product were collected and the solvent was distilled off under reduced pressure. The target compound was dissolved in dichloromethane, and the organic layer was washed with saturated aqueous sodium bicarbonate solution and saturated brine, dried over anhydrous sodium sulfate, filtered, and the solvent was distilled off under reduced pressure to obtain an amorphous target compound 30B (4.5 g, yield 49%).

[0901] 1 H-NMR (CDCl 3 )δ:7.38-7.24(5H,m),6.72(1H,d,J=8.5Hz),5.29(1H,d,J=3.0Hz),5.00(1H,dd,J=11.2,3.3Hz),4.76(1H ,d,J=8.5Hz),4.56(2H,s),4.31-4.21(1H,m),4.18-3.46(24H,m),2.15(3H,s),2.04(3H,s),1.97(6H,s).

[0902] C 32 H 49 NO 15 :[M+H] + Calculated value: 688, measured value: 688.

[0903] (30C)

[0904] Acetate [(2R,3R,4R,5R,6R)-5-acetamide-3,4-diacetoxy-6-[2-[2-[2-[(2R)-2,3-dihydroxypropoxy]ethoxy]ethoxy]ethoxy]ethoxy]tetrahydropyran-2-yl]methyl ester (Synthesis of 30C)

[0905] [Chemistry 122]

[0906]

[0907] Compound 30B (4.5 g, 6.5 mmol) synthesized in step (30B) was dissolved in ethanol (80 mL). 10% palladium / carbon (wet basis) (4 g) was added and stirred vigorously at room temperature for 8 hours under a hydrogen atmosphere. After the reaction was completed, the mixture was filtered and the obtained liquid was removed by vacuum distillation to obtain a crude product of the target compound 30C (3.9 g, quantitative yield). No further purification was performed and the mixture was used in the next reaction.

[0908] 1 H-NMR (CDCl 3 )δ:6.85(1H,d,J=9.7Hz),5.32(1H,d,J=3.0Hz),5.10(1H,dd,J=11.2,3.0Hz),4.74(1H,d,J=8.5 Hz),4.32-4.09(2H,m),3.99-3.56(23H,m),2.16(3H,s),2.05(3H,s),2.00(3H,s),1.99(3H,s).

[0909] C 25 H 43 NO 15 :[M+H] + Calculated value 598, measured value 598.

[0910] (30D)

[0911] Acetate [(2R,3R,4R,5R,6R)-5-acetamide-3,4-diacetoxy-6-[2-[2-[2-[2-[(2S)-3-[bis(4-methoxyphenyl)-phenyl-methoxy]-2-hydroxy-propoxy]ethoxy]ethoxy]ethoxy]ethoxy]tetrahydropyran-2-yl]methyl ester (Synthesis of Compound 30D)

[0912] [Chemistry 123]

[0913]

[0914] Compound 30C (3.9 g, 6.5 mmol) synthesized in step (30C) was added with an appropriate amount of pyridine and azeotroped under reduced pressure. Dissolved in pyridine (22 mL), 4,4'-dimethoxytriphenylchloromethane (4,4'-DimethoxytritylChloride) (2.4 g, 7.2 mmol) was added, and stirred at room temperature for 2 hours. After the reaction was completed, the solvent was distilled off under reduced pressure. After azeotroping with an appropriate amount of toluene, a crude product was obtained. It was purified by silica gel column chromatography (ethyl acetate: ethyl acetate / methanol (5 / 1) = 100:0-50:50, v / v, containing 1% triethylamine) to obtain an amorphous target 30D (3.12 g, yield 53%).

[0915] 1 H-NMR (CDCl 3 )δ:7.44-7.40(2H,m),7.33-7.18(7H,m),6.85-6.79(4H,m),6.63(1H,d,J=9.1Hz ),5.29(1H,d,J=3.0Hz),4.96(1H,dd,J=11.2,3.3Hz),4.75(1H,d,J=8.5Hz),4.29 -4.21(1H,m),4.17-4.07(2H,m),4.00-3.81(4H,m),3.79(6H,s),3.71-3.58(14H ,m),3.53-3.43(2H,m),3.21-3.10(2H,m),2.14(3H,s),2.02(3H,s),1.95(6H,s).

[0916] C 46 H 61 NO 17 :[M+Na] + Calculated value 922, measured value 922.

[0917] (30E)

[0918] Acetate [(2R,3R,4R,5R,6R)-5-acetamide-3,4-diacetoxy-6-[2-[2-[2-[2-[(2S)-3-[bis(4-methoxyphenyl)-phenyl-methoxy]-2-[2-cyanoethoxy-(diisopropylamino)phosphino]oxy-propoxy]ethoxy]ethoxy]ethoxy]ethoxy]tetrahydropyran-2-yl]methyl ester (Synthesis of Compound 30E)

[0919] [Chemistry 124]

[0920]

[0921] An appropriate amount of pyridine was added to the compound 30D (3.12 g, 3.47 mmol) synthesized in step (30D), and the mixture was dissolved in dichloromethane (35 mL) after azeotroping under reduced pressure. N,N-diisopropylethylamine (2.42 mL, 13.9 mmol) and 2-cyanoethyldiisopropylchlorophosphoramidite (0.93 mL, 4.16 mmol) were added, and the mixture was stirred at room temperature for 1 hour. After the reaction was completed, the solvent was distilled off under reduced pressure. An appropriate amount of toluene was added, and the crude product was obtained after azeotroping under reduced pressure. It was purified by silica gel column chromatography (hexane: ethyl acetate = 100:0-0:100, v / v, containing 1% triethylamine) to obtain the amorphous target 30E (2.98 g, yield 78%).

[0922] 1 H-NMR (CDCl 3 )δ:7.46-7.40(2H,m),7.35-7.17(7H,m),6.84-6.78(4H,m),6.43(1H,d,J= 9.1Hz),5.31(1H,br),5.00-4.93(1H,m),4.78(1H,d,J=8.5Hz),4.29-4.08 (3H,m),3.91-3.48(30H,m),3.27-3.04(2H,m),2.67-2.62(1H,m),2.46-2. 41(1H,m),2.15(3H,s),2.04(3H,s),1.97-1.94(6H,m),1.30-0.99(12H,m).

[0923] (Example 105)

[0924] HO-X 10 -X 10 -X 10 -A m1s -A e2s -U m1s -C m1s -C e2s -G m1s -A m1s -T e2s -G m1s -G m1s -C e2s -G m1s -A m1s -A e2s -G m1t -H (Sequence number 40)

[0925] Replace the part of X with X 10 , and synthesized in the same manner as in Example 91. 10The compound 30E synthesized in Reference Example 30 was used for the condensation three times.

[0926] The base sequence of the compound is a sequence complementary to the 92nd to 106th sequence from the 5' end of exon 5 of the c.648G>T variant G6PC gene in which the G at nucleotide number 728 is mutated to T in the mRNA of glucose-6-phosphatase catalytic subunit (G6PC) of Homo sapiens, transcript variant 1 (NCBI-GenBank accession number NM_000151.3). The compound was identified by negative ion ESI mass spectrometry (measured value: 6971.21).

[0927] (Reference Example 31)

[0928] (31A)

[0929] (2R)-1-Benzyloxy-3-[2-(2-hydroxyethoxy)ethoxy]propan-2-ol (Synthesis of Compound 31A)

[0930] [Chemistry 125]

[0931]

[0932] Diethylene glycol (5.0 g, 30.5 mmol) and benzyl (S)-(+)-glycidyl ether (6.5, 60.9 mmol) were dissolved in dichloromethane (75 mL), and boron trifluoride-diethyl ether complex (0.76 mL, 6.1 mmol) was added and stirred at room temperature for 2 hours. After the reaction was completed, a saturated aqueous sodium bicarbonate solution was added. After the organic layer was washed with a saturated aqueous sodium bicarbonate solution and a saturated saline solution, it was dried and filtered with anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure to obtain a crude product. The product was separated and purified by reverse phase HPLC (GL Science, Inertsil ODS-3) using a 0.1% trifluoroacetic acid aqueous solution and a 0.1% trifluoroacetic acid acetonitrile solution as eluents, and the components containing the target product were collected and the solvent was distilled off under reduced pressure. The target product was dissolved in dichloromethane, and the organic layer was washed with saturated aqueous sodium bicarbonate solution and saturated brine, dried over anhydrous sodium sulfate, and filtered. The solvent was distilled off under reduced pressure to obtain the target product 31A (5.3 g, yield 64%) as an oil.

[0933] 1 H-NMR (CDCl 3 )δ:7.38-7.27(5H,m),4.56(2H,s),4.05-3.97(1H,m),3.77-3.48(12H,m),2.85-2.82(1H,m),2.47-2.42(1H,m).

[0934] C14 H 22 O 5 :[M+Na] + Calculated value 293, measured value 293.

[0935] (31B)

[0936] Acetate [(2R,3R,4R,5R,6R)-5-acetamide-3,4-diacetoxy-6-[2-[2-[(2R)-3-benzyloxy-2-hydroxy-propoxy]ethoxy]ethoxy]tetrahydropyran-2-yl]methyl ester (Synthesis of Compound 31B)

[0937] [Chemistry 126]

[0938]

[0939] Trifluoromethanesulfonic acid (0.2 mL, 2.3 mmol) was added to a suspension of the compound synthesized in step 31A (4.0 g, 15 mmol) and the known compound in the literature [(2R, 3R, 4R, 5R)-5-acetamide-3,4,6-triacetoxy-tetrahydropyran-2-yl] methyl acetate (WO2011053614) (5.2 g, 13 mmol) in dichloromethane (100 mL), and the mixture was stirred at 45°C for 22 hours. After the reaction was completed, the solvent was distilled off under reduced pressure and concentrated, and ethyl acetate was added. The organic layer was washed with saturated sodium bicarbonate aqueous solution and saturated brine, dried with anhydrous sodium sulfate, filtered, and the solvent was distilled off under reduced pressure to obtain a crude product. The product was separated and purified by reverse phase HPLC (GLScience, Inertsil ODS-3) using 0.1% trifluoroacetic acid aqueous solution and 0.1% trifluoroacetic acid acetonitrile solution as eluents, and the fractions containing the target product were collected and the solvent was distilled off under reduced pressure. The target product was dissolved in dichloromethane, and the organic layer was washed with saturated sodium bicarbonate aqueous solution and saturated brine, dried over anhydrous sodium sulfate, filtered, and the solvent was distilled off under reduced pressure to obtain an amorphous target product 31B (4.15 g, yield 52%).

[0940] 1 H-NMR (CDCl 3 )δ:7.37-7.28(5H,m),6.71(1H,d,J=9.7Hz),5.30(1H,d,J=3.0Hz),5.12(1H,dd,J=11.2,3.3Hz),4.77(1H,d,J= 8.5Hz),4.57(2H,s),4.27-4.05(1H,m),3.93-3.45(16H,m),2.15(3H,s),2.05(3H,s),1.98(3H,s),1.95(3H,s).

[0941] C 28 H 41 NO 13 :[M+Na] + Calculated value 622, measured value 622.

[0942] (31C)

[0943] [(2R,3R,4R,5R,6R)-5-acetamide-3,4-diacetoxy-6-[2-[2-[(2R)-2,3-dihydroxypropoxy]ethoxy]ethoxy]tetrahydropyran-2-yl]methyl acetate (Synthesis of Compound 31C)

[0944] [Chemistry 127]

[0945]

[0946] The compound 31B (4.15 g, 6.9 mmol) synthesized in step (31B) was dissolved in ethanol (80 mL). 10% palladium on carbon (wet basis) (4 g) was added and stirred vigorously at room temperature for 8 hours under a hydrogen environment. 10% palladium on carbon (wet basis) (1 g) was further added and stirred vigorously at 50°C for 4 hours under a hydrogen environment. Furthermore, 10% palladium on carbon (wet basis) (1 g) was added and stirred vigorously at 50°C for 8 hours under a hydrogen environment. After the reaction was completed, the reaction mixture was filtered and the obtained liquid was removed by vacuum distillation to obtain a crude product of the target product 31C (3.0 g, yield 86%). No further purification was performed and it was used in the next reaction.

[0947] 1 H-NMR (CDCl 3 )δ:6.58(1H,d,J=9.1Hz),5.32-5.30(1H,m),5.16(1H,dd,J=11.2,3.3Hz),4.78(1H,d,J=8.5H z),4.26-4.09(2H,m),3.95-3.58(15H,m),2.16(3H,s),2.06(3H,s),2.01(3H,s),1.98(3H,s).

[0948] C 21 H 35 NO 13 :[M+H] + Calculated value 510, measured value 510.

[0949] (31D)

[0950] Synthesis of [(2R,3R,4R,5R,6R)-5-acetamide-3,4-diacetoxy-6-[2-[2-[(2S)-3-[bis(4-methoxyphenyl)-phenylmethoxy]-2-hydroxy-propoxy]ethoxy]ethoxy]tetrahydropyran-2-yl]methyl acetate (Compound 31D)

[0951] [Chemistry 128]

[0952]

[0953] Add an appropriate amount of pyridine to the compound 31C (3.0 g, 5.9 mmol) synthesized in step (31C) and perform azeotropy under reduced pressure. Dissolve in pyridine (25 mL), add 4,4'-dimethoxytriphenylchloromethane (2.2 g, 6.5 mmol), and stir at room temperature for 2 hours. After the reaction is completed, distill the solvent under reduced pressure. Add an appropriate amount of toluene and perform azeotropy to obtain a crude product. Purify it by silica gel column chromatography (ethyl acetate: ethyl acetate / methanol (5 / 1) = 100:0-50:50, v / v, containing 1% triethylamine) to obtain an amorphous target 31D (1.7 g, yield 36%).

[0954] 1 H-NMR (CDCl 3 )δ:7.47-7.42(2H,m),7.35-7.18(7H,m),6.86-6.80(4H,m),6.73(1H,d,J=9.7H z), 5.31 (1H, d, J = 3.0Hz), 5.14 (1H, dd, J = 11.2, 3.0Hz), 4.76 (1H, d, J = 8.5Hz), 4. 28-4.09(3H,m),4.07-3.83(4H,m),3.79(6H,s),3.72-3.49(8H,m),3.26(1H,d,J =3.6Hz),3.18-3.13(2H,m),2.14(3H,s),2.04(3H,s),1.94(3H,s),1.90(3H,s).

[0955] C 42 H 53 NO 15 :[M+Na] + Calculated value 834, measured value 834.

[0956] (31E)

[0957] Synthesis of [(2R,3R,4R,5R,6R)-5-acetamide-3,4-diacetoxy-6-[2-[2-[(2S)-3-[bis(4-methoxyphenyl)-phenylmethoxy]-2-[2-cyanoethoxy-(diisopropylamino)phosphino]oxypropoxy]ethoxy]ethoxy]tetrahydropyran-2-yl]methyl acetate (Compound 31E)

[0958] [Chemistry 129]

[0959]

[0960] The compound 31D (1.7 g, 2.1 mmol) synthesized in step (31D) was dissolved in dichloromethane (20 mL) after azeotroping with an appropriate amount of pyridine. N,N-diisopropylethylamine (1.5 mL, 8.4 mmol) and 2-cyanoethyldiisopropylchlorophosphoramidite (0.56 mL, 2.5 mmol) were added and stirred at room temperature for 3 hours. After the reaction was completed, the solvent was distilled off under reduced pressure. A crude product was obtained by adding an appropriate amount of toluene for azeotroping. It was purified by silica gel column chromatography (hexane: ethyl acetate = 50:50-0:100, v / v, containing 1% triethylamine) to obtain the amorphous target 31E (1.5 g, yield 71%).

[0961] 1 H-NMR (CDCl 3 )δ:7.47-7.40(2H,m),7.37-7.17(7H,m),6.86-6.77(4H,m),6.08-5.95(1H, m),5.34-5.29(1H,m),5.16-5.06(1H,m),4.76-4.69(1H,m),4.20-4.02(3H, m),3.95-3.47(22H,m),3.31-3.08(2H,m),2.69-2.40(2H,m),2.14(3H,s),2 .04-2.02(3H,m),1.97-1.94(3H,m),1.91-1.88(3H,m),1.21-1.01(12H,m).

[0962] (Example 106)

[0963] HO-X 11 -X 11 -X 11 -A m1s -A e2s -U m1s -C m1s -C e2s -G m1s -A m1s -Te2s -G m1s -G m1s -C e2s -G m1s -A m1s -A e2s -G m1t -H (Sequence number 40)

[0964] Replace the part of X with X 11 , and synthesized in the same manner as in Example 91. 11 The compound 31E synthesized in Reference Example 31 was used for the condensation three times.

[0965] The base sequence of the compound is complementary to the sequence from 92 to 106 from the 5' end of exon 5 of the c.648G>T variant G6PC gene in which the G at nucleotide number 728 is mutated to T in the catalytic subunit of glucose-6-phosphatase (G6PC) of Homo sapiens, transcript variant 1, mRNA (NCBI-GenBank accession number NM_000151.3). The compound was identified by negative ion ESI mass spectrometry (measured value: 6708.09).

[0966] (Reference Example 32)

[0967] (32A)

[0968] Synthesis of (2S)-1-benzyloxy-3-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]propan-2-ol (Compound 32A)

[0969] [Chemistry 130]

[0970]

[0971] Triethylene glycol (9.2 g, 61 mmol) and benzyl (R)-(-)-glycidyl ether (5.0 g, 30 mmol) were dissolved in dichloromethane (75 mL), and boron trifluoride-diethyl ether complex (0.77 mL, 6.1 mmol) was added and stirred at room temperature for 4 hours. After the reaction was completed, a saturated aqueous sodium bicarbonate solution was added and extracted with dichloromethane. After the organic layer was washed with saturated saline / saturated aqueous sodium bicarbonate solution, it was dried and filtered with anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure to obtain a crude product. The product was separated and purified by reverse phase HPLC (GLScience, Inertsil ODS-3) using 0.1% trifluoroacetic acid aqueous solution and 0.1% trifluoroacetic acid acetonitrile solution as eluents, and the components containing the target were collected and the solvent was distilled off under reduced pressure. The target product was dissolved in dichloromethane, and the organic layer was washed with saturated brine and saturated brine / saturated sodium bicarbonate aqueous solution, dried over anhydrous sodium sulfate, filtered, and the solvent was distilled off under reduced pressure to obtain an oily target product 32A (6.4 g, yield 67%).

[0972] 1 H-NMR (CDCl 3 )δ:7.38-7.25(5H,m),4.56(2H,s),4.19-3.45(17H,m).

[0973] C 16 H 26 O 6 :[M+H] + Calculated value 315, measured value 315.

[0974] (32B)

[0975] Synthesis of [(2R,3R,4R,5R,6R)-5-acetamide-3,4-diacetoxy-6-[2-[2-[2-[(2S)-3-benzyloxy-2-hydroxy-propoxy]ethoxy]ethoxy]ethoxy]tetrahydropyran-2-yl]methyl acetate (Compound 32B)

[0976] [Chemistry 131]

[0977]

[0978] Compound 32A (6.4 g, 20 mmol) synthesized in step (32A) and a known compound in the literature, acetic acid [(2R, 3R, 4R, 5R) -5-acetamide-3, 4, 6-triacetoxy-tetrahydropyran-2-yl] methyl ester (WO2011053614) (7.2 g, 18 mmol) in dichloromethane (200 mL) were suspended, and trifluoromethanesulfonic acid (0.28 mL, 3.1 mmol) was added, and stirred at 45 ° C for 20 hours. After the reaction was completed, a saturated sodium bicarbonate aqueous solution was added, and extraction was performed with dichloromethane. After washing the organic layer with saturated saline / saturated sodium bicarbonate aqueous solution, it was dried with anhydrous sodium sulfate, filtered, and the solvent was distilled off under reduced pressure to obtain a crude product. The target product was separated and purified by reverse phase HPLC (GL Science, Inertsil ODS-3) using 0.1% trifluoroacetic acid aqueous solution and 0.1% trifluoroacetic acid acetonitrile solution as eluents, and the fractions containing the target product were collected and the solvent was distilled off under reduced pressure. The target product was dissolved in ethyl acetate, and the organic layer was washed with saturated saline and saturated saline / saturated sodium bicarbonate aqueous solution, and then dried with anhydrous sodium sulfate, filtered, and the solvent was distilled off under reduced pressure to obtain an amorphous target product 32B (5.6 g, yield 47%).

[0979] 1 H-NMR (CDCl 3 )δ:7.39-7.25(5H,m),6.68(1H,d,J=9.1Hz),5.33-5.27(1H,m),5.07-4.95(1H,m),4.78(1H,d, J=8.5Hz),4.55(2H,s),4.25-3.24(21H,m),2.15(3H,s),2.04(3H,s),1.97(3H,s),1.96(3H,s).

[0980] C 30 H 45 NO 14 :[M+H] + Calculated value 644, measured value 644.

[0981] (32C)

[0982] Synthesis of [(2R,3R,4R,5R,6R)-5-acetamide-3,4-diacetoxy-6-[2-[2-[2-[(2S)-2,3-dihydroxypropoxy]ethoxy]ethoxy]ethoxy]tetrahydropyran-2-yl]methyl acetate (Compound 32C)

[0983] [Chemistry 132]

[0984]

[0985] The compound 32B (5.6 g, 8.7 mmol) synthesized in step (32B) was dissolved in ethanol (100 mL). 20% palladium hydroxide / carbon (wet basis) (1.5 g) was added and stirred vigorously at 50°C for 8 hours under a hydrogen atmosphere. After the reaction was completed, the mixture was filtered and the obtained liquid was removed by vacuum distillation. An appropriate amount of toluene was added for azeotropic distillation to obtain the target oil 32C (4.1 g, yield 85%).

[0986] 1 H-NMR (CDCl 3 )δ:6.80(1H,d,J=9.1Hz),5.34-5.30(1H,m),5.15-5.03(1H,m),4.81(1H,d,J=8.5Hz),4. 33-4.08(3H,m),3.99-3.48(18H,m),2.16(3H,s),2.06(3H,s),2.00(3H,s),1.98(3H,s).

[0987] C 23 H 39 NO 14 :[M+H] + Calculated value 554, measured value 554.

[0988] (32D)

[0989] Synthesis of [(2R,3R,4R,5R,6R)-5-acetamide-3,4-diacetoxy-6-[2-[2-[2-[(2R)-3-[bis(4-methoxyphenyl)-phenylmethoxy]-2-hydroxy-propoxy]ethoxy]ethoxy]ethoxy]tetrahydropyran-2-yl]methyl acetate (Compound 32D)

[0990] [Chemistry 133]

[0991]

[0992] Add an appropriate amount of pyridine to the compound 32C (4.1 g, 7.4 mmol) synthesized in step (32C) and perform azeotropy under reduced pressure. Dissolve in pyridine (30 mL), add 4,4'-dimethoxytriphenylchloromethane (2.8 g, 8.2 mmol), and stir at room temperature for 3 hours. After the reaction is completed, distill the solvent under reduced pressure to remove it. Add an appropriate amount of toluene to perform azeotropy to obtain a crude product. Purify it by silica gel column chromatography (ethyl acetate: ethyl acetate / methanol (5 / 1) = 100:0-50:50, v / v, containing 1% triethylamine) to obtain an amorphous target 32D (1.9 g, yield 30%).

[0993] 1 H-NMR (CDCl 3 )δ:7.44-7.40(2H,m),7.35-7.18(7H,m),6.86-6.79(4H,m),6.67(1H,d ,J=9.1Hz),5.29(1H,d,J=3.0Hz),5.03(1H,dd,J=11.2,3.0Hz),4.77(1H ,d,J=9.1Hz),4.26-4.06(3H,m),4.00-3.81(4H,m),3.79(6H,s),3.75- 3.50(12H,m),3.23-3.11(2H,m),2.15(3H,s),2.01(3H,s),1.94(6H,s).

[0994] C 44 H 57 NO 16 :[M+Na] + Calculated value 878, measured value 878.

[0995] (32E)

[0996] Synthesis of [(2R,3R,4R,5R,6R)-5-acetamide-3,4-diacetoxy-6-[2-[2-[2-[(2R)-3-[bis(4-methoxyphenyl)-phenylmethoxy]-2-[2-cyanoethoxy-(diisopropylamino)phosphino]oxypropoxy]ethoxy]ethoxy]ethoxy]tetrahydropyran-2-yl]methyl acetate (Compound 32E)

[0997] [Chemistry 134]

[0998]

[0999] The compound 32D (1.9 g, 2.2 mmol) synthesized in step (32D) was dissolved in dichloromethane (22 mL) after azeotroping with an appropriate amount of toluene. N,N-diisopropylethylamine (1.6 mL, 9.0 mmol) and 2-cyanoethyldiisopropylchlorophosphoramidite (0.59 mL, 2.7 mmol) were added and stirred at room temperature for 1 hour. After the reaction was completed, the solvent was distilled off under reduced pressure to obtain a crude product. It was purified by silica gel column chromatography (hexane: ethyl acetate = 50:50-0:100, v / v, containing 1% triethylamine) to obtain an amorphous target 32E (1.5 g, yield 64%).

[1000] 1 H-NMR (CDCl 3)δ:7.47-7.40(2H,m),7.36-7.16(7H,m),6.85-6.78(4H,m),6.45-6. 38(1H,m),5.33-5.28(1H,m),5.03-4.94(1H,m),4.80-4.75(1H,m),4. 27-4.08(3H,m),3.93-3.45(26H,m),3.28-3.04(2H,m),2.69-2.40(2H ,m),2.15(3H,s),2.04(3H,s),1.97-1.92(6H,m),1.21-1.01(12H,m).

[1001] (Example 107)

[1002] HO-X 12 -X 12 -X 12 -A m1s -A e2s -U m1s -C m1s -C e2s -G m1s -A m1s -T e2s -G m1s -G m1s -C e2s -G m1s -A m1s -A e2s -G m1t -H (Sequence number 40)

[1003] Replace the part of X with X 12 , and synthesized in the same manner as in Example 91. 12 The compound 32E synthesized in Reference Example 32 was used for the condensation three times.

[1004] The base sequence of the compound is a sequence complementary to the 92nd to 106th sequence from the 5' end of exon 5 of the c.648G>T variant G6PC gene in which the G at nucleotide number 728 is mutated to T in the mRNA of glucose-6-phosphatase catalytic subunit (G6PC) of Homo sapiens, transcript variant 1 (NCBI-GenBank accession number NM_000151.3). The compound was identified by negative ion ESI mass spectrometry (measured value: 6839.16).

[1005] (Example 108)

[1006] HO-X 9 -X 9 -X 9 -X 9 -X9 -A m1s -A e2s -U m1s -C m1s -C e2s -G m1s -A m1s -T e2s -G m1s -G m1s -C e2s -G m1s -A m1s -A e2s -G m1t -H (Sequence number 40)

[1007] Replace the part of X with X 9 , and synthesized in the same manner as in Example 91. 9 The compound 29E synthesized in Reference Example 29 was used for the condensation reaction 5 times.

[1008] The base sequence of the compound is complementary to the sequence from 92 to 106 from the 5' end of exon 5 of the c.648G>T variant G6PC gene in which the G at nucleotide number 728 is mutated to T in the catalytic subunit of glucose-6-phosphatase (G6PC) of Homo sapiens, transcript variant 1, mRNA (NCBI-GenBank accession number NM_000151.3). The compound was identified by negative ion ESI mass spectrometry (measured value: 7817.33).

[1009] (Example 109)

[1010] HO-X 9 -X 9 -X 9 -X 9 -X 9 -X 9 -X 9 -A m1s -A e2s -U m1s -C m1s -C e2s -G m1s -A m1s -T e2s -G m1s -G m1s -C e2s -G m1s -A m1s -A e2s -G m1t -H (Sequence number 40)

[1011] Replace the part of X with X 9, and synthesized in the same manner as in Example 91. 9 The compound 29E synthesized in Reference Example 29 was used for 7 condensation reactions.

[1012] The base sequence of the compound is complementary to the sequence from 92 to 106 from the 5' end of exon 5 of the c.648G>T variant G6PC gene in which the G at nucleotide number 728 is mutated to T in the mRNA of glucose-6-phosphatase catalytic subunit (G6PC) of Homo sapiens, transcript variant 1 (NCBI-GenBank accession number NM_000151.3). The compound was identified by negative ion ESI mass spectrometry (measured value: 8796.73).

[1013] (Reference Example 33)

[1014] (33A)

[1015] Synthesis of 9H-fluoren-9-ylmethyl N-[2-[2-[(2R)-3-benzyloxy-2-hydroxy-propoxy]ethoxy]ethyl]carbamate (Compound 33A)

[1016] [Chemistry 135]

[1017]

[1018] The known compound N-[2-(2-hydroxyethoxy)ethyl]carbamic acid 9H-fluoren-9-ylmethyl ester (Bioconjugate Chemistry, 2000, 11, 755-761) (3.11 g, 10 mmol) and benzyl (S)-(+)-glycidyl ether (0.8 g, 5 mmol) were dissolved in dichloromethane (30 mL), and boron trifluoride-diethyl ether complex (0.1 mL, 1 mmol) was added, and stirred at room temperature for 16 hours. After the reaction was completed, saturated sodium bicarbonate aqueous solution was added. The organic layer was washed with saturated sodium bicarbonate aqueous solution and saturated saline, dried with anhydrous sodium sulfate, filtered, and the solvent was distilled off under reduced pressure to obtain a crude product. The product was purified by silica gel column chromatography (hexane:ethyl acetate = 80:20-0:100, v / v) to obtain the target product 33A (0.74 g, yield 30%) as an oil.

[1019] 1 H-NMR (CDCl 3)δ:7.76(2H,d,J=7.9Hz),7.60(2H,d,J=7.3Hz),7.42-7.37(2H,m),7.35-7.27(7H,m),5.40(1H, s), 4.53 (2H, s), 4.39 (2H, d, J = 6.7Hz), 4.25-4.19 (1H, m), 4.04-3.97 (1H, m), 3.69-3.35 (12H, m).

[1020] C 29 H 33 NO 6 :[M+H] + Calculated value 492, measured value 492.

[1021] (33B)

[1022] Synthesis of (2R)-1-[2-(2-aminoethoxy)ethoxy]-3-benzyloxy-propane-2-ol, trifluoroacetate (Compound 33B)

[1023] [Chemistry 136]

[1024]

[1025] The compound 33A (13.8 g, 28.1 mmol) synthesized in step (33A) was dissolved in ethanol / tetrahydrofuran (50 mL / 50 mL), 1N sodium hydroxide aqueous solution (100 mL) was added, and the mixture was stirred at room temperature for 2 hours. After the reaction was completed, the organic solvent was distilled off under reduced pressure, and 1N hydrochloric acid (100 mL) was added. The solid matter produced was removed by filtration. The by-products contained in the aqueous layer as the passing liquid were extracted with ethyl acetate, and acetonitrile was added to the obtained residue after distillation under reduced pressure and filtered. The passing liquid was distilled off under reduced pressure to obtain a crude product. The components containing the target were collected by reverse phase HPLC (GL Science, Inertsil ODS-3) with 0.1% trifluoroacetic acid aqueous solution and 0.1% trifluoroacetic acid acetonitrile solution as eluents, and the oily target 33B (7.1 g, yield 66%) was obtained.

[1026] 1 H-NMR (CDCl 3 )δ:7.38-7.27(5H,m),4.55(2H,s),4.07-3.99(1H,m),3.77-3.48(10H,m),2.96-2.88(2H,m).

[1027] (33C)

[1028] Synthesis of [(2R,3R,4R,5R,6R)-5-acetamide-3,4-diacetoxy-6-[2-[2-[2-[(2R)-3-benzyloxy-2-hydroxy-propoxy]ethoxy]ethylamino]-2-oxo-ethoxy]tetrahydropyran-2-yl]methyl acetate (Compound 33C)

[1029] [Chemistry 137]

[1030]

[1031] The known compound 2-[(2R,3R,4R,5R,6R)-3-acetamide-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxyacetic acid (WO2012037254) (6 g, 14.8 mmol), 1-(-3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (3.4 g, 17.8 mmol), 3H-1,2,3-triazolo[4,5-b]pyridine-3-ol (2.0 g, 14.8 mmol), N,N-diisopropylethylamine (9.0 mL, 51.8 mmol), and compound 33B (6.7 g, 17.5 mmol) synthesized in step (33B) were dissolved in dichloromethane (130 mL) and stirred at room temperature for 4 hours. After the reaction, the organic layer was washed with 1N hydrochloric acid, saturated sodium bicarbonate aqueous solution, and saturated saline, and then dried with anhydrous sodium sulfate, filtered, and the solvent was distilled off under reduced pressure to obtain a crude product. The solvent was distilled off under reduced pressure to obtain a crude product. The product was purified by silica gel column chromatography (ethyl acetate: methanol = 100: 0-90: 10, v / v) to obtain an amorphous target product 33C (7.9 g, yield 81%).

[1032] 1 H-NMR (CDCl 3 )δ:7.39-7.28(5H,m),7.00-6.93(1H,m),6.50-6.44(1H,m),5.33-5.29(1H,m),5.16(1H,dd,J=11.5,3.6Hz),4.63- 4.53(3H,m),4.34(1H,d,J=15.1Hz),4.22-3.83(5H,m),3.73-3.26(13H,m),2.15(3H,s),2.05(3H,s),1.99(6H,s).

[1033] C 30 H 44 N 2 O 14 :[M+H] + Calculated value 657, measured value 657.

[1034] (33D)

[1035] Synthesis of [(2R,3R,4R,5R,6R)-5-acetamide-3,4-diacetoxy-6-[2-[2-[(2R)-2,3-dihydroxypropoxy]ethoxy]ethylamino]-2-oxo-ethoxy]tetrahydropyran-2-yl]methyl acetate (Compound 33D)

[1036] [Chemistry 138]

[1037]

[1038] The compound 33C (7.9 g, 12 mmol) synthesized in step (33C) was dissolved in ethanol (80 mL). 20% palladium hydroxide / carbon (wet basis) (2.0 g) was added and stirred vigorously at 50°C for 8 hours under a hydrogen atmosphere. After the reaction was completed, the mixture was filtered and the obtained liquid was removed by vacuum distillation. The amorphous target 33D (6.7 g, yield 98%) was obtained.

[1039] 1 H-NMR (CDCl 3 )δ:7.03-6.98(1H,m),6.43(1H,d,J=9.1Hz),5.35(1H,d,J=3.3Hz),5.15(1H,dd,J=11.2,3.3Hz),4.58(1H,d,J=7.9Hz ),4.35(1H,d,J=15.7Hz),4.28-3.82(5H,m),3.77-3.34(13H,m),2.18(3H,s),2.06(3H,s),2.03(3H,s),2.01(3H,s).

[1040] C 23 H 38 N 2 O 14 :[M+H] + Calculated value 568, measured value 568.

[1041] (33E)

[1042] Synthesis of [(2R,3R,4R,5R,6R)-5-acetamide-3,4-diacetoxy-6-[2-[2-[2-[(2S)-3-[bis(4-methoxyphenyl)-phenyl-methoxy]-2-hydroxy-propoxy]ethoxy]ethylamino]-2-oxo-ethoxy]tetrahydropyran-2-yl]methyl acetate (Compound 33E)

[1043] [Chemistry 139]

[1044]

[1045] Add an appropriate amount of pyridine to the compound 33D (6.8 g, 12 mmol) synthesized in step (33D) and perform azeotropy under reduced pressure. Dissolve in pyridine (40 mL), add 4,4'-dimethoxytriphenylmethane (4.5 g, 13 mmol), and stir at room temperature for 2 hours. After the reaction is completed, distill the solvent under reduced pressure. Add an appropriate amount of toluene and perform azeotropy to obtain a crude product. Purify it by silica gel column chromatography (ethyl acetate: ethyl acetate / methanol (5 / 1) = 100:0-50:50, v / v, containing 1% triethylamine) to obtain an amorphous target 33E (6.9 g, yield 66%).

[1046] 1 H-NMR (CDCl 3 )δ:7.42(2H,d,J=7.9Hz),7.33-7.19(7H,m),7.01-6.94(1H,m),6.83(4H,d,J =9.1Hz), 6.37(1H,d,J=8.5Hz), 5.33(1H,d,J=3.3Hz), 5.15(1H,dd,J=11.2,3. 3Hz),4.58(1H,d,J=7.9Hz),4.32(1H,d,J=15.7Hz),4.22-3.86(5H,m),3.79(6 H,s),3.66-3.12(13H,m),2.13(3H,s),2.03(3H,s),1.97(3H,s),1.95(3H,s).

[1047] C 44 H 56 N 2 O 16 :[M+Na] + Calculated value 893, measured value 892.

[1048] (33F)

[1049] Synthesis of [(2R,3R,4R,5R,6R)-5-acetamide-3,4-diacetoxy-6-[2-[2-[2-[(2S)-3-[bis(4-methoxyphenyl)-phenyl-methoxy]-2-[2-cyanoethoxy-(diisopropylamino)phosphino]oxy-propoxy]ethoxy]ethylamino]-2-oxo-ethoxy]tetrahydropyran-2-yl]methyl acetate (Compound 33F)

[1050] [Chemistry 140]

[1051]

[1052] The compound 33E (6.9 g, 7.9 mmol) synthesized in step (33E) was dissolved in dichloromethane (80 mL) after azeotroping with an appropriate amount of toluene. N,N-diisopropylethylamine (5.5 mL, 32 mmol) and 2-cyanoethyldiisopropylchlorophosphoramidite (2.1 mL, 9.5 mmol) were added and stirred at room temperature for 1 hour. After the reaction was completed, the solvent was distilled off under reduced pressure to obtain a crude product. It was purified by silica gel column chromatography (ethyl acetate: ethyl acetate / methanol (5 / 1) = 100:0-50:50, v / v, containing 1% triethylamine) to obtain an amorphous target 33F (6.6 g, yield 78%).

[1053] 1 H-NMR (CDCl 3 )δ:7.47-7.40(2H,m),7.35-7.17(7H,m),6.98-6.91(1H,m),6.85-6.78(4 H,m),6.05-5.89(1H,m),5.37-5.33(1H,m),5.17-5.07(1H,m),4.59-4.49( 1H,m),4.37-4.30(1H,m),4.23-3.06(28H,m),2.69-2.42(2H,m),2.15(3H, s),2.05(3H,s),2.02-1.98(3H,m),1.97-1.94(3H,m),1.30-1.00(12H,m).

[1054] (Example 110)

[1055] HO-X 13 -X 13 -X 13 -A m1s -A e2s -U m1s -C m1s -C e2s -G m1s -A m1s -T e2s -G m1s -G m1s -C e2s -G m1s -A m1s -A e2s -G m1t -H (Sequence number 40)

[1056] Replace the part of X with X 13 , and synthesized in the same manner as in Example 91. 13 The compound 33F synthesized in Reference Example 33 was used for the condensation three times.

[1057] The base sequence of the compound is complementary to the sequence from 92 to 106 from the 5' end of exon 5 of the c.648G>T variant G6PC gene in which the G at nucleotide number 728 is mutated to T in the mRNA of glucose-6-phosphatase catalytic subunit (G6PC) of Homo sapiens, transcript variant 1 (NCBI-GenBank accession number NM_000151.3). The compound was identified by negative ion ESI mass spectrometry (measured value: 6878.11).

[1058] (Example 111)

[1059] HO-X 13 -X 13 -X 13 -X 13 -X 13 -A m1s -A e2s -U m1s -C m1s -C e2s -G m1s -A m1s -T e2s -G m1s -G m1s -C e2s -G m1s -A m1s -A e2s -G m1t -H (Sequence number 40)

[1060] Replace the part of X with X 13 , and synthesized in the same manner as in Example 91. 13 The compound 33F synthesized in Reference Example 33 was used for condensation 5 times.

[1061] The base sequence of the compound is a sequence complementary to the 92nd to 106th sequence from the 5' end of exon 5 of the c.648G>T variant G6PC gene in which the G at nucleotide number 728 is mutated to T in the mRNA of glucose-6-phosphatase catalytic subunit (G6PC) of Homo sapiens, transcript variant 1 (NCBI-GenBank accession number NM_000151.3). The compound was identified by negative ion ESI mass spectrometry (measured value: 7882.43).

[1062] (Example 112)

[1063] HO-X 13 -X 13 -X 13 -X 13 -X 13 -X13 -X 13 -A m1s -A e2s -U m1s -C m1s -C e2s -G m1s -A m1s -T e2s -G m1s -G m1s -C e2s -G m1s -A m1s -A e2s -G m1t -H (Sequence number 40)

[1064] Replace the part of X with X 13 , and synthesized in the same manner as in Example 91. 13 The compound 33F synthesized in Reference Example 33 was used for 7 condensation reactions.

[1065] The base sequence of the present compound is a sequence complementary to the 92nd to 106th sequence from the 5' end of exon 5 of the c.648G>T variant G6PC gene in which the G at nucleotide number 728 is mutated to T in the mRNA (NCBI-GenBank accession number NM_000151.3) of the catalytic subunit of glucose-6-phosphatase of Homo sapiens (G6PC), transcript variant 1. The compound was analyzed under the following HPLC conditions. The analytical column used was Clarity Oligo-MS C18 (manufactured by Phenomenex) (2.6 μm, 50×2.1 mm), the mobile phase A used 100 mM hexafluoroisopropanol, 8 mM triethylamine aqueous solution, the mobile phase B used methanol, and the analysis was performed at a gradient of 10% to 25% in 4 minutes in the mobile phase B, a flow rate of 0.5 mL / min, and 60°C. The HPLC retention time of the compound was 3.0 minutes.

[1066] (Reference Example 34)

[1067] (34A)

[1068] Synthesis of tert-butyl N-[(5S)-6-[2-[2-[(2R)-3-benzyloxy-2-hydroxy-propoxy]ethoxy]ethylamino]-5-(tert-butoxycarbonylamino)-6-oxo-hexyl]carbamate (Compound 34A)

[1069] [Chemistry 141]

[1070]

[1071] (2S)-2,6-bis(tert-butoxycarbonylamino)hexanoic acid (1.7 g, 4.9 mmol), 1-(-3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.3 g, 6.9 mmol), 3H-1,2,3-triazolo[4,5-b]pyridin-3-ol (0.67 g, 4.9 mmol), N,N-diisopropylethylamine (3.0 mL, 17 mmol), and compound 33B (2.1 g, 5.4 mmol) synthesized in step (33B) were dissolved in dichloromethane (40 mL) and stirred at room temperature for 4 hours. Furthermore, 1-(-3-dimethylaminopropyl)-3-ethylcarbodiamide hydrochloride (1.3 g, 6.9 mmol) and N,N-diisopropylethylamine (1.0 ml, 5.7 mmol) were added and stirred overnight. After the reaction, the organic layer was washed with 1N hydrochloric acid, saturated sodium bicarbonate aqueous solution, and saturated saline, dried over anhydrous sodium sulfate, filtered, and the solvent was distilled off under reduced pressure to obtain a crude product (3.1 g) containing compound 34A. The entire amount was used in the next reaction without further purification.

[1072] C 30 H 51 N 3 O 9 :[M+H] + Calculated value 599, measured value 599.

[1073] (34B)

[1074] Synthesis of (2S)-2,6-diamino-N-[2-[2-[(2R)-3-benzyloxy-2-hydroxy-propoxy]ethoxy]ethyl]hexanamide TFA salt (trifluoroacetate) (Compound 34B)

[1075] [Chemistry 142]

[1076]

[1077] The crude product (3.1g) containing compound 34A synthesized in step (34A) is dissolved in dichloromethane (24mL) / trifluoroacetic acid (6mL) and stirred at room temperature for 90 minutes. After the reaction is completed, the solvent is distilled off under reduced pressure, and an appropriate amount of dichloromethane is added, and the solvent is distilled off under reduced pressure again. Then, an appropriate amount of acetonitrile / distilled water (4 / 1) is added, and the solvent is distilled off under reduced pressure again. Repeat the operation until the excess trifluoroacetic acid can be removed. Obtain a crude product (3.8g) containing compound 34B. No further refinement is performed, and the entire amount is used in the next reaction.

[1078] C 20 H 35 N3 O 5 :[M+H] + Calculated value 399, measured value 398.

[1079] (34C)

[1080] Synthesis of [(2R,3R,4R,5R,6R)-5-acetamide-6-[2-[[(5S)-5-[[2-[(2R,3R,4R,5R,6R)-3-acetamide-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxyacetyl]amino]-6-[2-[2-[(2R)-3-benzyloxy-2-hydroxy-propoxy]ethoxy]ethylamino]-6-oxo-hexyl]amino]-2-oxo-ethoxy]-3,4-diacetoxy-tetrahydropyran-2-yl]methyl acetate (Compound 34C)

[1081] [Chemistry 143]

[1082]

[1083] 2-[(2R,3R,4R,5R,6R)-3-acetamide-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxyacetic acid (WO2012037254) (3.5 g, 8.6 mmol), 1-(-3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (2.3 g, 12 mmol), 3H-1,2,3-triazolo[4,5-b]pyridine-3-ol (1.3 g, 9.6 mmol), N,N-diisopropylethylamine (10 mL, 59 mmol), and the crude product containing compound 34B synthesized in step (34B) were dissolved in dichloromethane (80 mL) and stirred overnight at room temperature. After the reaction was completed, the organic layer was washed with 1N hydrochloric acid, saturated sodium bicarbonate aqueous solution, and saturated saline, and then dried and filtered with anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure to obtain a crude product. The solvent was distilled off under reduced pressure to obtain a crude product, which was purified by silica gel column chromatography (ethyl acetate:methanol=100:0-70:30, v / v) to obtain an amorphous target compound 34C (3.5 g, yield 62% (3 steps)).

[1084] 1 H-NMR (CDCl 3)δ:7.50-7.44(1H,m),7.39-7.28(5H,m),7.17-7.11(2H,m),6.94-6.88(1H,m),6.30-6.26(1H,m),5.37-5.32(2H,m),5.09- 4.99(2H,m),4.57(2H,s),4.56-3.11(30H,m),2.18(3H,s),2.16(3H,s),2.07-1.99(15H,m),1.97(3H,s),1.96-1.34(6H,m).

[1085] C 52 H 77 N 5 O 25 :[M+H] + Calculated value 1172, measured value 1173.

[1086] (34D)

[1087] Synthesis of [(2R,3R,4R,5R,6R)-5-acetamide-6-[2-[[(5S)-5-[[2-[(2R,3R,4R,5R,6R)-3-acetamide-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxyacetyl]amino]-6-[2-[2-[(2R)-2,3-dihydroxypropoxy]ethoxy]ethylamino]-6-oxo-hexyl]amino]-2-oxo-ethoxy]-3,4-diacetoxy-tetrahydropyran-2-yl]methyl acetate (Compound 34D)

[1088] [Chemistry 144]

[1089]

[1090] Compound 34C (3.5 g, 3.0 mmol) synthesized in step (34C) was dissolved in ethanol (40 mL). 20% palladium hydroxide / carbon (wet basis) (1.2 g) was added and stirred vigorously at 50°C for 8 hours under a hydrogen atmosphere. After the reaction was completed, the mixture was filtered and the obtained liquid was removed by vacuum distillation. The amorphous target compound 34D (3.14 g, yield 97%) was obtained.

[1091] 1 H-NMR (CDCl 3)δ:7.72-7.67(1H,m),7.16(2H,d,J=9.1Hz),7.04-6.98(1H,m),6.27(1H,d,J=9.1Hz),5.36(2H,d,J=3.0Hz),5.1 0-4.99(2H,m),4.67-3.07(30H,m),2.18(3H,s),2.17(3H,s),2.08-2.00(15H,m),1.98(3H,s),1.93-1.33(6H,m).

[1092] C 45 H 71 N 5 O 25 :[M+H] + Calculated value 1082, measured value 1083.

[1093] (34E)

[1094] Synthesis of [(2R,3R,4R,5R,6R)-5-acetamide-6-[2-[[(5S)-5-[[2-[(2R,3R,4R,5R,6R)-3-acetamide-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxyacetyl]amino]-6-[2-[2-[(2S)-3-[bis(4-methoxyphenyl)-phenyl-methoxy]-2-hydroxy-propoxy]ethoxy]ethylamino]-6-oxo-hexyl]amino]-2-oxo-ethoxy]-3,4-diacetoxy-tetrahydropyran-2-yl]methyl acetate (Compound 34E)

[1095] [Chemistry 145]

[1096]

[1097] An appropriate amount of pyridine was added to the compound 34D (3.14 g, 2.9 mmol) synthesized in step (34D), and azeotroped under reduced pressure. Dissolved in pyridine (10 ml), 4,4'-dimethoxytriphenylmethane (1.1 g, 3.2 mmol) was added, and stirred at room temperature for 2 hours. After the reaction was completed, toluene (20 mL), ethanol (1 mL), and N,N-diisopropylethylamine (1 mL) were added, and the solvent was distilled off under reduced pressure to obtain a crude product. It was purified by silica gel column chromatography (ethyl acetate: ethyl acetate / methanol (5 / 1) = 100:0-50:50, v / v, containing 1% triethylamine) to obtain an amorphous target 34E (2.5 g, yield 62%).

[1098] 1 H-NMR (CDCl 3)δ:7.60-7.55(1H,m),7.43(2H,d,J=7.3Hz),7.35-7.20(7H,m),7.17-7.10(2H,m),6.92-6.87(1H,m),6.83(4H,d,J=9.1Hz),6.30(1H,d,J =9.1Hz),5.36-5.30(2H,m),5.09-4.95(2H,m),4.60-3.07(36H,m),2 .19-2.14(6H,m),2.06-1.99(15H,m),1.97(3H,s),1.70-1.34(6H,m).

[1099] C 66 H 89 N 5 O 27 :[M+Na] + Calculated value 1407, measured value 1407.

[1100] (34F)

[1101] Synthesis of [(2R,3R,4R,5R,6R)-5-acetamide-6-[2-[[(5S)-5-[[2-[(2R,3R,4R,5R,6R)-3-acetamide-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxyacetyl]amino]-6-[2-[2-[(2S)-3-[bis(4-methoxyphenyl)-phenyl-methoxy]-2-[2-cyanoethoxy-(diisopropylamino)phosphino]oxy-propoxy]ethoxy]ethylamino]-6-oxo-hexyl]amino]-2-oxo-ethoxy]-3,4-diacetoxy-tetrahydropyran-2-yl]methyl acetate (Compound 34F)

[1102] [Chemistry 146]

[1103]

[1104] The compound 34E (2.5 g, 1.8 mmol) synthesized in step (34E) was dissolved in dichloromethane (18 mL) after azeotroping with an appropriate amount of toluene / dichloromethane (3 / 1). N,N-diisopropylethylamine (1.3 mL, 7.2 mmol) and 2-cyanoethyldiisopropylchlorophosphoramidite (0.48 mL, 2.2 mmol) were added and stirred at room temperature for 1 hour. After the reaction was completed, the solvent was distilled off under reduced pressure to obtain a crude product. It was purified by silica gel column chromatography (ethyl acetate: ethyl acetate / methanol (5 / 1) = 100:0-50:50, v / v, containing 1% triethylamine) to obtain an amorphous target 34F (2.1 g, yield 73%).

[1105] 1 H-NMR (CDCl 3 )δ:7.47-7.41(2H,m),7.35-7.05(10H,m),6.89-6.78(5H,m),6.35-6.25(1H,m),5.37-5.34(2H,m),5.11-4.98(2H,m),4.55-3.08( 40H,m),2.70-2.45(2H,m),2.18(3H,s),2.17(3H,s),2.07-1.99(15H,m),1.98-1.94(3H,m),1.91-1.31(6H,m),1.21-1.02(12H,m).

[1106] (Example 113)

[1107] HO-X 14 -A m1s -A e2s -U m1s -C m1s -C e2s -G m1s -A m1s -T e2s -G m1s -G m1s -C e2s -G m1s -A m1s -A e2s -G m1t -H (Sequence number 40)

[1108] Replace the part of X with X 14 , and synthesized in the same manner as in Example 91. 14 The compound 34F synthesized in Reference Example 34 was used for the first condensation.

[1109] The base sequence of the compound is a sequence complementary to the 92nd to 106th sequence from the 5' end of exon 5 of the c.648G>T variant G6PC gene in which the G at nucleotide number 728 is mutated to T in the mRNA of glucose-6-phosphatase catalytic subunit (G6PC) of Homo sapiens, transcript variant 1 (NCBI-GenBank accession number NM_000151.3). The compound was identified by negative ion ESI mass spectrometry (measured value: 6263.04).

[1110] (Example 114)

[1111] HO-X 14 -X 14 -A m1s -A e2s -Um1s -C m1s -C e2s -G m1s -A m1s -T e2s -G m1s -G m1s -C e2s -G m1s -A m1s -A e2s -G m1t -H (Sequence number 40)

[1112] Replace the part of X with X 14 , and synthesized in the same manner as in Example 91. 14 The compound 34F synthesized in Reference Example 34 was used for the condensation reaction twice.

[1113] The base sequence of the compound is a sequence complementary to the 92nd to 106th sequence from the 5' end of exon 5 of the c.648G>T variant G6PC gene in which the G at nucleotide number 728 is mutated to T in the mRNA of glucose-6-phosphatase catalytic subunit (G6PC) of Homo sapiens, transcript variant 1 (NCBI-GenBank accession number NM_000151.3). The compound was identified by negative ion ESI mass spectrometry (measured value: 7154.36).

[1114] (Example 115)

[1115] HO-X 14 -X 14 -X 14 -A m1s -A e2s -U m1s -C m1s -C e2s -G m1s -A m1s -T e2s -G m1s -G m1s -C e2s -G m1s -A m1s -A e2s -G m1t -H (Sequence number 40)

[1116] Replace the part of X with X 14 , and synthesized in the same manner as in Example 91. 14 The compound 34F synthesized in Reference Example 34 was used for the condensation three times.

[1117] The base sequence of the compound is a sequence complementary to the 92nd to 106th sequence from the 5' end of exon 5 of the c.648G>T variant G6PC gene in which the G at nucleotide number 728 is mutated to T in the mRNA of glucose-6-phosphatase catalytic subunit (G6PC) of Homo sapiens, transcript variant 1 (NCBI-GenBank accession number NM_000151.3). The compound was identified by negative ion ESI mass spectrometry (measured value: 8046.64).

[1118] (Examples 116 to 127) Preparation of oligonucleotide-encapsulated nucleic acid lipid particles

[1119] A lipid solution was prepared using distearoylphosphatidylcholine (1,2-distearoyl-sn-glycero-3-phosphocholine: DSPC, NOF CORPORATION), cholesterol (Cholesterol: Chol, Sigma-Aldrich, Inc.), the compound of Example 8 described in International Publication No. 2015 / 005253 (referred to as LP), and N-[methoxypoly(ethylene glycol) 2000]carbamoyl]-1,2-dimyristyloxypropyl-3-amine (PEG-C-DMA, NOF CORPORATION) at a molar ratio of DSPC:Chol:LP:PEG-C-DMA = 10:48:40:2 so that the total lipid concentration in ethanol became 5 mM.

[1120] Oligonucleotides (21e_002, 18e_005, 21m_002, 18e_005, 18m_022, 15e_001, 15ed_001, 18e_008, 18e_025, 18m_008, 15e_002, and 15ed_002) were respectively dissolved in citrate buffer (20 mM, citrate buffer, pH 4.0) to prepare oligonucleotide solutions.

[1121] The lipid solution and the oligonucleotide solution were mixed in a microfluidic cartridge attached to the NanoAssemblr Benchtop (registered trademark, PrecisionNanosystems) in such a manner that the ratio (N / P) of nitrogen atoms (N) from LP to phosphorus atoms (P) from oligonucleotides was 3 (flow rate: 12 mL / min, mixing volume ratio: lipid solution: oligonucleotide solution = 1:3, temperature: room temperature) to obtain a dispersion of nucleic acid lipid particles. Subsequently, the dispersion of nucleic acid lipid particles was dialyzed for 12-18 hours (Float-A-Lyzer G2, MWCO: 1000 kD, Spectra / Por) using about 2 L of phosphate buffer (pH 7.4) to remove ethanol and neutralize to obtain a purified dispersion of nucleic acid lipid particles encapsulating oligonucleotides. In order to adjust the concentration, the dispersion of nucleic acid lipid particles can be appropriately concentrated by ultrafiltration (Amicon-Ultra, MWCO: 100 kD, MILLIPORE).

[1122] The properties of the oligonucleotide-encapsulating nucleic acid lipid particles of Examples 116 to 127 were evaluated. The methods for evaluating each property are described below.

[1123] (1) Oligonucleotide incorporation rate

[1124] The oligonucleotide incorporation rate was measured using the Quant-iT RiboGreen RNA Assay Kit (Invitrogen) according to the attached document. That is, the oligonucleotide in the dispersion of nucleic acid lipid particles was quantified in the presence and absence of 0.015% Triton X-100 surfactant, and the incorporation rate was calculated by the following formula.

[1125] {[Amount of oligonucleotide in the presence of surfactant] - [Amount of oligonucleotide in the absence of surfactant]} / [Amount of oligonucleotide in the presence of surfactant] × 100 (%)

[1126] (2) Ratio of oligonucleotide to lipid

[1127] The phospholipid mass in the dispersion of nucleic acid lipid particles was measured using Phospholipid C-Test Wako (Wako Pure Chemical Industries, Ltd.) according to the attached document. That is, the phospholipid in the sample was quantified in the presence of 1% Triton X-100 surfactant.

[1128] The cholesterol and LP amounts in the dispersion of nucleic acid lipid particles were determined by reverse phase chromatography (system: Agilent 1100 series, column: Chromolith Performance RP-18 capped 100-3 integrated HPLC-column (Merck), buffer A: 0.01% trifluoroacetic acid, buffer B: 0.01% trifluoroacetic acid, methanol, gradient (B%): 82-97% (0-17 min), flow rate: 2 mL / min, temperature: 50°C, detection: 205 nm).

[1129] The total lipid mass was calculated based on the composition ratio of the phospholipid mass, cholesterol mass, and LP mass to the lipid components constituting the liposomes, and the ratio of oligonucleotide to lipid was calculated by the following formula based on the "amount of oligonucleotide in the presence of surfactant" in (1) above.

[1130] [Oligonucleotide concentration in the presence of surfactant] / [Total lipid concentration] (wt / wt)

[1131] (3) Average particle size

[1132] The particle size of the liposome was measured by zeta potential / particle size NICOMP™ 380ZLS (PARTICLE SIZING SYSTEMS). The average particle size in the table represents the volume average particle size, and ± represents the deviation.

[1133] The results are shown in Table 11.

[1134] (Table 11)

[1135]

[1136] *ASO / lipid (wt / wt): weight ratio of oligonucleotide to lipid.

[1137] The above results clearly show that the oligonucleotides are encapsulated in the lipid particles, and these nucleic acid lipid particles have an average particle size of about 50 nm to about 70 nm.

[1138] (Example 128)

[1139] HO-X 14 -A m1s -T e2s -C m1s -C m1s -G e2s -A m1s -U m1s -G e2s -G m1s -C m1s -G e2s -A m1s -A m1s-G e2s -C m1t -H (Sequence number 44)

[1140] The sequence 15e_005 was used instead of the sequence used in Example 113, and synthesis was carried out in the same manner as in Example 113.

[1141] The base sequence of the compound is a sequence complementary to the 91st to 105th sequence from the 5' end of exon 5 of the c.648G>T variant G6PC gene in which the G at nucleotide number 728 is mutated to T in the mRNA of glucose-6-phosphatase catalytic subunit (G6PC) of Homo sapiens, transcript variant 1 (NCBI-GenBank accession number NM_000151.3). The compound was identified by negative ion ESI mass spectrometry (measured value: 6210.94).

[1142] (Example 129)

[1143] HO-X 14 -U m1s -C e2s -C m1s -G m1s -A e2s -U m1s -G m1s -G e2s -C m1s -G m1s -A e2s -A m1s -G m1s -C e2s -U m1t -H (Sequence number 45)

[1144] The sequence 15e_006 was used instead of the sequence used in Example 113, and synthesis was carried out in the same manner as in Example 113.

[1145] The base sequence of the compound is a sequence complementary to the 90th to 104th sequence from the 5' end of exon 5 of the c.648G>T variant G6PC gene in which the G at nucleotide number 728 is mutated to T in the mRNA of glucose-6-phosphatase catalytic subunit (G6PC) of Homo sapiens, transcript variant 1 (NCBI-GenBank accession number NM_000151.3). The compound was identified by negative ion ESI mass spectrometry (measured value: 6201.95).

[1146] (Example 130)

[1147] HO-X 14 -C m1s -C e2s -G m1s -Am1s -T e2s -G m1s -G m1s -C e2s -G m1s -A m1s -A e2s -G m1s -C m1s -T e2s -G m1t -H (Sequence number 41)

[1148] The sequence 15e_002 was used instead of the sequence used in Example 113, and synthesis was carried out in the same manner as in Example 113.

[1149] The base sequence of the compound is complementary to the sequence from 89 to 103 from the 5' end of exon 5 of the c.648G>T variant G6PC gene in which the G at nucleotide number 728 is mutated to T in the mRNA of glucose-6-phosphatase catalytic subunit (G6PC) of Homo sapiens, transcript variant 1 (NCBI-GenBank accession number NM_000151.3). The compound was identified by negative ion ESI mass spectrometry (measured value: 6268.98).

[1150] (Reference Example 35)

[1151] (35A) Synthesis of [(2R,3R,4R,5R,6R)-5-acetamide-3,4-diacetoxy-6-[2-[3-(tert-butoxycarbonylamino)propylamino]-2-oxo-ethoxy]tetrahydropyran-2-yl]methyl acetate (Compound 35A)

[1152] [Chemistry 147]

[1153]

[1154] To a solution of 2-[(2R,3R,4R,5R,6R)-3-acetamide-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxyacetic acid (WO2012037254) (10 g, 24.7 mmol) and tert-butyl N-(3-aminopropyl)carbamate (4.7 ml, 27.1 mmol) in N,N-dimethylformamide (70 ml) were added 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridine 3-oxide hexafluorophosphate (11.3 g, 29.6 mmol) and N,N-diisopropylethylamine (8.6 mL, 49.3 mmol), and the mixture was stirred at room temperature for 40 minutes. After the reaction, ethyl acetate was added, and the organic layer was washed with 15% saline, 0.5N hydrochloric acid, saturated saline, saturated sodium bicarbonate aqueous solution, and saturated saline, and then dried with anhydrous sodium sulfate, filtered, and the solvent was distilled off under reduced pressure to obtain a crude product. The solvent was distilled off under reduced pressure to obtain about 14g of a crude product containing the target compound. It was not further purified and used in the next reaction.

[1155] (35B) Synthesis of [(2R,3R,4R,5R,6R)-5-acetamide-3,4-diacetoxy-6-[2-(3-aminopropylamino)-2-oxo-ethoxy]tetrahydropyran-2-yl]methyl acetate TFA salt (Compound 35B)

[1156] [Chemistry 148]

[1157]

[1158] The crude product (14 g) synthesized in step (35A) was dissolved in dichloromethane (40 ml), and trifluoroacetic acid was added to a total of 16 ml until the reaction was completed. After the reaction was completed, the solvent was distilled off under reduced pressure to obtain a crude product. An appropriate amount of diethyl ether was added and decantation was performed. An appropriate amount of acetonitrile was added and distilled off under reduced pressure. Furthermore, an appropriate amount of dichloromethane was added and distilled off under reduced pressure. About 15.9 g of a crude product containing the target substance was obtained. No further purification was performed and it was used for the next reaction.

[1159] C 19 H 31 N 3 O 10 :[M+H] + Calculated value 462, measured value 462.

[1160] (35C) Synthesis of [(2R,3R,4R,5R,6R)-5-acetamide-3,4-diacetoxy-6-[2-[3-[[(4R)-2,2-dimethyl-1,3-dioxolan-4-yl]methoxycarbonylamino]propylamino]-2-oxo-ethoxy]tetrahydropyran-2-yl]methyl acetate (Compound 35C)

[1161] [Chemistry 149]

[1162]

[1163] (S)-(+)-1,2-isopropylidene glycerol (6.53 g, 49.4 mmol) and 4-nitrophenyl chloroformate (9.96 g, 49.4 mmol) were dissolved in dichloromethane (200 ml), pyridine (7.95 ml, 98.8 mmol) was added, and the mixture was stirred at room temperature for 30 minutes. A dichloromethane (100 ml) solution of the crude product (15.9 g) synthesized in step (35B) and triethylamine (20.5 ml, 148 mmol) were added to the reaction solution, and the mixture was stirred overnight at room temperature. After the reaction was completed, the organic layer was washed with 0.5 N hydrochloric acid, saturated saline, saturated sodium bicarbonate aqueous solution, and saturated saline, and then dried and filtered with anhydrous sodium sulfate. The solvent was distilled off under reduced pressure to obtain a crude product. The product was purified by silica gel column chromatography (ethyl acetate:methanol=100:0-65:35, v / v) to obtain the target compound 35C (9.0 g, yield 59% (3 steps)) as a solid.

[1164] 1 H-NMR (CDCl 3 )δ:7.09-7.01(1H,m),6.16(1H,d,J=8.5Hz),5.53-5.46(1H,m),5.37(1H,d ,J=3.6Hz),5.22-5.14(1H,m),4.53(1H,d,J=8.5Hz),4.41-3.99(9H,m),3.9 6-3.89(1H,m),3.79-3.71(1H,m),3.44-3.15(4H,m),2.17(3H,s),2.06(3H ,s),2.04(3H,s),1.98(3H,s),1.80-1.58(2H,m),1.43(3H,s),1.37(3H,s).

[1165] C 26 H 41 N 3 O 14 :[M+Na] + Calculated value 642, measured value 642.

[1166] (35D) Synthesis of [(2R,3R,4R,5R,6R)-5-acetamide-3,4-diacetoxy-6-[2-[3-[[(2R)-2,3-dihydroxypropoxy]carbonylamino]propylamino]-2-oxo-ethoxy]tetrahydropyran-2-yl]methyl acetate (Compound 35D)

[1167] [Chemistry 150]

[1168]

[1169] To a methanol solution (160 ml) of the compound synthesized in step (35C) (7.87 g, 12.7 mmol) was added p-toluenesulfonic acid (0.242 g, 1.27 mmol), and the mixture was stirred overnight. After the reaction was completed, the solvent was distilled off under reduced pressure to obtain 7.36 g of a crude product containing the target compound. The product was used in the next reaction without further purification.

[1170] C 23 H 37 N 3 O 14 :[M+H] + Calculated value 580, found value 580, [M+Na] + Calculated value 602, measured value 602.

[1171] (35E) Synthesis of [(2R,3R,4R,5R,6R)-5-acetamide-3,4-diacetoxy-6-[2-[3-[[(2R)-3-[bis(4-methoxyphenyl)-phenyl-methoxy]-2-hydroxy-propoxy]carbonylamino]propylamino]-2-oxo-ethoxy]tetrahydropyran-2-yl]methyl acetate (Compound 35E)

[1172] [Chemistry 151]

[1173]

[1174] Dichloromethane (150 ml) and pyridine (10 ml) were added to the crude product (7.36 g) synthesized in step (35D), and the solvent was distilled off under reduced pressure. Thereafter, the product was dissolved in pyridine (60 ml), 4,4'-dimethoxytriphenylmethane (5.59 g, 16.5 mmol) was added, and the mixture was stirred at room temperature for 30 minutes. After the reaction was completed, methanol (1.03 ml, 25.4 mmol) and N,N-diisopropylethylamine (4.42 mL, 25.4 mmol) were added, and the solvent was distilled off under reduced pressure. The product was purified by silica gel column chromatography (ethyl acetate: ethyl acetate / methanol (5 / 1) = 100:0-0:100, v / v, containing 1% triethylamine), and the target product 35E (4.51 g, yield 40%) was obtained as a solid.

[1175] 1 H-NMR (CDCl 3 )δ:7.45-7.39(2H,m),7.34-7.28(6H,m),7.24-7.18(1H,m),7.07-6.99(1H,m),6.86-6 .80(4H,m),6.21(1H,d,J=8.5Hz),5.48-5.41(1H,m),5.36(1H,d,J=3.6Hz),5.19-5.13( 1H,m),4.52(1H,d,J=8.5Hz),4.39-3.88(9H,m),3.79(6H,s),3.50-3.12(6H,m),2.92( 1H,d,J=4.8Hz),2.16(3H,s),2.05(3H,s),1.99(3H,s),1.95(3H,s),1.80-1.54(2H,m).

[1176] C 44 H 55 N 3 O 16 :[M+Na] + Calculated value 904, measured value 904.

[1177] Synthesis of (35F) Acetate [(2R,3R,4R,5R,6R)-5-acetamide-3,4-diacetoxy-6-[2-[3-[[(2R)-3-[bis(4-methoxyphenyl)-phenyl-methoxy]-2-[2-cyanoethoxy-(diisopropylamino)phosphino]oxy-propoxy]carbonylamino]propylamino]-2-oxo-ethoxy]tetrahydropyran-2-yl]methyl ester (Compound 35F)

[1178] [Chemistry 152]

[1179]

[1180] The compound 35E (4.51 g, 5.11 mmol) synthesized in step (35E) was dissolved in dichloromethane (90 ml) after azeotroping with an appropriate amount of ethyl acetate / toluene (1 / 4). N,N-diisopropylethylamine (3.56 ml, 20.5 mmol) and 2-cyanoethyldiisopropylchlorophosphoramidite (1.57 ml, 5.63 mmol) were added and stirred at room temperature for 1 hour. After the reaction was completed, the solvent was distilled off under reduced pressure to obtain a crude product. It was purified by silica gel column chromatography (ethyl acetate: ethyl acetate / methanol (5 / 1) = 100:0-50:50, v / v, containing 1% triethylamine) to obtain the target compound 35F (3.97 g, yield 72%) as a solid.

[1181] 1 H-NMR (CDCl 3 )δ:7.47-7.38(2H,m),7.35-7.16(7H,m),7.12-7.04(1H,m),6.86-6.76( 4H,m),6.25-6.12(1H,m),5.58-5.18(3H,m),4.61-4.53(1H,m),4.46-3. 42(19H,m),3.39-3.02(6H,m),2.68-2.41(2H,m),2.19-2.13(3H,m),2.0 8-2.02(3H,m),2.01-1.91(6H,m),1.78-1.54(2H,m),1.31-0.94(12H,m).

[1182] (Reference Example 36)

[1183] (36A) Synthesis of [(4R)-2,2-dimethyl-1,3-dioxolan-4-yl]methyl N-[2-(tert-butoxycarbonylamino)ethyl]carbamate (Compound 36A)

[1184] [Chemistry 153]

[1185]

[1186] (S)-(+)-1,2-isopropylidene glycerol (5.0 g, 37.5 mmol) and 4-nitrophenyl chloroformate (7.55 g, 37.5 mmol) were dissolved in dichloromethane (150 ml), pyridine (5.0 ml, 61.8 mmol) was added, and the mixture was stirred at room temperature for 1 hour. A dichloromethane solution (40 ml) of tert-butyl N-(2-aminoethyl)carbamate (6 g, 37.5 mmol) and triethylamine (8.8 ml, 63.7 mmol) were added to the reaction solution, and the mixture was stirred at room temperature for 1 hour. After the reaction was completed, the organic layer was washed with 0.5 N hydrochloric acid, saturated saline, saturated sodium bicarbonate aqueous solution, and saturated saline, and then dried and filtered with anhydrous sodium sulfate. The solvent was distilled off under reduced pressure to obtain a crude product (10.6 g) containing the target substance as a solid. It was not further purified and used in the next reaction.

[1187] (36B) Synthesis of N-(2-aminoethyl)carbamic acid [(2R)-2,3-dihydroxypropyl] ester TFA salt (Compound 36B)

[1188] [Chemistry 154]

[1189]

[1190] Trifluoroacetic acid (16.6 ml) and pure water (5.55 ml) were added to the crude product (10.6 g) synthesized in step (36A), and the mixture was stirred at room temperature for 1 hour. After the reaction was completed, the solvent was distilled off under reduced pressure. Then, acetonitrile was added and the solvent was distilled off under reduced pressure. Furthermore, acetonitrile / pure water (4 / 1) was added, and the solvent was distilled off under reduced pressure, and then dried under reduced pressure. An oily crude product (14.3 g) containing the target was obtained. It was not further refined and used for the next reaction.

[1191] (36C) Synthesis of Benzyl N-[(5S)-5-(Benzyloxycarbonylamino)-6-[2-[[(2R)-2,3-dihydroxypropoxy]carbonylamino]ethylamino]-6-oxo-hexyl]carbamate (Compound 36C)

[1192] [Chemistry 155]

[1193]

[1194] 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridine 3-oxide hexafluorophosphate (15.0 g, 40 mmol) and N,N-diisopropylethylamine (12 mL, 66 mmol) were added to a solution of N,N'-di-Cbz-L-lysine (13.7 g) in N,N-dimethylformamide (100 ml). Furthermore, a solution of the crude product (9.7 g) synthesized in step (36B) and N,N-diisopropylethylamine (12 mL, 66 mmol) in N,N-dimethylformamide (20 ml) was added, and the mixture was stirred at room temperature for 40 minutes. After the reaction was completed, ethyl acetate was added, and the organic layer was washed with 15% saline, 0.5N hydrochloric acid, saturated saline, saturated sodium bicarbonate aqueous solution, and saturated saline, and then dried over anhydrous sodium sulfate, filtered, and the solvent was distilled off under reduced pressure to obtain a solid. The solid was washed with 1N hydrochloric acid and pure water, and then washed with dichloromethane and diethyl ether, and then dried under reduced pressure to obtain a crude product (10.5 g) containing the target compound.

[1195] C 28 H 38 N 4 O 9 :[M+H] + Calculated value 575, measured value 575.

[1196] (36D) Synthesis of N-[2-[[(2S)-2,6-diaminohexanoyl]amino]ethyl]carbamic acid [(2R)-2,3-dihydroxypropyl] ester hydrochloride (Compound 36D)

[1197] [Chemistry 156]

[1198]

[1199] The crude product (9.5 g) synthesized in step (36C) was dissolved in tetrahydrofuran / 1N hydrochloric acid (36 ml), 10% palladium carbon (2.0 g) was added, and stirred at room temperature for 2.5 hours under a hydrogen atmosphere. After the reaction was completed, it was filtered. After the solvent was removed by vacuum distillation, it was dried under reduced pressure using a high-speed concentrator V-10 (Biotage). The crude product (6.4 g) of the target compound was obtained. It was not further refined and used in the next reaction.

[1200] (36E) Synthesis of [(2R,3R,4R,5R,6R)-5-acetamide-6-[2-[[(5S)-5-[[2-[(2R,3R,4R,5R,6R)-3-acetamide-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxyacetyl]amino]-6-[2-[[(2R)-2,3-dihydroxypropoxy]carbonylamino]ethylamino]-6-oxo-hexyl]amino]-2-oxo-ethoxy]-3,4-diacetoxy-tetrahydropyran-2-yl]methyl acetate (Compound 36E)

[1201] [Chemistry 157]

[1202]

[1203] 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridine 3-oxide hexafluorophosphate (3.0 g, 8.0 mmol) and N,N-diisopropylethylamine (3.8 mL, 21.7 mmol) were added to a solution of 2-[(2R,3R,4R,5R,6R)-3-acetamide-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxyacetic acid (WO2012037254) (2.64 g, 6.5 mmol) of a known compound in the literature and the crude product synthesized in step (36D) (1.37 g, 3.6 mmol) in N,N-dimethylformamide (30 ml), and stirred at room temperature for 40 minutes. After the reaction was completed, diethyl ether (500 ml) was added to obtain a precipitated residue. The product was separated and purified by reverse phase HPLC (GL Science, Inertsil ODS-3) using 0.1% trifluoroacetic acid aqueous solution and 0.1% trifluoroacetic acid acetonitrile solution as eluents, and the fractions containing the target product were pooled and the solvent was distilled off under reduced pressure to obtain an amorphous target product (2.23 g, yield 57%).

[1204] 1 H-NMR (DMSO-D 6)δ:8.14-8.08(1H,m),8.03-7.91(2H,m),7.33-7.22(2H,m),7.16-7.10(1H,m),5. 27-5.23(2H,m),5.01-4.93(2H,m),4.59-4.52(2H,m),4.26-3.55(17H,m),3.36-3 .30(2H,m),3.18-2.86(7H,m),2.14-2.06(6H,m),2.04-1.98(6H,m),1.94-1.89(6 H,m),1.88-1.79(6H,m),1.71-1.48(2H,m),1.47-1.35(2H,m),1.32-1.15(2H,m).

[1205] C 44 H 68 N 6 O 25 :[M+H] + Calculated value 1081, measured value 1081.

[1206] (36F) Synthesis of [(2R,3R,4R,5R,6R)-5-acetamide-6-[2-[[(5S)-5-[[2-[(2R,3R,4R,5R,6R)-3-acetamide-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxyacetyl]amino]-6-[2-[[(2R)-3-[bis(4-methoxyphenyl)-phenyl-methoxy]-2-hydroxy-propoxy]carbonylamino]ethylamino]-6-oxo-hexyl]amino]-2-oxo-ethoxy]-3,4-diacetoxy-tetrahydropyran-2-yl]methyl acetate (Compound 36F)

[1207] [Chemistry 158]

[1208]

[1209] Pyridine (10 ml) was added to the compound (7.36 g, 2.1 mmol) synthesized in step (36E), and the solvent was distilled off under reduced pressure. This operation was repeated 3 times. Thereafter, it was dissolved in pyridine (10 ml), 4,4'-dimethoxytriphenylmethane (0.77 g, 2.3 mmol) was added, and the mixture was stirred at room temperature for 30 minutes. After the reaction was completed, ethanol (1.0 ml) and N,N-diisopropylethylamine (1.0 mL) were added, and the solvent was distilled off under reduced pressure. It was purified by silica gel column chromatography (ethyl acetate: ethyl acetate / methanol (5 / 1) = 100:0-0:100, v / v, containing 1% triethylamine) to obtain an amorphous target product (1.17 g, yield 41%).

[1210] 1 H-NMR (CDCl 3 )δ:7.47-7.38(2H,m),7.36-6.95(10H,m),6.86-6.79(4H,m),6.33-6.27(1H,m),5.53-4. 99(4H,m),4.57-3.07(30H,m),2.21-2.14(6H,m),2.08-1.94(18H,m),1.92-1.07(6H,m).

[1211] C 65 H 86 N 6 O 27 :[M+Na] + Calculated value 1406, measured value 1406.

[1212] (36G) Synthesis of [(2R,3R,4R,5R,6R)-5-acetamide-6-[2-[[(5S)-5-[[2-[(2R,3R,4R,5R,6R)-3-acetamide-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxyacetyl]amino]-6-[2-[[(2R)-3-[bis(4-methoxyphenyl)-phenyl-methoxy]-2-[2-cyanoethyl-(diisopropylamino)phosphino]oxy-propoxy]carbonylamino]ethylamino]-6-oxo-hexyl]amino]-2-oxo-ethoxy]-3,4-diacetoxy-tetrahydropyran-2-yl]methyl acetate (Compound 36G)

[1213] [Chemistry 159]

[1214]

[1215] The compound 36F (1.17 g, 0.85 mmol) synthesized in step (36F) was dissolved in dichloromethane (8 ml) after azeotroping with an appropriate amount of dichloromethane / toluene (1 / 4). N,N-diisopropylethylamine (0.59 ml, 3.4 mmol) and 2-cyanoethyldiisopropylchlorophosphoramidite (0.23 ml, 1.0 mmol) were added and stirred at room temperature for 1 hour. After the reaction was completed, the solvent was distilled off under reduced pressure to obtain a crude product. It was purified by silica gel column chromatography (ethyl acetate: ethyl acetate / methanol (5 / 1) = 100:0-0:100, v / v, containing 1% triethylamine) to obtain an amorphous target 36G (0.81 g, yield 61%).

[1216] 1 H-NMR (CDCl 3)δ:7.47-7.40(2H,m),7.36-7.10(11H,m),6.95-6.86(1H,m),6.85-6.78 (4H,m),6.34-6.28(1H,m),5.56-5.33(2H,m),5.12-4.98(2H,m),4.52-3. 04(36H,m),2.72-2.46(2H,m),2.20-2.15(6H,m),2.08-1.94(18H,m),1.9 3-1.81(2H,m),1.67-1.52(2H,m),1.46-1.34(2H,m),1.22-0.98(12H,m).

[1217] (Reference Example 37)

[1218] (37A) Synthesis of [(2R,3R,4R,5R)-5-acetamide-3,4-diacetoxy-6-[3-(benzyloxycarbonylamino)propoxy]tetrahydropyran-2-yl]methyl acetate (Compound 37A)

[1219] [Chemistry 160]

[1220]

[1221] Trifluoromethanesulfonic acid (450 μl, 5.1 mmol) was added to a suspension of N-(3-hydroxypropyl) benzyl carbamate (6.4 g, 30.8 mmol) and acetic acid [(2R, 3R, 4R, 5R)-5-acetamide-3, 4, 6-triacetoxy-tetrahydropyran-2-yl] methyl ester (WO2011053614) (10 g, 25.7 mmol) of a known compound in the literature in dichloromethane (200 ml), and stirred at 45 degrees for 4 hours. After the reaction was completed, half of the solvent was distilled off under reduced pressure and concentrated. After washing the organic layer with saturated sodium bicarbonate aqueous solution and saturated brine, it was dried and filtered with anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure to obtain a crude product. Isopropyl ether (100 m) was added and stirred for 3 hours. The obtained solid was filtered to obtain the target 37A (11.9 g). No further purification was performed and it was used for the next reaction.

[1222] 1 H-NMR (CDCl 3)δ:7.43-7.29(5H,m),6.26(1H,d,J=8.5Hz),5.36-5.29(1H,m),5.12(1H,d,J=12.1Hz),5 .08(1H,d,J=12.1Hz),5.03-4.95(2H,m),4.33(1H,d,J=9.1Hz),4.21-4.06(3H,m),4.01- 3.93(1H,m),3.80-3.73(1H,m),3.62-3.49(1H,m),3.44-3.35(1H,m),3.14-3.04(1H,m), 2.15(3H,s),2.05(3H,s),2.01(3H,s),1.95(3H,s),1.88-1.75(1H,m),1.69-1.56(1H,m).

[1223] C 25 H 34 N 2 O 11 :[M+H] + Calculated value 539, measured value 539.

[1224] (37B) Synthesis of [(2R,3R,4R,5R)-5-acetamido-3,4-diacetoxy-6-(3-aminopropoxy)tetrahydropyran-2-yl]methyl acetate hydrochloride (Compound 37B)

[1225] [Chemistry 161]

[1226]

[1227] Compound 37A (10 g, 18.6 mmol) synthesized in step (37A) was dissolved in tetrahydrofuran (200 ml) / 1N hydrochloric acid (20 ml). 10% palladium / carbon (wet basis) (1 g) was added and stirred vigorously at room temperature for 1 hour under a hydrogen atmosphere. After the reaction was completed, the mixture was filtered and the obtained liquid was removed by vacuum distillation to obtain a crude product (8.97 g) containing the target compound 37B. No further purification was performed and the mixture was used in the next reaction.

[1228] C 17 H 28 N 2 O 9 :[M+H] + Calculated value 405, measured value 405.

[1229] (37C) Synthesis of [(2R,3R,4R,5R,6R)-5-acetamide-6-[3-[[(3S)-4-[3-[(2R,3R,4R,5R,6R)-3-acetamide-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypropylamide]-3-(9H-fluoren-9-ylmethoxycarbonylamino)-4-oxo-butyryl]amino]propoxy]-3,4-diacetoxy-tetrahydropyran-2-yl]methyl acetate (Compound 37C)

[1230] [Chemistry 162]

[1231]

[1232] 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridine 3-oxide hexafluorophosphate (4.7 g, 12.4 mmol) and N,N-diisopropylethylamine (5.8 mL, 33.5 mmol) were added to a solution of N-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-aspartic acid (1.7 g, 4.78 mmol) and the compound synthesized in step (37B) (5.48 g, 12.4 mmol) in N,N-dimethylformamide (15 ml), and the mixture was stirred at room temperature for 1 hour. Ethyl acetate was added, and the organic layer was washed with 15% saline, 0.5N hydrochloric acid, saturated saline, saturated sodium bicarbonate aqueous solution, and saturated saline, and then dried over anhydrous sodium sulfate, filtered, and the solvent was distilled off under reduced pressure to obtain a solid. The solid was washed with isopropyl ether and then dried under reduced pressure to obtain a crude product (5.4 g) containing the target compound 37C. The solid was used in the next reaction without further purification.

[1233] C 53 H 69 N 5 O 22 :[M+H] + Calculated value 1128, found value 1129, [M+Na] + Calculated value 1151, measured value 1151.

[1234] (37D) Synthesis of [(2R,3R,4R,5R,6R)-5-acetamide-6-[3-[[(3S)-4-[3-[(2R,3R,4R,5R,6R)-3-acetamide-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypropylamino]-3-amino-4-oxo-butyryl]amino]propoxy]-3,4-diacetoxy-tetrahydropyran-2-yl]methyl acetate TFA salt (Compound 37D)

[1235] [Chemistry 163]

[1236]

[1237] To a solution of the compound (5.4 g, 4.8 mmol) synthesized in step (37C) in N,N-dimethylformamide (25 ml), piperidine (0.57 ml, 5.7 mmol) was added and stirred at room temperature for 1.5 hours. After the reaction was completed, trifluoroacetic acid (0.31 ml) was added. The reaction solution was added to an appropriate amount of diethyl ether to obtain a precipitated residue. The product was separated and purified by reverse phase HPLC (GL Science, Inertsil ODS-3) using 0.1% trifluoroacetic acid aqueous solution and 0.1% trifluoroacetic acid acetonitrile solution as eluents, and the components containing the target product were collected and the solvent was removed by reduced pressure distillation. The amorphous target product (2.7 g, yield 55%) was obtained.

[1238] 1 H-NMR (DMSO-D 6 )δ:8.34-8.26(1H,m),8.14-8.05(4H,m),7.90-7.82(2H,m),5.25-5.21(2H, m),5.00-4.92(2H,m),4.50-4.45(2H,m),4.08-3.82(5H,m),3.80-3.63(3H,m ),3.51-3.39(3H,m),3.19-3.02(4H,m),2.70-2.53(2H,m),2.14-2.05(6H,m) ,2.04-1.96(6H,m),1.93-1.86(6H,m),1.84-1.75(6H,m),1.69-1.57(4H,m).

[1239] C 38 H 59 N 5 O 20 :[M+H] + Calculated value 905, measured value 906.

[1240] (37E) Synthesis of [(2R,3R,4R,5R,6R)-5-acetamide-6-[3-[[(3S)-4-[3-[(2R,3R,4R,5R,6R)-3-acetamide-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypropylamino]-3-[[(4R)-2,2-dimethyl-1,3-dioxolan-4-yl]methoxycarbonylamino]-4-oxo-butyryl]amino]propoxy]-3,4-diacetoxy-tetrahydropyran-2-yl]methyl acetate (Compound 37E)

[1241] [Chemistry 164]

[1242]

[1243] Using the compound synthesized in step (37D) (2.7 g, 2.6 mmol), the target amorphous product (0.77 g, yield 27%) was obtained in the same manner as step (35C).

[1244] 1 H-NMR (CDCl 3 )δ:7.19-7.12(1H,m),6.96-6.85(2H,m),6.83-6.75(1H,m),6.16(1H,d,J=9.1Hz ),5.41-5.34(2H,m),5.25-5.08(2H,m),4.66(1H,d,J=8.5Hz),4.51-3.87(16H,m ),3.79-3.72(1H,m),3.64-3.14(6H,m),3.01-2.91(1H,m),2.68-2.57(1H,m),2. 22-2.14(6H,m),2.10-1.93(18H,m),1.90-1.63(4H,m),1.42(3H,s),1.35(3H,s).

[1245] C 45 H 69 N 5 O 24 :[M+Na] + Calculated value 1086, measured value 1086.

[1246] (37F) Synthesis of [(2R,3R,4R,5R,6R)-5-acetamide-6-[3-[[(3S)-4-[3-[(2R,3R,4R,5R,6R)-3-acetamide-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypropylamino]-3-[[(2R)-2,3-dihydroxypropoxy]carbonylamino]-4-oxo-butyryl]amino]propoxy]-3,4-diacetoxy-tetrahydropyran-2-yl]methyl acetate (Compound 37F)

[1247] [Chemistry 165]

[1248]

[1249] The compound synthesized in step (37E) (0.77 g, 0.72 mmol) was used in the same manner as step (35D) to obtain a crude product (0.74 g) containing the target compound 37F. The product was used in the next reaction without further purification.

[1250] C 42 H 65 N 5 O 24 :[M+H] + Calculated value is 1024, measured value is 1024.

[1251] (37G) Synthesis of [(2R,3R,4R,5R,6R)-5-acetamide-6-[3-[[(3S)-4-[3-[(2R,3R,4R,5R,6R)-3-acetamide-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypropylamino]-3-[[(2R)-3-[bis(4-methoxyphenyl)-phenyl-methoxy]-2-hydroxy-propoxy]carbonylamino]-4-oxo-butyryl]amino]propoxy]-3,4-diacetoxy-tetrahydropyran-2-yl]methyl acetate (Compound 37G)

[1252] [Chemistry 166]

[1253]

[1254] Using the compound synthesized in step (37F) (0.74 g), the target amorphous product (0.63 g, yield 66% (2 steps)) was obtained in the same manner as in step (35E).

[1255] 1 H-NMR (CDCl 3 )δ:7.44-7.38(2H,m),7.33-7.09(9H,m),6.86-6.69(6H,m),6.22(1H,d,J=7. 9Hz),5.38-5.33(2H,m),5.23-5.09(2H,m),4.66(1H,d,J=7.9Hz),4.54-4.42( 2H,m),4.24-3.87(16H,m),3.79(6H,s),3.57-3.14(8H,m),2.97-2.87(1H,m) ,2.66-2.50(1H,m),2.19-2.13(6H,m),2.08-1.91(10H,m),1.87-1.65(4H,m).

[1256] C 63 H 83 N 5 O 26 :[M+Na] + Calculated value 1349, measured value 1349.

[1257] Synthesis of (37H) Acetate [(2R,3R,4R,5R,6R)-5-acetamide-6-[3-[[(3S)-4-[3-[(2R,3R,4R,5R,6R)-3-acetamide-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypropylamino]-3-[[(2R)-3-[bis(4-methoxyphenyl)-phenyl-methoxy]-2-[2-cyanoethoxy-(diisopropylamino)phosphino]oxy-propoxy]carbonylamino]-4-oxo-butyryl]amino]propoxy]-3,4-diacetoxy-tetrahydropyran-2-yl]methyl ester (Compound 37H)

[1258] [Chemistry 167]

[1259]

[1260] Using the compound synthesized in step (37G) (0.63 g, 0.48 mmol), the target amorphous product (0.52 g, yield 71%) was obtained in the same manner as step (35F).

[1261] 1 H-NMR (CDCl 3 )δ:7.45-7.38(2H,m),7.34-6.95(9H,m),6.88-6.62(6H,m),6.18-6.10(1H,m),5.39-5.32(2H,m),5.24-5.05(2H,m),4.69-4.60(1H,m), 4.52-4.41(2H,m),4.34-2.90(32H,m),2.68-2.42(3H,m),2.20-2.14(6H,m),2.09-1.91(18H,m),1.87-1.66(4H,m),1.21-0.97(12H,m).

[1262] (Reference Example 38)

[1263] (38A) Synthesis of Benzyl N-[3-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxypropyl]carbamate (Compound 38A)

[1264] [Chemistry 168]

[1265]

[1266] 28% sodium methoxide methanol solution (0.85 ml) was added to a suspension of the compound (12.4 g, 23 mmol) synthesized in step (37A) in ethanol (200 ml), and stirred at room temperature. As the reaction proceeds, the solution becomes transparent. After standing for 60 minutes, a precipitate is obtained. The precipitate is filtered, washed with ethanol and diisopropyl ether, and dried under reduced pressure. A crude product (10 g) containing the target compound 38A is obtained in the form of a solid. No further purification is performed and it is used in the next reaction.

[1267] 1 H-NMR (DMSO-D 6 )δ:7.60(1H,d,J=9.1Hz),7.40-7.17(5H,m),5.01(2H,s),4.62-4.55(2H,m),4.49(1H,d,J=4.2H z), 4.20 (1H, d, J = 8.5Hz), 3.76-3.27 (6H, m), 3.08-2.99 (2H, m), 1.81 (3H, s), 1.66-1.56 (2H, m).

[1268] C 19 H 28 N 2 O 8 :[M+Na] + Calculated value 435, measured value 435.

[1269] (38B) Synthesis of N-[(2R,3R,4R,5R,6R)-2-(3-aminopropoxy)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-3-yl]acetamide hydrochloride (Compound 38B)

[1270] [Chemistry 169]

[1271]

[1272] The compound synthesized in step (38A) (22.8 g, 55.3 mmol) was dissolved in tetrahydrofuran (280 ml) / 1N hydrochloric acid (61 ml). 10% palladium / carbon (wet basis) (5.56 g) was added and stirred vigorously at room temperature for 3.5 hours under a hydrogen environment. After the reaction was completed, the mixture was filtered and the obtained liquid was distilled under reduced pressure to remove it. An appropriate amount of ethanol was added, and the solids produced were filtered, washed with ethanol and diethyl ether, and dried under reduced pressure. A crude product (17 g) containing the target compound 38B was obtained. No further purification was performed and it was used in the next reaction.

[1273] 1 H-NMR (D 2O)δ: 4.27 (1H, d, J = 8.5Hz), 3.90-3.50 (8H, m), 2.93 (2H, t, J = 7.0Hz), 1.89 (3H, s), 1.82-1.75 (2H, m).

[1274] (38C) Synthesis of 9H-fluoren-9-ylmethyl N-[3-[(2R,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)tetrahydropyran-2-yl]oxypropyl]carbamate (Compound 38C)

[1275] [Chemistry 170]

[1276]

[1277] The compound synthesized in step (38B) (16.4 g, 52.1 mmol) was dissolved in tetrahydrofuran (200 ml) / pure water (50 ml), and N,N-diisopropylethylamine (12.7 mL, 72.9 mmol) was added. N-[(9H-fluoren-9-ylmethoxy)carbonyloxy]succinimide (21.1 g, 62.5 mmol) was added under ice-cooling, and the mixture was stirred at room temperature for 1.5 hours. After the reaction was completed, the solvent was distilled off under reduced pressure. Diethyl ether (100 ml) was added and stirred for 1 hour. The solid was washed with ethanol, ethyl acetate, and diethyl ether in sequence, and then dried under reduced pressure to obtain a crude product (22.9 g) containing the target compound 38C.

[1278] 1 H-NMR (DMSO-D 6 )δ:7.89(2H,d,J=7.3Hz),7.69(2H,d,J=7.3Hz),7.61(1H,d,J=9.1Hz),7.42(2H,t,J=7.3Hz),7.33(2H,t,J=7.3Hz),7.24(1H,t,J=5.7 Hz),4.60-4.57(2H,m),4.49(1H,d,J=4.2Hz),4.31-4.18(4H,m),3.76-3.28(8H,m),3.05-2.97(2H,m),1.81(3H,s),1.62-1.57(2H,m).

[1279] C 26 H 32 N 2 O 8 :[M+Na] + Calculated value 523, measured value 523.

[1280] (38D) Synthesis of [(2R,3R,4R,5R,6R)-5-acetamido-4-acetoxy-2-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-6-[3-(9H-fluoren-9-ylmethoxycarbonylamino)propoxy]tetrahydropyran-3-yl]acetate (Compound 38D)

[1281] [Chemistry 171]

[1282]

[1283] 4,4'-dimethoxytriphenylmethane (1.27 g, 3.7 mmol) was added to a suspension of the compound (1.56 g, 3.1 mmol) synthesized in step (38C) in pyridine (20 ml), and the mixture was stirred at room temperature for 30 minutes. Subsequently, acetic anhydride (1.18 ml, 12.5 mmol) was added, and the mixture was stirred at room temperature for 20 hours. After the reaction was completed, N,N-diisopropylethylamine (1.1 mL, 6.2 mmol) and ethanol (2 ml) were added, and the solvent was distilled off under reduced pressure to obtain a crude product. It was purified by silica gel column chromatography (hexane:ethyl acetate = 75:25-0:100, v / v, containing 1% triethylamine) to obtain an amorphous target product 38D (2.8 g, quantitative).

[1284] 1 H-NMR (CDCl 3 )δ:7.77(2H,d,J=7.3Hz),7.62(2H,d,J=7.3Hz),7.43-7.18(13H,m),6.83-6.79(4H,m ),6.03(1H,d,J=9.1Hz),5.55(1H,d,J=3.0Hz),5.14-5.03(2H,m),4.46-4.43(3H,m), 4.24-4.04(2H,m),3.97-3.91(1H,m),3.85-3.82(1H,m),3.77(6H,s),3.72-3.71(1H, m),3.54-3.33(3H,m),3.09-3.04(2H,m),2.03(3H,s),1.89(6H,s),1.65-1.56(2H,m).

[1285] C 52 H 55 NO 12 :[M+Na] + Calculated value 908, measured value 909.

[1286] (38E) Synthesis of [(2R,3R,4R,5R,6R)-5-acetamido-4-acetoxy-6-(3-aminopropoxy)-2-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]tetrahydropyran-3-yl]acetate (Compound 38D)

[1287] [Chemistry 172]

[1288]

[1289] To a solution of the compound (2.49 g, 2.8 mmol) synthesized in step (38D) in dichloromethane (8 ml), piperidine (0.45 ml, 4.5 mmol) was added and stirred at room temperature. If the reaction was not complete, piperidine was added as needed. After the reaction was completed, the solvent was distilled off under reduced pressure to obtain a crude product. It was purified by NH-silica gel column chromatography (dichloromethane:methanol=100:0-85:15, v / v) to obtain an amorphous target product 38E (1.48 g, yield 79%).

[1290] 1 H-NMR (CDCl 3 )δ:7.39-7.18(9H,m),6.83-6.80(4H,m),5.55(1H,d,J=3.0Hz),5.51(1H,d,J=9.1Hz),5. 27(1H,dd,J=11.2,3.3Hz),4.64(1H,d,J=8.5Hz),3.98-3.90(2H,m),3.84-3.82(1H,m),3 .79(6H,s),3.75-3.74(1H,m),3.58-3.56(1H,m),3.35(1H,dd,J=9.1,5.4Hz),3.07(1H,t ,J=8.5Hz),2.82-2.74(4H,m),2.01(3H,s),1.96(3H,s),1.89(3H,s),1.74-1.69(2H,m).

[1291] C 37 H 45 NO 10 :[M+Na] + Calculated value 687, measured value 687.

[1292] (Reference Example 39)

[1293] (39A) Synthesis of Benzyl 2-[[2-Benzyloxy-3-[(2-benzyloxy-2-oxo-ethyl)carbamoyloxy]propyloxy]carbonylamino]acetate (Compound 39A)

[1294] [Chemistry 173]

[1295]

[1296] 2-Benzyloxy-1,3-propanediol (2.5 g, 14 mmol) and 4-nitrophenyl chloroformate (5.8 g, 29 mmol) were dissolved in tetrahydrofuran (100 ml), pyridine (5.0 ml, 61.8 mmol) was added, and the mixture was stirred at room temperature for 20 minutes. After filtering the precipitated solid, tetrahydrofuran (70 ml) was added. Subsequently, glycine benzyl ester TFA salt (9.2 g, 33 mmol) and N,N-diisopropylethylamine (9.6 mL, 55 mmol) were added, and the mixture was stirred at room temperature overnight. After the reaction was completed, the solvent was distilled off under reduced pressure to obtain a crude product. It was purified by silica gel column chromatography (hexane: ethyl acetate = 100: 0-40: 60, v / v) to obtain the rubbery target 39A (6.5 g, yield 84%).

[1297] 1 H-NMR (CDCl 3 )δ:7.37-7.33(15H,m),5.48(2H,t,J=5.4Hz),5.18(4H,s),4.64(2H,s),4.23(4H,d,J=5.4Hz), 3.99(4H,d,J=5.4Hz), 3.84(1H,t,J=5.4Hz).

[1298] C 30 H 32 N 2 O 9 :[M+H] + Calculated value 565, found value 565, [M+Na] + Calculated value 587, measured value 587

[1299] (39B) Synthesis of 2-[[3-(carboxymethylaminoformyloxy)-2-hydroxy-propyloxy]carbonylamino]acetic acid (Compound 39B)

[1300] [Chemistry 174]

[1301]

[1302] Compound 39A (7.3 g, 2.8 mmol) synthesized in step (39A) was dissolved in tetrahydrofuran (200 ml) / pure water (50 ml). 20% palladium hydroxide-activated carbon (about 50% water) (3 g) was added and stirred vigorously at room temperature for 5 hours under a hydrogen atmosphere. After the reaction was completed, the mixture was filtered and the obtained liquid was removed by vacuum distillation to obtain a crude product (3.2 g) containing the target compound 39B. No further purification was performed and the mixture was used for the next reaction.

[1303] 1 H-NMR (D 2 O)δ:4.21-4.14(5H,m),3.86(4H,s).

[1304] (39C) Synthesis of [(2R,3R,4R,5R,6R)-5-acetamide-6-[3-[[2-[[3-[[2-[3-[(2R,3R,4R,5R,6R)-3-acetamide-4,5-diacetoxy-6-[[bis(4-methoxyphenyl)-phenyl 1-methoxy]methyl]tetrahydropyran-2-yl]oxypropylamino]-2-oxo-ethyl]carbamoyloxy]-2-hydroxy-propyloxy]carbonylamino]acetyl]amino]propyloxy]-4-acetoxy-2-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]tetrahydropyran-3-yl]acetate (Compound 39C)

[1305] [Chemistry 175]

[1306]

[1307] To a solution of compound 39B (0.31 g, 1.05 mmol) synthesized in step (39B), compound 38E (1.47 g, 2.21 mmol) synthesized in step (38E), and N,N-diisopropylethylamine (0.91 mL, 5.27 mmol) in N,N-dimethylformamide (9 ml) was added 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridine 3-oxide hexafluorophosphate (11.3 g, 29.6 mmol), and the mixture was stirred at room temperature for 3 hours. After the reaction was completed, the reaction solution was added dropwise to diethyl ether (185 ml in total). After removing the solvent from the supernatant, the obtained gel-like crude product was purified by silica gel column chromatography (dichloromethane:methanol=100:0-15:85, v / v, containing 1% triethylamine) to obtain the target compound 39C (1.05 g, yield 63%) as a solid.

[1308] 1 H-NMR (CDCl 3 )δ:7.39-7.17(18H,m),6.83-6.80(8H,m),6.70-6.27(2H,m),5.56-5.54(2H,m),5.10(2H,d ,J=10.9Hz),4.49-3.02(44H,m),2.01(6H,s),1.96(6H,s),1.90(6H,s),1.80-1.63(4H,m).

[1309] C81 H 98 N 6 O 27 :[M+Na] + Calculated value 1611, measured value 1611

[1310] (39D) Synthesis of [(2R,3R,4R,5R,6R)-5-acetamide-6-[3-[[2-[[3-[[2-[3-[(2R,3R,4R,5R,6R)-3-acetamide-4,5-diacetoxy-6-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]tetrahydropyran-2-yl]oxypropylamino]-2-oxo-ethyl]carbamoyloxy]-2-[2-cyanoethoxy-(diisopropylamino)phosphino]oxy-propoxy]carbonylamino]acetyl]amino]propoxy]-4-acetoxy-2-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]tetrahydropyran-3-yl]acetate (Compound 39D)

[1311] [Chemistry 176]

[1312]

[1313] Compound 39C (0.47 g, 0.30 mmol) synthesized in step (39C) was dissolved in dichloromethane (3 ml) after azeotroping with an appropriate amount of ethyl acetate / toluene (1 / 1). N,N-diisopropylethylamine (210 μl, 1.18 mmol) and 2-cyanoethyldiisopropylchlorophosphoramidite (79 μl, 0.36 mmol) were added, and the mixture was stirred at room temperature for 1 hour. Furthermore, 2-cyanoethyldiisopropylchlorophosphoramidite (20 μl, 0.09 mmol) was added, and the mixture was stirred at room temperature for 15 minutes. After the reaction was completed, the solvent was distilled off under reduced pressure to obtain a crude product. It was purified by silica gel column chromatography (ethyl acetate:methanol=100:0-85:15, v / v, containing 1% triethylamine) to obtain an amorphous target compound 39D (0.34 g, yield 64%).

[1314] 1 H-NMR (CDCl 3 )δ:7.39-7.17(18H,m),6.87-6.70(8H,m),6.29-6.06(2H,m),5.55(2H,d,J=3.0Hz),5.11(2H,d,J=10.9Hz),4.47-3. 02(47H,m),2.63(2H,t,J=6.3Hz),2.02(6H,s),1.96(6H,s),1.90(6H,s),1.80-1.64(4H,m),1.17(12H,d,J=6.7Hz).

[1315] (40A) Synthesis of Benzyl 2-[[3-Benzyloxy-2-[(2-benzyloxy-2-oxo-ethyl)carbamoyloxy]propyloxy]carbonylamino]acetate (Compound 40A)

[1316] [Chemistry 177]

[1317]

[1318] Using (+ / -)-3-benzyloxy-1,2-propanediol (1.0 g, 5.5 mmol) instead of 2-benzyloxy-1,3-propanediol, the target compound 40A (2.8 g, yield 90%) was obtained in the same manner as step (39A).

[1319] 1 H-NMR (CDCl 3 )δ:7.39-7.30(15H,m),5.37-5.35(2H,m),5.17-5.12(5H,m),4.56-4.51(2H,m),4.33-4.30(2H,m),4.03-3.94(4H,m),3.61(2H,d,J=5.4Hz).

[1320] C 30 H 32 N 2 O 9 :[M+Na] + Calculated value 587, measured value 587

[1321] (40B) Synthesis of 2-[[2-(carboxymethylaminoformyloxy)-3-hydroxy-propyloxy]carbonylamino]acetic acid (Compound 40B)

[1322] [Chemistry 178]

[1323]

[1324] Using compound 40A (2.8 g, 5.0 mmol) instead of compound 39A, the same procedure as in step (39B) was carried out to obtain a crude product (1.2 g) containing the target compound 40B.

[1325] 1 H-NMR (D 2 O)δ:4.32-4.19(3H,m),3.94-3.87(4H,m),3.81-3.71(2H,m).

[1326] (40C) Synthesis of [(2R,3R,4R,5R,6R)-5-acetamide-6-[3-[[2-[[2-[[2-[3-[(2R,3R,4R,5R,6R)-3-acetamide-4,5-diacetoxy-6-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]tetrahydropyran-2-yl]oxypropylamino]-2-oxo-ethyl]carbamoyloxy]-3-hydroxy-propyloxy]carbonylamino]acetyl]amino]propyloxy]-4-acetoxy-2-[[bis(4-methoxyphenyl)-phenyl 1-methoxy]methyl]tetrahydropyran-3-yl]acetate (Compound 40C)

[1327] [Chemistry 179]

[1328]

[1329] Using compound 40B (0.31 g, 1.05 mmol) instead of compound 39B, the target compound 40C (0.74 g, yield 44%) was obtained in the same manner as step (39C).

[1330] 1 H-NMR (CDCl 3 )δ:7.40-7.18(18H,m),6.81(8H,dd,J=9.1,3.0Hz),6.70-6.50(1H,m),6.16-6.02(1H,m),5.56-5.54(2 H,br),5.11-5.00(2H,m),4.53-2.99(44H,m),2.01(6H,s),1.95(6H,s),1.88(6H,s),1.77-1.68(4H,m).

[1331] C 81 H 98 N 6 O 27 :[M+Na] + Calculated value 1611, measured value 1611

[1332] (40D) Synthesis of [(2R,3R,4R,5R,6R)-5-acetamide-6-[3-[[2-[[2-[[2-[3-[(2R,3R,4R,5R,6R)-3-acetamide-4,5-diacetoxy-6-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]tetrahydropyran-2-yl]oxypropylamino]-2-oxo-ethyl]carbamoyloxy]-3-[2-cyanoethoxy-(diisopropylamino)phosphino]oxy-propoxy]carbonylamino]acetyl]amino]propoxy]-4-acetoxy-2-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]tetrahydropyran-3-yl]acetate (Compound 40D)

[1333] [Chemistry 180]

[1334]

[1335] Using compound 40C (0.31 g, 1.05 mmol) instead of compound 39C, the target compound 40D (0.60 g, yield 72%) was obtained in the same manner as step (39D).

[1336] 1 H-NMR (CDCl 3 )δ:7.38-7.18(18H,m),7.13-6.97(1H,m),6.83-6.81(8H,m),6.58-6.36(1H,m),5.56-5.52(2H,m),5.10-5.04(2H,m),4.46-2.97( 47H,m),2.65-2.63(2H,m),2.02(6H,s),1.95(3H,s),1.94(3H,s),1.90(3H,s),1.89(3H,s),1.77-1.67(4H,m),1.20-1.16(12H,m).

[1337] (Example 131)

[1338] HO-X 16 -A m1s -A e2s -U m1s -C m1s -C e2s -G m1s -A m1s -T e2s -G m1s -G m1s -C e2s -G m1s -A m1s -A e2s -G m1t-H (Sequence number 40)

[1339] Replace the part of X with X 16 , and synthesized in the same manner as in Example 91. 16 The compound 36G synthesized in Reference Example 36 was used for the first condensation.

[1340] The base sequence of this compound is complementary to the sequence from 92 to 106 from the 5' end of exon 5 of the c.648G>T variant G6PC gene in which the G at nucleotide number 728 is mutated to T in the catalytic subunit of glucose-6-phosphatase (G6PC) of Homo sapiens, transcript variant 1, mRNA (NCBI-GenBank accession number NM_000151.3). The compound was identified by negative ion ESI mass spectrometry (measured value: 6261.97)

[1341] (Example 132)

[1342] HO-X 15 -X 15 -X 15 -A m1s -A e2s -U m1s -C m1s -C e2s -G m1s -A m1s -T e2s -G m1s -G m1s -C e2s -G m1s -A m1s -A e2s -G m1t -H (Sequence number 40)

[1343] Replace the part of X with X 15 , and synthesized in the same manner as in Example 91. 15 The compound 35F synthesized in Reference Example 35 was used for the condensation three times.

[1344] The base sequence of this compound is complementary to the sequence from 92 to 106 from the 5' end of exon 5 of the c.648G>T variant G6PC gene in which the G at nucleotide number 728 is mutated to T in the catalytic subunit of glucose-6-phosphatase (G6PC) of Homo sapiens, transcript variant 1, mRNA (NCBI-GenBank accession number NM_000151.3). The compound was identified by negative ion ESI mass spectrometry (measured value: 6917.09)

[1345] (Example 133)

[1346] X 18 -A m1s -A e2s -U m1s -C m1s -C e2s -G m1s -A m1s -T e2s -G m1s -G m1s -C e2s -G m1s -A m1s -A e2s -G m1t -H (Sequence number 40)

[1347] Replace the part of X with X 18 , and synthesized in the same manner as in Example 91. 18 The compound 39D synthesized in Reference Example 39 was used for the first condensation.

[1348] The base sequence of this compound is complementary to the sequence from 92 to 106 from the 5' end of exon 5 of the c.648G>T variant G6PC gene in which the G at nucleotide number 728 is mutated to T in the catalytic subunit of glucose-6-phosphatase (G6PC) of Homo sapiens, transcript variant 1, mRNA (NCBI-GenBank accession number NM_000151.3). The compound was identified by negative ion ESI mass spectrometry (measured value: 6247.96)

[1349] (Example 134)

[1350] HO-X 15 -X 15 -X 15 -A e2s -U m1s -C m1s -C e2s -G m1s -A m1s -T e2s -G m1s -G m1s -C e2s -G m1s -A m1s -A e2s -G m1s -C e2t -H (Sequence number 44)

[1351] The sequence 15e_005.1 was used instead of the sequence used in Example 132, and synthesis was carried out in the same manner as in Example 132.

[1352] The base sequence of this compound is complementary to the sequence from 91 to 105 from the 5' end of exon 5 of the c.648G>T variant G6PC gene in which the G at nucleotide number 728 is mutated to T in the catalytic subunit of glucose-6-phosphatase (G6PC) of Homo sapiens, transcript variant 1, mRNA (NCBI-GenBank accession number NM_000151.3). The compound was identified by negative ion ESI mass spectrometry (measured value: 6919.09)

[1353] (Example 135)

[1354] HO-X 15 -X 15 -X 15 -U m1s -C m1s -C e2s -G m1s -A m1s -T e2s -G m1s -G m1s -C e2s -G m1s -A m1s -A e2s -G m1t -C e2s -U m1t -H (Sequence number 45)

[1355] The sequence 15e_006.1 was used instead of the sequence used in Example 132, and synthesis was carried out in the same manner as in Example 132.

[1356] The base sequence of this compound is complementary to the sequence from 90 to 104 from the 5' end of exon 5 of the c.648G>T variant G6PC gene in which the G at nucleotide number 728 is mutated to T in the catalytic subunit of glucose-6-phosphatase (G6PC) of Homo sapiens, transcript variant 1, mRNA (NCBI-GenBank accession number NM_000151.3). The compound was identified by negative ion ESI mass spectrometry (measured value: 6884.07)

[1357] (Example 136)

[1358] HO-X 15 -X 15 -X 15 -A m1s -A m1s -A e2s -U m1s -C m1s -C e2s -G m1s -A m1s -Te2s -G m1s -G m1s -C e2s -G m1s -A m1s -A e2s -G m1t -H (Sequence number 42)

[1359] The sequence 16e_001 was used instead of the sequence used in Example 132, and synthesis was carried out in the same manner as in Example 132.

[1360] The base sequence of this compound is complementary to the sequence from 92 to 107 from the 5' end of exon 5 of the c.648G>T variant G6PC gene in which the G at nucleotide number 728 is mutated to T in the catalytic subunit of glucose-6-phosphatase (G6PC) of Homo sapiens, transcript variant 1, mRNA (NCBI-GenBank accession number NM_000151.3). The compound was identified by negative ion ESI mass spectrometry (measured value: 7276.10)

[1361] (Example 137)

[1362] HO-X 15 -X 15 -X 15 -A m1s -A m1s -A m1s -A e2s -U m1s -C m1s -C e2s -G m1s -A m1s -T e2s -G m1s -G m1s -C e2s -G m1s -A m1s -A e2s -G m1t -H (Sequence number 46)

[1363] The sequence 17e_001 was used instead of the sequence used in Example 132, and synthesis was carried out in the same manner as in Example 132.

[1364] The base sequence of this compound is complementary to the sequence from 92 to 108 from the 5' end of exon 5 of the c.648G>T variant G6PC gene in which the G at nucleotide number 728 is mutated to T in the catalytic subunit of glucose-6-phosphatase (G6PC) of Homo sapiens, transcript variant 1, mRNA (NCBI-GenBank accession number NM_000151.3). The compound was identified by negative ion ESI mass spectrometry (measured value: 7636.19)

[1365] (Example 138)

[1366] X 18 -A e2s -U m1s -C m1s -C e2s -G m1s -A m1s -T e2s -G m1s -G m1s -C e2s -G m1s -A m1s -A e2s -G m1s -C e2t -H (Sequence number 44)

[1367] The sequence 15e_005.1 was used instead of the sequence used in Example 133, and synthesis was carried out in the same manner as in Example 133.

[1368] The base sequence of this compound is complementary to the sequence from 91 to 105 from the 5' end of exon 5 of the c.648G>T variant G6PC gene in which the G at nucleotide number 728 is mutated to T in the catalytic subunit of glucose-6-phosphatase (G6PC), transcript variant 1, mRNA (NCBI-GenBank accession number NM_000151.3) of Homo sapiens. The compound was identified by negative ion ESI mass spectrometry (measured value: 6250.00)

[1369] (Example 139)

[1370] X 18 -U m1s -C m1s -C e2s -G m1s -A m1s -T e2s -G m1s -G m1s -C e2s -G m1s -A m1s -A e2s -G m1s-C e2s -U m1t -H (Sequence number 45)

[1371] The sequence 15e_006.1 was used instead of the sequence used in Example 133, and synthesis was carried out in the same manner as in Example 133.

[1372] The base sequence of this compound is complementary to the sequence from 90 to 104 from the 5' end of exon 5 of the c.648G>T variant G6PC gene in which the G at nucleotide number 728 is mutated to T in the catalytic subunit of glucose-6-phosphatase (G6PC) of Homo sapiens, transcript variant 1, mRNA (NCBI-GenBank accession number NM_000151.3). The compound was identified by negative ion ESI mass spectrometry (measured value: 6214.96)

[1373] (Example 140)

[1374] X 18 -A m1s -A m1s -A e2s -U m1s -C m1s -C e2s -G m1s -A m1s -T e2s -G m1s -G m1s -C e2s -G m1s -A m1s -A e2s -G m1t -H (Sequence number 42)

[1375] The sequence 16e_001 was used instead of the sequence used in Example 133, and synthesis was carried out in the same manner as in Example 133.

[1376] The base sequence of this compound is complementary to the sequence from 92 to 107 from the 5' end of exon 5 of the c.648G>T variant G6PC gene in which the G at nucleotide number 728 is mutated to T in the catalytic subunit of glucose-6-phosphatase (G6PC) of Homo sapiens, transcript variant 1, mRNA (NCBI-GenBank accession number NM_000151.3). The compound was identified by negative ion ESI mass spectrometry (measured value: 6607.02)

[1377] (Example 141)

[1378] X 18 -A m1s -A e2s -U m1s-C m1s -C e2s -G m1s -A m1s -T e2s -G m1s -G m1s -C e2s -G m1s -A m1s -A e2s -G m1s -C e2t -H (Sequence number 47)

[1379] The sequence 16e_002 was used instead of the sequence used in Example 133, and synthesis was carried out in the same manner as in Example 133.

[1380] The base sequence of this compound is complementary to the sequence from 91 to 106 from the 5' end of exon 5 of the c.648G>T variant G6PC gene in which the G at nucleotide number 728 is mutated to T in the catalytic subunit of glucose-6-phosphatase (G6PC) of Homo sapiens, transcript variant 1, mRNA (NCBI-GenBank accession number NM_000151.3). The compound was identified by negative ion ESI mass spectrometry (measured value: 6609.03)

[1381] (Example 142)

[1382] X 18 -A e2s -U m1s -C m1s -C e2s -G m1s -A m1s -T e2s -G m1s -G m1s -C e2s -G m1s -A m1s -A e2s -G m1s -C e2s -U m1t -H (Sequence number 48)

[1383] The sequence 16e_003 was used instead of the sequence used in Example 133, and synthesis was carried out in the same manner as in Example 133.

[1384] The base sequence of this compound is complementary to the sequence from 90 to 105 from the 5' end of exon 5 of the c.648G>T variant G6PC gene in which the G at nucleotide number 728 is mutated to T in the catalytic subunit of glucose-6-phosphatase (G6PC), transcript variant 1, mRNA (NCBI-GenBank accession number NM_000151.3) of Homo sapiens. The compound was identified by negative ion ESI mass spectrometry (measured value: 6586.00)

[1385] (Example 143)

[1386] X 18 -A m1s -A e2s -U m1s -C m1s -C e2s -G m1s -A e2s -U m1s -G m1s -G m1s -C e2s -G m1s -A m1s -A e2s -G m1t -H (Sequence number 40)

[1387] The sequence 15e_001.5 was used instead of the sequence used in Example 133, and synthesis was carried out in the same manner as in Example 133.

[1388] The base sequence of this compound is complementary to the sequence from 92 to 106 from the 5' end of exon 5 of the c.648G>T variant G6PC gene in which the G at nucleotide number 728 is mutated to T in the catalytic subunit of glucose-6-phosphatase (G6PC) of Homo sapiens, transcript variant 1, mRNA (NCBI-GenBank accession number NM_000151.3). The compound was identified by negative ion ESI mass spectrometry (measured value: 6233.97)

[1389] (Example 144)

[1390] X 18 -A e2s -U m1s -C m1s -C e2s -G m1s -A e2s -U m1s -G m1s -G m1s -C e2s -G m1s -A m1s -A e2s-G m1s -C e2t -H (Sequence number 44)

[1391] The sequence 15e_005.5 was used instead of the sequence used in Example 133, and synthesis was carried out in the same manner as in Example 133.

[1392] The base sequence of this compound is complementary to the sequence from 91 to 105 from the 5' end of exon 5 of the c.648G>T variant G6PC gene in which the G at nucleotide number 728 is mutated to T in the catalytic subunit of glucose-6-phosphatase (G6PC) of Homo sapiens, transcript variant 1, mRNA (NCBI-GenBank accession number NM_000151.3). The compound was identified by negative ion ESI mass spectrometry (measured value: 6235.97)

[1393] (Example 145)

[1394] X 18 -U m1s -C m1s -C e2s -G m1s -A e2s -U m1s -G m1s -G m1s -C e2s -G m1s -A m1s -A e2s -G m1s -C e2s -U m1t -H (Sequence number 45)

[1395] The sequence 15e_006.5 was used instead of the sequence used in Example 133, and synthesis was carried out in the same manner as in Example 133.

[1396] The base sequence of this compound is complementary to the sequence from 90 to 104 from the 5' end of exon 5 of the c.648G>T variant G6PC gene in which the G at nucleotide number 728 is mutated to T in the catalytic subunit of glucose-6-phosphatase (G6PC) of Homo sapiens, transcript variant 1, mRNA (NCBI-GenBank accession number NM_000151.3). The compound was identified by negative ion ESI mass spectrometry (measured value: 6200.94)

[1397] (Example 146)

[1398] X 18 -A m1s -A m1s -A e2s -Um1s -C m1s -C e2s -G m1s -A e2s -U m1s -G m1s -G m1s -C e2s -G m1s -A m1s -A e2s -G m1t -H (Sequence number 42)

[1399] The sequence 16e_001.5 was used instead of the sequence used in Example 133, and synthesis was carried out in the same manner as in Example 133.

[1400] The base sequence of this compound is complementary to the sequence from 92 to 107 from the 5' end of exon 5 of the c.648G>T variant G6PC gene in which the G at nucleotide number 728 is mutated to T in the catalytic subunit of glucose-6-phosphatase (G6PC) of Homo sapiens, transcript variant 1, mRNA (NCBI-GenBank accession number NM_000151.3). The compound was identified by negative ion ESI mass spectrometry (measured value: 6593.02)

[1401] (Example 147)

[1402] X 18 -A m1s -A e2s -U m1s -C m1s -C e2s -G m1s -A e2s -U m1s -G m1s -G m1s -C e2s -G m1s -A m1s -A e2s -G m1s -C e2t -H (Sequence number 47)

[1403] The sequence 16e_002.5 was used instead of the sequence used in Example 133, and synthesis was carried out in the same manner as in Example 133.

[1404] The base sequence of this compound is complementary to the sequence from 91 to 106 from the 5' end of exon 5 of the c.648G>T variant G6PC gene in which the G at nucleotide number 728 is mutated to T in the catalytic subunit of glucose-6-phosphatase (G6PC) of Homo sapiens, transcript variant 1, mRNA (NCBI-GenBank accession number NM_000151.3). The compound was identified by negative ion ESI mass spectrometry (measured value: 6595.01)

[1405] (Example 148)

[1406] X 18 -A e2s -U m1s -C m1s -C e2s -G m1s -A e2s -U m1s -G m1s -G m1s -C e2s -G m1s -A m1s -A e2s -G m1s -C e2s -U m1t -H (Sequence number 48)

[1407] The sequence 16e_003.5 was used instead of the sequence used in Example 133, and synthesis was carried out in the same manner as in Example 133.

[1408] The base sequence of this compound is complementary to the sequence from 90 to 105 from the 5' end of exon 5 of the c.648G>T variant G6PC gene in which the G at nucleotide number 728 is mutated to T in the catalytic subunit of glucose-6-phosphatase (G6PC) of Homo sapiens, transcript variant 1, mRNA (NCBI-GenBank accession number NM_000151.3). The compound was identified by negative ion ESI mass spectrometry (measured value: 6571.98)

[1409] (Example 149)

[1410] HO-X 16 -A m1s -A e2s -U m1s -C m1s -C e2s -G m1s -A e2s -U m1s -G m1s -G m1s -C e2s -Gm1s -A m1s -A e2s -G m1t -H (Sequence number 40)

[1411] The sequence 15e_001.5 was used instead of the sequence used in Example 131, and synthesis was carried out in the same manner as in Example 131.

[1412] The base sequence of this compound is complementary to the sequence from 92 to 106 from the 5' end of exon 5 of the c.648G>T variant G6PC gene in which the G at nucleotide number 728 is mutated to T in the catalytic subunit of glucose-6-phosphatase (G6PC) of Homo sapiens, transcript variant 1, mRNA (NCBI-GenBank accession number NM_000151.3). The compound was identified by negative ion ESI mass spectrometry (measured value: 6247.99)

[1413] (Example 150)

[1414] X 19 -A m1s -A e2s -U m1s -C m1s -C e2s -G m1s -A m1s -T e2s -G m1s -G m1s -C e2s -G m1s -A m1s -A e2s -G m1t -H (Sequence number 40)

[1415] Replace the part of X with X 19 , and synthesized in the same manner as in Example 91. 19 The compound 40D synthesized in Reference Example 40 was used for the first condensation.

[1416] The base sequence of this compound is complementary to the sequence from 92 to 106 from the 5' end of exon 5 of the c.648G>T variant G6PC gene in which the G at nucleotide number 728 is mutated to T in the catalytic subunit of glucose-6-phosphatase (G6PC) of Homo sapiens, transcript variant 1, mRNA (NCBI-GenBank accession number NM_000151.3). The compound was identified by negative ion ESI mass spectrometry (measured value: 6247.99)

[1417] (Example 151)

[1418] X17 -A m1s -A e2s -U m1s -C m1s -C e2s -G m1s -A m1s -T e2s -G m1s -G m1s -C e2s -G m1s -A m1s -A e2s -G m1t -H (Sequence number 40)

[1419] Replace the part of X with X 17 , and synthesized in the same manner as in Example 91. 17 The compound 37H synthesized in Reference Example 37 was used for the first condensation.

[1420] The base sequence of this compound is complementary to the sequence from 92 to 106 from the 5' end of exon 5 of the c.648G>T variant G6PC gene in which the G at nucleotide number 728 is mutated to T in the catalytic subunit of glucose-6-phosphatase (G6PC) of Homo sapiens, transcript variant 1, mRNA (NCBI-GenBank accession number NM_000151.3). The compound was identified by negative ion ESI mass spectrometry (measured value: 6204.97)

[1421] (Reference Example 41)

[1422] (41A) Synthesis of Benzyl 3-[[2-Benzyloxy-3-[(3-benzyloxy-3-oxo-propyl)carbamoyloxy]propyloxy]carbonylamino]propionic acid ester (Compound 41A)

[1423] [Chemistry 181]

[1424]

[1425] 3-aminopropionic acid benzyl ester TFA salt (2.4 g, 8.17 mmol) was used instead of glycine benzyl ester TFA salt used in step (39A), and the target compound 41A (1.62 g, yield 80%) was obtained in the same manner as step (39A).

[1426] 1 H-NMR (CDCl 3)δ:7.39-7.27(15H,m),5.21(2H,br s),5.14(4H,s),4.63(2H,s),4.24-4.10(4H,m),3.79-3.74(1H,m),3.48-3.44(4H,m),2.59(4H,t,J=6.0Hz).

[1427] C 32 H 36 N 2 O 9 :[M+H] + Calculated value 593, found value 593, [M+Na] + Calculated value 615, measured value 615

[1428] (41B) Synthesis of 3-[[3-(2-carboxyethylcarbamoyloxy)-2-hydroxy-propyloxy]carbonylamino]propionic acid (Compound 41B)

[1429] [Chemistry 182]

[1430]

[1431] The target compound 41B (0.89 g, quantitative) was obtained in the same manner as in step (39B) using compound 41A (1.62 g, 2.73 mmol) instead of compound 39A used in step (39B).

[1432] 1 H-NMR (D 2 O)δ:4.22-4.04(5H,m),3.46-3.32(4H,m),2.57(4H,t,J=6.7Hz).

[1433] (41C) Synthesis of [(2R,3R,4R,5R,6R)-5-acetamide-6-[3-[3-[[3-[3-[3-[(2R,3R,4R,5R,6R)-3-acetamide-4,5-diacetoxy-6-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]tetrahydropyran-2-yl]oxypropylamino]-3-oxo-propyl]carbamoyloxy]-2-hydroxy-propyloxy]carbonylamino]propionylamino]propoxy]-4-acetoxy-2-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]tetrahydropyran-3-yl]acetate (Compound 41C)

[1434] [Chemistry 183]

[1435]

[1436] Using compound 41B (300 mg, 0.93 mmol) instead of compound 39B used in step (39C), the target compound 41C (950 mg, yield 63%) was obtained in the same manner as in step (39C).

[1437] 1 H-NMR (CDCl 3 )δ:7.32-7.24(18H,m),6.82-6.80(8H,m),6.64-6.61(1H,br),6.27-6.24(1H,br),5.91-5.88(1H,br),5.56(2H,d,J=3.0Hz ),5.10(2H,d,J=11.5Hz),4.46-3.05(43H,m),2.47-2.37(4H,m),2.02(6H,s),1.98(6H,s),1.90(6H,s),1.78-1.67(4H,m).

[1438] C 83 H 102 N 6 O 27 :[M+Na] + Calculated value 1638, measured value 1638

[1439] (41D) Synthesis of [(2R,3R,4R,5R,6R)-5-acetamide-6-[3-[3-[[3-[3-[3-[(2R,3R,4R,5R,6R)-3-acetamide-4,5-diacetoxy-6-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]tetrahydropyran-2-yl]oxypropylamino]-3-oxo-propyl]carbamoyloxy]-2-[2-cyanoethoxy-(diisopropylamino)phosphino]oxy-propoxy]carbonylamino]propanoylamino]propoxy]-4-acetoxy-2-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]tetrahydropyran-3-yl]acetate (Compound 41D)

[1440] [Chemistry 184]

[1441]

[1442] Using compound 41C (945 mg, 0.58 mmol) instead of compound 39C used in step (39D), the target compound 41D (638 mg, yield 60%) was obtained in the same manner as in step (39D).

[1443] 1 H-NMR (CDCl 3)δ:7.39-7.18(18H,m),6.85-6.78(8H,m),6.73-6.64(1H,m),6.40-6.23(1H,m), 5.83-5.72(1H,m),5.56(2H,d,J=3.0Hz),5.13-5.04(2H,m),4.45-4.35(2H,m),4. 22-3.30(40H,m),3.22-3.03(4H,m),2.64(2H,t,J=6.0Hz),2.54-2.32(4H,m),2. 02(6H,s),2.00-1.95(6H,m),1.90(6H,s),1.83-1.59(4H,m),1.20-1.10(12H,m).

[1444] (Reference Example 42)

[1445] (42A) Synthesis of Benzyl 4-[[2-Benzyloxy-3-[(4-benzyloxy-4-oxo-butyl)carbamoyloxy]propyloxy]carbonylamino]butyrate (Compound 42A)

[1446] [Chemistry 185]

[1447]

[1448] The target compound 42A (0.85 g, yield 78%) was obtained in the same manner as in step (39A) using 4-aminobutyric acid benzyl ester TFA salt (1.30 g, 4.21 mmol) instead of glycine benzyl ester TFA salt.

[1449] 1 H-NMR (CDCl 3 )δ:7.38-7.27(15H,m),5.11(4H,s),4.80(2H,br s),4.63(2H,s),4.19-4.15(4H,m),3.77-3.75(1H,m),3.23-3.18(4H,m),2.40(4H,t,J=7.3Hz),1.88-1.81(4H,m).

[1450] C 34 H 40 N 2 O 9 :[M+H] + Calculated value 621, found value 622, [M+Na] + Calculated value 643, measured value 644

[1451] (42B) Synthesis of 4-[[3-(3-carboxypropylcarbamoyloxy)-2-hydroxy-propyloxy]carbonylamino]butyric acid (Compound 42B)

[1452] [Chemistry 186]

[1453]

[1454] The target compound 42B (0.49 g, quantitative) was obtained in the same manner as in step (39B) using compound 42A (0.85 g, 1.4 mmol) instead of compound 39A used in step (39B).

[1455] 1 H-NMR (D 2 O)δ:4.19-4.05(5H,m),3.19-3.12(4H,m),2.36-2.27(4H,m),1.83-1.73(4H,m).

[1456] (42C) Synthesis of [(2R,3R,4R,5R,6R)-5-acetamide-6-[3-[4-[[3-[[4-[3-[(2R,3R,4R,5R,6R)-3-acetamide-4,5-diacetoxy-6-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]tetrahydropyran-2-yl]oxypropylamino]-4-oxo-butyl]carbamoyloxy]-2-hydroxy-propyloxy]carbonylamino]butyrylamino]propyloxy]-4-acetoxy-2-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]tetrahydropyran-3-yl]acetate (Compound 42C)

[1457] [Chemistry 187]

[1458]

[1459] Using compound 42B (300 mg, 0.86 mmol) instead of compound 39B used in step (39C), the target compound 42C (1.16 g, yield 82%) was obtained in the same manner as in step (39C).

[1460] 1 H-NMR (CDCl 3)δ:7.31-7.24(18H,m),6.82-6.80(8H,m),6.68-6.65(3H,m),5.91-5.84(1H,m),5.56(2H,d,J=3.0Hz),5.10(2H,d,J=10.9H z),4.45(2H,d,J=8.5Hz),4.23-3.12(40H,m),2.31-2.22(4H,m),2.01(6H,s),1.98(6H,s),1.90(6H,s),1.81-1.68(8H,m).

[1461] C 85 H 106 N 6 O 27 :[M+Na] + Calculated value 1666, measured value 1667

[1462] (42D) Synthesis of [(2R,3R,4R,5R,6R)-5-acetamide-6-[3-[3-[[3-[3-[3-[(2R,3R,4R,5R,6R)-3-acetamide-4,5-diacetoxy-6-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]tetrahydropyran-2-yl]oxypropylamino]-4-oxo-butyl]carbamoyloxy]-2-[2-cyanoethoxy-(diisopropylamino)phosphino]oxy-propyloxy]carbonylamino]butyrylamino]propyloxy]-4-acetoxy-2-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]tetrahydropyran-3-yl]acetate (Compound 42D)

[1463] [Chemistry 188]

[1464]

[1465] Using compound 42C (1.16 g, 0.71 mmol) instead of compound 39C used in step (39D), the target compound 42D (795 mg, yield 61%) was obtained in the same manner as in step (39D).

[1466] 1 H-NMR (CDCl 3)δ:7.40-7.17(18H,m),6.85-6.77(8H,m),6.72-5.65(5H,m),5.56(2H,d,J=3.0Hz),5.11-5.03(2H,m),4.46-4.35(2H,m),4.25-3.02(4 2H,m),2.65(2H,t,J=6.0Hz),2.39-2.16(4H,m),2.02(6H,s),1.99-1.94(6H,m),1.90(6H,s),1.87-1.59(8H,m),1.18(12H,d,J=6.7Hz).

[1467] (Reference Example 43)

[1468] (43A) Synthesis of [(2R,3R,4R,5R,6R)-5-acetamide-3,4-diacetoxy-6-(5-aminopentyloxy)tetrahydropyran-2-yl]methyl acetate hydrochloride (Compound 43A)

[1469] [Chemistry 189]

[1470]

[1471] Compound 21A (3.87 g, 6.83 mmol) synthesized in step (21A) was dissolved in tetrahydropyran (32 ml) / 1N hydrochloric acid (8 ml). 10% palladium / carbon (wet basis) (2 g) was added and stirred vigorously at room temperature under a hydrogen atmosphere. After the reaction was completed, the mixture was filtered and the obtained liquid was removed by vacuum distillation to obtain a crude product of the target compound 43A (3.1 g, yield 97%). No further purification was performed and the mixture was used in the next reaction.

[1472] C 19 H 32 N 2 O 9 :[M+H] + Calculated value 433, measured value 433.

[1473] (43B) Synthesis of [(2R,3R,4R,5R,6R)-5-acetamide-6-[5-[[3-[5-[(2R,3R,4R,5R,6R)-3-acetamide-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentylcarbamoyloxy]-2-benzyloxy-propoxy]carbamoylamino]pentyloxy]-3,4-diacetoxy-tetrahydropyran-2-yl]methyl acetate (Compound 43B)

[1474] [Chemistry 190]

[1475]

[1476] 2-Benzyloxy-1,3-propanediol (600 mg, 3.29 mmol) and 4-nitrophenyl chloroformate (1.39 g, 6.91 mmol) were dissolved in tetrahydrofuran (22 ml), pyridine (1.1 ml, 13.2 mmol) was added, and the mixture was stirred at room temperature for 15 minutes. The precipitated solid was filtered, and tetrahydrofuran (16 ml) was added. Then, a tetrahydrofuran (15 ml) / dichloromethane (5 ml) solution of the compound (43A) (3.09 g, 6.59 mmol) synthesized in step (43A) and N,N-diisopropylethylamine (3.4 mL, 19.8 mmol) were added, and the mixture was stirred at 40°C for 2 hours. After the reaction was completed, the solvent was distilled off under reduced pressure. Dichloromethane was added, and the organic layer was washed with 0.5N hydrochloric acid, saturated saline, 0.2N sodium hydroxide aqueous solution, and saturated saline, and then dried with anhydrous sodium sulfate, filtered, and the solvent was distilled off under reduced pressure to obtain a crude product. It was purified by silica gel column chromatography (dichloromethane:methanol=100:0-90:10, v / v) to obtain a mixture (2.1 g) mainly containing the target compound 43B.

[1477] C 50 H 74 N 4 O 23 :[M+H] + Calculated value 1099, found value 1099.[M+Na] + Calculated value 1121, measured value 1121.

[1478] (43C) Synthesis of [(2R,3R,4R,5R,6R)-5-acetamide-6-[5-[[3-[5-[(2R,3R,4R,5R,6R)-3-acetamide-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentylcarbamoyloxy]-2-hydroxy-propoxy]carbonylamino]pentyloxy]-3,4-diacetoxy-tetrahydropyran-2-yl]methyl acetate (Compound 43C)

[1479] [Chemistry 191]

[1480]

[1481] The mixture containing 43B obtained in step (43B) (2.1 g) was dissolved in tetrahydropyran (20 ml) / pure water (1 ml). 1N hydrochloric acid (0.38 ml) and 10% palladium / carbon (wet basis) (1 g) were added, and the mixture was stirred vigorously at room temperature under a hydrogen atmosphere. After the reaction was completed, the mixture was filtered and the obtained liquid was removed by vacuum distillation to obtain a crude product. It was purified by silica gel column chromatography (ethyl acetate: methanol = 100: 0-90: 15, v / v) to obtain the target product 43C (1.38 g, yield 72%) as a solid.

[1482] 1 H-NMR (CDCl 3 )δ:6.10-5.96(1H,m),5.38-4.94(6H,m),4.70-4.64(2H,m),4.23-3.44(19H,m),3.20 -3.15(4H,m),2.15(6H,s),2.05(6H,s),2.01(6H,s),1.97(6H,s),1.67-1.38(12H,m).

[1483] C 43 H 68 N 4 O 23 :[M+H] + Calculated value 1009, found value 1009.[M+Na] + Calculated value 1031, measured value 1031.

[1484] (43D) Synthesis of [(2R,3R,4R,5R,6R)-5-acetamide-6-[5-[[3-[5-[(2R,3R,4R,5R,6R)-3-acetamide-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypentylcarbamoyloxy]-2-[2-cyanoethoxy-(diisopropylamino)phosphino]oxy-propoxy]carbamoylamino]pentyloxy]-3,4-diacetoxy-tetrahydropyran-2-yl]methyl acetate (Compound 43D)

[1485] [Chemistry 192]

[1486]

[1487] Using compound 43C (1.38 g, 1.37 mmol) instead of compound 39C used in step (39D), the target compound 43D (850 mg, yield 51%) was obtained in the same manner as in step (39D).

[1488] 1 H-NMR (CDCl 3)δ:5.95-5.89(1H,m),5.38-5.26(4H,m),5.20-5.16(1H,m),5.00-4.95(1H,m),4.71-4.68(2H,m),4.36-3.44(22H,m),3.15(4H,q ,J=6.4Hz),2.67(2H,t,J=6.3Hz),2.15(6H,s),2.05(6H,s),2.01(6H,s),1.96(6H,s),1.67-1.33(12H,m),1.18(12H,d,J=6.7Hz).

[1489] (Reference Example 44)

[1490] (44A) Synthesis of [(2R,3R,4R,5R,6R)-5-acetamide-6-[3-[3-[[3-[3-[3-[(2R,3R,4R,5R,6R)-3-acetamide-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypropylamino]-3-oxo-propyl]carbamoyloxy]-2-hydroxy-propoxy]carbonylamino]propionylamino]propoxy]-3,4-diacetoxy-tetrahydropyran-2-yl]methyl acetate (Compound 44A)

[1491] [Chemistry 193]

[1492]

[1493] To a solution of compound 41B (200 mg, 0.62 mmol) synthesized in step (41B) in N, N-dimethylformamide (2 ml), O-(N-succinimidyl)-N, N, N', N'-tetramethyluronium tetrafluoroborate (411 mg, 1.37 mmol) and N, N-diisopropylethylamine (0.32 ml, 1.86 mmol) were added, and the mixture was stirred at room temperature for 20 minutes. The reaction solution was added to a solution of compound 37B (657 mg, 1.49 mmol) synthesized in step (37B) in N, N-dimethylformamide (2 ml), and N, N-diisopropylethylamine (0.32 ml, 1.86 mmol) was added, and the mixture was stirred at room temperature for 60 minutes. After the reaction was completed, the reaction solution was added dropwise to diethyl ether (80 ml in total). After removing the solvent from the supernatant, the obtained gel-like crude product was purified by silica gel column chromatography (dichloromethane:methanol=100:0-75:25, v / v) to obtain an amorphous target compound 44A (408 mg, yield 60%).

[1494] 1 H-NMR (CDCl 3)δ:6.76(2H,br),6.69-6.63(2H,m),5.96(1H,br),5.36(2H,d,J=3.0Hz),5.13-5.08(2H,m),4.53(2H,d,J=8.5Hz ),4.25-3.07(27H,m),2.51-2.44(4H,m),2.17(6H,s),2.06(6H,s),2.02(6H,s),2.00(6H,s),1.86-1.73(4H,m).

[1495] C 45 H 70 N 6 O 25 :[M+H] + Calculated value 1095, found value 1096. [M+Na] + Calculated value 1117, measured value 1117.

[1496] (44B) Synthesis of [(2R,3R,4R,5R,6R)-5-acetamide-6-[3-[3-[[3-[3-[3-[(2R,3R,4R,5R,6R)-3-acetamide-4,5-diacetoxy-6-(acetoxymethyl)tetrahydropyran-2-yl]oxypropylamino]-3-oxo-propyl]carbamoyloxy]-2-[2-cyanoethoxy-(diisopropylamino)phosphino]oxy-propoxy]carbonylamino]propionylamino]propoxy]-3,4-diacetoxy-tetrahydropyran-2-yl]methyl acetate (Compound 44B)

[1497] [Chemistry 194]

[1498]

[1499] Using compound 44A (1.53 g, 1.40 mmol) instead of compound 39C used in step (39D), the target compound 44B (510 mg, yield 28%) was obtained in the same manner as in step (39D).

[1500] 1 H-NMR (CDCl 3)δ:6.60-6.44(4H,m),5.76-5.68(1H,m),5.36(2H,d,J=3.0Hz),5.09-5.05(2H,m),4.50-4.45(2H,m),4.26-3.37(28H,m),3.17(2H,br), 2.67(2H,t,J=6.3Hz),2.52-2.42(4H,m),2.17(6H,s),2.05(6H,s),2.02(6H,s),1.99(6H,s),1.81-1.70(4H,m),1.18(12H,d,J=6.7Hz).

[1501] (Example 152)

[1502] X 20 -A m1s -A e2s -U m1s -C m1s -C e2s -G m1s -A e2s -U m1s -G m1s -G m1s -C e2s -G m1s -A m1s -A e2s -G m1t -H (Sequence number 40)

[1503] X 18 Partial replacement of X 20 , and was synthesized in the same manner as in Example 143. The sequence used was the sequence 15e_001.5.

[1504] The base sequence of this compound is complementary to the sequence from 92 to 106 from the 5' end of exon 5 of the c.648G>T variant G6PC gene in which the G at nucleotide number 728 is mutated to T in the catalytic subunit of glucose-6-phosphatase (G6PC) of Homo sapiens, transcript variant 1, mRNA (NCBI-GenBank accession number NM_000151.3). The compound was identified by negative ion ESI mass spectrometry (measured value: 6261.98)

[1505] (Example 153)

[1506] X 20 -A e2s -U m1s -C m1s -C e2s -G m1s -A e2s -Um1s -G m1s -G m1s -C e2s -G m1s -A m1s -A e2s -G m1s -C e2t -H (Sequence number 44)

[1507] X 18 Partial replacement of X 20 , and was synthesized in the same manner as in Example 144. The sequence used was the sequence 15e_005.5.

[1508] The base sequence of this compound is complementary to the sequence from 91 to 105 from the 5' end of exon 5 of the c.648G>T variant G6PC gene in which the G at nucleotide number 728 is mutated to T in the catalytic subunit of glucose-6-phosphatase (G6PC) of Homo sapiens, transcript variant 1, mRNA (NCBI-GenBank accession number NM_000151.3). The compound was identified by negative ion ESI mass spectrometry (measured value: 6264.00)

[1509] (Example 154)

[1510] X 20 -A m1s -A m1s -A e2s -U m1s -C m1s -C e2s -G m1s -A e2s -U m1s -G m1s -G m1s -C e2s -G m1s -A m1s -A e2s -G m1t -H (Sequence number 42)

[1511] X 18 Partial replacement of X 20 , and was synthesized in the same manner as in Example 146. The sequence used was the sequence 16e_001.5.

[1512] The base sequence of this compound is complementary to the sequence from 92 to 107 from the 5' end of exon 5 of the c.648G>T variant G6PC gene in which the G at nucleotide number 728 is mutated to T in the catalytic subunit of glucose-6-phosphatase (G6PC) of Homo sapiens, transcript variant 1, mRNA (NCBI-GenBank accession number NM_000151.3). The compound was identified by negative ion ESI mass spectrometry (measured value: 6621.04)

[1513] (Example 155)

[1514] X 20 -A m1s -A e2s -U m1s -C m1s -C e2s -G m1s -A e2s -U m1s -G m1s -G m1s -C e2s -G m1s -A m1s -A e2s -G m1s -C e2t -H (Sequence number 47)

[1515] X 18 Partial replacement of X 20 , and was synthesized in the same manner as in Example 147. The sequence used was the sequence 16e_002.5.

[1516] The base sequence of this compound is complementary to the sequence from 91 to 106 from the 5' end of exon 5 of the c.648G>T variant G6PC gene in which the G at nucleotide number 728 is mutated to T in the catalytic subunit of glucose-6-phosphatase (G6PC) of Homo sapiens, transcript variant 1, mRNA (NCBI-GenBank accession number NM_000151.3). The compound was identified by negative ion ESI mass spectrometry (measured value: 6623.05)

[1517] (Example 156)

[1518] X 21 -A m1s -A e2s -U m1s -C m1s -C e2s -G m1s -A e2s -U m1s -G m1s -G m1s -Ce2s -G m1s -A m1s -A e2s -G m1t -H (Sequence number 40)

[1519] X 18 Partial replacement of X 21 , and was synthesized in the same manner as in Example 143. The sequence used was the sequence 15e_001.5.

[1520] The base sequence of this compound is complementary to the sequence from 92 to 106 from the 5' end of exon 5 of the c.648G>T variant G6PC gene in which the G at nucleotide number 728 is mutated to T in the catalytic subunit of glucose-6-phosphatase (G6PC) of Homo sapiens, transcript variant 1, mRNA (NCBI-GenBank accession number NM_000151.3). The compound was identified by negative ion ESI mass spectrometry (measured value: 6290.02)

[1521] (Example 157)

[1522] X 22 -A m1s -A e2s -U m1s -C m1s -C e2s -G m1s -A e2s -U m1s -G m1s -G m1s -C e2s -G m1s -A m1s -A e2s -G m1t -H (Sequence number 40)

[1523] X 18 Partial replacement of X 22 , and was synthesized in the same manner as in Example 143. The sequence used was the sequence 15e_001.5.

[1524] The base sequence of this compound is complementary to the sequence from 92 to 106 from the 5' end of exon 5 of the c.648G>T variant G6PC gene in which the G at nucleotide number 728 is mutated to T in the catalytic subunit of glucose-6-phosphatase (G6PC) of Homo sapiens, transcript variant 1, mRNA (NCBI-GenBank accession number NM_000151.3). The compound was identified by negative ion ESI mass spectrometry (measured value: 6175.96)

[1525] (Example 158)

[1526] X 22 -A e2s -U m1s -C m1s -C e2s -G m1s -A e2s -U m1s -G m1s -G m1s -C e2s -G m1s -A m1s -A e2s -G m1s -C e2t -H (Sequence number 44)

[1527] X 18 Partial replacement of X 22 , and was synthesized in the same manner as in Example 144. The sequence used was the sequence 15e_005.5.

[1528] The base sequence of this compound is complementary to the sequence from 91 to 105 from the 5' end of exon 5 of the c.648G>T variant G6PC gene in which the G at nucleotide number 728 is mutated to T in the catalytic subunit of glucose-6-phosphatase (G6PC), transcript variant 1, mRNA (NCBI-GenBank accession number NM_000151.3) of Homo sapiens. The compound was identified by negative ion ESI mass spectrometry (measured value: 6177.98)

[1529] (Example 159)

[1530] X 22 -A m1s -A m1s -A e2s -U m1s -C m1s -C e2s -G m1s -A e2s -U m1s -G m1s -G m1s -C e2s -G m1s -A m1s -A e2s -G m1t -H (Sequence number 42)

[1531] X 18 Partial replacement of X 22 , and was synthesized in the same manner as in Example 146. The sequence used was the sequence 16e_001.5.

[1532] The base sequence of this compound is complementary to the sequence from 92 to 107 from the 5' end of exon 5 of the c.648G>T variant G6PC gene in which the G at nucleotide number 728 is mutated to T in the catalytic subunit of glucose-6-phosphatase (G6PC), transcript variant 1, mRNA (NCBI-GenBank accession number NM_000151.3) of Homo sapiens. The compound was identified by negative ion ESI mass spectrometry (measured value: 6535.03)

[1533] (Example 160)

[1534] X 22 -A m1s -A e2s -U m1s -C m1s -C e2s -G m1s -A e2s -U m1s -G m1s -G m1s -C e2s -G m1s -A m1s -A e2s -G m1s -C e2t -H (Sequence number 47)

[1535] X 18 Partial replacement of X 22 , and was synthesized in the same manner as in Example 147. The sequence used was the sequence 16e_002.5.

[1536] The base sequence of this compound is complementary to the sequence from 91 to 106 from the 5' end of exon 5 of the c.648G>T variant G6PC gene in which the G at nucleotide number 728 is mutated to T in the catalytic subunit of glucose-6-phosphatase (G6PC) of Homo sapiens, transcript variant 1, mRNA (NCBI-GenBank accession number NM_000151.3). The compound was identified by negative ion ESI mass spectrometry (measured value: 6537.05)

[1537] (Test Example 1) Evaluation of Repair of Abnormal Splicing Using Example Compounds Using Cultured Cells

[1538] Culture of 293A cells (human embryonic kidney cells)

[1539] 293A cells (R705-07 invitrogen) were cultured as follows.

[1540] 293A cells were maintained in a maintenance medium (DMEM (Dulbecco's Modified Eagle Medium), 10% FBS (Fetal Bovine Serum)). 5 Cells were seeded in 6-well plates at a density of 1×10 cells / well. 5 The cells were seeded in a 12-well plate at a density of 1:1 cells / well, and on the next day, the compounds of the examples and the plasmid vector were simultaneously introduced in the manner described below for evaluation.

[1541] Preparation of human G6PC full-length plasmid vector

[1542] The human G6PC full-length plasmid vector (pcDNA hG6PC, pcDNA hG6PC (c.648G>T)+Int4) was prepared as follows.

[1543] Using the human multiple tissue cDNA plate as a template, the G6PC cDNA was amplified using the following G6PC cDNA amplified primers, and then further amplified using the G6PC cDNA IF primers. The amplified fragment was inserted into the BamHI site of pcDNA3.1 (pcDNA hG6PC) using the InFusion system.

[1544] G6PC cDNA amplification primers:

[1545] Forward primer 5'-ATAGCAGAGCAATCACCACCAAGCC-3' (SEQ ID NO: 49)

[1546] Reverse primer 5'-ATTCCACGACGGCAGAATGGATGGC-3' (SEQ ID NO: 50)

[1547] G6PC IF Primers:

[1548] Forward primer 5'-TACCGAGCTCGGATCCACCACCAAGCCTGGAATAACTGC-3' (SEQ ID NO: 51)

[1549] Reverse primer 5'-CTGGACTAGTGGATCCTGGCATGGTTGTTGACTTTAAAC-3' (SEQ ID NO: 52)

[1550] Using human genomic DNA as a template, the following G6PC Int4 amplification primer was used to amplify the G6PC intron 4 and a region containing a portion of exon 5, and then the G6PC Int4 IF primer was used to amplify the G6PC intron 4. In addition, the pcDNA hG6PC prepared in STEP1-1 was amplified using the following hG6PC vector IF primer. Furthermore, the c.648G>T mutation in exon 5 was introduced into the InFusion primer. The two fragments were linked by the InFusion system, and the G6PC intron 4 sequence was inserted into the pcDNA hG6PC (pcDNA hG6PC (c.648G>T) + Int4).

[1551] G6PC Int4 Amplification Primers:

[1552] Forward primer 5'-TCTGGGCTGTGCAGCTGAATGTCTG-3' (SEQ ID NO: 53)

[1553] Reverse primer 5'-GTAGGGGATGACACTGACGGATGCC-3' (SEQ ID NO: 54)

[1554] G6PC Int4 IF Primers:

[1555] Forward primer 5'-CTGGAGTCCTGTCAGGTATGGGC-3' (SEQ ID NO: 55)

[1556] Reverse primer 5'-AGCTGAAAAGGAAGAAGGTAATGAG-3' (SEQ ID NO: 56)

[1557] hG6PC vector IF primers:

[1558] Forward primer 5'-TCTTCCTTTTCAGCTTCGCCATCGG-3' (SEQ ID NO: 57)

[1559] Reverse primer 5'-CTGACAGGACTCCAGCAACAAC-3' (SEQ ID NO: 58)

[1560] Co-transfection of the compounds of the examples and plasmid vectors

[1561] The compound of the example and the plasmid vector were co-transfected in the following manner.

[1562] The following liquid A and liquid B were prepared and mixed.

[1563] When the method is carried out in a 6-well plate, 250 μL of Opti-MEM medium (gibco), 0.50 μL of plasmid vector (1 mg / mL), and 4.0 μL (final: 20 nM) of the compound prepared in the example (12.5 μM) were prepared as solution A for each well, and 250 μL of Opti-MEM medium (gibco) and 6.0 μL of Lipofectamine 2000 (Invitrogen) were prepared a...

Claims

1. A complex, or a pharmacologically acceptable salt or solvate thereof, wherein the complex is a complex of an oligonucleotide represented by any one of the formulas of Examples 133, 143 to 148, and 152 to 155 below and a GalNAc unit: Embodiment 133: X 18 -A m1s -A e2s -U m1s -C m1s -C e2s -G m1s -A m1s -T e2s -G m1s -G m1s -C e2s -G m1s -A m1s -A e2s -G m1t -H (Sequence number 40) Embodiment 143: X 18 -A m1s -A e2s -U m1s -C m1s -C e2s -G m1s -A e2s -U m1s -G m1s -G m1s -C e2s -G m1s -A m1s -A e2s -G m1t -H (Sequence number 40) Embodiment 144: X 18 -A e2s -U m1s -C m1s -C e2s -G m1s -A e2s -U m1s -G m1s -G m1s -C e2s -G m1s -A m1s -A e2s -G m1s -C e2t -H (Sequence number 44) Embodiment 145: X 18 -U m1s -C m1s -C e2s -G m1s -A e2s -U m1s -G m1s -G m1s -C e2s -G m1s -A m1s -A e2s -G m1s -C e2s -U m1t -H (Sequence number 45) Embodiment 146: X 18 -A m1s -A m1s -A e2s -U m1s -C m1s -C e2s -G m1s -A e2s -U m1s -G m1s -G m1s -C e2s -G m1s -A m1s -A e2s -G m1t -H (Sequence number 42) Embodiment 147: X 18 -A m1s -A e2s -U m1s -C m1s -C e2s -G m1s -A e2s -U m1s -G m1s -G m1s -C e2s -G m1s -A m1s -A e2s -G m1s -C e2t -H (Sequence number 47) Embodiment 148: X 18 -A e2s -U m1s -C m1s -C e2s -G m1s -A e2s -U m1s -G m1s -G m1s -C e2s -G m1s -A m1s -A e2s -G m1s -C e2s -U m1t -H (Sequence number 48) Embodiment 152: X 20 -A m1s -A e2s -U m1s -C m1s -C e2s -G m1s -A e2s -U m1s -G m1s -G m1s -C e2s -G m1s -A m1s -A e2s -G m1t -H (Sequence number 40) Embodiment 153: X 20 -A e2s -U m1s -C m1s -C e2s -G m1s -A e2s -U m1s -G m1s -G m1s -C e2s -G m1s -A m1s -A e2s -G m1s -C e2t -H (Sequence number 44) Embodiment 154: X 20 -A m1s -A m1s -A e2s -U m1s -C m1s -C e2s -G m1s -A e2s -U m1s -G m1s -G m1s -C e2s -G m1s -A m1s -A e2s -G m1t -H (Sequence number 42) Embodiment 155: X 20 -A m1s -A e2s -U m1s -C m1s -C e2s -G m1s -A e2s -U m1s -G m1s -G m1s -C e2s -G m1s -A m1s -A e2s -G m1s -C e2t -H (Sequence number 47) [In the formula, the left side represents the 5' end, the right side represents the 3' end, A m1t , G m1t , C m1t , 5meC m1t , U m1t , A m1p , G m1p , C m1p , 5meC m1p , U m1p , A m1s , G m1s , C m1s , 5meC m1s , U m1s , A e2t , G e2t , C e2t , T e2t , A e2p , G e2p , C e2p , T e2p , A e2s , G e2s , C e2s , T e2s , X 18 , X 20 is a group having the structure shown below, X 18 and X 20 The bond to the phosphate group refers to the bond to the carbon atom at the 5' end of the oligonucleotide to form a phosphodiester bond.] [Chemistry 1] [Chemistry 2] [Chemistry 3] [Chemistry 4] 2. A therapeutic drug for glycogenopathies type Ia, comprising the complex according to claim 1, or a pharmacologically acceptable salt or solvate thereof, and is administered to a glycogenopathies type Ia patient having a c.648G>T mutation in the G6PC gene.

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