WO2019027009A1

WO2019027009A1 - Nucleic acid complex

Info

Publication number: WO2019027009A1
Application number: PCT/JP2018/029079
Authority: WO
Inventors: 陽史山田; 宏徒岩井; 上原　啓嗣; 康裕鈴木; 裕一福田; 達人木内
Original assignee: 協和発酵キリン株式会社
Priority date: 2017-08-02
Filing date: 2018-08-02
Publication date: 2019-02-07
Also published as: TW201920668A

Abstract

The present invention provides a nucleic acid complex represented by formula 1, the nucleic acid complex being capable of suppressing the expression of β2GPI. Formula 1: (in formula 1, X is a double-stranded nucleic acid that comprises a sense strand and an antisense strand and includes a duplex region of at least 11 base pairs; the double-stranded nucleic acid is complementary with any of the β2GPI mRNA sequences of interest described in Tables 1-1 to 1-16 in an oligonucleotide strand having a strand length of 17-30 nucleotides in the antisense strand, and the 3' end or 5' end of the sense strand binds to S3; L1 and L2 each independently a sugar ligand; and S1, S2, and S3 each independently represent a linker.)

Description

Nucleic acid complex

The present invention relates to a nucleic acid complex, a pharmaceutical composition containing the nucleic acid complex, and the like.

As nucleic acid drugs, aptamers, antisenses, decoy nucleic acids, ribozymes, siRNAs, miRNAs and antimiRNAs are known. Nucleic acid drugs are expected to be clinically applicable to various diseases that have been considered difficult to treat up to now, because of their high versatility in which all genes in cells can be controlled.
In addition, nucleic acid drugs are expected as next-generation drugs next to antibodies and small molecule drugs because of high target selectivity and activity in cells.
However, the problem with nucleic acid medicines is that delivery to target tissues is difficult.

The use of a nucleic acid complex (conjugate) of a targeting compound and a nucleic acid has been reported as one of the effective nucleic acid drug delivery methods in vivo. Targeting compounds include ligands capable of binding to extracellularly expressed receptors. Among them, in particular, a plurality of nucleic acid complexes using N-acetyl-D-galactosamine (GalNAc) or the like as a ligand capable of binding to asialoglycoprotein receptor (ASGPR) which is extremely expressed in liver cells is reported. There is. In recent years, it has been reported that nucleic acid complexes in which these ligands and siRNAs are bound are efficiently delivered to hepatocytes (Non-patent Document 1).

Patent Documents 1 and 2 disclose, for example, nucleic acid complexes shown below as complexes of a targeting compound and an oligonucleotide.

(In the formula, Ac represents an acetyl group. Hereinafter, the same applies in the present specification.)

Patent Document 3 discloses a nucleic acid complex having the following structure having the same sugar ligand-tether unit as Patent Documents 1 and 2.

Patent Document 4 discloses a nucleic acid complex having a structure shown below as a sugar ligand-tether unit.

On the other hand, β2-glycoprotein 1 (β2GPI) (aka apolipoprotein H, also referred to as apoH) is a glycoprotein composed of 326 amino acids, and has a higher-order structure in which five domain structures are linked. β2GPI is considered to have various physiological actions, and is reported to be involved in platelet aggregation reaction, coagulation / fibrinolytic reaction, and uptake of oxidized LDL into macrophages (Non-patent Document 2).

Regarding the association with disease, β2GPI is known to be the main corresponding antigen of antiphospholipid antibodies appearing in autoimmune diseases such as antiphospholipid antibody syndrome (APS) and systemic lupus erythematosus (SLE). Anti-β2GPI antibody is also deeply involved in the pathogenesis of diseases, and the complex formed by β2GPI and anti-β2GPI antibody is on the membrane of various cells such as vascular endothelial cells, monocytes, platelets and trophoblasts. It has become clear from studies using animal models and clinical research that it can generate activation signals to the receptor and, as a result, it can cause pathologies characteristic of APS such as thrombosis and pregnancy abnormalities (Non-patent Document 3) ). Although it can be expected that the above-mentioned diseases can be prevented or treated by specifically inhibiting the formation of an immune complex consisting of β2GPI and anti-β2GPI antibodies, β2GPI is relatively high in blood at 50-500 μg / mL. It is not easy to continue to inhibit all these β2GPI, for example, by general antibody drugs, which are present (Non-patent Document 4).

International Publication No. 2009/073809 International Publication No. 2013/075035 International Publication No. 2015/105083 International Publication No. 2014/179620

An object of the present invention is to provide a nucleic acid complex capable of suppressing the expression of β2GPI.

The present invention relates to the following.
[1]
A nucleic acid complex represented by the following formula 1.
Formula 1:

(In the formula 1,
X is a double stranded nucleic acid consisting of a sense strand and an antisense strand, comprising a duplex region of at least 11 base pairs,
The double-stranded nucleic acid is an oligonucleotide chain of 17 to 30 nucleotides in length in the antisense strand, and any one of the target β2GPI mRNA sequences described in Tables 1-1 to 1-16. Complementary and
The 3 'or 5' end of the sense strand is linked to S3,
L1 and L2 are each independently a sugar ligand,
S1, S2 and S3 are each independently a linker. )
[2]
The nucleic acid complex according to [1], which has a structure represented by the following formula 2.
Formula 2:

(In the formula 2,
X, L1, L2 and S3 are as defined above,
P1, P2, P3, P4, P5 and P6, and T1 and T2 are each independently absent, or -CO-, -NH-, -O-, -S-, -O-CO- , -S-CO-, -NH-CO-, -CO-O-, -CO-S- or -CO-NH-,
Q1, Q2, Q3 and Q4 are each independently absent, or substituted or unsubstituted alkylene having 1 to 12 carbon atoms or-(CH ₂ CH ₂ O) _n -CH ₂ CH _2- , N is an integer of 0 to 99,
B1 and B2 are each independently a bond or a structure represented by the following formula 2-1, and the terminal black dot in each structure is P2 or P3 or P5, respectively. Or a point of attachment to P6, and m1, m2, m3 and m4 are each independently an integer of 0 to 10,
Formula 2-1:

p1 and p2 are each independently an integer of 1, 2 or 3;
q1, q2, q3 and q4 are each independently an integer of 0 to 10,
However, when p1 and p2 are integers of 2 or 3, respectively, P3 and P6, Q2 and Q4, T1 and T2 and L1 and L2 may be the same or different, and q1 to q4 are 2 to 10 And each of the combinations of-[P2-Q1]-,-[Q2-P3]-,-[P5-Q3]-and-[Q4-P6]-may be the same or different. )
[3]
The nucleic acid complex according to [2], wherein P1 and P4 are each independently -CO-NH-, -NH-CO- or -O-.
[4]
-[P2-Q1] _q1 - _and- [P5-Q3] _q3 -each independently do not exist, or any one of structures represented by the following formulas 3-1 to 3-3: The nucleic acid complex as described in 2] or [3].
Formula 3-1:

Formula 3-2:

Formula 3-3:

(In Formula 3-1 to Formula 3-3,
m5 and m6 are each independently an integer of 0 to 10, and the terminal black dot in the structure of Formula 3-1 to Formula 3-3 is a bonding point to B1 or B2 or P1 or P4, respectively is there. )
[5]
The nucleic acid complex according to any one of [2] to [4], which has any of the structures represented by the following formulas 4-1 to 4-9.
Formula 4-1:

Equation 4-2:

Equation 4-3:

Formula 4-4:

Formula 4-5:

Formula 4-6:
Equation 4-7:

Formula 4-8:

Formula 4-9:

(In the formulas 4-1 to 4-9,
X, L1, L2, S3, P3, P6, T1, T2, Q2, Q4, q2 and q4 are as defined above. )
[6]
The nucleic acid complex according to [1], having a structure represented by the following formula 5.
Formula 5:

(In the equation 5,
X, S3, P1, P2, P3, Q1, Q2, B1, T1, L1, p1, q1 and q2 are as defined above. )
[7]
The nucleic acid complex according to [6], wherein P1 is -CO-NH-, -NH-CO- or -O-.
[8]
The nucleic acid complex according to [6] or [7], which has any one of structures represented by the following formulas 6-1 to 6-9.
Formula 6-1:

Formula 6-2:

Equation 6-3:

Equation 6-4:

Formula 6-5:

Formula 6-6:

Formula 6-7:

Formula 6-8:

Formula 6-9:

(In the formulas 6-1 to 6-9,
X, S3, P3, Q2, T1, L1 and q2 are as defined above. )
[9]
The nucleic acid complex according to any one of [2] to [5], which has any one of structures represented by the following formulas 7-1 to 7-9.
Formula 7-1:

Equation 7-2:

Formula 7-3:

Equation 7-4:

Formula 7-5:

Formula 7-6:

Formula 7-7:

Formula 7-8:

Formula 7-9:

(In the formulas 7-1 to 7-9,
X, S3, L1 and L2 are as defined above. )
[10]
The nucleic acid complex according to any one of [1] to [9], wherein the sugar ligand is N-acetylgalactosamine.
[11]
The nucleic acid complex according to any one of [1] to [10], wherein the double stranded nucleic acid comprises a modified nucleotide.
[12]
The nucleic acid complex according to any one of [1] to [11], wherein the 3 'end of the sense strand and the 5' end of the antisense strand form a blunt end.
[13]
The nucleic acid complex according to any one of [1] to [12], wherein the double stranded nucleic acid comprises a sugar moiety-modified nucleotide.
[14]
X is a pair of sense strand / antisense strand selected from the group consisting of sense strand / antisense strand described in Table M1-1 to Table M1-18 or Table 1-1 to Table 1-16, The nucleic acid complex according to any one of [1] to [13].
[15]
The nucleic acid complex according to any one of [1] to [14], which has a structure represented by the following formula 7-8-1.
Formula 7-8-1:

(In formula 7-8-1, X is as defined above.)
[16]
The nucleic acid complex according to [15], wherein X is a double stranded nucleic acid selected from the group consisting of CHOO96, CHOO99, CH0125, CH0126, CH0318, CH0943 and CH1139 in Tables M1-1 to M1-18.
[17]
A pharmaceutical composition comprising the nucleic acid complex according to any one of [1] to [16].
[18]
The pharmaceutical composition according to [17], for introduction into cells.
[19]
The pharmaceutical composition according to [17] or [18], which is administered intravenously or subcutaneously.
[20]
Administering the nucleic acid complex according to any one of [1] to [16] or the pharmaceutical composition according to any one of [17] to [19] to a patient in need thereof And methods of treating or preventing the disease.
[21]
Introducing a double stranded nucleic acid into a cell using the nucleic acid complex according to any one of [1] to [16] or the pharmaceutical composition according to any one of [17] to [19] A method for suppressing the expression of the β2 GPI gene, including
[22]
[Beta] 2GPI-related diseases comprising administering the nucleic acid complex according to any one of [1] to [16] or the pharmaceutical composition according to any one of [17] to [19] to a mammal Treatment method.
[23]
A medicament for use in the treatment of a β2GPI related disease, comprising the nucleic acid complex according to any one of [1] to [16] or the pharmaceutical composition according to any one of [17] to [19] .
[24]
A therapeutic agent for a β2GPI related disease, comprising the nucleic acid complex according to any one of [1] to [16] or the pharmaceutical composition according to any one of [17] to [19].
[25]
The therapeutic method according to [22], wherein the β2GPI related disease is an autoimmune disease or thrombosis.
[26]
The medicament according to [23], wherein the β2 GPI related disease is an autoimmune disease or thrombosis.
[27]
The therapeutic agent according to [24], wherein the β2 GPI related disease is an autoimmune disease or thrombosis.

For example, a pharmaceutical composition containing the nucleic acid complex of the present invention can be administered to a mammal to treat various related diseases in vivo.

The nucleic acid complex of the present invention is a nucleic acid complex represented by the following formula 1.
Formula 1:

In formula 1,
X is a double stranded nucleic acid consisting of a sense strand and an antisense strand, comprising a duplex region of at least 11 base pairs,
The double-stranded nucleic acid is an oligonucleotide chain of 17 to 30 nucleotides in length in the antisense strand, and any one of the target β2GPI mRNA sequences described in Tables 1-1 to 1-16. Complementary and
The 3 'or 5' end of the sense strand is linked to S3,
L1 and L2 are each independently a sugar ligand,
S1, S2 and S3 are each independently a linker.

In the present invention, S1 and S2 can bind to the benzene ring in the ortho position, meta position and para position respectively with respect to the substitution position on the benzene ring of S3, but the nucleic acid complex represented by the following formula 1-1 It is preferred that it is a body. It is meant that the bond of S1 and S2 to the benzene ring in Formula 1 may be at any position other than the substitution position of S3 on the benzene ring.
Formula 1-1:

In Formula 1-1,
X, L1, L2, S1, S2 and S3 are as defined above.
In the present specification, the same meaning as described above will be described by exemplifying Formula 1-1: X, L1, L2, S1, and S2 in Formula 1-1, X, L1, It means that it may be the same group as the definition for each of L2, S1 and S2.

In the present invention, X is a double stranded nucleic acid consisting of a sense strand and an antisense strand and comprising a double stranded region of at least 11 base pairs.
In addition, the double-stranded nucleic acid is a target β2GPI mRNA described in Tables 1-1 to 1-16 described later in the oligonucleotide strand having a chain length of 17 to 30 nucleotides in the antisense strand. Complementary to any of the sequences.
Furthermore, the 3 'or 5' end of the sense strand is linked to S3.

L1 and L2 are each independently a sugar ligand.
In the present invention, a sugar ligand means a group derived from a saccharide (such as monosaccharide, disaccharide, trisaccharide and polysaccharide) capable of binding to a receptor expressed in a target cell. In the present invention, when the sugar ligand is bound to the S1 and S2 linkers by O-bond, the moiety excluding the hydroxyl group involved in the binding of the saccharide constituting the sugar ligand is a group derived from the saccharide. By sugar ligand is meant.
In the present invention, a sugar ligand to be a target cell of an oligonucleotide may be selected.
Examples of monosaccharides include allose, altose, arabinose, cladinose, erythrose, erythrulose, fructose, D-fusitol, L-fusitol, fucosamine, fucose, fucose, galactosamine, D-galactosaminitol, N-acetyl-galactosamine, Galactose, glucosamine, N-acetyl-glucosamine, glucosaminitol, glucose, glucose-6-phosphate, gulose, glyceraldehyde, L-glycero-D-manno-heptose, glycerol, glycerone, gulose, idoses, liquisose, mannosamine , Mannose, mannose-6-phosphate, psicose, quinobose, quinovosamine, rhamnitol, rhamnosamine, rhamnose, ribose, ribulose, sedoheptulose, sorbo Scan, tagatose, talose, tartaric acid, threose, and xylose, and xylulose and the like.
Examples of disaccharides, trisaccharides and polysaccharides include avequase, aclavose, amysetose, amylopectin, amylose, apiose, alkanose, alkanose, alkanose, alkanose, ascorbic acid, boibinose, cellobiose, cellotriose, cellulose, cacotriose, chalcose, chitin, colitos, Cyclodextrin, Dextrose, Dextrin, 2-Deoxyribose, 2-Deoxyglucose, Diginose, Digitulose, Digitose, Evalose, Evemitrose, Fructooligosaccharide, Galto-oligosaccharide (galto-oligosaccharide), Gentianose, Gentiobiose, Glucan, Glucogen, Glycogen , Hammellose, heparin, inulin, isolevoglucosenone, isoma Tose, Isomaltotriose, Isopanose, Codibiose, Lactose, Lactosamine, Lactosamine, Laminarabiose, Levoglucosan, Levoglucosenone, Beta-Maltose, Martriose, Mannan-Oligosaccharide, Manninotriose, Meleticose, Melibiose, Muramic Acid, Micarose, Misinose , Neuraminic acid, sialic acid-containing sugar chain, nigerose, nojirimycin, nobiose, oleandrosose, panose, paratose, plumoseose, primosevelose, raffinose, rhainose, rutinose, salmentose, sedoheptulose, sedohepturosan, solatriose, sophorose, stachyose, streptose , Α, α-Trehalose, trahalosamine, turanose, tivelose, xylobio Vinegar, umbelliferose, and the like.

Each monosaccharide in the saccharide may be in D-form or L-form, or may be a mixture of D-form and L-form in any proportion.
The saccharides may be deoxy sugars (alcohol hydroxy group substituted with hydrogen atom), amino sugars (alcohol hydroxy group substituted with amino group), thio sugars (alcohol hydroxy group substituted with thiol), or C = O Is substituted by C = S, or ring oxygen is substituted by sulfur), seleno sugar, telluro sugar, aza sugar (ring carbon is substituted by nitrogen), imino sugar (ring oxygen is substituted by nitrogen) ), Phosphanosugars (in which ring oxygen is substituted by phosphorus), phosphasaccharides (in which ring carbon is substituted by phosphorus), C-substituted monosaccharides (in which hydrogen atoms at non-terminal carbon atoms are substituted by carbon atoms), Unsaturated monosaccharide, alditol (the carbonyl group is substituted by CHOH group), aldonic acid (the aldehyde group is substituted by carboxy group), ketoaldonic acid, uronic acid, Etc. may contain an Dal acid.
As an amino sugar in saccharides, galactosamine, glucosamine, glucosamine, mannosamine, fucosamine, quinosamine, neuraminic acid, muramic acid, lactose acid, lactose diamine, acosamine, dacrosamine, daunosamine, desosamine, forosamine, garosamine, canosamine, kansosamine (kansamine) Mikaminose, mycosamine, perosamine, pneuimosamine, purpurosamine, rhodosamine etc. are mentioned. In addition, the amino group of the amino sugar may be substituted with an acetyl group or the like.
Examples of sialic acid-containing sugar chains include sugar chains containing NeuAc at the non-reducing end of the sugar chain, such as NeuAc-Gal-GlcNAc-containing sugar chains, Neu5Acα (2-6) Galβ (1-3) GlcNAc, etc. Can be mentioned.
Each monosaccharide in the saccharide may be substituted by a substituent, as long as it can bind to the receptor expressed in the target cell, for example, a hydroxyl group may be substituted, and each monosaccharide The hydrogen atom in may be substituted one or more with an azide and / or an aryl group which may be substituted.

As a sugar ligand, it is preferable to select a sugar ligand that binds to a receptor expressed on the surface of a target cell corresponding to each target organ, for example, when the target cell is a hepatocyte, on the surface of a hepatocyte Sugar ligands to the expressed receptor are preferred, and sugar ligands to the asialoglycoprotein receptor (ASGPR) are more preferred.

As a sugar ligand for ASGPR, mannose or N-acetylgalactosamine is preferable, and N-acetylgalactosamine is more preferable.
As sugar ligands having higher affinity to ASGPR, for example, sugar derivatives described in Bioorganic Medicinal Chemistry, 17, 7254 (2009), and Journal of American Chemical Society, 134, 1978 (2012), etc. are known. These may be used.

In the present invention, S1, S2 and S3 are linkers.
S1 and S2 are not particularly limited as long as they are structures that link the sugar ligands L1 and L2 with the benzene ring, and a known structure used in a nucleic acid complex may be adopted. S1 and S2 may be the same or different.
The sugar ligands L1 and L2 are preferably linked to S1 and S2 by glycosidic bonds, and S1 and S2 may be combined with a benzene ring, for example, -CO-, -NH-, -O-, -S- And -O-CO-, -S-CO-, -NH-CO-, -CO-O-, -CO-S-, or -CO-NH- bond.

S3 is not particularly limited as long as it is a structure linking X, which is a double-stranded nucleic acid, and a benzene ring, and a known structure used in a nucleic acid complex may be adopted.
The oligonucleotide X is preferably linked to S3 by a phosphodiester bond, and S3 is linked to a benzene ring, for example, -CO-, -NH-, -O-, -S-, -O-CO. -, -S-CO-, -NH-CO-, -CO-O-, -CO-S- or -CO-NH- bond may be linked.

As linkers of S1, S2 and S3, for example, WO 2009/073809, WO 2013/075035, WO 2015/105083, WO 2014/179620, WO 2015/006740 The structure disclosed in the above may be adopted.

In the present invention, the nucleic acid complex is preferably a nucleic acid complex having a structure represented by the following formula 2.
Formula 2:

In formula 2,
X, L1, L2 and S3 are as defined above,
P1, P2, P3, P4, P5 and P6, and T1 and T2 are each independently absent, or -CO-, -NH-, -O-, -S-, -O-CO- , -S-CO-, -NH-CO-, -CO-O-, -CO-S- or -CO-NH-,
Q1, Q2, Q3 and Q4 are each independently absent, or substituted or unsubstituted alkylene having 1 to 12 carbon atoms or-(CH ₂ CH ₂ O) _n -CH ₂ CH _2- , N is an integer of 0 to 99.

P1 and P4 are each independently absent, or -CO-, -NH-, -O-, -S-, -O-CO-, -S-CO-, -NH-CO-, -CO-O-, -CO-S- or -CO-NH-, but preferably -O-, -O-CO-, -NH-CO- or -CO-NH-, -O -, -NH-CO- or -CO-NH- is more preferable, and -NH-CO- is more preferable.
When P1 or P4 is, for example, -NH-CO-, it has a partial structure of -NH-CO-benzene ring.

Q1, Q2, Q3 and Q4 are each independently absent, or substituted or unsubstituted alkylene having 1 to 12 carbon atoms or-(CH ₂ CH ₂ O) _n -CH ₂ CH _2- And n is an integer of 0 to 99, preferably substituted or unsubstituted alkylene having 1 to 12 carbon atoms, more preferably unsubstituted alkylene having 1 to 12 carbon atoms, and unsubstituted More preferably, it is an alkylene having 1 to 6 carbon atoms, and still more preferably an unsubstituted alkylene having 1 to 4 carbon atoms.

P2 and P5 are each independently absent, or -CO-, -NH-, -O-, -S-, -O-CO-, -S-CO-, -NH-CO-, -CO-O-, -CO-S- or -CO-NH-, but not present, preferably -CO-O- or -CO-NH-, preferably absent, -CO-NH It is more preferable that it is-. When P2 and P5 are, for example, -CO-NH-, they have partial structures of B1-CO-NH-Q1 and B2-CO-NH-Q3.

-[P2-Q1] _q1 - _and- [P5-Q3] _q3 -each independently do not exist or have any of the structures represented by the following formulas 3-1 to 3-3 It is suitable.
Formula 3-1:

Formula 3-2:

Formula 3-3:

In formulas 3-1 to 3-3,
m5 and m6 are each independently an integer of 0 to 10, and the terminal black dot in the structure of Formula 3-1 to Formula 3-3 is a bonding point to B1 or B2 or P1 or P4, respectively is there.

B1 and B2 are each independently a bond or a structure represented by the following formula, and the terminal black dot in each structure is P2 or P3 or P5 or P6, respectively. And m1, m2, m3 and m4 are each independently an integer of 0 to 10.

B1 and B2 are preferably groups derived from amino acids including non-natural amino acids such as glutamic acid, aspartic acid, lysine and iminodiacetic acid, or amino alcohols such as 2-amino-1,3-propanediol, And when B2 is a group derived from glutamic acid and aspartic acid, it is preferable that the amino groups of glutamic acid and aspartic acid are respectively bonded to form -NH-CO- as P2 and P5, and B1 and B2 are lysine When it is a group derived from, it is preferable that the carboxyl group of lysine is respectively bonded to form -CO-NH- bond as P2 and P5, and when B1 and B2 are groups derived from iminodiacetic acid, The amino group of iminodiacetic acid is bonded to form -CO- bonded as P2 and P5, respectively. It is preferable to be. Specifically, B1 and B2 preferably have the following structures.

When p1 and p2 are integers of 2 or 3, respectively, P3 and P6, Q2 and Q4, T1 and T2 and L1 and L2 may be the same or different.
When q1 to q4 are 2 to 10, the combinations of-[P2-Q1]-,-[Q2-P3]-,-[P5-Q3]-and-[Q4-P6]-are the same or different. May be The combinations of-[P2-Q1]-,-[Q2-P3]-,-[P5-Q3]-, and-[Q4-P6]-are identical or different from each other as-[P2-Q1]- It means that each 2 to 10 unit represented by,-[Q2-P3]-,-[P5-Q3]-, and-[Q4-P6]-may be identical or different.

In the present invention, the nucleic acid complex is preferably a nucleic acid complex having a structure represented by the following formulas 4-1 to 4-9.
Formula 4-1:

Equation 4-2:

Equation 4-3:

Formula 4-4:

Formula 4-5:

Formula 4-6:

Equation 4-7:

Formula 4-8:

Formula 4-9:

In formulas 4-1 to 4-9,
X, L1, L2, S3, P3, P6, T1, T2, Q2, Q4, q2 and q4 are as defined above.

P3 and P6 each independently do not exist, or -CO-, -NH-, -O-, -S-, -O-CO-, -S-CO-, -NH-CO-, -CO-O-, -CO-S- or -CO-NH-, but preferably -O-CO- or -NH-CO-, and more preferably -NH-CO-. When P3 and P6 are, for example, -NH-CO-, they have partial structures of B1-NH-CO-Q2 and B2-NH-CO-Q4, respectively.

T1 and T2 are each independently absent, or -CO-, -NH-, -O-, -S-, -O-CO-, -S-CO-, -NH-CO-, -CO-O-, -CO-S- or -CO-NH- is preferably -O- or -S-, more preferably -O-.

In the present invention, the nucleic acid complex is preferably a nucleic acid complex having a structure represented by the following formula 5.
In Equation 5, P1 and P4, P2 and P5, P3 and P6, Q1 and Q3, Q2 and Q4, B1 and B2, T1 and T2, L1 and L2, p1 and p2, q1 and q3, and q2 and q4 in Equation 2. Are the same.
Formula 5:

In the equation 5,
X, S3, P1, P2, P3, Q1, Q2, B1, T1, L1, p1, q1 and q2 are as defined above.
Further, X, S3, P1, P2, P3, Q1, Q2, B1, T1, L1, p1, q1 and q2 in Formula 5 may be the above-mentioned suitable groups, but P1 is —CO— It is preferable that it is NH-, -NH-CO- or -O-.
It is preferable that — (P 2 _−Q 1) _q 1 − in the formula 5 is absent or has any structure represented by the above formulas 3-1 to 3-3.

In the present invention, the nucleic acid complex is preferably a nucleic acid complex having a structure represented by the following formulas 6-1 to 6-9.
Formula 6-1:

Formula 6-2:

Equation 6-3:

Equation 6-4:

Formula 6-5:

Formula 6-6:

Formula 6-7:

Formula 6-8:

Formula 6-9:

In Formula 6-1 to Formula 6-9,
X, S3, P3, Q2, T1 and L1 are as defined above.

In the present invention, the nucleic acid complex is preferably a nucleic acid complex having a structure represented by any one of the following formulas 7-1 to 7-9.
Formula 7-1:

Equation 7-2:

Formula 7-3:

Equation 7-4:

Formula 7-5:

Formula 7-6:

Formula 7-7:

Formula 7-8:

Formula 7-9:

In formulas 7-1 to 7-9,
X, L1, L2 and S3 are as defined above. L1 and L2 may be identical or different, and are preferably identical.
In formulas 7-1 to 7-9, each alkylene group moiety is introduced with an alkylene chain having a different chain length, or an amide bond or the like is substituted with another bond to form formulas 7-1 to 7 Nucleic acid derivatives other than the nucleic acid complex having the structure represented by -9 can also be produced.

In the present invention, the nucleic acid complex is preferably a nucleic acid complex having a structure represented by the following formula 11.
Formula 11:

In formula 11,
L1, L2, S1 and S2 are each as defined above,
P7 and P8 each independently do not exist, or -CO-, -NH-, -O-, -S-, -O-CO-, -S-CO-, -NH-CO-, -CO-O-, -CO-S- or -CO-NH-,
Q5, Q6 and Q7 are each independently absent or alkylene substituted or unsubstituted C 1-12 or _{_{_{- (CH 2 CH 2 O)}}} n8 -CH 2 CH 2 - and is, n8 Is an integer from 0 to 99,
B3 is herein referred to as a blanker unit and is any structure represented by the following formula 11-1 and means a bond with Q5 and Q6, respectively, in a broken line:
Formula 11-1:

In Formula 11-1, substitution at a group having a triazole ring is any of the nitrogen atoms at positions 1 and 3 of the triazole ring.
q5 and q6 are each independently an integer of 0 to 10.

P7 is absent, or -CO-, -NH-, -O-, -S-, -O-CO-, -S-CO-, -NH-CO-, -CO-O-,- CO-S- or -CO-NH-, preferably -O-, -NH-CO- or -CO-NH-, more preferably -O- or -NH-CO- . . When P7 is, for example, -O-, it has a partial structure of a benzene ring -O-.

P8 is absent, or -CO-, -NH-, -O-, -S-, -O-CO-, -S-CO-, -NH-CO-, -CO-O-,- CO—S— or —CO—NH—, when present, is preferably —CO—O— or —CO—NH—, more preferably —CO—NH—. When P8 is, for example, -CO-NH-, it has a partial structure of Q6-CO-NH-.

Q5, Q6 and Q7 are each independently absent or alkylene substituted or unsubstituted C 1-12 or _{_{_{- (CH 2 CH 2 O)}}} n8 -CH 2 CH 2 - and is, n8 Is an integer of 0 to 99, preferably substituted or unsubstituted alkylene having 1 to 12 carbon atoms, more preferably unsubstituted alkylene having 1 to 12 carbon atoms, and unsubstituted unsubstituted carbon atoms An alkylene of 1 to 6 is more preferable, and an unsubstituted alkylene of 1 to 4 carbon atoms is still more preferable.

- _(P7-Q5) q5 - _is, -O- _{(CH 2)} m15 -NH- and _-NH-CO- _{(CH 2)} a m16 -NH, m15 and m16 are each independently 1 to 10 It is preferable that it is an integer of

In the present invention, the nucleic acid complex is preferably a nucleic acid complex having a structure represented by any one of the following formulas 12-1 to 12-12.
Formula 12-1:

Formula 12-2:

Formula 12-3:

Formula 12-4:

Formula 12-5:

Formula 12-6:

Formula 12-7:

Formula 12-8:

Formula 12-9:

Formula 12-10:

Formula 12-11:

Formula 12-12:

In formulas 12-1 to 12-12,
X, L1, L2, S1 and S2 are as defined above, and n1 'to n12' are each independently an integer of 1 to 10.

The nucleic acid complex of the present invention is a nucleic acid complex obtained by combining the structure described in formula 2 corresponding to S1 and S2 with the structure described in formula 11 corresponding to S3 in the nucleic acid complex represented by formula 1 Is preferred. Formula 2 may be Formula 4-1 to Formula 4-9, Formula 6-1 to Formula 6-9, Formula 7-1 to Formula 7-9, and Formula When 2 is Expression 4-1 to Expression 4-9, Expression 6-1 to Expression 6-9, or Expression 7-1 to Expression 7-9, Expression 11 has Expression 12-1 to Expression 12-. It may be twelve. The nucleic acid complex of the present invention is a nucleic acid complex represented by formula 1, which has a structure according to any one of formulas 4-1 to 4-9 corresponding to S1 and S2, and a formula 12 corresponding to S3. A nucleic acid complex combining any one of the structures described in any one of the structures 1 to 12-12, any one of the structures described in the formulas 6-1 to 6-9 corresponding to S1 and S2, and A nucleic acid complex combining any one of the structures described in the corresponding formula 12-1 to the formula 12-12, any one of the structures described in the formula 7-1 to the formula 7-9 corresponding to S1 and S2 More preferably, it is a nucleic acid complex obtained by combining any one of the structures described in formulas 12-1 to 12-12 corresponding to S3.

The nucleic acid complex of the present invention is preferably represented by the following formula 7-8-1.
Formula 7-8-1:

(In formula 7-8-1, X is as defined above.)

X in Formula 1 is a double-stranded nucleic acid consisting of a sense strand and an antisense strand and containing a double-stranded region of at least 11 base pairs, said double-stranded nucleic acid being in the antisense strand 17 An oligonucleotide chain of a chain length of 30 to 30 nucleotides is complementary to any of the target β2GPI mRNA sequences listed in Table 1-1 to Table 1-16. Since the 3 'end or the 5' end of the sense strand is linked to S3, in the formula 1, X binding to S3 is a sense strand constituting a double-stranded nucleic acid, which will be described later in Table 1-1 to Table 1- 16 or the sense strand represented by the sense strand sequence in Table M1-1 to Table M1-18.

In the present invention, a nucleic acid containing a base sequence complementary to β2GPI mRNA is referred to as antisense strand nucleic acid, and a nucleic acid containing a base sequence complementary to the base sequence of antisense strand nucleic acid is also referred to as sense strand nucleic acid.

The double-stranded nucleic acid constituting the nucleic acid complex used in the present invention is a double-stranded nucleic acid having the ability to reduce or stop the expression of the β2GPI gene when introduced into mammalian cells, which comprises a sense strand and an anti It is a double stranded nucleic acid having a sense strand. In addition, the sense strand and the antisense strand have at least 11 base pairs, and at least 17 nucleotides and at most 30 nucleotides, ie, 17 to 30 nucleotides in the antisense strand. An oligonucleotide strand of the following length is complementary to a target β2GPI mRNA sequence selected from the group described in Tables 1-1 to 1-16.

The double-stranded nucleic acid constituting the nucleic acid complex used in the present invention may be any molecule obtained by polymerizing a nucleotide or a molecule having the same function as the nucleotide, for example, a polymer of ribonucleotide And RNA which is a polymer of deoxyribonucleotide, a chimeric nucleic acid consisting of RNA and DNA, and a nucleotide polymer in which at least one nucleotide of these nucleic acids is substituted with a molecule having the same function as the nucleotide. Be In addition, derivatives containing at least one molecule having a function equivalent to a nucleotide in these nucleic acids are also included in the double stranded nucleic acid as a drug used in the present invention. Moreover, uracil (U) can be read unambiguously as thymine (T).

Examples of molecules having the same function as nucleotides include nucleotide derivatives and the like. The nucleotide derivative may be any molecule as long as the nucleotide is modified. For example, in order to improve or stabilize nuclease resistance as compared to RNA or DNA, the affinity to the complementary strand nucleic acid is selected. In order to increase, to increase cell permeability, or to visualize, a ribonucleotide or a deoxyribonucleotide modified molecule is preferably used.

Examples of molecules in which nucleotides have been modified include sugar-modified nucleotides, phosphodiester bond-modified nucleotides, base-modified nucleotides, and nucleotides in which at least one of a sugar moiety, a phosphodiester bond and a base has been modified.

The sugar moiety-modified nucleotide may be any one in which part or all of the chemical structure of the nucleotide sugar is modified or substituted with an arbitrary substituent or substituted with an arbitrary atom, but '-Modified nucleotides are preferably used.

The 2'-modified nucleotides, such as 2'-OH group of the ribose is H, OR, R, R'OR, SH, SR, NH 2, NHR, NR 2, N 3, CN, F, Cl, Br and Substituted with a substituent selected from the group consisting of I, wherein R is alkyl or aryl, preferably alkyl of 1 to 6 carbons, and R ′ is alkylene, preferably alkylene of 1 to 6 carbons It is a nucleotide, more preferably a nucleotide in which the 2'-OH group is substituted with H, F or a methoxy group, still more preferably a nucleotide in which the 2'-OH group is substituted with F or a methoxy group. In addition, 2′-OH group is 2- (methoxy) ethoxy group, 3-aminopropoxy group, 2-[(N, N-dimethylamino) oxy] ethoxy group, 3- (N, N-dimethylamino) propoxy group, 2- A substituent selected from the group consisting of [2- (N, N-dimethylamino) ethoxy] ethoxy group, 2- (methylamino) -2-oxoethoxy group, 2- (N-methylcarbamoyl) etoxy group and 2-cyanoetoxy group Also included are substituted nucleotides and the like.
The content of 2'-modified nucleotides is preferably 50 to 100%, more preferably 70 to 100%, still more preferably 90 to 100% with respect to nucleotides in the double stranded nucleic acid region. . The content of 2′-modified nucleotides is preferably 20 to 100%, more preferably 40 to 100%, still more preferably 60 to 100%, based on the nucleotides of the sense strand. The 2'-modified nucleotide is preferably contained in an amount of 20 to 100%, more preferably 40 to 100%, still more preferably 60 to 100%, with respect to the nucleotides of the antisense strand.

As a phosphodiester bond modified nucleotide, any one of those obtained by modifying or substituting a part or all of the chemical structure of phosphodiester bond of the nucleotide with an arbitrary substituent, or substituting with an arbitrary atom For example, a nucleotide in which a phosphodiester bond is substituted with a phosphorothioate bond, a nucleotide in which a phosphodiester bond is substituted with a phosphorodithioate bond, a nucleotide in which a phosphodiester bond is substituted with an alkyl phosphonate bond, phosphoric acid The nucleotide etc. by which the diester bond was substituted by the phosphoroamidate bond etc. are mentioned.

As the base-modified nucleotide, any part or all of the chemical structure of the base of the nucleotide may be modified or substituted with any substituent, or any one substituted with any atom, for example, a base In which the oxygen atom is substituted by a sulfur atom, the hydrogen atom is substituted by an alkyl group having 1 to 6 carbon atoms, halogen or the like, the methyl group is hydrogen, hydroxymethyl, an alkyl group having 2 to 6 carbon atoms, etc. And those in which the amino group is substituted by an alkyl group having 1 to 6 carbon atoms, an alkanoyl group having 1 to 6 carbon atoms, an oxo group, a hydroxy group or the like.

As a nucleotide derivative, a nucleotide or a nucleotide derivative in which at least one of sugar moiety, phosphodiester bond or base is modified, peptide, protein, sugar, lipid, phospholipid, phenazine, folate, phenanthridine, anthraquinone, acridine, Fluorescein, rhodamine, coumarin, dyes, etc., which are added with another chemical substance directly or through a linker, can also be mentioned, and specifically, 5'-polyamine adduct nucleotide derivative, cholesterol adduct nucleotide derivative, steroid adduct nucleotide derivative And bile acid-added nucleotide derivatives, vitamin-added nucleotide derivatives, Cy5-added nucleotide derivatives, Cy3-added nucleotide derivatives, 6-FAM-added nucleotide derivatives, biotin-added nucleotide derivatives, and the like.
The nucleotide derivative may form a cross-linked structure, such as an alkylene structure, a peptide structure, a nucleotide structure, an ether structure, an ester structure, and a structure combining at least one of these with other nucleotides or nucleotide derivatives in the nucleic acid.

In the present specification, "complementary" means a relationship capable of base pairing between two bases, for example, a loose hydrogen such as a relationship between adenine and thymine or uracil, and a relationship between guanine and cytosine. It refers to one that takes on a double helical structure as a whole double stranded region via a bond.

In the present specification, "complementary" means not only perfect complementation of two nucleotide sequences but also having 0 to 30%, 0 to 20% or 0 to 10% of mismatched bases between the nucleotide sequences. For example, an antisense strand complementary to β2GPI mRNA means that one or more bases may be substituted in the base sequence completely complementary to the partial base sequence of the mRNA. Specifically, the antisense strand is 1 to 8, preferably 1 to 6, preferably 1 to 4, 1 to 3, particularly 2 or 1 mismatched bases relative to the target sequence of the target gene. You may have. For example, when the antisense strand is 21 bases long, it may have 6, 5, 4, 3, 2, 2 or 1 mismatched bases with respect to the target sequence of the target gene, The position of the mismatch may be at the 5 'end or 3' end of each sequence.
In addition, “complementary” includes a case where one nucleotide sequence is a sequence in which one or more bases are added and / or deleted in a base sequence completely complementary to the other nucleotide sequence. For example, β2GPI mRNA and the antisense strand nucleic acid of the present invention have one or two bulge bases in the antisense strand and / or target β2GPI mRNA region by addition and / or deletion of bases in the antisense strand. You may

The double-stranded nucleic acid as a drug used in the present invention is a nucleic acid containing a base sequence complementary to a part of the base sequence of β2GPI mRNA and / or a base sequence complementary to the base sequence of the nucleic acid As long as it contains the nucleic acid, it may be composed of any nucleotide or its derivative. In the double-stranded nucleic acid of the present invention, a nucleic acid containing a base sequence complementary to a target β2GPI mRNA sequence and a nucleic acid containing a base sequence complementary to the base sequence of the nucleic acid have at least 11 bases. Although any length may be used as long as it can form paired duplexes, the length of the sequence capable of forming duplexes is usually 11 to 27 bases, preferably 15 to 25 bases, and 17 to 23 bases. Are more preferred, and 19 to 23 bases are even more preferred.

As the antisense strand of the nucleic acid complex of the present invention, a nucleic acid containing a base sequence complementary to a target β2GPI mRNA sequence is used, and among the nucleic acids, 1 to 3 bases, preferably 1 to 2 bases, Preferably, one in which one base is deleted, substituted or added may be used.

The nucleic acid which suppresses the expression of β2GPI is a nucleic acid containing a base sequence complementary to the target β2GPI mRNA sequence, and is a single-stranded nucleic acid which suppresses the expression of β2GPI, or is complementary to the target β2GPI mRNA sequence A double-stranded nucleic acid consisting of a nucleic acid containing a specific base sequence and a nucleic acid containing a base sequence complementary to the base sequence of the nucleic acid, and suppressing expression of β2GPI is preferably used.

The single-stranded antisense strand nucleic acid and the single-stranded antisense strand nucleic acid constituting the double-stranded nucleic acid may be identical or different, usually 11 to 30 bases, but identical or different, 17 to 27 bases Preferably, it consists of 17-25 bases, more preferably consists of 19-25 bases, still more preferably consists of 21-23 bases.

In the double-stranded nucleic acid as a drug used in the present invention, the double-stranded region, if having an additional nucleotide or nucleotide derivative which does not form a double strand at the 3 'or 5' side following the double-stranded region, the overhang Call it (overhang). In the case of having an overhang, the nucleotides constituting the overhang may be ribonucleotides, deoxyribonucleotides or derivatives thereof.

As a double-stranded nucleic acid having an overhang, one having an overhang consisting of 1 to 6 bases, usually 1 to 3 bases, at the 3 'end or 5' end of at least one strand is used, but from 2 bases Those having a protruding portion are preferably used, for example, those having a protruding portion consisting of dTdT (dT represents deoxythymidine) or UU (U represents uridine). The overhang can be present only in the antisense strand, only in the sense strand, and in both the antisense and sense strands, but in the present invention, a double-stranded nucleic acid having an overhang in the antisense strand is preferably used. The antisense strand is selected from the group described in Tables 1-1 to 1-16 in an oligonucleotide strand consisting of 17 to 30 nucleotides, which comprises a double-stranded region followed by an overhang. It is sufficiently complementary to the target β2GPI mRNA sequence to be Furthermore, as the double-stranded nucleic acid of the present invention, for example, a nucleic acid molecule (WO 2005/089287) that produces a double-stranded nucleic acid by the action of ribonuclease such as Dicer or the like, or a protrusion at the 3 'end or the 5' end A double stranded nucleic acid which forms a blunt end, a double stranded nucleic acid (US2012 / 0040459) etc. which only the sense strand protruded can also be used.

As the double-stranded nucleic acid constituting the nucleic acid complex used in the present invention, a nucleic acid consisting of the same sequence as the base sequence of the target gene or the base sequence of its complementary strand may be used. A double-stranded nucleic acid consisting of a nucleic acid in which the 5 ′ end or 3 ′ end of the strand has been deleted by 1 to 4 bases and a nucleic acid containing a base sequence complementary to the base sequence of the nucleic acid may be used.

The double-stranded nucleic acid constituting the nucleic acid complex used in the present invention is a double-stranded RNA (dsRNA) in which the RNAs form a duplex, and a double-stranded DNA (dsDNA) in which the DNAs form a duplex. Or a hybrid nucleic acid in which RNA and DNA form a duplex. Alternatively, one or both strands of the double strand may be a chimeric nucleic acid of DNA and RNA. Preferably, it is a double stranded RNA (dsRNA).

The second nucleotide from the 5 'end of the antisense strand of the nucleic acid complex of the present invention is preferably complementary to the second deoxyribonucleotide from the 3' end of the target β2GPI mRNA sequence, and the 5 'end of the antisense strand More preferably, the 2nd to 7th nucleotides are completely complementary to the 2nd to 7th deoxyribonucleotides from the 3 'end of the target β2 GPI mRNA sequence, and the 2nd to 11th nucleotides from the 5' end of the antisense strand It is further preferred that is completely complementary to the 2nd to 11th deoxyribonucleotides from the 3 'end of the target β2GPI mRNA sequence. In addition, it is preferable that the 11th nucleotide from the 5 'end of the antisense strand in the nucleic acid of the present invention is complementary to the 11th deoxyribonucleotide from the 3' end of the target β2GPI mRNA sequence, and the 5 'end of the antisense strand It is more preferable that the 9th to 13th nucleotides are completely complementary to the 9th to 13th deoxyribonucleotides from the 3 'end of the target β2GPI mRNA sequence, and the 7th to 15th nucleotides from the 5' end of the antisense strand More preferably, it is completely complementary to the 7th to 15th deoxyribonucleotides from the 3 'end of the target β2GPI mRNA sequence.

The antisense and sense strands of the nucleic acid complex of the present invention are based on, for example, the nucleotide sequence (SEQ ID NO: 3541) of the cDNA (sense strand) of full-length mRNA of human β2GPI registered as Genbank Accession No. NM — 000042 It can be designed.

Double stranded nucleic acids can be designed to interact with target sequences within the β2 GPI gene sequence.

The sequence of one strand of double stranded nucleic acid is complementary to the target site sequence described above. Double stranded nucleic acids can be chemically synthesized using the methods described herein.

The RNA may be produced enzymatically or by partial / total organic synthesis, and modified ribonucleotides can be introduced in vitro by enzymatic or organic synthesis. In one embodiment, each strand is chemically prepared. Methods for chemically synthesizing RNA molecules are known in the art [see Nucleic Acids Research, 1998, Volume 32, p. 936-948]. In general, double stranded nucleic acids can be synthesized by using solid phase oligonucleotide synthesis methods (e.g., Usman et al., U.S. Patent 5,804,683; U.S. Patent 5,831,071; U.S. Patent No. 5,998,203; U.S. Patent No. 6,117,657; U.S. Patent No. 6,353,098; U.S. Patent No. 6,362,323; U.S. Patent No. 6,437,117; U.S. Patent No. 6,469,158; Scaringe et al. U.S. Patent No. 6,111,086; U.S. Patent No. 6,008,400; U.S. Patent No. 6,111,086).

Single-stranded nucleic acids are synthesized, deprotected, and prepared using the solid phase phosphoramidite method (see Nucleic Acids Research, 1993, Vol. 30, p. 2435-2443). Desalted on a NAP-5 column (Amersham Pharmacia Biotech, Piscataway, NJ). Oligomers are Amersham Source 15Q columns using a 15 minute linear gradient-1.0 cm. Purified using ion exchange high performance liquid chromatography (IE-HPLC) at .25 cm height (Amersham Pharmacia Biotech, Piscataway, NJ). The gradient changes from 90: 10 buffer A: B to 52: 48 buffer A: B, buffer A is 100 mmol / L Tris pH 8.5, and buffer B is 100 mmol / L Tris pH It is 8.5 (1 mol / L NaCl). Samples are monitored at 260 nm and peaks corresponding to full-length oligonucleotide species are collected, pooled, desalted on a NAP-5 column, and lyophilized.

The purity of each single stranded nucleic acid is determined by capillary electrophoresis (CE) on a Beckman PACE 5000 (Beckman Coulter, Inc., Fullerton, Calif.). The CE capillary has an internal diameter of 100 μm and contains ssDNA 100R Gel (Beckman-Coulter). Typically, about 0.6 nmole of oligonucleotide is injected into the capillary and run at an electric field of 444 V / cm, detected by UV absorbance at 260 nm. Denatured Tris-Borate-7 mol / L-Urea running buffer is purchased from Beckman-Coulter. Single stranded nucleic acids that are at least 90% pure as assessed by CE are obtained for use in the experiments described below. Compound identity is determined using the matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass on Voyager DE.TM. Biospectrometry workstation (Applied Biosystems, Foster City, Calif.) According to the manufacturer's recommended protocol. Verified by spectroscopy. The relative molecular mass of single stranded nucleic acid can be obtained within 0.2% of the expected molecular mass.

Single stranded nucleic acid is resuspended at a concentration of 100 μmol / L in a buffer consisting of 100 mmol / L potassium acetate, 30 mmol / L HEPES, pH 7.5. The complementary sense and antisense strands are mixed in equal molar amounts to obtain a final solution of 50 μmol / L double stranded nucleic acid. The sample is heated to 95 ° C. for 5 minutes and allowed to cool to room temperature before use. Double stranded nucleic acids are stored at -20 ° C. Single stranded nucleic acids are lyophilized or stored at -80 ° C. in nuclease free water.

The sense strand and the antisense strand according to the present invention, which comprises a duplex region of at least 11 base pairs, in the antisense strand, an oligonucleotide of 11 to 30 nucleotides in length, 1-1 from the group consisting of the antisense strand described in Tables 1-1 to 1-16 as a double-stranded nucleic acid complementary to the target β2GPI mRNA sequence selected from the group described in 1-1 to Table 1-16 A double-stranded nucleic acid comprising the selected sequence, or a sequence selected from the group consisting of the sense strands described in Table M1-1 to Table M1-18, and Tables 1-1 to 1-16 described below Single-stranded nucleic acid or a pair of sense strands selected from the group consisting of sense strands / antisense strands described in Table M1-1 to Table M1-18 and Tables 1-1 to 1-16 described later Double stranded nucleic acids comprising the sequence of the antisense strand may be used. That is, specific examples of the double stranded nucleic acid constituting the nucleic acid complex used in the present invention are the sense strand and the anti in the table M1-1 to the table M1-18, and the table 1-1 to the table 1-16 described later. It is a double stranded nucleic acid consisting of a sense strand. In Tables M1-1 to M1-18, N (M) represents 2'-O-methyl-modified RNA, N (F) represents 2'-fluorine-modified RNA, and ^ represents phosphorothioate. In addition, the 5 'terminal nucleotide of the antisense strand sequences described in Tables 1-1 to 1-16 and Tables M1-1 to M1-18 may be phosphorylated at the 5' end, It does not have to be, but is preferably phosphorylated.
The double-stranded nucleic acid containing the sequence of the sense strand / antisense strand described in Tables 1-1 to 1-16 described later has a relative expression amount of β2GPI of 0.15 or less in the measurement of its knockdown activity desirable.

The method for producing the nucleic acid complex of the present invention will be described. In the preparation methods shown below, when the defined group changes under the conditions of the preparation method or is unsuitable for carrying out the preparation method, introduction and removal of protecting groups commonly used in synthetic organic chemistry Methods [eg, Protective Groups in Organic Synthesis, third edition, by T. Greene, John Wiley & Sons Inc. The target compound can be produced by using the method described in (1999), etc.]. Moreover, the order of reaction processes, such as substituent introduction, can also be changed as needed.
The nucleic acid polymer represented by Formula 1 can also be synthesized by solid phase synthesis.

The nucleic acid polymer represented by the formula 1 can be synthesized with reference to a synthesis method of a linker structure known as a nucleic acid complex.
The synthesis of the L1-benzene ring unit using S1 as a linker and the L2-benzene ring unit using S2 as a linker in the nucleic acid complex represented by formula 1 can be performed, for example, using the nucleic acid complex represented by formula 2 as an example. explain.
The L1-benzene ring unit and the L2-benzene ring unit in the nucleic acid complex represented by the formula 2 are linked by P1, P2, P3, P4, P5, and P6, and T1 and T2.
P1, P2, P3, P4, P5, and P6 and T1 and T2 of -CO-, -NH-, -O-, -S-, -O-CO-, -S-CO-, -NH-CO- , -CO-O-, -CO-S- or -CO-NH- bond, for example, 4th edition Experimental Chemistry Lecture 19 "Synthesis of organic compounds I" Maruzen (1992), 4th edition Experimental Chemistry Lecture 20 Based on the method of coupling reaction described in “Synthesis of organic compounds II”, Maruzen (1992), etc., synthesis is appropriately carried out by selecting appropriate raw materials for forming the structure represented by Formula 2. be able to.
In addition, the partial structure of the L1-benzene ring unit can be produced by combining a compound having Q1 as a partial structure and a compound having B1 as a partial structure sequentially from the benzene ring.
Separately synthesizing a compound having L1 and Q2 as a partial structure, and binding a compound having L1 and Q2 as a partial structure to a compound having a partial structure of an L1-benzene ring unit having a benzene ring and Q1 and B1 as a partial structure Thus, L 1 -benzene ring unit structure can be produced.
Similarly for L2-benzene ring unit, the partial structure of L2-benzene ring unit can be produced by sequentially binding a compound having Q3 as a partial structure and a compound having B2 as a partial structure sequentially from the benzene ring .
Separately synthesizing a compound having L2 and Q4 as a partial structure, and combining a compound having L2 and Q4 as a partial structure with a compound having a partial structure of L2-benzene ring unit having a benzene ring and Q3 and B2 as partial structures Thus, an L2-benzene ring unit structure can be produced.

As a compound having Q1 as a partial structure and a compound having Q3 as a partial structure, an alkylene having 1 to 10 carbon atoms or a — (CH ₂ CH ₂ O) _n —CH ₂ CH ₂ — terminal group is a hydroxyl group or a carboxyl group at both ends And compounds having an amino group and a thiol group.
The compound having B1 as a partial structure and the compound having B2 as a partial structure have one of the structures represented by the following formula 2-1, and a hydroxyl group and a carboxyl group are represented by the black dots at the end of each structure, respectively. And compounds having an amino group or a thiol group.
Formula 2-1:

Specific examples of the compound having B1 as a partial structure and the compound having B2 as a partial structure include glycol, glutamic acid, aspartic acid, lysine, Tris, iminodiacetic acid, 2-amino-1,3-propanediol and the like. Glutamic acid, aspartic acid, lysine and iminodiacetic acid are preferred. Specifically, B1 and B2 preferably have the following structures.

A compound having L1, Q2 and B1 as a partial structure may be synthesized and then coupled to a compound having Q1 and a benzene ring to produce an L1-benzene ring unit structure.
A compound having L2, Q4 and B2 as a partial structure may be synthesized and then combined with Q3 and a compound having a benzene ring to produce an L2-benzene ring unit structure.
In the present _{_{invention, [L1-T1- (Q2-}} P3) q2 -] p1 -B1- and substructure is _(P2-Q1) q1 a -P1-, [L2-T2- (Q3 -P6) q4 -] _The partial structure which is _p2- B2- (P5-Q3) _q3- P2- may be identical or different, but is preferably identical.

Examples of the unit corresponding to L1-T1-Q2 of the sugar ligand include L3-T1-Q2-COOH, L3-T1- (Q2-P3) _q2-1 -Q2-NH _{2 and the} like. Specifically, L3-O-alkylene-COOH having 1 to 12 carbon atoms, L-alkylene-CO-NH having 1 to 12 carbons, alkylene-NH ₂ having 2 to 12 carbons, etc. may be mentioned.
L3 is not particularly limited as long as it is a sugar ligand derivative that becomes L1 by deprotecting. The substituent of the sugar ligand is not particularly limited as long as it is a substituent generally used in the field of carbohydrate chemistry, but an Ac group is preferable.

Specifically, referring to the method described in the examples, the number of carbon atoms of the alkylene chain is appropriately increased or decreased, with reference to the method described in the Examples, and synthesis of L1-benzene ring unit using S1 as a linker and L2 benzene ring unit using S2 as a linker In addition, the terminal amino group and the terminal carboxyl group can be selected from -CO-, -NH-, -O-, -S-, -O-CO-, -S-CO-, -NH-CO-, -CO-O- The compound can be synthesized by using a compound converted into a group capable of forming a -CO-S- or -CO-NH- bond. In addition, mannose or N-acetylgalactosamine is exemplified in the examples also for L1 sugar ligands, but other sugar ligands can be used instead.

The synthesis of the X-benzene ring unit using the S3 as a linker in the nucleic acid complex represented by the formula 1 will be described, for example, by taking the nucleic acid complex represented by the formula 12 as an example.
The X-benzene ring unit in the nucleic acid complex represented by Formula 12 has a bond represented by P7 and P8 in addition to the bond of the oligonucleotide.
-CO-, -NH-, -O-, -S-, -O-CO-, -S-CO-, -NH-CO-, -CO-O-, -CO-S- or P7 and P8 -CO-NH- bond, for example, 4th edition Experimental chemistry course 19 "Synthesis of organic compounds I" Maruzen (1992), 4th edition experimental chemistry course 20 "Synthesis of organic compounds II", Maruzen (1992) The compound can be appropriately synthesized by selecting an appropriate raw material for forming the structure represented by Formula 12 with reference to the method of the binding reaction described in 1.).
In addition, the partial structure of the X-benzene ring unit can be produced by combining a compound having Q5 as a partial structure and a compound having B3 as a partial structure sequentially from the benzene ring.

A compound having X and Q7 as a partial structure or a compound having X and Q6 as a partial structure is separately synthesized, and a compound having X and Q7 as a partial structure or a compound having X and Q6 as a partial structure is a benzene ring and Q5 An X-benzene ring unit structure can be produced by combining with a compound having a partial structure of the X-benzene ring unit having a partial structure to construct a B3 portion.
Specifically, in the case of having an azido group at the end of a compound having a partial structure of a benzene ring and an X-benzene ring unit having a partial structure of Q5, for example, a terminal bonding functional as disclosed in the Examples. An X-benzene ring unit structure can be produced by reacting a formed oligonucleotide to cause cycloaddition to form a triazole ring to construct a B3 moiety.

As a compound having Q5 as a partial structure, a compound having Q6 as a partial structure, and a compound having Q7 as a partial structure, an alkylene having 1 to 10 carbon atoms _or- (CH ₂ CH ₂ O) _{n 8} -CH ₂ CH _2- The compound which has a hydroxyl group, a carboxyl group, an amino group, and a thiol group is mentioned at both ends.

The L1-benzene ring unit structure, the L2-benzene ring unit structure, and the X-benzene ring unit structure can be sequentially produced, but the L1-benzene ring unit structure and the L2-benzene ring unit structure are synthesized. Then, it is preferable to combine the X-benzene ring unit structure. In particular, X having an oligonucleotide moiety is preferably introduced into the compound near the final step of sugar ligand complex synthesis.

In the present invention, compounds represented by the following formulas 8 to 10 are obtained as synthesis intermediates.
Formula 8:

(In the equation 8,
R1 and R2 are each independently a hydrogen atom, t-butoxycarbonyl group (Boc group), benzyloxycarbonyl group (Z group), 9-fluorenylmethyloxycarbonyl group (Fmoc group), -CO-R4 Or -CO-B4-[(P9-Q8) _q7- T3-L3] _p3 ,
P9 and T3 are each independently absent, or -CO-, -NH-, -O-, -S-, -O-CO-, -S-CO-, -NH-CO-, -CO-O-, -CO-S- or -CO-NH-,
Q8 is absent or substituted or unsubstituted alkylene having 1 to 12 carbon atoms, or-(CH ₂ CH ₂ O) _{n 1} -CH ₂ CH _2- , and n 1 is an integer of 0 to 99,
B4 is each independently a bond or a structure represented by Formula 8-1 below, and the terminal black dot in each structure is a bond to a carbonyl group or P9, respectively And m7, m8, m9 and m10 are each independently an integer of 0 to 10,
Formula 8-1:

p3 is an integer of 1, 2 or 3 and
q7 is an integer of 0 to 10,
L3 is a sugar ligand,
Y is —O— (CH ₂ ) _{m 11} —NH— and —NH—CO— (CH ₂ ) _{m 12} —NH, and m 11 and m 12 are each independently an integer of 1 to 10,
R3 is a hydrogen atom, t-butoxycarbonyl group, benzyloxycarbonyl group, 9-fluorenylmethyloxycarbonyl _{_{_{group, -CO-R4, -CO- (CH}}} 2 CH 2 O) n2 -CH 2 CH 2 -N ₃ or -CO-Q9- _B5- (Q10-P10) _q8- X1, and n2 is an integer of 0 to 99,
P10 is absent, or -CO-, -NH-, -O-, -S-, -O-CO-, -S-CO-, -NH-CO-, -CO-O-,- CO-S- or -CO-NH-,
Q9 and Q10 are each independently absent, or substituted or unsubstituted alkylene having 1 to 12 carbon atoms _or- (CH ₂ CH ₂ O) _{n 3} -CH ₂ CH _2- , and n3 is 0 It is an integer of ~ 99,
B5 is any structure represented by the following formula 8-2, and in broken lines, means a bond with Q9 and Q10, respectively
Formula 8-2:

In Formula 8-2, substitution at a group having a triazole ring is any of the nitrogen atoms at positions 1 and 3 of the triazole ring.
q8 is an integer of 0 to 10,
X 1 is a hydrogen atom or a solid support,
R 4 is selected from the group consisting of t-butoxycarbonyl group, benzyloxycarbonyl group, amino group substituted or unsubstituted with 9-fluorenylmethyloxycarbonyl group, carboxy group, maleimide group, and aralkyloxycarbonyl group It is an alkyl group having 2 to 10 carbon atoms which is substituted by 1 or 2 substituents. )

In the present invention, a compound represented by the following formula 9 is obtained as a synthetic intermediate.
Formula 9:

(In the equation 9,
R5 and R6 each independently represent a hydrogen atom, t-butoxycarbonyl group, benzyloxycarbonyl group, 9-fluorenylmethyloxycarbonyl group, -CO-R4 ', or -CO-Q11- (P11-Q11) ') _Q9 -T4-L4,
P11 and T4 are each independently absent, or -CO-, -NH-, -O-, -S-, -O-CO-, -S-CO-, -NH-CO-, -CO-O-, -CO-S- or -CO-NH-,
Q11 and Q11 ′ are absent or substituted or unsubstituted alkylene having 1 to 12 carbon atoms _or- (CH ₂ CH ₂ O) _{n 4} -CH ₂ CH _2- , and n 4 is an integer of 0 to 99 And
q9 is an integer of 0 to 10,
L4 is a sugar ligand,
Y 'is -O- _(CH _{2) _m11' 'is} -NH, m11' -NH- and -NH-CO- _(CH _{2) m12} and m12 'are each independently an integer from 1 to 10 Yes,
R3 'is a hydrogen atom, t-butoxycarbonyl group, benzyloxycarbonyl group, 9-fluorenylmethyloxycarbonyl _{group, -CO-R4', - CO-} (CH 2 CH 2 O) n2 '-CH 2 CH ₂ -N ₃ or -CO-Q9'-B5 '-(Q10'-P10') _q8'- X1 ', and n2' is an integer of 0 to 99,
P10 'is absent, or -CO-, -NH-, -O-, -S-, -O-CO-, -S-CO-, -NH-CO-, -CO-O-, -CO-S- or -CO-NH-,
Q9 'and Q10' are each independently absent or alkylene substituted or unsubstituted 1 to 12 carbon atoms or _{_{_{- (CH 2 CH 2 O)}}} n3 '-CH 2 CH 2 - and is, n3 'is an integer of 0 to 99,
B5 'is any structure represented by the following formula 9-1 and means a bond with Q9' and Q10 ', respectively, in a broken line,
Formula 9-1:

In Formula 9-1, substitution at a group having a triazole ring is any of the nitrogen atoms at positions 1 and 3 of the triazole ring.
q8 'is an integer of 0 to 10,
X1 'is a hydrogen atom or a solid support,
R 4 ′ is selected from the group consisting of t-butoxycarbonyl group, benzyloxycarbonyl group, amino group substituted or unsubstituted with 9-fluorenylmethyloxycarbonyl group, carboxy group, maleimide group, and aralkyloxycarbonyl group An alkyl group of 2 to 10 carbon atoms substituted with one or two substituents. )

In the present invention, a compound represented by the following formula 10 is obtained as a synthetic intermediate.
Formula 10:

In formula 10,
R7 and R8 are each independently a hydroxy group, a t-butoxy group, a benzyloxy group, -NH-R10 or -NH-Q12- (P12-Q12 ') _q10- T4-L4,
P12 and T4 are each independently absent, or -CO-, -NH-, -O-, -S-, -O-CO-, -S-CO-, -NH-CO-, -CO-O-, -CO-S- or -CO-NH-,
Q12 and Q12 ′ are absent or substituted or unsubstituted alkylene having 1 to 12 carbon atoms or-(CH ₂ CH ₂ O) n ₂ -CH ₂ CH _2- , and n 2 is an integer of 0 to 99 And
L4 is a sugar ligand,
Y 2 is —O— (CH ₂ ) m 9 —NH— and —NH—CO— (CH ₂ ) m 10 —NH, and m 9 and m 10 are each independently an integer of 1 to 10,
q10 is an integer of 0 to 10,
R9 is hydrogen, t-butoxycarbonyl group, benzyloxycarbonyl group, 9-fluorenylmethyloxycarbonyl _{_{_{group, -CO-R10, -CO- (CH}}} 2 CH 2 O) n6 -CH 2 CH 2 -N ₃ or -CO-Q13-B6- (Q14-P13) _q11- X2, and n6 is an integer of 0 to 99,
P13 is absent, or -CO-, -NH-, -O-, -S-, -O-CO-, -S-CO-, -NH-CO-, -CO-O-,- CO-S- or -CO-NH-,
Q13 and Q14 are each independently absent, or substituted or unsubstituted alkylene having 1 to 12 carbon atoms _or- (CH ₂ CH ₂ O) _{n 7} -CH ₂ CH _2- , and n 7 is 0 It is an integer of ~ 99,
B6 is any structure represented by the following formula 10-1 and means a bond with Q13 and Q14, respectively, in a broken line,
Formula 10-1:

In Formula 10-1, substitution at a group having a triazole ring is any of the nitrogen atoms at positions 1 and 3 of the triazole ring.
q11 is an integer of 0 to 10,
X2 is a hydrogen atom or a solid support,
R 10 is selected from the group consisting of t-butoxycarbonyl group, benzyloxycarbonyl group, amino group substituted or unsubstituted with 9-fluorenylmethyloxycarbonyl group, carboxy group, maleimide group, and aralkyloxycarbonyl group It is an alkyl group having 2 to 10 carbon atoms which is substituted by 1 or 2 substituents. )

Hereinafter, the manufacturing method will be shown as an example in connection with the present invention. In the following description relating to Production Method 1 to Production Method 17, a compound represented by Formula 1 to Formula 12 in a nucleic acid derivative etc. of the present invention, wherein the same symbol is used as a symbol representing a group Although both are understood separately, the description of the groups described for Production Method 1 to Production Method 12 does not limit the present invention. In addition, with regard to the nucleic acid derivative in the present invention, X representing an oligonucleotide is described as —O—X in production methods 1 to 17.

Manufacturing method 1
The nucleic acid derivative in the present invention can be exemplified as a method for producing a compound having a partial structure represented by the formula (I ′).

(Wherein, P 1 represents a protecting group which can be deprotected by a base such as Fmoc, DMTr represents p, p′-dimethoxytrityl group, R represents a sugar ligand-tether unit, R ′ represents R in Each hydroxyl group of the sugar ligand is a group protected by a protecting group which can be deprotected by a base such as an acetyl group, Polymer is a solid phase carrier, and Q 'is -CO-.

Step 1
Compound (I-B) is a compound (IA) and p, p'-dimethoxytrityl chloride in a solvent such as pyridine, optionally in the presence of a cosolvent, at a temperature between 0 ° C. and 100 ° C. Can be produced by reacting for 5 minutes to 100 hours.
As a co-solvent, for example, methanol, ethanol, dichloromethane, chloroform, 1,2-dichloroethane, toluene, ethyl acetate, acetonitrile, diethyl ether, tetrahydrofuran, 1,2-dimethoxyethane, dioxane, N, N-dimethylformamide (DMF) And N, N-dimethylacetamide, N-methylpyrrolidone, pyridine, water and the like, and these may be used alone or in combination.

Step 2
Compound (IC) is reacted with Compound (IB) without solvent or in the presence of 1 to 1000 equivalents of a secondary amine in a solvent at a temperature between room temperature and 200 ° C. for 5 minutes to 100 hours. It can be manufactured by
As the solvent, for example, methanol, ethanol, dichloromethane, chloroform, 1,2-dichloroethane, toluene, ethyl acetate, acetonitrile, diethyl ether, tetrahydrofuran, 1,2-dimethoxyethane, dioxane, N, N-dimethylformamide (DMF), Examples thereof include N, N-dimethylacetamide, N-methylpyrrolidone, pyridine, water and the like, which may be used alone or in combination.
Examples of secondary amines include diethylamine, piperidine and the like.

Step 3
Compound (1-E) is a compound (IC) and compound (ID), 1 to 30 equivalents of a base, a condensing agent, and optionally 0.01 to 30 equivalents without solvent or in a solvent C. for 5 minutes to 100 hours at a temperature between room temperature and 200.degree. C. in the presence of an additive.
As the solvent, those exemplified in step 2 can be mentioned.
As a base, for example, cesium carbonate, potassium carbonate, potassium hydroxide, sodium hydroxide, sodium methoxide, potassium tert-butoxide, triethylamine, diisopropylethylamine, N-methylmorpholine, pyridine, 1,8-diazabicyclo [5.4. 0] -7-Undecene (DBU), N, N-dimethyl-4-aminopyridine (DMAP) and the like.
As the condensing agent, for example, 1,3-dicyclohexanecarbodiimide (DCC), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide · hydrochloride (EDC), carbonyldiimidazole, benzotriazol-1-yloxytris (Dimethylamino) phosphonium hexaflurophosphate, (benzotriazol-1-yloxy) tripyrrolidinophosphonium hexafluorophosphate, O- (7-azabenzotriazol-1-yl) -N, N, N ′, N '-Tetramethyluronium hexafluorophosphate (HATU), O- (benzotriazol-1-yl) -N, N, N', N'-tetramethyluronium hexafluorophosphate (HBTU), iodide 2 And -chloro-1-methylpyridinium and the like.
Examples of the additive include 1-hydroxybenzotriazole (HOBt), 4-dimethylaminopyridine (DMAP) and the like.
Compound (ID) can be obtained by a known method (see, for example, Journal of American Chemical Society, 136, 16958, (2014)) or a method analogous thereto.

Step 4
Compound (IF) is prepared by reacting Compound (IE) and succinic anhydride in a solvent in the presence of 1 to 30 equivalents of a base at a temperature between room temperature and 200 ° C. for 5 minutes to 100 hours. Can be manufactured by

As the solvent, those exemplified in step 2 can be mentioned.

As the base, those exemplified in Step 3 can be mentioned.

Step 5
The compound (IG) is obtained by using a compound (IF) and a terminally aminated solid phase carrier, 1 to 30 equivalents of a base, a condensing agent, and optionally 0. After reacting for 5 minutes to 100 hours at a temperature between room temperature and 200 ° C. in the presence of 01 to 30 equivalents of additive, for 5 minutes to 100 hours at a temperature between room temperature and 200 ° C. It can be produced by reaction.
As the solvent, those exemplified in step 2 can be mentioned.
Examples of the base, the condensing agent and the additive include those exemplified in Step 3.
Examples of the aminated solid phase carrier include long-chain alkylamine pore glass (LCAA-CPG) and the like, which can be obtained as commercial products.

Step 6
The nucleic acid complex having a sugar ligand-tether-brancher unit represented by the formula (I ′) is a compound (IG) after extending the corresponding nucleotide chain by a known oligonucleotide chemical synthesis method. It can be produced by removal from the solid phase, deprotection of the protective group and purification.

Examples of known oligonucleotide chemical synthesis methods include the phosphoroamidite method, the phosphorothioate method, the phosphotriester method, the CEM method (see Nucleic Acids Research, 35, 3287 (2007)), and the like, for example, ABI 3900 high. It can be synthesized by a throughput nucleic acid synthesizer (manufactured by Applied Biosystems).

Removal from solid phase, deprotection may be prepared by treatment with a base for 10 seconds to 72 hours after chemical synthesis of oligonucleotide in a solvent or in the absence of solvent, at a temperature between -80 ° C and 200 ° C. it can.

As the base, for example, ammonia, methylamine, dimethylamine, ethylamine, diethylamine, isopropylamine, diisopropylamine, piperidine, triethylamine, ethylenediamine, 1,8-diazabicyclo [5.4.0] -7-undecene (DBU), carbonate Potassium and the like can be mentioned.

Examples of the solvent include water, methanol, ethanol, THF and the like.

Purification of the oligonucleotide is possible by combining a C18 reverse phase column or an anion exchange column, preferably the two methods described above. The nucleic acid complex purity after purification is desirably 90% or more, preferably 95% or more.

In addition, in the above step 3, if necessary, the compound (ID) can be divided into two units, divided into two steps, and condensed with the compound (IC). Specifically, for example, when R-Q 'is R-NH-CO-Q4'-CO- (Q4' is substituted or unsubstituted alkylene having 1 to 12 carbon atoms), the compound in step 3 Ethyl ester of the compound obtained by condensing (IC) and CH ₃ CH ₂ -O-CO-Q 4 '-CO-OH (Q 4' is as defined above) in the same manner as in step 3. The compound is hydrolyzed with a base such as lithium hydroxide in a solvent such as ethanol or water, and then it is condensed with R′—NH ₂ (R ′ is as defined above) to give the desired compound. _{_{Incidentally, CH 3 CH 2 -O-CO}} -Q4'-CO-OH (Q4 ' is as defined above) and _R'-NH 2 (R' is as defined above), a known method (for example, Journal of American Chemical Society, 136, 16958 (2014)) or a method analogous thereto. Here, in Q 4 ′, the substituted or unsubstituted C 1 to C 12 alkylene substituent and alkylene moiety are as defined above.

Although the case where Q is -CO- is illustrated as an example, the same method as in the above, known methods or these are also applied to compounds where Q is not -CO- by appropriately changing the structure of Q. Can be prepared by combining and changing the reaction conditions appropriately.

Manufacturing method 2
The nucleic acid derivative in the present invention can be exemplified as a method for producing a compound having a partial structure represented by formula (II ′).

(Wherein, DMTr, R, R ′, X, Q ′, and Polymer are as defined above. TBDMS represents a t-butyldimethylsilyl group, and Fmoc represents a 9-fluorenylmethyloxycarbonyl group)

Step 7
Compound (II-A) can be prepared by reacting compound (IA), t-butyldimethylsilyl chloride and dimethylaminopyridine in a solvent such as N, N-dimethylformamide (DMF), preferably in the presence of 2 equivalents of a base The reaction can be carried out at a temperature between 0 ° C. and 100 ° C. for 5 minutes to 100 hours.
As the base, those exemplified in Step 3 of Production Method 1 can be mentioned.

Step 8
Compound (II-B) can be produced using compound (II-A) under the same conditions as in step 1 of production method 1.

Step 9
Compound (II-C) is produced by reacting compound (II-B) with n-tetrabutylammonium fluoride (TBAF) in a solvent at a temperature between room temperature and 200 ° C. for 5 minutes to 100 hours. can do.
As the solvent, those exemplified in step 2 can be mentioned.

Step 10
Compound (II-D) can be produced using compound (II-C) under the same conditions as in step 2 of production method 1.

Step 11
Compound (II-E) can be produced using compound (II-D) and compound (ID) under the same conditions as in step 3 of production method 1.

Steps 12 to 14
Compound (II ′) can be produced using compound (II-E) under the same conditions as steps 4 to 6 of production method 1.

In addition, in the above-mentioned step 11, if necessary, the compound (ID) can be divided into two units, divided into two steps, and condensed with the compound (II-C). Specifically, for example, when R-Q 'is -NH-CO-Q4'-CO- (Q4' is substituted or unsubstituted alkylene having 1 to 12 carbon atoms), compound (II- C) and CH ₃ CH ₂ -O-CO-Q 4 '-CO-OH (Q 4' is as defined above) are condensed in the same manner as in step 11, and the ethyl ester of the obtained compound is ethanol or After hydrolysis with a base such as lithium hydroxide in a solvent such as water, the compound is further condensed with R′—NH ₂ (R ′ is as defined above) to give the desired compound. _{_{Incidentally, CH 3 CH 2 -O-CO}} -Q4'-CO-OH (Q4 ' is as defined above) and _R'-NH 2 (R' is as defined above), a known method (for example, Journal of American Chemical Society, 136, 16958 (2014)) or a method analogous thereto.

Manufacturing method 3
The nucleic acid derivative in the present invention can be exemplified as a method for producing a compound having a partial structure represented by formula (III ′).

(Wherein, DMTr, Fmoc, R, R ′, Q ′, X and Polymer are as defined above)

Compound (III ′) can be produced using compound (III-A) under the same conditions as steps 1 to 6 of production method 1. Compound (III-A) can be obtained as a commercial product.

Step 15
Compound (III-B) can be produced using compound (III-A) under the same conditions as in step 1 of production method 1.
Compound (III-A) can be purchased as a commercial product.

Step 16
Compound (III-C) can be produced using compound (III-B) under the same conditions as in step 2 of production method 1.

Step 17
Compound (III-E) can be produced using compound (III-C) under the same conditions as in step 3 of production method 1.

Steps 18 to 20
Compound (III ′) can be produced using compound (III-E) under the same conditions as in Steps 4 to 6 of Production Method 1.

In addition, in the above-mentioned step 17, if necessary, the compound (ID) can be divided into two units, divided into two steps, and condensed with the compound (III-C). Specifically, for example, when R-Q 'is -NH-CO-Q4'-CO- (Q4' is a substituted or unsubstituted alkylene having 1 to 12 carbon atoms), the compound (III-) is C) and CH ₃ CH ₂ -O-CO-Q 4 '-CO-OH (wherein Q 4' is as defined above) are condensed in the same manner as in step 17 to obtain ethyl ester of the obtained compound as ethanol or After hydrolysis with a base such as lithium hydroxide in a solvent such as water, the compound is further condensed with R′—NH ₂ (R ′ is as defined above) to give the desired compound. _{_{Incidentally, CH 3 CH 2 -O-CO}} -Q4'-CO-OH (Q4 ' is as defined above) and _R'-NH 2 (R' is as defined above), a known method (for example, Journal of American Chemical Society, 136, 16958 (2014)) or a method analogous thereto.

Manufacturing method 4
The nucleic acid derivative in the present invention can be exemplified as a method for producing a compound having a partial structure represented by formula (IV ′).

(Wherein, DMTr, Fmoc, R, R ′, Q ′, X and Polymer are as defined above)

Compound (IV ′) can be produced using compound (IV-A) under the same conditions as steps 1 to 6 of production method 1. Compound (IV-A) can be obtained as a commercial product.

In addition, in the above-mentioned step 23, if necessary, compound (ID) can be divided into two units, divided into two steps, and condensed with compound (IV-C). Specifically, for example, when R'-Q 'is -NH-CO-Q4'-CO- (Q4' is substituted or unsubstituted alkylene having 1 to 12 carbon atoms), the compound in step 23 (IV-C) and CH ₃ CH ₂ —O—CO—Q 4 ′ —CO—OH (Q 4 ′ is as defined above) are condensed in the same manner as in step 23 to obtain ethyl ester of the compound obtained The compound is hydrolyzed with a base such as lithium hydroxide in a solvent such as ethanol or water, and then it is condensed with R′—NH ₂ (R ′ is as defined above) to give the desired compound. _{_{Incidentally, CH 3 CH 2 -O-CO}} -Q4'-CO-OH (Q4 ' is as defined above) and R'-NH2 (R' is as defined above), a known method (for example, Journal of American Chemical Society, 136, 16958 (2014)) or a similar method.

Manufacturing method 5
The nucleic acid derivative in the present invention can be exemplified as a method for producing a compound having a partial structure represented by the formula (V ′).

(Wherein, DMTr, R, R ′, X, Q ′, TBDMS, Fmoc and Polymer are as defined above)

Compound (V ′) can be produced using compound (IV-A) under the same conditions as steps 1 to 7 of production method 2. Compound (IV-A) can be obtained as a commercial product.

In addition, in the above-mentioned step 31, the compound (ID) can also be divided into two units if necessary, divided into two steps, and condensed with the compound (VD). Specifically, for example, when R'-Q 'is -NH-CO-Q4'-CO- (Q4' is substituted or unsubstituted alkylene having 1 to 12 carbon atoms), the compound in step 31 (VD) and CH ₃ CH ₂ —O—CO—Q 4 ′ —CO—OH (Q 4 ′ is as defined above) are condensed in the same manner as in step 31 to obtain ethyl ester of the compound obtained The compound is hydrolyzed with a base such as lithium hydroxide in a solvent such as ethanol or water, and then it is condensed with R′—NH ₂ (R ′ is as defined above) to give the desired compound. _{_{Incidentally, CH 3 CH 2 -O-CO}} -Q4'-CO-OH (Q4 ' is as defined above) and _R'-NH 2 (R' is as defined above), a known method (for example, Journal of American Chemical Society, 136, 16958 (2014)) or a method analogous thereto.

Although the case where Q is -CO- is illustrated as an example, the method similar to the above, the known method or the method described above is also applied to the compound where Q is not -CO- by appropriately changing the structure of Q-OH. It can prepare by combining these and changing reaction conditions suitably.

Manufacturing method 6
The method for producing the nucleic acid complex of the present invention in which a sugar ligand-tether-brancher unit is linked to the 5 'end of the oligonucleotide is exemplified below.

(Wherein, R, R ′, Q ′, DMTr and X are as defined above)

Step 35
Compound (I-H) comprises compound (II-E) and 2-cyanoethyl-N, N, N ′, N′-tetraisopropylphosphodiamidite in the presence of a base and a reaction accelerator either in the absence or in the presence of a solvent. C., at a temperature between room temperature and 200.degree. C., for 5 minutes to 100 hours.
As the solvent, those exemplified in step 2 of production method 1 can be mentioned.
As the base, those exemplified in step 3 of production method 1 can be mentioned.
Examples of reaction accelerators include 1H-tetrazole, 4,5-dicyanoimidazole, 5-ethylthiotetrazole, 5-benzylthiotetrazole and the like, and they can be purchased as commercial products.

Step 36
The oligonucleotide chain is extended, and finally the compound (IH) is used to modify the 5 'end of the oligonucleotide with a sugar ligand-tether-brancher unit, followed by removal from the solid phase, deprotection of the protecting group Compound (I ′ ′) can be produced by performing and purification. Here, the removal from the solid phase, the deprotection of the protective group and the purification can be carried out in the same manner as in step 7 of production method 1, respectively.

Manufacturing method 7
The method for producing the nucleic acid complex of the present invention in which a sugar ligand-tether-brancher unit is linked to the 5 'end of the oligonucleotide is exemplified below.

It can manufacture on the conditions similar to the processes 35 and 36 of the manufacturing method 6.

(Wherein, R, R ', Q', DMTr and X are as defined above)

Manufacturing method 8
The method for producing the nucleic acid complex of the present invention in which a sugar ligand-tether-brancher unit is linked to the 5 'end of the oligonucleotide is exemplified below.

(Wherein, R, R ', Q', DMTr and X are as defined above)

Manufacturing method 9
The method for producing the nucleic acid complex of the present invention in which a sugar ligand-tether-brancher unit is linked to the 5 'end of the oligonucleotide is exemplified below.

(Wherein, R, R ', Q', DMTr and X are as defined above)

Manufacturing method 10
The method for producing the nucleic acid complex of the present invention in which a sugar ligand-tether-brancher unit is linked to the 5 'end of the oligonucleotide is exemplified below.

(Wherein, R, R ', Q', DMTr and X are as defined above)

Manufacturing method 11
Water or a suitable buffer of the sense strand having a sugar ligand-tether-brancher unit at the 3 'end or 5' end of the sense strand constituting the double stranded nucleic acid, and the antisense strand constituting the double stranded nucleic acid A nucleic acid complex having double-stranded nucleic acid can be obtained by dissolving each in a liquid and mixing.
Examples of the buffer include acetate buffer, Tris buffer, citrate buffer, phosphate buffer, water and the like, and these may be used alone or in combination.

The mixing ratio of the sense strand to the antisense strand is preferably 0.5 to 2 equivalents, more preferably 0.9 to 1.1 equivalents, and 0.95 equivalents to 1 equivalent of the antisense strand per equivalent of the sense strand. More preferred is .05 equivalents.

In addition, after mixing the sense strand and the antisense strand, an annealing treatment may be appropriately performed. The annealing treatment is carried out by heating the mixture of sense strand and antisense strand to preferably 50 to 100 ° C., more preferably 60 to 100 ° C., still more preferably 80 to 100 ° C., and then gradually cooling to room temperature. Can.

The antisense strand can be obtained according to the known oligonucleotide synthesis method described above.

Manufacturing method 12
The nucleic acid derivative in the present invention can be exemplified as a method for producing a compound having a partial structure represented by the formula (VI ′).

(Wherein, DMTr, R, R ′, X, Q ′, Polymer, and Fmoc are as defined above, TBS represents a t-butyldimethylsilyl group, R 0 and R x are the same or different, and are hydrogen atoms , A C1-C10 alkylene or a C3-C8 cycloalkylene, W is a C1-C10 alkylene, a C3-C8 cycloalkylene, or together with R0, a C4-C8 nitrogen-containing heterocyclic ring You may form.)

Step 45
Compound (VI-B) can be produced using compound (VI-A) under the same conditions as in step 1 of production method 1.
Compound (VI-A) can be obtained as a commercially available product, or by a known method (eg, Bioorganic & Medical Chemistry Letters, 11: 383-386) or a method analogous thereto.

Step 46
Compound (VI-C) can be produced using compound (VI-B) under the same conditions as in step 2 of production method 1.

Step 47
Compound (VI-D) can be produced using compound (VI-C) under the same conditions as in step 3 of production method 1.

Step 48
Compound (VI-E) can be produced using compound (VI-D) under the same conditions as in step 2 of production method 1.

Step 49
Compound (VI-G) can be produced under the same conditions as in step 3 of production method 1 using compound (VI-E) and compound (VI-F).

Step 50
Compound (VI-H) can be produced using compound (VI-G) under the same conditions as in step 9 of production method 2.

Steps 51-53
Compound (VI ′) can be produced using compound (VI-H), compound (VI-I) and compound (VI-J) under the same conditions as in steps 4 to 6 of production method 1.

Steps 45 to 53 can be carried out by a known method (for example, the method described in WO 2015/105083) or a method analogous thereto.
Compound (VI-F) can be obtained by a known method (for example, a method described in Journal of American Chemical Society, 136, 16958, Trio, 2014), or a method according thereto be able to.

Manufacturing method 13
The sugar ligand-tether unit in formula 2 wherein P1 and P4 are -NH-CO-, -O-CO- or -S-CO- can be produced by the following method.

(Wherein, Q 1, Q 2, Q 3, Q 4, Q 5, P 2, P 3, P 5, P 6, P 7, T 1, T 2, L 1, L 2, q 1, q 2, q 3 and q 4 are as defined above, and q 2 ′ is represents an integer smaller than 1 by q2, q4 'represents an integer smaller by 1 than q4, P1' and P4 'each independently represent -NH-CO-, -O-CO- or -S-CO- , Z represents H, OH, NH ₂ , SH, a chlorine atom, a bromine atom, an iodine atom, methanesulfonyloxy, p-toluenesulfonyloxy or a carboxylic acid, and B1 'and B2' are any of the structures of the following formulas And PG1, PG2, PG3, PG4, PG5, PG6 and PG7 each represent a suitable protecting group.)
formula:

m1, m2, m3 or m4 each independently represent an integer of 0 to 10.

Step 54
Compound (VII-C) is obtained by adding Compound (VII-A) and Compound (VII-B) in a solvent such as tetrohydrofuran etc, triphenylphosphine polymer support, and under ice-cooling, diisopropylazodicarboxylate toluene It can be produced by reacting a solution.
As the solvent, those exemplified in step 2 of production process 1 can be mentioned.
Compound (VII-A) can be obtained as a commercial product.

Step 55
Compound (VII-D) can be produced by reacting Compound (VII-C) in a solvent such as methanol under ice-cooling in the presence of a base.
As the solvent, those exemplified in step 2 of production process 1 can be mentioned.
As the base, those exemplified in Step 3 of Production Process 1 can be mentioned.

Step 56
Compound (VII-F) can be produced using compound (VII-D) and compound (VII-E) under the same conditions as in step 3 of production process 1.

Step 57
Compound (VII-H) can be produced using compound (VII-F) and compound (VII-G) under the same conditions as in step 3 of production process 1.

Step 58
Compound (VII-J) can be produced using compound (VII-H) and compound (VII-I) under the same conditions as in step 3 of production process 1.
Also, by repeatedly performing the DP step and the step 58, a compound (VII-J) having a desired value of q1 can be produced.

Step 59
Compound (VII-L) can be produced using compound (VII-J) and compound (VII-K) under the same conditions as in step 3 of production process 1.

Step 60
Compound (VII-N) can be produced using compound (VII-L) and compound (VII-M) under the same conditions as in step 3 of production process 1.

Steps 61 to 63
Compound (VII ′) can be produced using compound (VII-O), compound (VII-P) and compound (VII-Q) under the same conditions as in step 3 of production process 1.
Also, by repeatedly performing the DP step and the step 61, a compound (VII ′) having a desired q3 value can be produced.

Process DP
Methods commonly used in organic synthesis chemistry [eg Protective Groups in Organic Synthesis, third edition), by T. W. Greene, John Wiley & Sons Inc. (1999), etc.] can be used appropriately.
Compound (VII-B), Compound (VII-E), Compound (VII-G), Compound (VII-I), Compound (VII-K), Compound (VII-M), Compound (VII-O), Compound (VII-P) and the compound (VII-Q) are commercially available, or “Experimental Chemistry Lecture 4th Edition Organic Synthesis, p. 258, Maruzen (1992)”, “March's Advanced Organic Chemistry: Reactions, Mechanisms , And Structure, 7 ^th Edition "can be obtained by combining the methods described in the above or similar methods.

Manufacturing method 14
A unit in which P7 is -O- in Formula 4 can be manufactured by the following method.

(Wherein, Q5, P7, q5 are as defined above, q5 ′ ′ is an integer smaller than 2 by q5, q5 ′ is an integer smaller than 1 by 5, and Z2 is H, OH, NH ₂ or SH , PG8 and PG9 each represent a suitable protecting group, LC represents a sugar ligand-tether unit, and E represents a carboxylic acid or maleimide.)

Step 64
Compound (VIII-C) can be produced using compound (VIII-A) and compound (VIII-B) under the same conditions as in step 3 of production process 1.
Also, by repeatedly performing the DP step and the step 64, a compound (VIII-C) having a desired q5 ′ ′ value can be produced. Compound (VIII-B) is commercially available, or “Experimental Chemistry Lecture 4th Edition Organic Synthesis, p. 258, Maruzen (1992)”, “March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 7 ^th It can obtain by combining the method as described in Edition, or the method according to it.

Step 65
Compound (VIII ′) can be produced using compound (VIII-C) and compound (VIII-D) under the same conditions as in step 3 of production process 1.
Compound (VIII-D) is commercially available, or “Experimental Chemistry Lecture 4th Edition Organic Synthesis, p. 258, Maruzen (1992)”, “March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 7 ^th It can obtain by combining the method as described in Edition, or the method according to it.

Process DP
Methods commonly used in organic synthesis chemistry [eg Protective Groups in Organic Synthesis, third edition), by T. W. Greene, John Wiley & Sons Inc. (1999), etc.] can be used appropriately.

Manufacturing method 15
A sugar ligand-tether unit in which P1 and P4 in the formula 2 are -O- can be produced by the following method.

(Wherein, Q1, Q2, Q3, Q4, P2, P3, P5, P6, T1, T2, L1, L2, q1, q2, q3, q4, q2 ', q4', Z, B1 ', B2' are And PG10, PG11, PG12, PG13, PG14 and PG15 each represent a suitable protecting group.
formula:

m1, m2, m3 and m4 are as defined above.

Step 66
Compound (IX-C) is prepared by dissolving Compound (IX-A) and Compound (IX-B) in a solvent such as N, N′-dimethylformamide, adding a base such as potassium hydrogen carbonate, and the like to room temperature to 200 ° C. The reaction can be carried out by reacting for 5 minutes to 100 hours.
As the solvent, those exemplified in step 2 of production process 1 can be mentioned.
As the base, those exemplified in Step 3 of Production Process 1 can be mentioned.

Step 67
Compound (IX-E) is prepared by dissolving Compound (IX-C) and Compound (IX-D) in a solvent such as N, N′-dimethylformamide, adding a base such as potassium hydrogen carbonate, and the like to room temperature to 200 ° C. The reaction can be carried out by reacting for 5 minutes to 100 hours.
As the solvent, those exemplified in step 2 of production process 1 can be mentioned.
As the base, those exemplified in Step 3 of Production Process 1 can be mentioned.
Compound (IX-A) can be obtained as a commercial product.

Step 68
Compound (IX-G) can be produced using compound (IX-E) and compound (IX-F) under the same conditions as in step 3 of production process 1.

Step 69
Compound (IX-I) can be produced using compound (IX-G) and compound (IX-H) under the same conditions as in step 3 of production process 1.
Also, by repeatedly performing the DP step and the step 69, it is possible to produce the compound (VII-J) having a desired value of q1.

Step 70
Compound (IX-K) can be produced using compound (IX-I) and compound (IX-J) under the same conditions as in step 3 of production process 1.

Step 71
Compound (IX-M) can be produced using compound (IX-K) and compound (IX-L) under the same conditions as in step 3 of production process 1.

Steps 72-74
The compound (IX ′) can be produced using compound (IX-M), compound (IX-N), compound (IX-O) and compound (IX-P) under the same conditions as in step 3 of production process 1. Can.
Also, by repeatedly performing the DP step and the step 72, the compound (IX ′) having a desired value of q3 can be produced.

Compound (IX'-B), Compound (IX'-D), Compound (IX'-F), Compound (IX'-H), Compound (IX'-J), Compound (IX'-L), Compound (IX) IX'-N), compound (IX'-O) and compound (IX'-P) are commercially available products, or "Experimental Chemistry Lecture 4th Edition Organic Synthesis, p. 258, Maruzen (1992)", "March 's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 7 th Edition "combining the methods described, or can be obtained by a method analogous thereto.

Manufacturing method 16
The following method can also be used as a method for producing the nucleic acid complex of Formula 1 to Formula 8.

(Wherein, LC, Q5, Q6, Q7, P7, P8, q5 ', q6 and X are as defined above)

Step 75
Compound (XB) can be produced by reacting compound (XIII ′ ′) with compound (XA) in a solvent at 0 ° C. to 100 ° C. for 10 seconds to 100 hours.
Examples of the solvent include water, phosphate buffer, sodium acetate buffer, dimethyl sulfoxide and the like, and these can be used alone or in combination.
Compound (VIII ′) can be obtained by using production method 14.

The compound (X-A) can be prepared by a known method (for example, Bioconjugate Chemistry, Vol. 21, pp. 187-202, 2010, or Current Protocols in Nucleic Acid Chemistry (Current Protocols) in Nucleic Acid Chemistry), September, 2010; CHAPTER: Unit 4.41) or an equivalent method.

Step 76
The compound (X ′) is produced by reacting the compound (X-B) at a temperature of room temperature and 200 ° C. for 5 minutes to 100 hours under conditions of pH 8 or more such as aqueous sodium carbonate solution or ammonia water Can.

Manufacturing method 17
The following method can also be used as a method for producing the nucleic acid complex of Formula 1 to Formula 8.

(Wherein, LC, Q5, Q6, Q7, P7, P8, q5 ', q6 and X are as defined above)

Step 77
The compound (XI-A) can be produced by a known method (for example, the method described in Bioconjugate Chemistry, Vol. 26, pp. 1451-1455, 2015) using the compound (VIII ′ ′ ′) It can obtain by the method according to it.
Compound (VIII ′ ′ ′) can be obtained by using production method 14.

Step 78
The compound (XI ′) can be produced using a compound (XI-A) and a compound (XI-B) according to a known method (eg, Bioconjugate Chemistry, vol. 26, p. 1451-1455, 2015) The methods described) or can be obtained by methods analogous thereto.
Compound (XI-B) can be obtained by the method described in Bioconjugate Chemistry, vol. 26, p. 1451-1455 (2015) or a method analogous thereto.

Step 79
As another method, compound (XI ′) can be obtained by a known method (see, for example, Bioconjugate Chemistry, 22: 1723-1728, 2011) or a method according thereto. It can be obtained directly from XI-A).

The nucleic acid complex in the present specification can also be obtained as a salt such as an acid addition salt, a metal salt, an ammonium salt, an organic amine addition salt, an amino acid addition salt and the like.

Examples of acid addition salts include inorganic acid salts such as hydrochlorides, sulfates and phosphates, and organic acid salts such as acetates, maleates, fumarates, citrates and methanesulfonates. Examples of metal salts include alkali metal salts such as sodium salts and potassium salts, alkaline earth metal salts such as magnesium salts and calcium salts, aluminum salts, zinc salts and the like, and examples of ammonium salts include ammonium and tetramethyl Salts such as ammonium may be mentioned, organic amine addition salts may include addition salts such as morpholine and piperidine, and amino acid addition salts may include addition salts such as lysine, glycine and phenylalanine.

When it is desired to prepare a salt of the nucleic acid complex of the present invention, the complex may be purified as it is when it is obtained in the form of the desired salt, or when it is obtained in free form, the complex Is dissolved or suspended in an appropriate solvent, the corresponding acid or base is added, and isolation and purification may be performed. In addition, when converting the counter ion that forms the complex salt to a different counter ion, the complex salt is dissolved or suspended in an appropriate solvent, and then an acid, a base and / or a salt (sodium chloride, chloride It may be isolated and purified by adding several equivalent to a large excess amount of an inorganic salt such as ammonium).

Although some of the nucleic acid complexes of the present invention may have geometric isomers, stereoisomers such as optical isomers, tautomers etc., all possible isomers and their mixtures are also possible. Included in the present invention.

Also, the nucleic acid complex of the present invention may exist in the form of an adduct with water or various solvents, and these adducts are also included in the present invention.

Furthermore, the nucleic acid complex of the present invention also includes those in which a part or all of the atoms in the molecule are substituted by atoms (isotopes) having different mass numbers (for example, deuterium atoms etc.).

The pharmaceutical composition of the present invention comprises the nucleic acid complex represented by Formula 1. The nucleic acid complex of the present invention is recognized by the target cell by having the L1 and L2 sugar ligands, and is introduced into the cell.
The nucleic acid complex of the present invention can be administered to a mammal to suppress or reduce the expression of a target gene in vivo, and can be used to treat a disease associated with the target gene.
When the nucleic acid complex of the present invention is used as a therapeutic agent or a prophylactic agent, it is desirable to use the most effective administration route for treatment as a therapeutic agent or a prophylactic agent, and it is not particularly limited. Administration, subcutaneous administration, intramuscular administration and the like can be mentioned, preferably subcutaneous administration.
Although the dose varies depending on the condition, age, administration route and the like of the administration subject, it may be administered, for example, such that the daily dose converted to double-stranded oligonucleotide is 0.1 μg to 1000 mg, daily administration More preferably, the amount is 1 to 100 mg.

Examples of preparations suitable for intravenous administration or intramuscular administration include injections, and it is possible to use the prepared solution as it is, for example, in the form of injections, etc. The solvent is removed and used by lyophilization, the solution is used by lyophilization, and / or the solution with an excipient such as mannitol, lactose, trehalose, maltose or glycine is used by lyophilization. It can also be done.
In the case of injection, it is preferable to prepare an injection by mixing, for example, water, acid, alkali, various buffers, physiological saline, amino acid infusion, etc. into a solution or a composition from which a solvent or solvent has been removed or lyophilized. For example, an injection can be prepared by adding an antioxidant such as citric acid, ascorbic acid, cysteine or EDTA, or an isotonic agent such as glycerin, glucose or sodium chloride. For example, a cryopreservative such as glycerin can be added and cryopreserved.

The double stranded nucleic acid in the composition of the present invention can be introduced into cells by administering the composition of the present invention to mammalian cells.

The introduction of the nucleic acid complex of the present invention into mammalian cells in vivo may be carried out according to known transfection procedures which can be carried out in vivo. The composition of the present invention can be delivered to the liver by intravenous administration to mammals including humans, and the double stranded nucleic acid in the composition of the present invention can be introduced into the liver or hepatocytes.

When the double stranded nucleic acid in the composition of the present invention is introduced into the liver or hepatocytes, the expression of the β2GPI gene in the hepatocytes can be reduced to treat or prevent β2GPI related diseases. The β2GPI-related diseases include autoimmune diseases or thrombosis, and more specifically include systemic lupus erythematosus (SLE), antiphospholipid antibody syndrome, hemodialysis complications in patients with end-stage renal failure and arteriosclerosis.
The administration subject is a mammal, preferably a human.

In addition, the nucleic acid complex in the present invention can also be used as a tool for verifying the efficacy of suppressing the β2GPI gene in an in vivo drug efficacy evaluation model for the therapeutic or preventive agent for the above-mentioned diseases. As an in vivo efficacy evaluation model, lupus anticoagulant (LA) test and the like can be mentioned. Here, LA is a kind of antiphospholipid antibody like anti-β2GPI antibody, and exhibits an activity of inhibiting phospholipid-dependent coagulation reaction of collected blood in vitro. LA mainly appears in diseases such as SLE and APS, and many have been reported to be correlated with the onset or pathogenesis of thrombosis and infertility like anti-β2GPI antibodies (“blood”, 2003, Vol. 101, No. 5, p1827-1832). And, it is also reported that the anti-β2GPI antibody exerts β2GPI-dependent LA activity because it acquires phospholipid binding activity via β2GPI (“Thrombosis and Haemostasis”, 1998, 79, No. 1, p 79-86). Furthermore, it has been reported that this β2GPI-dependent LA strongly correlates with the onset rate of the pathological condition (“blood”, 2004, Vol. 104, No. 12, p 3598-3602).
If it is possible to release LA activity by suppressing the expression of β2GPI in blood, it is considered possible to provide a novel and effective treatment for β2GPI related diseases. In clinical tests LA can be detected by measuring activated partial thromboplastin time, kaolin clotting time and / or diluted Russell's snake venom time (dRVVT).

Although the dose varies depending on the condition, age, administration route and the like of the administration subject, it may be administered, for example, such that the daily dose converted to double-stranded nucleic acid is 0.1 μg to 1000 mg. It is preferable to administer to 1 to 100 mg.

The invention also relates to nucleic acid complexes for use in the treatment of diseases; pharmaceutical compositions for use in the treatment of diseases; use of nucleic acid complexes for the treatment of diseases; in the manufacture of a medicament for the treatment of diseases A nucleic acid complex for use in the manufacture of a medicament for treating a disease; a method for treating or preventing a disease comprising administering an effective amount of the nucleic acid complex to a subject in need thereof; provide.

Next, the present invention will be specifically described by way of reference examples, examples and test examples. However, the present invention is not limited to these examples and test examples. The proton nuclear magnetic resonance spectra ( ¹ H NMR) shown in Examples and Reference Examples were measured at 270 MHz, 300 MHz or 400 MHz, and exchangeable protons were clearly observed depending on the compound and measurement conditions. It may not be. Moreover, although the thing normally used is used as a description of the multiplicity of a signal, br means broad, and it represents that it is an apparently wide signal.
The following conditions were used for UPLC analysis.
Mobile phase A: aqueous solution containing 0.1% formic acid, B: acetonitrile solution gradient: linear gradient (10 minutes-90%) of mobile phase B (3 minutes)
Column: Waters ACQUITY UPLC BEH C18 (1.7 μm, internal diameter 2.1 x 50 mm)
Flow rate: 0.8 mL / min
PDA detection wavelength: 254 nm (detection range 190-800 nm)

Reference Example compounds are shown in Table X-1 to Table X-4.

Reference Example 1

Synthesis of Compound RE1-2 Compound RE1-1 synthesized by the method described in Journal of American Chemical Society, Volume 136, pp. 16958-16961, 2014 (0.8755 g, Dissolve 1.9567 mmol) in tetrahydrofuran (10 mL) and stir 1, 3-dicyclohexanecarbodiimide (DCC, 0.4247 g, 2.0584 mmol) and N-hydroxysuccinimide (0.2412 g, 2.0958 mmol) at room temperature overnight did. The solid precipitated from the reaction mixture was removed, and the solvent was evaporated under reduced pressure. The resulting mixture is dissolved in N, N'-dimethylformamide (DMF), and 2-aminoethylmaleimide hydrobromide (0.6479 g, 2.5491 mmol) and diisopropylethylamine (1.7 mL, 9.7835 mmol) are added, and then room temperature is obtained. Stir overnight. The solvent of the reaction mixture was evaporated under reduced pressure, and elution was performed with reverse phase column chromatography (water / methanol = 80/20) to give compound RE1-2 (0.8502 g, yield 76%).
ESI-MS m / z: 570 (M + H) ⁺ ;
¹ H-NMR (DMSO-D ₆ ) δ: 1.45-1.56 (4H, m), 1.78 (3H, s), 1.90 (3H, s), 1.97 (2H, t, J = 7.0 Hz), 2.00 (3H , s), 2.11 (3H, s), 3.18-3.19 (2H, m), 3.38-3.45 (3H, m), 3.64-3.71 (1H, m), 3.85-3.89 (1H, m), 4.01-4.04 (3H, m), 4.48 (1H, d, J = 8.6 Hz), 4.95-4.98 (1H, m), 5.21 (1 H, d, J = 3.5 Hz), 6.99 (2H, s), 7.81-7.87 ( 2H, m).

Synthesis step 1 of compound RE1-4
The compound RE1-1 (0.9602 g, 2.1460 mmol) is dissolved in N, N'-dimethylformamide (10 mL), N-Boc-ethylenediamine (manufactured by Sigma-Aldrich, 0.6877 g, 4.292 mmol), diisopropylethylamine (1.90 mL) , 10.87 mmol), and 2- (1H-benzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate (manufactured by Wako Pure Chemical Industries, Ltd., 1.6437 g, 4.3229 mmol) Was added and stirred overnight at room temperature. Water was added to the reaction solution, and the mixture was extracted twice with chloroform, and then the organic layer was washed with saturated brine and dried over anhydrous magnesium sulfate. The solvent was evaporated under reduced pressure to give a crude purified product of compound RE1-3.
ESI-MS m / z: 590 (M + H) ⁺

Step 2
Compound RE1-4 Compound RE1-3 (1.2654 g, 2.1460 mmol) synthesized in step 1 was dissolved in dichloromethane (15 mL), trifluoroacetic acid (4 mL) was added, and the mixture was stirred overnight at room temperature. . Water was added to the reaction solution, and the mixture was extracted with ethyl acetate, and then the organic layer was washed with saturated brine and dried over anhydrous magnesium sulfate. The solvent was evaporated under reduced pressure and the residue was eluted with reverse phase column chromatography (water / methanol = 80/20) to obtain Compound RE1-4 (0.3879 g, yield 37%).
ESI-MS m / z: 490 (M + H) ⁺ ;
¹ H-NMR (DMSO-D ₆ ) δ: 1.46 to 1.52 (4H, m), 1.78 (3H, s), 1.90 (3H, s), 2.00 (3H, s), 2.08 (2H, t, J = 7.4 Hz), 2.11 (3 H, s), 2. 85 (2 H, t, J = 6.3 Hz), 3. 27 (2 H, dd, J = 12.3, 6.2 Hz), 3.67-3.69 (1 H, m), 3.68-3. 73 (3. 1H, m), 3.86-3.90 (1H, m), 4.01-4.04 (3H, m), 4.49 (1H, d, J = 8.4 Hz), 4.97 (1 H, dd, J = 11.3, 3.4 Hz), 5.22 (1H, d, J = 3.5 Hz), 7.86 (1 H, d, J = 9.1 Hz), 7.95-8.02 (1 H, m).

Reference Example 2

Reference Example 2 Step 1
(9H-fluoren-9-yl) methyl ((2R, 3R) -1,3-dihydroxybutane-2-yl) carbamate (Compound RE2-1, Chem-Impex International, Inc., 1.50 g, 4.58 mmol) The residue was dissolved in pyridine (20 mL), and 4,4′-dimethoxytrityl chloride (manufactured by Tokyo Chemical Industry Co., Ltd., 1.71 g, 5.04 mmol) was added under ice-cooling, followed by stirring at room temperature for 2 hours. The reaction mixture was ice-cooled, 10% aqueous citric acid solution was added, and the mixture was extracted with ethyl acetate. The organic layer was washed with saturated brine and dried over anhydrous magnesium sulfate. The solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography (hexane / ethyl acetate = 90/10) to give compound RE2-2 (1.07 g, yield 37%).
ESI-MS m / z: 630 (M + H) ⁺

Reference Example 2 Step 2
The compound RE2-2 (1.07 g, 1.699 mmol) synthesized in step 1 of Reference Example 2 is dissolved in N, N-dimethylformamide (10 mL), and piperidine (0.336 mL, 3.40 mmol) is added at room temperature to obtain 3 Stir for hours. Water was added to the reaction solution, and the mixture was extracted with ethyl acetate, and then the organic layer was washed with saturated brine and dried over anhydrous magnesium sulfate. The solvent was evaporated under reduced pressure, and the residue was purified by amino silica gel column chromatography (chloroform / methanol = 90/10) to give compound RE2-3 (0.59 g, yield 85%).
ESI-MS m / z: 408 (M + H) ⁺

Reference Example 3

Reference Example 3 Step 1
Dimethyl 5-hydroxyisophthalate (Compound RE3-1, manufactured by Wako Pure Chemical Industries, Ltd., 5.0443 g, 24 mmol) is dissolved in tetrahydrofuran (25 mL manufactured by Wako Pure Chemical Industries, Ltd.), and 2- (tert-butoxycarbonylamino) After adding -1-ethanol (manufactured by Tokyo Chemical Industry Co., Ltd., 4.0343 g, 25.03 mmol), and triphenylphosphine polymer support (manufactured by Sigma Aldrich, 6.61 g, 25.2 mmol), 40% diisopropylazo under ice cooling Dicarboxylate (DIAD) toluene solution (manufactured by Tokyo Chemical Industry Co., Ltd., 13.26 mL, 25.2 mmol) was added, and the mixture was stirred overnight at room temperature. The reaction solution is filtered, the filtrate is evaporated under reduced pressure, and the residue is purified by amino silica gel column chromatography (hexane / ethyl acetate = 95 / 5-80 / 20) to give compound RE3-2 (5.3071). g, yield 63%).
ESI-MS m / z: 254 (M + H) ⁺ , detected as a de-Boc derivative

Reference Example 3 Step 2
Reference Example 3 The compound RE3-2 (5.3071 g, 15.02 mmol) synthesized in step 1 was dissolved in methanol (25 mL), and under ice-cooling, 2 mol / L aqueous sodium hydroxide solution (Wako Pure Chemical Industries, 13 mL) ) Was added and stirred at room temperature for 4 hours. The reaction mixture was ice-cooled, 10% aqueous citric acid solution was added, and the mixture was extracted with ethyl acetate. The organic layer was washed with saturated brine and dried over anhydrous magnesium sulfate. Compound RE3-3 was quantitatively obtained by distilling off the solvent under reduced pressure.
ESI-MS m / z: 324 (M-H) ^-

Reference Example 3 Step 3
Reference Example 3 The compound RE3-3 (1.9296 g, 5.93 mmol) synthesized in step 2 was dissolved in N, N'-dimethylformamide (70 mL) to give N-1- (9H-fluoren-9-ylmethoxycarbonyl). -Ethylenediamine hydrochloride (3.3493 g, 11.86 mmol), diisopropylethylamine (5.18 mL, 29.7 mmol), and 2- (1H-benzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluoro Phosphate (4.5168 g, 11.88 mmol) was added and stirred at room temperature for 4 hours. The reaction mixture was ice-cooled, 10% aqueous citric acid solution was added, and extracted with chloroform. The organic layer was washed with saturated aqueous sodium hydrogen carbonate solution and saturated brine, and dried over anhydrous magnesium sulfate. The solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography (chloroform / methanol) to give compound RE3-4 (3.4407 g, yield 68%).
ESI-MS m / z: 898 (M + HCOO) ^-

Reference Example 3 Step 4
Reference Example 3 The compound RE3-4 (1.6087 g, 1.884 mmol) synthesized in step 3 is dissolved in dichloromethane (20 mL), trifluoroacetic acid (5 mL, 64.9 mmol) is added under ice cooling, and the reaction is carried out at room temperature 4 Stir for hours. The reaction mixture was evaporated under reduced pressure and the solvent was evaporated to quantitatively obtain Compound RE3-5 (2.4079 g).
ESI-MS m / z: 798 (M + HCOO) ^-

Reference Example 3 Step 5
Reference Example 3 The compound RE3-5 (386 mg, 0.512 mmol) synthesized in step 4 is dissolved in tetrahydrofuran (10 mL), benzoyl chloride (175 mg, 1.024 mmol) is added under ice-cooling, and 1 at room temperature. Stir for hours. The solvent was distilled off under reduced pressure of the reaction liquid, and the residue was purified by silica gel column chromatography (chloroform / methanol) to obtain compound RE3-6 (373 mg, yield 82%).
ESI-MS m / z: 888 (M + H) ⁺

Reference Example 3 Step 6
The compound RE3-6 (108 mg, 0.122 mmol) synthesized in Reference Example 3 step 5 was dissolved in dichloromethane (5 mL), diethylamine (0.5 mL, 4.8 mmol) was added at room temperature, and the mixture was stirred for 1 hour. The solvent was evaporated under reduced pressure to quantitatively obtain Compound RE3-7 (54.9 mg).
ESI-MS m / z: 444 (M + H) ⁺

Reference Example 3 Step 7
Reference Example 3 Compound RE 3-7 (180 mg, 0.406 mmol) synthesized in step 6 Nα, Nε-bis (tert-butoxycarbonyl) -L-lysine (manufactured by Nova Biochem, 295 mg, 0.852 mmol), diisopropylethylamine ( Add 0.354 mL, 2.029 mmol) and 2- (1H-benzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate (324 mg, 0.852 mmol) at room temperature And stirred overnight. The reaction mixture was ice-cooled, 10% aqueous citric acid solution was added, and the mixture was extracted with chloroform. The organic layer was washed with saturated brine and dried over anhydrous magnesium sulfate. The solvent was evaporated under reduced pressure, and the residue was purified by amino silica gel column chromatography (chloroform / methanol) to quantitatively obtain Compound RE3-8 (450 mg).
ESI-MS m / z: 1101 (M + H) ⁺

Reference Example 3 Step 8
Reference Example 3 Compound RE3-8 (2.1558 g, 1.9593 mmol) synthesized in step 7 was dissolved in dichloromethane (20 mL), trifluoroacetic acid (5 mL) was added under ice cooling, and the mixture was stirred at room temperature for 4 hours did. The reaction mixture was evaporated under reduced pressure to distill off the solvent, whereby compound RE3-9 was quantitatively obtained.
ESI-MS m / z: 700 (M + H) ⁺

Reference Example 4

(In the figure, Bn represents a benzyl group.)

Reference Example 4 Step 1
Compound RE4-1 synthesized by the method described in Reference Example 3 (compound RE3-5, 0.5716 g, 0.7582 mmol in Reference Example 3), Bioconjugate Chemistry, Volume 22, pp. 690-699, Dodecanoic acid monobenzyl ester (0.4859 g, 1.5164 mmol), diisopropylethylamine (0.662 mL, 3.79 mmol) synthesized by the method described in 2011, and 2- (1H-benzotriazol-1-yl) -1,1 3,3,3-tetramethyluronium hexafluorophosphate (0.5766 g, 1.516 mmol) was dissolved in N, N dimethylformamide (12 mL) and stirred at room temperature for 1 hour. The reaction mixture was ice-cooled, saturated aqueous citric acid solution was added, and the mixture was extracted with chloroform. The organic layer was washed with saturated brine and dried over anhydrous magnesium sulfate. The solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography (chloroform / methanol) to give compound RE4-2 (0.88 g, yield 84%).
ESI-MS m / z: 1057 (M + H) ⁺

Reference Example 4 Step 2
The compound RE4-2 (0.7545 g, 0.714 mmol) synthesized in Reference Example 4 step 1 was dissolved in dichloromethane-tetrahydrofuran (20 mL), diethylamine (5 mL, 47.9 mmol) was added at room temperature, and the mixture was stirred overnight. Compound RE4-3 was quantitatively obtained by distilling off the solvent under reduced pressure.
ESI-MS m / z: 612 (M + H) ⁺

Reference Example 4 Step 3
Reference Example 4 Compound RE4-3 (0.437 g, 0.7143 mmol) synthesized in step 2, Nα, Nε-bis (tert-butoxycarbonyl) -L-lysine (manufactured by Nova Biochem, 0.5483 g, 1.583 mmol), diisopropylethylamine ( Add 0.624 mL, 3.57 mmol) and 2- (1H-benzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate (0.5703 g, 1.5 mmol) to room temperature. The mixture was stirred for 2 hours. To the reaction mixture was added 10% aqueous citric acid solution, and the mixture was extracted with ethyl acetate. The organic layer was washed with saturated brine and dried over anhydrous magnesium sulfate. The solvent was evaporated under reduced pressure to give a crude product of compound RE4-4.
ESI-MS m / z: 1269 (M + H) ⁺

Reference Example 4 Step 4
Reference Example 4 The compound RE4-4 (0.906 g, 0.7143 mmol) synthesized in step 3 was dissolved in dichloromethane (12 mL), trifluoroacetic acid (3 mL, 38.9 mmol) was added under ice-cooling, and 4 was added at room temperature. Stir for hours. The solvent of the reaction mixture was evaporated under reduced pressure to give a crude product of compound RE4-5.
ESI-MS m / z: 869 (M + H) ⁺

Reference Example 5

Reference Example 5 Step 1
The compound RE5-1 (RE 3-8, 100 mg, 0.091 mmol in Reference Example 3) synthesized by the method described in Reference Example 3 is dissolved in methanol (3 mL), acetic acid (2 μL) is added, and then palladium is added. / Catalytic hydrogen reduction by carbon was performed. The solvent of the obtained solution fraction was evaporated under reduced pressure to quantitatively obtain Compound RE5-2.
ESI-MS m / z: 967 (M + H) ⁺

Reference Example 5 Step 2
Compound RE5-2 (50 mg, 0.052 mmol) and N- (6-maleimidocaproyloxy) succinimide (48 mg, 0.155 mmol) are dissolved in tetrahydrofuran (2 mL) and diisopropylethylamine (0.045 mL, 0.259 mmol) is dissolved in In addition, it was stirred overnight at room temperature. The solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography (chloroform / methanol) to give compound RE5-3 (18 mg, yield 30%).
ESI-MS m / z: 1160 (M + H) ⁺

Reference Example 5 Step 3
Reference Example 5 The compound RE5-3 (18 mg, 0.016 mmol) synthesized in step 2 was dissolved in dichloromethane (2 mL), trifluoroacetic acid (0.2 mL, 2.6 mmol) was added under ice-cooling, and the mixture was stirred at room temperature. Stir overnight. The reaction mixture was evaporated under reduced pressure, the solvent was evaporated, and the residue was crystallized with diethyl ether to give compound RE5-4 (7.5 mg, yield 64%) as a white solid.
ESI-MS m / z: 759 (M + H) ⁺

Reference Example 6

Reference Example 6 Step 1
Compound RE6-1 synthesized by the method described in Reference Example 3 (compound RE3-2 in the reference example 3, 0.9372 g, 2.8809 mmol), β-alanine methyl ester hydrochloride (manufactured by Tokyo Chemical Industry Co., Ltd., 0.8082 g, 5.7902) Compound RE6-2 was quantitatively obtained in the same manner as in step 3 of Reference Example 3 using mmol).
ESI-MS m / z: 495 (M + H) ⁺

Reference Example 6 Step 2
Reference Example 6 Compound RE6-3 was quantitatively obtained in the same manner as in step 4 of reference example 3 using compound RE6-2 (0.9622 g, 1.952 mmol) synthesized in step 1.
ESI-MS m / z: 396 (M + H) ⁺

Reference Example 6 Step 3
Reference Example 6 Compound RE6-3 (0.1146 g, 0.290 mmol) synthesized in step 2 and N-succinimidyl 15-azido-4,7,10,13-tetraoxapentadecanoic acid (N3-PEG4-NHS, Tokyo Chemical Industry Co., Ltd. Compound RE6-4 was quantitatively obtained in the same manner as in step 2 of Reference Example 5, using company-made, 0.0750 g, 0.1931 mmol).
ESI-MS m / z: 669 (M + H) ⁺

Reference Example 6 Step 4
Reference Example 6 Compound RE6-5 (0.1291 g, 0.193 mmol) synthesized in step 3 was used to quantitatively obtain compound RE6-5 in the same manner as in step 2 of reference example 3.
ESI-MS m / z: 641 (M + H) ⁺

Reference Example 6 Step 5
Reference Example 6 The process of Reference Example 3 using compound RE6-5 (0.1252 g, 0.193 mmol) synthesized in step 4 and L-glutamic acid di-tert-butyl ester (manufactured by Watanabe Kagaku Co., Ltd., 0.1180 g, 0.399 mmol). Compound RE6-6 (0.0521 g, yield 24%) was obtained in the same manner as 3.
ESI-MS m / z: 1124 (M + H) ⁺

Reference Example 6 Step 6
Reference Example 6 Compound RE6-7 (36 mg, yield 86%) was obtained in the same manner as in Step 4 of Reference Example 3, using Compound RE6-6 (0.0521 g, 0.0464 mmol) synthesized in Step 5.
ESI-MS m / z: 899 (M + H) ⁺

Reference Example 7

Reference Example 7 Step 1
Compound RE7-1 (RE 3-9 in Example 3; 0.2586 g, 0.3695 mmol) synthesized by the method described in Reference Example 3 and Journal of American Chemical Society (Journal of American Chemical Society), Volume 136 Compound RE7- described in Reference Example 1 using the compound RE-1 (0.8559 g, 1.7927 mmol) described in Reference Example 1 synthesized by the method described in Chem. 2 (0.5272 g, yield 61%) were obtained.
ESI-MS m / z: 2418 (M + H) ⁺

Reference Example 7 Step 2
Reference Example 7 Compound RE7-2 (0.1524 g, yield 61%) was obtained in the same manner as in step 1 of reference example 5 using compound RE7-2 (0.2653 g, 0.1097 mmol) synthesized in step 1.
ESI-MS m / z: 2284 (M + H) ⁺

Reference Example 8: Synthesis of Compounds A1 and B1

Synthesis of Compound A1 Using compound RE8-1 (compound RE7-3 in the reference example 7, 0.0152 g, 0.006657 mmol) synthesized by the method described in reference example 7, compound A1 was prepared in the same manner as in step 2 of reference example 5. (0.0077 g, 47% yield) was obtained.
ESI-MS m / z: 1239 (M + 2 H) ²⁺
¹ H-NMR (DMSO-D ₆ ) δ: 1.11-1.66 (34H, m), 1.77 (12H, d, J = 1.5 Hz), 1.89 (12H, s), 2.01-2.14 (10H, m), 2.01 (12H, s), 2.10 (12H, s), 2.92-2.99 (4H, m), 3.16-3.54 (14H, m), 3.65-3.74 (4H, m), 3.81-3.91 (4H, m), 3.98 -4.08 (14H, m), 4.11-4.24 (4H, m), 4.48 (4H, dd, J = 8.4, 1.8 Hz), 4.93-5.00 (4H, m), 5.21 (4 H, d, J = 3.5 Hz) ), 6.99 (2H, s), 7.52 (2H, s), 7.66-7.75 (2H, m), 7.78-7.87 (6H, m), 7.91 (1H, br s), 8.01-8.08 (3H, br m) ), 8.54-8.60 (2H, br m).

Synthesis of Compound B1 Using compound RE8-1 (compound RE7-3 in Reference Example 7, 0.0150 g, 0.00657 mmol), Compound B1 (0.0062 g, yield 37%) was prepared in the same manner as in Step 3 of Reference Example 3. Obtained.
ESI-MS m / z: 1279 (M + 2 H) ²⁺
¹ H-NMR (DMSO-D ₆ ) δ: 1.11-1.66 (30H, m), 1.77 (12H, s), 1.89 (12H, s), 2.01-2.14 (8H, m), 2.01 (12H, s) , 2.10 (12H, s), 2.33-2.38 (2H, m), 2.92-2.99 (4H, m), 3.16-3.54 (14H, m), 3.58-3.63 (16H, m), 3.65-3.74 (4H, m) m), 3.81-3.91 (4H, m), 3.98-4.08 (12H, m), 4.11-4.24 (4H, m), 4.48 (4H, dd, J = 8.4, 1.8 Hz), 4.93-5.00 (4H, m) m), 5.21 (4H, d, J = 3.5 Hz), 7.52 (2H, s), 7.66-7.75 (2H, m), 7.78-7.87 (6H, m), 7.91 (1H, br s), 8.01- 8.08 (3H, br m), 8.54-8.60 (2H, br m).

Reference Example 9: Synthesis of Compounds B2 and B3

Synthesis of Compound B2 Compound RE9-1 synthesized by the method described in Reference Example 6 (compound RE6-5 in the reference example 6, 0.00436 g, 0.00681 mmol) and compound RE1-4 of the reference example 1 (0.010 g, 0.020 mmol) The crude product of Compound B2 was obtained in the same manner as in Step 3 of Reference Example 3 using
ESI-MS m / z: 1584 (M + H) ⁺

Synthesis of Compound B3 Using compound RE9-2 synthesized by the method described in Reference Example 6 (Compound RE 6-7 in Reference Example 6, 0.0100 g, 0.01112 mmol), Compound B3 was prepared in the same manner as in Step 3 of Reference Example 3. (0.0223 g, 72% yield) was obtained.
ESI-MS m / z: 1393 (M + 2 H) ²⁺

Reference Example 10: Synthesis of Compounds C1 and D1

Reference Example 10 Step 1
Compound RE10-1 (compound RE4-5, 0.1952 g, 0.225 mmol in Reference Example 4) synthesized by the method described in Reference Example 4 and Journal of American Chemical Society, 136 In the same manner as in step 3 of Reference Example 3, Compound RE10-2 was prepared using Compound RE1-1 (0.4162 g, 0.93 mmol) of Reference Example 1 synthesized by the method described in Volume, pp. 16958-16961, 2014. (334.8 mg, yield 58%) was obtained.
ESI-MS m / z: 1294 (M + 2 H) ²⁺

Reference Example 10 Step 2
Reference Example 10 Compound RE (112 mg, yield 80%) was obtained in the same manner as in step 1 of reference example 5 using compound RE10-2 (0.1459 g, 0.056 mmol) synthesized in step 1.
ESI-MS m / z: 1249 (M + 2 H) ²⁺
¹ H-NMR (DMSO-D ₆ ) δ: 1.11-1.66 (44H, m), 1.77 (12H, d, J = 1.5 Hz), 1.89 (12H, s), 2.01-2.20 (12H, m), 2.01 (12H, s), 2.10 (12H, s), 2.92-2.99 (4H, m), 3.16-3.54 (10H, m), 3.58-3.64 (4H, m), 3.65-3.74 (4H, m), 3.81 -3.91 (4H, m), 3.98-4.08 (12H, m), 4.11-4.24 (4H, m), 4.48 (4H, dd, J = 8.4, 1.8 Hz), 4.93-5.00 (4H, m), 5.21 (4H, d, J = 3.5 Hz), 6.99 (2H, s), 7.52 (2H, s), 7.66-7.75 (2H, m), 7.78-7.87 (6H, m), 7.91 (1H, br s) , 8.01-8.08 (3H, br m), 8.54-8.60 (2H, br m).

Reference Example 10 Step 3
Reference Example 10 Compound RE10- was prepared using Compound D1 (0.1091 g, 0.044 mmol) synthesized in Step 2 and Compound RE2-3 (0.0748 g, 0.184 mmol) of Reference Example 2 in the same manner as in Step 3 of Reference Example 3. 3 was obtained as a crude product.
ESI-MS m / z: 1292 (M + 2H) ²⁺ , detected as de-DMTr body

Reference Example 10 Step 4
Reference Example 10 Compound RE10-3 (0.161 g, 0.05586 mmol) synthesized in step 3 was dissolved in dichloromethane (5 mL), succinic anhydride (manufactured by Tokyo Chemical Industry Co., Ltd., 0.1182 g, 1.181 mmol), N, N -Dimethylaminopyridine (0.0224 g, 0.183 mmol) and triethylamine (0.55 mL, 3.95 mmol) were added and stirred overnight at room temperature. The reaction mixture was ice-cooled, water was added, and the mixture was extracted twice with ethyl acetate, and then the organic layer was washed with saturated brine and dried over anhydrous magnesium sulfate. The solvent was distilled off under reduced pressure to obtain a crude product of compound RE10-4.
ESI-MS m / z: 1342 (M + 2H) ²⁺ , detected as de-DMTr body

Reference Example 10 Step 5
Reference Example 10 Compound RE10-4 synthesized in step 4 (0.0816 g, 0.02734 mmol), O- (benzotriazol-1-yl) -N, N, N ′, N′-tetramethyluronium hexafluorophosphate (0.0221 g, 0.05827 mmol) and diisopropylethylamine (0.02 mL, 0.1094 mmol) are dissolved in N, N-dimethylformamide (4 mL), LCAA-CPG (Chem Gene, 0.4882 g) is added, and the mixture is allowed to reach room temperature. Stir overnight. The mixture was filtered off, washed successively with dichloromethane, 10% methanol solution in dichloromethane and diethyl ether, and then reacted with acetic anhydride / pyridine solution to obtain compound C1 (49.5 μmol / g, yield 89%) . The yield was calculated from the introduction ratio to the solid phase carrier which can be calculated from the absorption derived from the DMTr group by adding 1% trifluoroacetic acid / dichloromethane solution to the solid phase carrier.

Reference Example 11

Compound RE 11-2 (1.050) was prepared according to the method described in Compound RE 11-1 (1.200 g, 3.640 mmol), Journal of Medicinal Chemistry, Volume 59, 2718-2733 (2016). g, 50% yield) was synthesized.
ESI-MS m / z: 582 (M + H) ⁺

Reference Example 12

Compound RE12-1 (4.000 g, 10.27 mmol) is dissolved in dichloromethane (60 mL), benzyl-2- (2-hydroxyethoxy) ethyl carbamate (2.700 g, 11.30 mmol), and trifluoromethanesulfonic acid (0.1360 mL, 1.541 mmol) was added and stirred overnight under reflux conditions. To the reaction mixture was added 10 wt% aqueous potassium carbonate solution, and the mixture was partitioned with dichloromethane, and then the organic phase was washed with water and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the solution was concentrated by displacement to 2-methyltetrahydrofuran. The residue was added dropwise to heptane, and the obtained crystals were filtered to give compound RE12-2 (5.130 g, yield 88%).
ESI-MS m / z: 570 (M + H) ⁺

Reference Example 13

Compound RE 13-1 (Compound RE 1-1, 898.0 mg, 2.007 mmol in Reference Example 1) was dissolved in dichloromethane (15 mL), 1-hydroxybenzotriazole monohydrate (338.0 mg, 2.208 mmol), 1- (1 Add 3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (343 mg, 2.208 mmol), and N-1-Z-l, 3-diaminopropane hydrochloride (0.4910 mL, 2.208 mmol), and add 3 hours at room temperature. It stirred. Water was added to the reaction solution, and the mixture was partitioned with dichloromethane, and then the organic layer was washed with a saturated aqueous sodium hydrogen carbonate solution and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography (chloroform / methanol = 90/10) to obtain Compound RE13-2 (873.0 mg, yield 68%).
ESI-MS m / z: 639 (M + H) ⁺

Reference Example 14

Reference Example 14 Step 1
Compound RE 14-1 (Compound RE 1-1 in Reference Example 1, 3.00 g, 6.70 mmol) was dissolved in dichloromethane (60 mL), and L-lysine benzyl ester di-p-toluenesulfonate (1.75 g, 3.02) at room temperature. mmol), triethylamine (0.935 mL, 6.70 mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (1.29 g, 6.70 mmol), 1-hydroxybenzotriazole monohydrate (103 mg, 0.670) mmol) was added and stirred for 2.5 hours. The reaction solution was washed with water and saturated aqueous sodium hydrogen carbonate solution, and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography (chloroform / methanol = 90/10) to quantitatively obtain Compound RE14-2.
ESI-MS m / z: 1096 (M + H) ⁺

Reference Example 14 Step 2
Compound RE14-2 (2.30 g, 2.10 mmol) was dissolved in tetrahydrofuran (46 mL), 10% palladium carbon powder (hydrous product, 54.29%; 424 mg) was added at room temperature, and the mixture was stirred overnight under a hydrogen atmosphere. The reaction solution was filtered, and the solvent was evaporated under reduced pressure to quantitatively obtain Compound RE14-3.
ESI-MS m / z: 1006 (M + H) ⁺

Reference Example 15

Reference Example 15 Step 1
Dissolve iminodiacetic acid (manufactured by Tokyo Chemical Industry Co., Ltd., 1.5 g, 6.43 mmol,) in methylene chloride (30 mL), pentafluorotrifluoroacetic acid (manufactured by Tokyo Chemical Industry Co., Ltd., 2.75 mL, 16.08 mmol), triethylamine (4.48) mL, 32.2 mmol) was added and stirred for 4 hours. To the reaction solution was added 10% aqueous citric acid solution, and the mixture was extracted with chloroform, and then the organic layer was washed with saturated brine and dried over anhydrous magnesium sulfate. Therein, a mixture of compound RE15-1 (compound RE11-2 in Reference Example 11; 2 g, 3.45 mmol) synthesized by the method described in Reference Example 11 in ethyl acetate (10 mL) and acetonitrile (10 mL) And catalytic hydrogen reduction with palladium / carbon. The solvent of the obtained solution was distilled off under reduced pressure to obtain a crude product of compound RE15-2.
ESI-MS m / z: 1091 (M + H) ⁺

Reference Example 15 Step 2
Compound RE15-2 (1.5 g, 1.37 mmol) was used to quantitatively obtain Compound RE15-3 in the same manner as in Step 2 of the synthesis of Compound RE1-4.
ESI-MS m / z: 990 (M + H) ⁺

Reference Example 16

Reference Example 16 Step 1
Using Compound RE16-1 (1.855 g, 3.19 mmol) synthesized by the method described in Reference Example 11 using N- (t-butoxycarbonyl) -L-glutamic acid (manufactured by Tokyo Chemical Industry Co., Ltd.), Reference Example 15 A crude purified product of compound RE16-2 was obtained in the same manner as in the step 1).
ESI-MS m / z: 1105 (M + H) ⁺

Reference Example 16 Step 2
Reference Example 1 Compound RE16-3 was quantitatively obtained in the same manner as in step 2 of the synthesis of compound RE1-4, using compound RE16-2 (1.421 g, 1.2866 mmol).
ESI-MS m / z: 1004 (M + H) ⁺

Reference Example 17

Compound RE17-1 (90 mg, 0.173 mmol) synthesized by the method described in Journal of Organic Chemistry, 74, 6837-6842, 2009, in tetrahydrofuran (1 mL) The mixture was dissolved in water, added with triphenylphosphine and polymer supported (Sigma Aldrich, 63 mg, 0.189 mmol), and stirred under heating reflux for 3 hours. The reaction mixture was filtered, and the solvent was evaporated under reduced pressure to give compound RE17-2 (70 mg, yield 82%).
ESI-MS m / z: 516 (M + Na) ⁺
¹ H-NMR (400 MHz, CDCl ₃ ): δ 0.89 (3H, s), 1.42-1.48 (2H, m), 1.52-1.61 (2H, m), 1.85 (1H, br s), 2.68 (2H , t, J = 7.2 Hz), 3.06-3.07 (2H, m), 3.39-3.44 (3H, m), 3.51-3.55 (3H, m), 3.78 (6H, s), 6.80-6.85 (4H, m) ), 7.17-7.23 (1 H, m), 7. 27-7. 33 (6 H, m), 7.41-7. 43 (2 H, m).

Reference Example 18

Reference Example 18 Step 1
A crude product (1.5 g) of compound RE18-2 was obtained in the same manner as in step 1 of Reference Example 2 using compound RE18-1 (manufactured by Tokyo Chemical Industry Co., Ltd., 0.500 g, 3.73 mmoL).
ESI-MS m / z: 435 (MH) ^-

Reference Example 18 Step 2
A compound RE18-3 was prepared by using the crude product of compound RE18-2 (1.5 g) and 1,4-Diaminobutane (Tokyo Chemical Industry Co., Ltd., 3.29 g, 37.3 mmol) in the same manner as in step 3 of Reference Example 3. 0.18 g, 2 step yield 10%) was obtained.
ESI-MS m / z: 551 (M + HCOO) ^-
¹ H-NMR (400 MHz, MeOD): δ 1.09 (3 H, s), 1.45-1. 52 (4 H, m), 2. 80 (2 H, t, J = 7.2 Hz), 2. 91 (2 H, s), 3.05 ( 1H, d, J = 8.8 Hz), 3.12-3.16 (4H, m), 3.24 (1H, s), 3.43 (1H, d, J = 10.8 Hz), 3.62-3.66 (7H, m), 6.71-6.76 (4H, m), 7.05-7.11 (1H, m), 7.12-7.20 (6H, m), 7.28-7. 32 (2H, m).

Reference Example 19

Compound RE19-1 (110 mg, 0.212 mmol) synthesized by the method described in Journal of Organic Chemistry, Vol. 74, pp. 6837-6842, 2009, in tetrahydrofuran (2 mL) Dissolved in water, N- (1R, 8S, 9s) -Bicyclo [6.1.0] non-4-yn-9-ylmethyloxycarbonyl-1,8-diamino-3,6-dioxaoctane (manufactured by Tokyo Chemical Industry Co., Ltd., 72 mg) , 0.222 mmol) was added and stirred at room temperature for 1 hour. Water was added to the reaction mixture, and the mixture was extracted with chloroform. The organic layer was washed with saturated brine and dried over anhydrous magnesium sulfate. The solvent was evaporated under reduced pressure, and the residue was purified by amino silica gel column chromatography (chloroform / methanol = 90/10) to obtain Compound RE19-2 (160 mg, yield 90%). ESI-MS m / z: 845 (M + H) ⁺
¹ H-NMR (400 MHz, CDCl ₃ ): δ 0.88 (3H, s), 0.91-1.09 (3H, m), 1.20-1.25 (1H, m), 1.52-1.59 (4H, m), 1.80- 1.85 (2H, m), 2.19-2.25 (4H, m), 2.59-2.68 (1H, m), 2.84-2.90 (4H, m), 3.02-3.11 (3H, m), 3.35-3.44 (5H, m) ), 3.49-3.53 (5H, m), 3.54-3.58 (2H, m), 3.62 (5H, s), 3.78 (6H, s), 4.13 (2H, d, J = 6.4 Hz), 4.21 (2H, s) t, J = 7.2 Hz), 6.79-6.84 (4H, m), 7.18-7.21 (1H, m), 7.24-7.27 (2H, m), 7.28-7.32 (4H, m), 7.39-7.44 (2H, m) m).

Reference Example 20

Reference Example 20 Step 1
The same method as in step 3 of Reference Example 3 using compound RE20-1 (AstaTech, 100 mg, 1.148 mmol) and Fmoc-Ser (tBuMe2Si) -OH (Watanabe Chemical Industry, 532 mg, 1.205 mmol) Thus, compound RE20-2 (410 mg, yield 70%) was obtained.
ESI-MS m / z: 511 (M + H) ⁺
¹ H-NMR (400 MHz, CDCl ₃ ): δ 0.06 (6 H, s), 0.90 (9 H, s), 2. 76-2.85 (1 H, m), 3.65-3. 86 (5 H, m), 4.02-4. 23 (3 H , m), 4.32-4.40 (4H, m), 5.55 (1H, d, J = 8.0 Hz), 7.31 (2H, t, J = 7.6 Hz), 7.40 (2H, t, J = 7.6 Hz), 7.59 (2H, d, J = 7.6 Hz), 7.76 (2H, d, J = 7.6 Hz).

Reference Example 20 Step 2
A crude product (680 mg) of a compound RE20-3 was obtained in the same manner as in step 1 of Reference Example 2 using the compound RE20-2 (410 mg, 0.803 mmol).
ESI-MS m / z: 814 (M + H) ⁺

Reference Example 20 Step 3
Using the crude product (680 mg) of compound RE20-3, compound RE20-4 (330 mg, 70% two-step yield) was obtained in the same manner as in step 5 of Reference Example 2.
ESI-MS m / z: 519 (M + H) ⁺
¹ H-NMR (400 MHz, CDCl ₃ ): δ 0.02-0.09 (6 H, m), 0.89 (9 H, d, J = 28.8 Hz), 2.84-2.94 (1 H, m), 3.24-3. 30 (2 H, m), 3.46 (1H, t, J = 7.2 Hz), 3.52-3.68 (2H, m), 3.75-3.80 (1H, m), 3.82 (6H, d, J = 2.4 Hz), 3.89-3.96 (1H , m), 4.05-4.17 (1H, m), 4.27-4.37 (1H, m), 6.82-6.89 (4H, m), 7.22-7.27 (1H, m), 7.29-7.34 (6H, m), 7.41 -7.45 (2H, m)

Reference Example 21

Reference Example 21 Step 1
Dissolve N- (Tert-butoxycarbonyl) -1,3-diaminopropane (Tokyo Chemical Industry Co., Ltd., 1.788 g, 10.26 mmol) in dichloromethane (22.8 mL), add triethylamine (1.907 mL, 13.68 mmol), and room temperature The mixture was stirred for 15 minutes. To the reaction solution is added dropwise a dichloromethane solution (5 mL) of a compound RE 21-1 (1.126 g, 6.84 mmol) synthesized by the method described in Organic Letter, 16: 6318-6321, 2014. And stirred at room temperature for 2 hours. Water was added to the reaction mixture, and the mixture was extracted with chloroform. The organic layer was washed with saturated brine and dried over anhydrous magnesium sulfate. The solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography (heptane / ethyl acetate = 35/65) to obtain compound RE21-2 (1.65 g, yield 80%).
ESI-MS m / z: 303 (M + H) ⁺

Reference Example 21 Step 2
Reference Example 1 Compound RE21-2 (1.10 g, 100% yield) was obtained in the same manner as in step 2 of the synthesis of compound RE1-4, using compound RE21-2 (1.65 g, 5.46 mmol).
ESI-MS m / z: 203 (M + H) ⁺
¹ H-NMR (400 MHz, CDCl ₃ ): δ 1.74 (2 H, dt, J = 12.0, 6.0 Hz), 2.95 (2 H, t, J = 6.0 Hz), 3.18 (1 H, s), 3.60 (2 H) , td, J = 6.0, 5.2 Hz), 7.54 (2H, dt, J = 8.4, 1.8 Hz), 7.76 (2H, dt, J = 8.4, 1.8 Hz), 7.97 (1 H, br s).

Reference Example 22

Reference Example 22 step 1
A crude product of compound RE22-2 was obtained in the same manner as in step 1 of Reference Example 2 using compound RE22-1 (manufactured by Tokyo Chemical Industry Co., Ltd., 1.2 g, 4.24 mmol).
ESI-MS m / z: 608 (M + Na) ⁺

Reference Example 22 Step 2
The crude product of compound RE22-2 was used to obtain compound R22-3 (1.34 g, 52% yield in two steps) by the method described in Step 5 of Reference Example 2 or Step 11 of Example 1.
ESI-MS m / z: 386 (M + Na) ⁺
¹ H-NMR (400 MHz, CDCl ₃ ): δ 3.34 (2 H, t, J = 6.4 Hz), 3. 47 (2 H, t, J = 6.4 Hz), 3. 79 (6 H, s), 6. 78-6. 84 (4 H , m), 7.17-7.21 (1 H, m), 7.27-7.35 (6 H, m), 7.42-7.46 (2 H, m).

Reference Example 22 Process 3
Compound RE22- was prepared by the same method as in step 3 of Reference Example 3 using compound RE22-3 (1.15 g, 3.16 mmol) and Fmoc-Ser (tBuMe2Si) -OH (manufactured by Watanabe Chemical Industries, Ltd., 1.677 g, 3.8 mmol). 4 (560 mg, 31% yield) was obtained.
1 H-NMR (400 MHz, CDCl ³ ) δ: 0.00-0.07 (6 H, m), 0.83-0.89 (9 H, m), 3.18-3.26 (2 H, m), 3.39-3. 46 (2 H, m), 3.61- 3.68 (1 H, m), 3. 76 (6 H, s), 3. 89 (1 H, dd, J = 10.0, 4.0 Hz), 4.03 (1 H, dd, J = 10.0, 4.0 Hz), 4.15-4.20 (1 H, m) , 4.22-4.28 (1H, m), 4.32-4.40 (2H, m), 5.65-5.88 (1H, m), 6.76-6.85 (4H, m), 7.16-7.23 (1H, m), 7.25-7.34 ( 8H, m), 7.36-7.44 (4H, m), 7.50-7.64 (2H, m), 7.72-7.79 (2H, m) ^.

Reference Example 23
The compounds RE23-1 to RE23-5 listed in Table Y-1 and Fmoc-Ser (tBuMe ₂ Si) -OH were listed in Table Y-2 in the same manner as in Reference Example 22. Compounds RE23-6 to RE23-10 were obtained.
The compound RE23-11 described in Table Y-2 was obtained in the same manner as in Reference Example 22 using the compound RE22-3 in Reference Example 22 and Fmoc-Thr (tBuMe ₂ Si) -OH.
The compound RE23-12 described in Table Y-2 is obtained in the same manner as in Reference Example 22 using the compound RE23-1 and Fmoc-Thr (tBuMe ₂ Si) -OH described in Table Y-1. The
The NMR analysis data of the compound synthesized according to this example is shown in Table Y-3.

Reference Example 24

Compound RE24-2 was obtained in the same manner as in Step 2 of Reference Example 2 using Compound RE24-1 (compound RE22-4 in Reference Example 22; 2.487 g, 3.16 mmol) synthesized by the method described in Reference Example 22. (1.2 g, 67% yield).
ESI-MS m / z: 587 (M + Na) ⁺
¹ H-NMR (400 MHz, CDCl ₃ ): δ-0.01-0.07 (6H, m), 0.86-0.90 (9H, m), 3.15-3.21 (2H, m), 3.41-3.48 (3H, m), 3.72 (1H, dd, J = 10.0, 6.4 Hz), 3.79 (6H, s), 3.84 (1H, dd, J = 10.0, 4.8 Hz), 6.79-6.84 (4H, m), 7.18-7.23 (1H, 1) m), 7.27-7.33 (6H, m), 7.40-7.44 (2H, m), 7.72-7.75 (1H, br m).

Reference Example 25
Compounds RE25-1 to RE25-7 described in Table Z-1 were obtained in the same manner as in Reference Example 24 using compounds RE23-6 to RE23-12 described in Table Y-2.
The mass spectrometry results of the compound synthesized according to this example are shown in Table Z-2.

Reference Example 26

Reference example 26 process 1
Compound RE26-1 (2.00 g, 9.47 mmol) was dissolved in N, N'-dimethylformamide (40 mL), iminodiacetic acid di-tert-butyl ester (5.11 g, 20.84 mmol) at room temperature, 1- ( 3-Dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (4.00 g, 20.84 mmol) and 1-hydroxybenzotriazole monohydrate (145 mg, 0.947 mmol) were added and stirred for 2 hours. Water was added to the reaction solution, and the mixture was extracted with ethyl acetate, and then the organic layer was washed with saturated aqueous sodium hydrogen carbonate solution and saturated brine, and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography (heptane / ethyl acetate = 50/50). Furthermore, slurry purification with methanol was performed to obtain compound RE26-2 (4.07 g, yield 65%).
ESI-MS m / z: 664 (M-H) ^-

Reference Example 26 Step 2
Compound RE26-2 (2.66 g, 4.00 mmol) was dissolved in tetrahydrofuran (53 mL), 10% palladium carbon powder (hydrous product, 54.29%; 490 mg) was added at room temperature, and stirred for 3 hours under a hydrogen atmosphere . The reaction mixture was filtered, and the solvent was evaporated under reduced pressure to give compound RE26-3 (2.86 g, yield 113%).
ESI-MS m / z: 634 (M-H) ^-

Reference Example 26 Step 3
The compound RE26-3 (871.0 mg, 1.370 mmol) was dissolved in N, N'-dimethylformamide (17 mL), O- (7-azabenzotriazol-1-yne) -N, N, N ', N' -Tetramethyluronium hexafluorophosphate (625.0 mg, 1.644 mmol), diisopropylethylamine (0.5730 mL, 3.290 mmol), and dodecanedioic acid monobenzyl ester (527.0 mg, 1.644 mmol) were added and stirred overnight at room temperature . Water was added to the reaction solution, and the mixture was extracted with ethyl acetate, and then the organic layer was washed with saturated aqueous sodium hydrogen carbonate solution and saturated brine and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography (heptane / ethyl acetate = 60/40) to give compound RE26-4 (1.030 g, yield 80%).
ESI-MS m / z: 939 (M + H) ⁺

Reference Example 26 Step 4
Compound RE26-4 (1.030 g, 1.098 mmol) was dissolved in dichloromethane (10 mL), trifluoroacetic acid (10.00 mL, 130.0 mmol) was added, and the mixture was stirred at room temperature for 1 hour. The solvent was evaporated under reduced pressure to give a crude product of compound RE26-5.
ESI-MS m / z: 713 (M-H) ^-

Reference Example 27

Reference Example 27 Step 1
Compound RE 27-1 (2.000 g, 12.98 mmol) is dissolved in N, N'-dimethylformamide (30 mL), potassium hydrogen carbonate (1.559 g, 15.57 mmol) and benzyl chloride (2.328 mL, 19.47 mmol) are added. The mixture was stirred at room temperature for 4 hours. Saturated ammonium chloride was added to the reaction solution, extraction was performed with dichloromethane, and then the organic layer was washed with water and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography (heptane / ethyl acetate = 50/50) to give compound RE27-2 (2.850 g, yield 90%).
ESI-MS m / z: 243 (M-H) ^-

Reference Example 27 Step 2
Compound RE27-2 (2.500 g, 10.24 mmol) is dissolved in N, N'-dimethylformamide (30 mL), potassium carbonate (5.660 g, 40.90 mmol) and tert-butyl bromoacetic acid (3.300 mL, 22.52 mmol) are added The mixture was stirred at 90 ° C. for 4 hours. Saturated ammonium chloride was added to the reaction solution, extraction was carried out with dichloromethane, and the organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography (heptane / ethyl acetate = 75/25) to give compound RE27-3 (4.300 g, yield 89%).
ESI-MS m / z: 472 (M-H) ^-

Reference Example 27 Step 3
Compound RE27-3 (1.000 g, 2.116 mmol) was dissolved in dichloromethane (10 mL), trifluoroacetic acid (10.00 mL, 130.0 mmol) was added, and the mixture was stirred at room temperature for 6 hours. The solvent was evaporated under reduced pressure to give a crude product of compound RE27-4.
ESI-MS m / z: 359 (M-H) ^-

Reference Example 27 Step 4
The compound RE27-4 (350.0 mg, 0.9710 mmol) is dissolved in N, N'-dimethylformamide (7 mL), 1-hydroxybenzotriazole monohydrate (327.0 mg, 2.137 mmol), 1- (3-dimethyl-dimethyl) Aminopropyl) -3-ethylcarbodiimide hydrochloride (410.0 mg, 2.137 mmol) and iminodiacetic acid di-tert-butyl ester (524.0 mg, 2.137 mmol) were added and stirred at room temperature for 5 hours. Water was added to the reaction solution, and the mixture was extracted with ethyl acetate, and then the organic layer was washed with saturated aqueous sodium hydrogen carbonate solution and saturated brine and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography (heptane / ethyl acetate = 60/40) to give compound RE27-5 (617.0 mg, yield 78%).
ESI-MS m / z: 814 (M-H) ^-

Reference Example 27 Step 5
Compound RE27-5 (610.0 mg, 0.7490 mmol) was dissolved in dichloromethane (6 mL), trifluoroacetic acid (6 mL, 78.00 mmol) was added, and the mixture was stirred at room temperature for 1 hour. The solvent was distilled off under reduced pressure to obtain a crude product of compound RE27-6.
ESI-MS m / z: 590 (M + H) ⁺

Reference Example 28

Reference Example 28 step 1
Compound RE28-1 (Compound RE 26-3, 474 mg, 0.744 mmol in Reference Example 26) synthesized by the method described in Reference Example 26 is dissolved in N, N'-dimethylformamide (10 mL), and a journal is prepared at room temperature. Trans-cyclohexane-1,4-dicarboxylic acid monobenzyl ester (0.234 mg, 0.893) synthesized by the method described in Journal of Medicinal Chemistry, Vol. 54, p. 2433-2446, 2011. mmol), diisopropylethylamine (0.312 mL, 1.79 mmol), O- (7-azabenzotriazol-1-yl) -N, N, N ', N'-tetramethyluronium hexafluorophosphate (339 mg, 0.893) The mmol) was added and stirred for 6 hours. Water was added to the reaction solution, and the mixture was extracted with ethyl acetate, and then the organic layer was washed with saturated aqueous sodium hydrogen carbonate solution and saturated brine, and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography (heptane / ethyl acetate = 50/50) to obtain Compound RE28-2 (448 mg, yield 68%).
ESI-MS m / z: 879 (M-H) ^-

Reference Example 28 Step 2
Compound RE28-2 (341 mg, 0.387 mmol) was dissolved in dichloromethane (3.4 mL), trifluoroacetic acid (3.4 mL) was added at room temperature, and the mixture was stirred overnight. The reaction solution was concentrated under reduced pressure, azeotroped with ethyl acetate, and slurry-purified with heptane to give compound RE28-3 (254 mg, yield 100%).
ESI-MS m / z: 656 (M + H) ⁺

Reference Example 29

Reference Example 29 Step 1
Compound RE 29-1 (500 mg, 2.75 mmol) is dissolved in N, N'-dimethylformamide (10 mL), and iminodiacetic acid di-tert-butyl ester (1.48 g, 6.04 mmol) at room temperature, 1- (3 -Dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (1.16 g, 6.04 mmol) and 1-hydroxybenzotriazole monohydrate (42.0 mg, 0.275 mmol) were added and stirred for 4 hours. Water was added to the reaction solution, and the mixture was extracted with ethyl acetate, and then the organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography (heptane / ethyl acetate = 50/50) to obtain Compound RE29-2 (329 mg, yield 19%).
ESI-MS m / z: 635 (M-H) ^-

Reference Example 29 Step 2
Compound RE29-2 (323 mg, 0.507 mmol) was dissolved in N, N'-dimethylformamide (6 v 5 mL) and potassium carbonate (84.0 mg, 0.609 mmol) at room temperature, benzyl bromoacetate (139 mg, 0.609 mmol) Was added and stirred for 3 hours. Water was added to the reaction solution, and the mixture was extracted with ethyl acetate, and then the organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography (heptane / ethyl acetate = 50/50) to obtain Compound RE29-3 (313 mg, yield 79%).
ESI-MS m / z: 783 (M-H) ^-

Reference Example 29 Step 3
Compound RE29-3 (312 mg, 0.398 mmol) was dissolved in dichloromethane (3.1 mL), trifluoroacetic acid (3.1 mL) was added at room temperature, and the mixture was stirred overnight. The reaction mixture was concentrated under reduced pressure and azeotroped with ethyl acetate to give compound RE29-4 (252 mg, quantitative).
ESI-MS m / z: 561 (M + H) ⁺

Reference Example 30

Reference Example 30 Step 1
Compound RE30-1 (2.00 g, 9.47 mmol) is dissolved in N, N'-dimethylformamide (40 mL), and 2-amino-1,3-propanediol (1.90 g, 20.84 mmol) at room temperature, 1- (3-Dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (4.00 g, 20.84 mmol) and 1-hydroxybenzotriazole monohydrate (145 mg, 0.947 mmol) were added and stirred for 2 hours. The solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography (ethyl acetate / methanol = 70/30). Furthermore, slurry purification with ethyl acetate was performed to obtain compound RE30-2 (2.68 g, yield 79%).
ESI-MS m / z: 356 (M-H) ^-

Reference Example 30 Step 2
Compound RE30-2 (500 mg, 1.40 mmol) is suspended in acetonitrile (10 mL), and acrylic acid tert-butyl ester (3.59 g, 28.0 mmol) at room temperature, benzyltrimethylammonium hydroxide (40% aqueous solution; 1.76 mL) , 702 mmol) was added and stirred overnight. The solvent was evaporated under reduced pressure, water was added, extraction was performed with ethyl acetate, and then the organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography (heptane / ethyl acetate = 50/50) to obtain compound RE30-3 (300 mg, yield 24%).
ESI-MS m / z: 871 (M + H) ⁺

Reference example 30 step 3
Compound RE30-3 (340 mg, 0391 mmol) was dissolved in tetrahydrofuran (6.8 mL), 10% palladium carbon powder (hydrous product, 54.29%; 31.3 mg) was added at room temperature, and stirred for 6 hours under a hydrogen atmosphere . The reaction mixture is filtered, the solvent is evaporated under reduced pressure, and the residue is purified by silica gel column chromatography (heptane / ethyl acetate = 30/70) to give compound RE30-4 (235 mg, yield 72%) I got
ESI-MS m / z: 841 (M + H) ⁺

Reference Example 30 Step 4
Compound RE30-4 (232 mg, 0.276 mmol) was dissolved in N, N'-dimethylformamide (4.6 mL), and dodecanoic acid monobenzyl ester (0.133 mg, 0.414 mmol) at room temperature, diisopropylethylamine (0.145 mL, 0.829) The mixture was added with mmol), O- (7-azabenzotriazol-1-yl) -N, N, N ′, N′-tetramethyluronium hexafluorophosphate (158 mg, 0.414 mmol) and stirred overnight. Water was added to the reaction solution, and the mixture was extracted with ethyl acetate, and then the organic layer was washed with saturated aqueous sodium hydrogen carbonate solution and saturated brine, and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography (heptane / ethyl acetate = 30/70) to obtain compound RE30-5 (274 mg, yield 87%).
ESI-MS m / z: 1141 (M-H) ^-

Reference Example 30 Step 5
Compound RE30-5 (273 mg, 0.239 mmol) was dissolved in dichloromethane (2.7 mL), trifluoroacetic acid (2.7 mL) was added at room temperature, and the mixture was stirred overnight. The reaction mixture was concentrated under reduced pressure and azeotroped with ethyl acetate to give compound RE30-6 (231 mg, quantitative).
ESI-MS m / z: 919 (M + H) ⁺

Reference Example 31

Reference Example 31 step 1
Dissolve 4-nitroisophthalic acid RE31-1 (500 mg, 2.37 mmol) and N-Boc-ethylenediamine (808 mg, 5.21 mmol) in N, N'-dimethylformamide (10 mL) and use triethylamine (0.90) at room temperature. Add 1 mL (7.11 mmol), 1-hydroxybenzotriazole monohydrate (703 mg, 5.21 mmol) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (1.36 g, 7.11 mmol) to Stir for hours. The reaction mixture was worked up, and the crude product was purified by silica gel column chromatography to obtain compound RE31-2 (650 mg, yield 55%).

Reference Example 31 Step 2
Compound RE 31-2 (500 mg, 1.01 mmol) and zinc powder (330 mg, 5.05 mmol) are suspended in methanol (3.5 mL) and tetrahydrofuran (3.5 mL), and ammonium chloride (378 mg, 7.07 mmol) at 0 ° C. The aqueous solution was added dropwise and stirred at room temperature for 24 hours. The reaction mixture was worked up, and the crude product was purified by silica gel column chromatography to obtain compound RE31-3 (160 mg, 34% yield).

Reference Example 31 Step 3
Compound RE31-3 (200 mg, 0.430 mmol) and N-benzyloxycarbonyl-glycine (benzyloxycarbonyl is also described as Cbz) (90.0 mg, 0.430 mmol) with N, N'-dimethylformamide (2.0 mL) Dissolved in water, and at room temperature diisopropylethylamine (0.220 mL, 1.29 mmol), O- (7-azabenzotriazol-1-yl) -N, N, N ', N'-tetramethyluronium hexafluorophosphate ( Add 245 mg, 0.645 mmol) and stir for 16 hours. The reaction mixture was worked up, and the crude product was purified by silica gel column chromatography to obtain compound RE31-4 (180 mg, yield 64%).
ESI-MS m / z: 657 (M + H) ⁺

Reference Example 32

Reference example 32 process 1
Dissolve 3,5-dinitrobenzoic acid RE 32-1 (500 mg, 2.36 mmol) and N-Cbz-ethylenediamine (588 mg, 2.83 mmol) in N, N'-dimethylformamide (5.0 mL) and use triethylamine at room temperature Add (0.65 mL, 4.72 mmol), 1-Hydroxybenzotriazole monohydrate (380 mg, 2.83 mmol) and 1- (3-Dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (675 mg, 3.54 mmol) Stir for 16 hours. The reaction mixture was worked up, and the crude product was purified by silica gel column chromatography to obtain compound RE32-2 (445 mg, yield 48%).

Reference example 32 process 2
Compound RE32-2 (200 mg, 0.515 mmol) is dissolved in ethanol (5.0 mL), tin (II) chloride (584 mg, 3.09 mmol) and concentrated hydrochloric acid (0.2 mL) are added at room temperature, and Stir for hours. The reaction mixture was worked up to give compound RE32-3 (180 mg, quantitative).
ESI-MS m / z: 329 (M + H) ⁺

Reference Example 33

Reference Example 33, Step 1
A compound RE33-2 (3.7% by a method similar to step 4 of Reference Example 3) was synthesized using Compound RE33-1 (the compound RE3-2 in Reference Example 3 8.17 g, 23.12 mmol) synthesized by the method described in Reference Example 3. g, yield 63%).
ESI-MS m / z: 254 (M + H) ⁺

Reference Example 33 Step 2
The compound RE33-3 (3.82 g, yield 67%) was obtained in the same manner as in the step 5 of Reference Example 3 using the compound RE33-2 (3.7 g, 14.63 mmol).
ESI-MS m / z: 432 (M + HCOO) ^-

Reference Example 33 Step 3
The compound RE33-4 (3.08 g, yield 87%) was obtained in the same manner as in Step 2 of Reference Example 1 using the compound RE33-3 (3.82 g, 9.86 mmol).
ESI-MS m / z: 360 (M + H) ⁺

Reference Example 34

Reference Example 34 Step 1
Compound RE34-1 was prepared by the same method as in step 1 of Reference Example 3 using compound RE34-1 (2 g, 9.53 mmol) and tert-butoxycarbonylamino) -1-pentanol (manufactured by Tokyo Chemical Industry Co., Ltd., 2 g, 10 mmol). (2.40 g, 63% yield) was obtained.
ESI-MS m / z: 296 (M + H) ⁺ , detected as de-Boc derivative

Reference Example 34 Step 2
Compound RE34-2 (1.579 g, yield 21%) was obtained in the same manner as in Steps 2 to 4 of Reference Example 3 and Steps 1 to 4 of Reference Example 4 using Compound RE 34-2.
ESI-MS m / z: 910 (M + H) ⁺

Reference Example 35: Synthesis of Compound D2

Reference Example 35, Step 1
Compound RE 35-1 (Compound RE 11-2 in Reference Example 11, 1.015 g, 1.748 mmol) synthesized by the method described in Reference Example 11 was dissolved in N, N′-dimethylformamide (12 mL), % Palladium carbon powder (hydrated product, 54.29%; 187 mg) was added and stirred for 6 hours under hydrogen atmosphere. The reaction solution was filtered. Compound RE26-5 (250.0 mg, 0.350 mmol) synthesized in Reference Example 26, 1-hydroxybenzotriazole monohydrate (26.80 mg, 0.1750 mmol), and 1- (3-dimethylaminopropyl) -3 in the filtrate. -Ethyl carbodiimide hydrochloride (402.0 mg, 2.099 mmol) was added and stirred at room temperature overnight. Water was added to the reaction solution, and the mixture was extracted with ethyl acetate, and then the organic layer was washed with saturated aqueous sodium hydrogen carbonate solution and saturated brine and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography (chloroform / methanol = 87/13) to obtain compound RE35-2 (617.0 mg, yield 88%).
ESI-MS m / z: 1215 (M + 2 H) ²⁺

Reference Example 35 Step 2
Compound RE35-2 (0.7380 g, 0.3040 mmol) was dissolved in tetrahydrofuran (7 mL), 10% palladium carbon powder (hydrous product, 54.29%; 135.90 mg) was added at room temperature, and the mixture was stirred overnight under a hydrogen atmosphere. The reaction mixture was filtered, the solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography (chloroform / methanol = 87/13) to give compound D2 (581 mg, yield 82%) .
ESI-MS m / z: 1170 (M + 2 H) ²⁺
¹ H-NMR (400 MHz, DMSO-d6, δ): 1.12-2.36 (106H, m), 2.91-3.19 (8H, m), 3.23-3.55 (14H, m), 3.60-3.76 (4H, m) , 3.78-3.94 (8H, m), 3.95-4.10 (16H, m), 4.47 (4H, d, J = 8.8 Hz), 4.92-5.01 (4H, m), 5.17-5.24 (4H, m), 6.98 (1H, s), 7.64 (2H, s), 7.81-7.95 (4H, m), 8.28-8.38 (2H, m), 8.44-8.56 (2H, m), 10.13 (1H, s)

Reference Example 36: Synthesis of Compound D3

Reference Example 36, Step 1
Compound RE36-1 (Compound RE 12-2 in Reference Example 12, 500 mg, 0.879 mmol) synthesized by the method described in Reference Example 12 was dissolved in N, N'-dimethylformamide (6.5 mL), % Palladium carbon powder (hydrous, 54.29%; 94 mg) was added and stirred for 4 hours under hydrogen atmosphere. The reaction solution was filtered. The compound RE26-5 (126.0 mg, 0.176 mmol) in Reference Example 26, 1-hydroxybenzotriazole monohydrate (13.47 mg, 0.088 mmol), and 1- (3-dimethylaminopropyl) -3-ethyl were added to the filtrate. Carbodiimide hydrochloride (202.0 mg, 1.055 mmol) was added and stirred overnight at room temperature. The reaction solution was evaporated under reduced pressure, the solvent was evaporated, and the residue was purified by reverse phase column chromatography (water / acetonitrile) to obtain compound RE36-2 (249.7 mg, yield 60%).
ESI-MS m / z: 1191 (M + 2 H) ²⁺

Reference Example 36 Step 2
Compound RE36-2 (0.242 g, 0.102 mmol) is dissolved in tetrahydrofuran (3.6 mL) and water (1.2 mL), 10% palladium on carbon powder (hydrated product, 54.29%; 45 mg) is added at room temperature, and a hydrogen atmosphere is added. It stirred under 4 hours. The reaction mixture was filtered, and the solvent was evaporated under reduced pressure to give compound D3 (216 mg, yield 93%).
ESI-MS m / z: 1146 (M + 2 H) 2+
1 H-NMR (400 MHz, DMSO-d6, δ): 1.15-1.65 (20 H, m), 1.68-2.15 (52 H, m), 3.13-3.29 (6 H, m), 3.40-3.67 (16 H, m), 3.71-3.96 (11H, m), 3.98-4.14 (16H, m), 4.55 (4H, t, J = 8.8 Hz), 4.93-5.06 (4H, m), 5.12-5.28 (4H, m), 6.56 (6) 1H, s), 6.98 (1H. S), 7.64 (2H, s), 7.77-7.93 (4H, m), 8.26-8.49 (3H, m), 10.10 (1H, s)

Reference Example 37: Synthesis of Compound D4

Reference Example 37 Step 1
Compound RE 37-1 (Compound RE 13-2 in Reference Example 13, 430 mg, 0.674 mmol) synthesized by the method described in Reference Example 13 was dissolved in N, N′-dimethylformamide (6 mL), % Palladium carbon powder (water content, 54.29%; 79 mg) was added and stirred for 4 hours under hydrogen atmosphere. The reaction solution was filtered. The compound RE26-5 (105.0 mg, 0.148 mmol) in Reference Example 26, 1-hydroxybenzotriazole monohydrate (11. 31 mg, 0.074 mmol), and 1- (3-dimethylaminopropyl) -3-ethyl were added to the filtrate. Carbodiimide hydrochloride (170.0.0 mg, 0.887 mmol) was added and stirred overnight at room temperature. The reaction mixture was concentrated under reduced pressure, the solvent was evaporated, and the residue was purified by reverse phase column chromatography (water / acetonitrile) to give compound RE37-2 (218.1 mg, yield 56%).
ESI-MS m / z: 1329 (M + 2 H) ²⁺

Reference Example 37 Step 2
Compound RE37-2 (0.210 g, 0.079 mmol) is dissolved in tetrahydrofuran (3.1 mL) and water (1.0 mL), 10% palladium carbon powder (hydrous, 54.29%; 39 mg) is added at room temperature, and a hydrogen atmosphere is added. It stirred under 4 hours. The reaction mixture was filtered, and the solvent was evaporated under reduced pressure to give compound D4 (192.7 mg, yield 95%).
ESI-MS m / z: 1284 (M + 2 H) 2+
1 H-NMR (400 MHz, DMSO-d6, δ): 1.17-1.65 (42 H, m), 1.69-2.13 (61 H, m), 2.95-3.17 (16 H, m), 3.65-3. 77 (3 H, m), 3.79-3.94 (6H, m), 3.96-4.10 (16H, m), 4.48 (4H, d, J = 8.4 Hz), 4.96 (4H, dd, J = 2.4, 11.2 Hz), 5.21 (4H, d, J = 3.2 Hz), 7.01 (1 H, s), 7.64-7.92 (11 H, m), 8.26-8.48 (4 H, m), 10.14 (1 H, s)

Reference Example 38: Synthesis of Compound D5

Reference Example 38, Step 1
Compound RE38-1 (Compound RE 12-2 in Reference Example 12 450 mg, 0.791 mmol) synthesized by the method described in Reference Example 12 was dissolved in N, N′-dimethylformamide (6 mL), % Palladium carbon powder (water content, 54.29%; 85 mg) was added and stirred for 5 hours under hydrogen atmosphere. The reaction solution was filtered. The compound RE27-6 (94 mg, 0.158 mmol) in Reference Example 27, 1-hydroxybenzotriazole monohydrate (133.0 mg, 0.871 mmol), and 1- (3-dimethylaminopropyl) -3-ethyl were added to the filtrate. Carbodiimide hydrochloride (182.0 mg, 0.950 mmol) was added and stirred overnight at room temperature. The reaction solution was evaporated under reduced pressure, the solvent was evaporated, and the residue was purified by reverse phase column chromatography (water / acetonitrile) to obtain compound RE38-2 (99 mg, yield 28%).
ESI-MS m / z: 1129 (M + 2 H) ²⁺

Reference Example 38 Step 2
Compound RE38-2 (80 mg, 0.035 mmol) is dissolved in tetrahydrofuran (1.7 mL) and water (0.85 mL), 10% palladium carbon powder (hydrous, 54.29%; 26 mg) is added at room temperature, and a hydrogen atmosphere is added. It stirred under 2 hours. The reaction mixture was filtered, and the solvent was evaporated under reduced pressure to give compound D5 (57.5 mg, yield 75%).
ESI-MS m / z: 1084 (M + 2 H) ²⁺
¹ H-NMR (400 MHz, DMSO-d6, δ): 1.69-2.21 (46H, m), 3.14-3.65 (28H, m), 3.67-4.22 (27H, m), 4.44-4.66 (4H, m) , 4.69-4.88 (4H, m), 4.89-5.08 (4H, m), 5.12-5.32 (4H, m), 6.54-6.68 (1H, br), 7.01 (2H, s), 7.78-8.09 (3H, m), 8.13-8.31 (2H, m), 8.58-8.75 (2H, m)

Reference Example 39: Synthesis of Compound D6

Reference Example 39 Step 1
Compound RE 39-1 (Compound RE 13-2, 418 mg, 0.655 mmol in Reference Example 13) synthesized by the method described in Reference Example 13 was dissolved in N, N′-dimethylformamide (6 mL), % Palladium carbon powder (hydrous, 54.29%; 77 mg) was added and stirred for 5 hours under hydrogen atmosphere. The reaction solution was filtered. The compound RE27-6 (85 mg, 0.144 mmol), 1-hydroxybenzotriazole monohydrate (121.0 mg, 0.791 mmol), and 1- (3-dimethylaminopropyl) -3 which were synthesized in Reference Example 27 were added to the filtrate. -Ethyl carbodiimide hydrochloride (165.0 mg, 0.863 mmol) was added and stirred at room temperature overnight. The reaction solution was evaporated under reduced pressure, the solvent was evaporated, and the residue was purified by reverse phase column chromatography (water / acetonitrile) to give compound RE39-2 (99 mg, yield 28%).
ESI-MS m / z: 1268 (M + 2 H) ²⁺

Reference Example 39 Step 2
Compound RE39-2 (186 mg, 0.073 mmol) is dissolved in tetrahydrofuran (2.8 mL) and water (0.93 mL), 10% palladium carbon powder (hydrous, 54.29%; 40 mg) is added at room temperature, and a hydrogen atmosphere is added. It stirred under 2 hours. The reaction mixture was filtered, and the solvent was evaporated under reduced pressure to give compound D6 (156.7 mg, yield 87%).
ESI-MS m / z: 1222 (M + 2H) ²⁺
¹ H-NMR (400 MHz, DMSO-d6, δ): 1.36-1.62 (27 H, m), 1.67-2.17 (64 H, m), 2.92-3. 21 (15 H, m), 3.58-3. 77 (2 H, m) , 3.80-3.95 (7H, m), 3.97-4.13 (15H, m), 4.47 (4H, d, J = 8.8 Hz), 4.88-5.02 (7H, m), 5.10-5.24 (3H, m), 6.95 -7.00 (1H, m), 7.26-7.31 (2H, m), 7.72-7.88 (8H, m), 8.10-8.20 (2H, m), 8.51-8.60 (2H, m)

Reference Example 40

Reference Example 40, Step 1
Compound D5 (171 mg, 0.079 mmol) synthesized by the method described in Reference Example 38 is dissolved in N, N'-dimethylformamide (3.4 mL), and glycine benzyl p-toluenesulfonate (32.0 mg, 0.095 mmol) , 1-hydroxybenzotriazole monohydrate (12.09 mg, 0.079 mmol), and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (18.16 mg, 0.095 mmol) were added and stirred overnight at room temperature . The reaction solution was evaporated under reduced pressure, the solvent was evaporated, and the residue was purified by reverse phase column chromatography (water / acetonitrile) to obtain compound RE40-2 (55.7 mg, yield 31%).
ESI-MS m / z: 1158 (M + 2 H) 2+

Reference Example 40 Step 2
Compound RE40-2 (54 mg, 0.023 mmol) is dissolved in tetrahydrofuran (0.83 mL) and water (0.28 mL), 10% palladium carbon powder (hydrous, 54.29%; 18 mg) is added at room temperature, and a hydrogen atmosphere is added. It stirred under 2 hours. The reaction mixture was filtered, and the solvent was evaporated under reduced pressure to give compound RE40-3 (50.1 mg, yield 97%).
ESI-MS m / z: 1112 (M + 2 H) 2+
1 H-NMR (400 MHz, DMSO-d6, δ): 0.96-1.06 (3H, m), 1.71-2.20 (54H, m), 3.41-3.64 (15H, m), 3.68-4.20 (32H), 4.55 ( 4H, d, J = 8.4 Hz), 4.81 (4H, s), 4.94-5.02 (4H, m), 5.17-5.25 (4H, m), 6.63-6.76 (2H, m), 6.93-7.02 (2H, 2) m), 7.84-8.00 (3H, m), 8.17-8.30 (2H, m), 8.58-8.70 (2H, m)

Reference Example 41: Synthesis of Compound D7

Reference Example 41 Step 1
Compound RE 41-1 (500 mg, 0.861 mmol) is dissolved in N, N'-dimethylformamide (10 mL), 10% palladium carbon powder (hydrous, 54.29%; 92.1 mg) is added at room temperature, and a hydrogen atmosphere is added. It stirred under 2 hours. Compound RE27-3 (113 mg, 0.172 mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (198 mg, 1.03 mmol), synthesized in Reference Example 27 at room temperature under an argon atmosphere, 1 -Hydroxybenzotriazole monohydrate (13.2 mg, 0.086 mmol) was added and stirred overnight. The reaction solution is filtered through celite, the solvent is evaporated under reduced pressure, and the residue is purified by silica gel column chromatography (chloroform / methanol = 90/10) and further purified by reverse phase preparative HPLC (acetonitrile / water) As a result, compound RE41-2 (195 mg, yield 48%) was obtained.
ESI-MS m / z: 1186 (M + 2 H) ²⁺

Reference Example 41 Step 2
Compound RE 41-2 (194 mg, 0.082 mmol) is dissolved in tetrahydrofuran (2.9 mg) and water (1.0 mL), 10% palladium carbon powder (hydrous, 54.29%; 35.7 mg) is added at room temperature, and a hydrogen atmosphere is added. Stirred for 8 hours below. The reaction mixture was filtered, and the solvent was evaporated under reduced pressure to give compound D7 (183 mg, yield 98%).
ESI-MS m / z: 1141 (M + 2 H) ²⁺
¹ H-NMR (400 MHz, DMSO-d6, δ): 1.21-1.45 (m, 40H), 1.76-2.18 (m, 50H), 3.00-3.09 (m, 8H), 3.40-4.20 (m, 32H) , 4.47 (d, J = 8.5 Hz, 4 H), 4. 96 (dd, J = 3.1, 11.2 Hz, 4 H), 5.21 (d, J = 3.1 Hz, 4 H), 6. 98 (s, 1 H), 7. 65 (s, 2H), 7.84 (d, J = 9.0 Hz, 4H), 8.31 (brs, 1H), 8.44 (brs, 1H), 10.11 (s, 1H).

Reference Example 42: Synthesis of Compound D8

Reference Example 42 Step 1
Compound RE 42-1 (Compound RE 11-2 in Reference Example 11, 500 mg, 0.861 mmol) synthesized by the method described in Reference Example 11 was dissolved in N, N′-dimethylformamide (10 mL), and 10 % Palladium carbon powder (hydrous, 54.29%; 92.1 mg) was added and stirred for 2 hours under hydrogen atmosphere. Compound RE29-4 (97.0 mg, 0.172 mmol) synthesized in Reference Example 29 at room temperature under an argon atmosphere, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (198 mg, 1.03 mmol), 1 -Hydroxybenzotriazole monohydrate (13.2 mg, 0.086 mmol) was added and stirred overnight. The reaction solution is filtered through celite, the solvent is evaporated under reduced pressure, and the residue is purified by silica gel column chromatography (chloroform / methanol = 85/15) and further purified by reverse phase preparative HPLC (acetonitrile / water) Thus, compound RE42-2 (179 mg, yield 46%) was obtained.
ESI-MS m / z: 1138 (M + 2 H) ²⁺

Reference Example 42 Step 2
Compound RE42-2 (175 mg, 0.077 mmol) is dissolved in tetrahydrofuran (2.6 mg) and water (0.9 mL), 10% palladium on carbon powder (hydrated product, 54.29%; 32.4 mg) is added at room temperature, and a hydrogen atmosphere is added. It stirred below for 1 hour. The reaction mixture was filtered, and the solvent was evaporated under reduced pressure to give compound D8 (160 mg, yield 95%).
ESI-MS m / z: 1093 (M + 2 H) ²⁺
¹ H-NMR (400 MHz, DMSO-d6, δ): 1.23-1.45 (m, 32H), 1.77 (s, 12H), 1.89 (s, 12H), 1.99 (s, 12H), 2.10 (s, 12H) ), 3.01-3.11 (m, 8H), 3.69-4.02 (m, 34H), 4.47-4.50 (m, 4H), 4.94-4.98 (m, 4H), 5.21 (d, J = 3.1 Hz, 4H), 6.92 (s, 1 H), 6. 94 (s, 2 H), 7. 87 (d, J = 9.4 Hz, 2 H), 7. 92 (d, J = 8.5 Hz, 2 H), 8.33 (brs, 2 H), 8. 50 (brs, 2 H ).

Reference Example 43: Synthesis of Compound D9

Reference Example 43 Step 1
Compound RE43-1 (compound RE13-2 in Reference Example 13, 500 mg, 0.784 mmol) synthesized by the method described in Reference Example 13 was dissolved in N, N'-dimethylformamide (10 mL), % Palladium carbon powder (hydrous, 54.29%; 92.1 mg) was added and stirred for 2 hours under hydrogen atmosphere. Compound RE30-6 (144 mg, 0.157 mmol) synthesized in Reference Example 30 at room temperature under an argon atmosphere, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (180 mg, 0.941 mmol), 1 -Hydroxybenzotriazole monohydrate (12.0 mg, 0.078 mmol) was added and stirred overnight. The reaction solution is filtered through celite, the solvent is evaporated under reduced pressure, and the residue is purified by silica gel column chromatography (chloroform / methanol = 80/20) and further purified by reverse phase preparative HPLC (acetonitrile / water) As a result, compound RE43-2 (191 mg, yield 43%) was obtained.
ESI-MS m / z: 1431 (M + 2 H) ²⁺

Reference Example 43 Step 2
Compound RE43-2 (186 mg, 0.065 mmol) is dissolved in tetrahydrofuran (2.8 mg) and water (0.9 mL), 10% palladium carbon powder (hydrous, 54.29%; 34.3 mg) is added at room temperature, and a hydrogen atmosphere is added. It stirred below for 1 hour. The reaction mixture was filtered, and the solvent was evaporated under reduced pressure to give compound D9 (174 mg, yield 97%).
ESI-MS m / z: 1386 (M + 2 H) ²⁺
¹ H-NMR (400 MHz, DMSO-d6, δ): 1.25-4.02 (m, 164 H), 4.18-4.26 (m, 2 H), 4.47 (d, J = 8.1 Hz, 4 H), 4.96 (dd, J = 3.1, 11.2 Hz, 4 H), 5.21 (d, J = 3.6 Hz, 4 H), 7.74-9.91 (m, 13 H), 8. 18 (s, 2 H), 8. 36 (d, J = 7.2 Hz, 2 H), 10.27 (brs, 1H).

Reference Example 44: Synthesis of Compound A2

Reference Example 44, Step 1
Compound RE44-1 (Compound RE 31-4, 150 mg, 0.228 mmol in Reference Example 31) synthesized by the method described in Reference Example 31 was dissolved in dichloromethane (1.5 mL), and trifluoroacetic acid (1.5 mL) was added at room temperature. Was added and stirred for 4 hours. The reaction solution was concentrated under reduced pressure and azeotroped with ethyl acetate. The residue was dissolved in N, N'-dimethylformamide (3 mL), and Compound RE14-3 of Reference Example 14 (574 mg, 0.571 mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide was added at room temperature. The hydrochloride (109 mg, 0.571 mmol), triethylamine (0.159 mL, 1.14 mmol) and 1-hydroxybenzotriazole monohydrate (3.50 mg, 0.023 mmol) were added and stirred overnight. The reaction solution is concentrated under reduced pressure, and the residue is purified by silica gel column chromatography (chloroform / methanol = 90/10) and further purified by reverse phase preparative HPLC (acetonitrile / water) to give compound RE44-2 (242 mg) , Yield 44%).
ESI-MS m / z: 1216 (M + 2 H) ²⁺

Reference Example 44 Step 2
Compound RE44-2 (242 mg, 0.100 mmol) is dissolved in tetrahydrofuran / water (4/1; 12 mL), 10% palladium carbon powder (hydrous, 54.29%; 44.6 mg) is added at room temperature, and a hydrogen atmosphere is added. It stirred under 2 hours. 6-maleimidohexanoic acid (23.2 mg, 0.110 mmol), 4- (4,6-dimethoxy-1,3,5-triazin-2-yl) -4-methylmorpholinium chloride at room temperature under an argon atmosphere 55.2 mg, 0.199 mmol) was added and stirred overnight. The reaction mixture was filtered through celite, the solvent was evaporated under reduced pressure, and the residue was purified by HP20 resin (acetone / water) to give compound A2 (97.4 mg, yield 39%).
ESI-MS m / z: 1245 (M + 2 H) ²⁺
¹ H-NMR (400 MHz, DMSO-d6, δ): 1.14-2.16 (100H, m), 2.96-2.98 (4H, m), 3.24-3.41 (18H, m), 3.69-3.73 (4H, m) , 3.83-3.91 (6H, m), 4.14-4.16 (2H, m), 4.47 (4H, d, J = 8.6 Hz), 4.96 (4H, dd, J = 3.6, 11.3 Hz), 5.21 (4H, d) , J = 3.2 Hz), 7.01 (2H, s), 7.73-7.75 (2H, m), 7.83-7.94 (7H, m), 8.05-8.08 (2H, m), 8.14-8.20 (3H, m), 8.55-8.56 (2H, m), 10.24 (1H, brs).

Reference Example 45: Synthesis of Compound A3

Reference Example 45 Step 1
Compound RE 45-1 (Compound RE 32-3, 75.0 mg, 0.228 mmol in Reference Example 32) synthesized by the method described in Reference Example 32 is dissolved in N, N'-dimethylformamide (3.0 mL), and the reaction is performed at room temperature Compound RE 14-3 of Example 14 (574 mg, 0.571 mmol), diisopropylethylamine (0.199 mL, 1.14 mmol), O- (7-azabenzotriazol-1-yl) -N, N, N ′, N′-tetra Methyluronium hexafluorophosphate (217 mg, 0.571 mmol) was added and stirred overnight. The solvent is distilled off under reduced pressure, and the residue is purified by silica gel column chromatography (chloroform / methanol = 90/10) and further purified by reverse phase preparative HPLC (acetonitrile / water) to give compound RE 45-2 202 mg (yield 38%) were obtained.
ESI-MS m / z: 1152 (M + 2 H) ²⁺

Reference Example 45 Step 2
Compound RE 45-2 (196 mg, 0.085 mmol) is dissolved in tetrahydrofuran / water (4/1; 10 mL), 10% palladium carbon powder (hydrous, 54.29%; 36.1 mg) is added at room temperature, and a hydrogen atmosphere is added. It stirred under 2 hours. 6-maleimidohexanoic acid (19.8 mg, 0.094 mmol), 4- (4,6-dimethoxy-1,3,5-triazin-2-yl) -4-methylmorpholinium chloride at room temperature under an argon atmosphere 47.0 mg, 0.170 mmol) was added and stirred overnight. The reaction mixture was filtered through celite, the solvent was evaporated under reduced pressure, and the residue was purified by HP20 resin (acetone / water) to give compound A3 (42.4 mg, yield 21%).
ESI-MS m / z: 1182 (M + 2 H) ²⁺

Reference Example 46: Compound D10

Using compound RE46-1 (compound RE34-3 in Reference Example 34) synthesized by the method described in Reference Example 34, compound D10 (2.2 g, yield 58%) in the same manner as in Steps 1 and 2 of Reference Example 10 Got).
ESI-MS m / z: 2437 (M + H) ⁺

Reference Example 47: Synthesis of Compound A4

Reference Example 47 Step 1
Using compound RE47-1 (compound RE33-4 in Reference Example 33, 0.101 g, 0.282 mmol) synthesized by the method described in Reference Example 33, Compound RE15-2 (0.607 g, 0.613 mmol) of Reference Example 15 was synthesized. The compound RE47-2 (0.25 g, yield 39%) was obtained by the same method as used in Step 3 of Reference Example 3.
ESI-MS m / z: 2304 (M + H) ⁺

Reference Example 47 Step 2
Using compound RE47-2 (0.255 g, 0.111 mmol), compound RE47-3 (0.15 g, yield 63%) was obtained by the same method as in step 2 of Reference Example 3.
ESI-MS m / z: 2170 (M + H) ⁺

Reference Example 47 Step 3
Using compound RE47-3 (20.8 mg, 9.59 μmol), compound A4 (5.5 mg, 24% yield) was obtained by the same method as in step 2 of Reference Example 5.
ESI-MS m / z: 1182 (M + 2H) ²⁺

Reference Example 48: Synthesis of Compound A5

Reference Example 48, Step 1
Compound RE16-3 (0.618 g synthesized in the step 2 of Reference Example 16) using Compound RE48-1 synthesized by the method described in Reference Example 33 (Compound RE 33-4 in the Reference Example 33, 0.099 g, 0.277 mmol) Compound RE48-2 (0.343 g, yield 53%) was obtained by the same method as in step 3 of Reference Example 3 using ,, 0.615 mmol).
ESI-MS m / z: 2333 (M + H) ⁺

Reference Example 48 Step 2
Using compound RE48-2, compound A5 (6.9 mg, yield 28%) was obtained in the same manner as in Steps 1 and 2 of Reference Example 10.
ESI-MS m / z: 2392 (M + H) ⁺

Reference Example 49: Synthesis of Compound A6

A compound A6 was synthesized by the same method as the synthesis of the compound A1 of Reference Example 8 using the compound RE49-1 (0.048 g, 0.021 mmol) and N-succinimidyl 3-maleimidopropionate (manufactured by Tokyo Chemical Industry Co., Ltd., 0.017 g, 0.064 mmol). (0.040 g, 78% yield) was obtained.
ESI-MS m / z: 2480 (M + HCOO) ^-

Reference Example 50: Synthesis of Compound A7

Step 131
Reference Example using Compound D1 (23.6 mg, 9.46 μmol) synthesized by the method described in Reference Example 10 and N- (2-aminoethyl) maleimido trifluoroacetate (7.21 mg, 0.028 μmol, manufactured by Sigma-Aldrich Co.) Compound A7 (9.1 mg, yield 36%) was obtained in the same manner as in Step 3 of 3.
ESI-MS m / z: 1310 (M + 2H) ²⁺

Reference Example 51

Reference Example 51 step 1
Compound RE51-1 (0.122 g, 0.054 mmol) synthesized by the method described in Reference Example 7 and the method described in Bioconjugate Chemistry, Vol. 22, pp. 690-699, 2011 and A compound RE51-2 (0.076 g, yield 56%) was obtained in the same manner as in Reference Example 4, step 1, using hexanoic acid monobenzyl ester synthesized using a similar method.
ESI-MS m / z: 2503 (M + H) ⁺

Reference Example 51 Step 2
A compound RE51-3 (0.030 g, yield 40%) was obtained in the same manner as in step 1 of Reference Example 3 using the compound RE51-2 (0.076 g, 0.03 mmol).
ESI-MS m / z: 2412 (M + H) ⁺

Reference Example 52

The compound RE52-1 (the compound RE6-5, 4.36 mg, 0.006 mmol in Reference Example 6) synthesized by the method described in Reference Example 6 and the compound RE1-4 (10 mg, 0.02 mmol) of Reference Example 1 were used as a reference. The compound RE52-2 (7 mg, yield 65%) was obtained in the same manner as in Step 7 of Example 3.
ESI-MS m / z: 1581 (M-H) ^-

Reference Example 53: Synthesis of Compound C2

Reference Example 53 Step 1
Using compound D10 (0.2011 g, 0.079 mmol) synthesized by the method described in Reference Example 46, compound RE53-1 (0.129 g, yield 55%) was obtained using step 3 of reference example 10.
ESI-MS m / z: 2972 (M + HCOO) ^-

Reference Example 53 Step 2
Using compound RE 53-1 (0.129 g, 0.044 mmol), step 4 of Reference Example 10 was used to obtain a crude product of compound RE 53-2.
ESI-MS m / z: 1535 (M + HCOOH-2H) ^2-

Reference Example 53 Step 3
Compound C2 (19.4 μmol / g, yield 35%) was obtained using Compound RE53-2 (0.0467 g, 0.013 mmol) and using Step 5 of Reference Example 10.

Reference Example 54: Synthesis of Compound C3

Reference Example 54 Step 1
Compound RE54-1 (90 mg, 90 mg, using Compound D1 (100 mg, 0.040 mmol) synthesized by the method described in Reference Example 10 and Compound RE 17-2 of Reference Example 17 in the same manner as in step 3 of Reference Example 10 A yield of 76% was obtained.
ESI-MS m / z: 1335 (M-DMTr + 2H) ²⁺

Reference Example 54 Step 2
A crude product of compound RE54-2 was obtained in the same manner as in step 4 of Reference Example 10 using compound RE54-1 (90 mg, 0.030 mmol).
ESI-MS m / z: 1558 (M + HCOOH-2H) ^2-

Reference Example 54 Step 3
Compound C3 (21.5 μmol / g, two-step yield 32%) was obtained in the same manner as in Step 5 of Reference Example 10 using the crude product of compound RE54-2.

Reference Example 55: Synthesis of Compound C4

Reference Example 55 Step 1
Using compound D10 (100 mg, 0.039 mmol) synthesized by the method described in Reference Example 46 and compound RE 17-2 synthesized by Reference Example 17, compound RE 55-1 (90 mg, yield 76%) was obtained.
ESI-MS m / z: detected as 1356 (M + 2H) ²⁺ de-DM Tr body

Reference Example 55 Step 2
A crude product of compound RE55-2 was obtained in the same manner as in step 4 of Reference Example 10, using compound RE55-1 (90 mg, 0.030 mmol).
ESI-MS m / z: 1579 (M + HCOOH-2H) ^2-

Reference Example 55 Step 3
The crude product of compound RE55-2 was used to obtain compound C4 (17.0 μmol / g, two-step yield 26%) in the same manner as in step 5 of Reference Example 10.

Reference Example 56: Synthesis of Compound C5

Reference Example 56, Step 1
Using compound D10 (100 mg, 0.039 mmol) synthesized by the method described in Reference Example 46 and compound RE18-3 synthesized in Step 2 of Reference Example 18, Compound RE56- is prepared in the same manner as in Step 3 of Reference Example 10. 1 (50 mg, 42% yield) was obtained.
ESI-MS m / z: detected as 1363 (M + 2H) ²⁺ de-DM Tr body

Reference Example 56, Step 2
A crude product of compound RE56-2 was obtained in the same manner as in step 4 of Reference Example 10 using compound RE56-1 (50 mg, 0.017 mmol).
ESI-MS m / z: 1587 (M + HCOOH-2H) ^2-

Reference Example 56, Step 3
The crude product of compound RE56-2 was used to obtain compound C5 (0.5 μmol / g, two-step yield 1%) in the same manner as in step 5 of Reference Example 10.

Reference Example 57: Synthesis of Compound C6

Reference Example 57 Step 1
Compound RE57-1 (40 mg, using the compound D10 (100 mg, 0.039 mmol) synthesized by the method described in Reference Example 46 and the compound RE 19-2 of Reference Example 19 in the same manner as in step 3 of Reference example 10 30% yield) was obtained.
ESI-MS m / z: 1532 (M + 2 H) ²⁺ dedetected as DM Tr body

Reference Example 57 Step 2
Using compound RE 57-1 (40 mg, 0.012 mmol), a crude product of compound RE 57-2 was obtained by the same method as in step 4 of Reference Example 10.
ESI-MS m / z: 1582 (M + 2H) ²⁺
Detected as a non-DMTr body

Reference Example 57 Step 3
The crude product of compound RE57-2 was used to obtain compound C6 (0.2 μmol / g, two-step yield 1%) in the same manner as in step 5 of Reference Example 10.

Reference Example 58: Synthesis of Compound C7

Reference Example 58, step 1
Using compound D1 (70 mg, 0.028 mmol) synthesized by the method described in Reference Example 10 and the compound RE 21-3 of Reference Example 21, compound RE 58-1 (58 mg, collection) by the same method as in step 3 of Reference Example 10. The rate is 77%).
ESI-MS m / z: 1341 (M + 2H) ²⁺

Reference Example 58 Step 2
Compound RE 58-1 (58 mg, 0.022 mmol) was dissolved in methanol (0.15 mL) and synthesized according to the method described in Journal of Organic Chemistry, Vol. 74, pages 6837-6842, 2009. Compound RE 17-1 of Reference Example 17 (16.9 mg, 0.032 mmol), 1 mol / L SODIUM L-AS CORBAtee aqueous solution (0.022 mL, 0.022 mmol), 20 mmol / L aqueous copper (II) sulfate solution (0.011 mL, 0.22 μmol) And 10 mmol / L Tris (2-benzimidazolylmethyl) amine / DMSO solution (0.022 mL, 0.22 μmol) were added, and the mixture was stirred at room temperature for 3 hours. The reaction solution was purified by silica gel column chromatography (chloroform / methanol = 80/20) to obtain compound RE58-2 (7 mg, yield 10%).
ESI-MS m / z: detected as 1450 (M + 2H) ²⁺ de-DM Tr body

Reference Example 58 Step 3
A crude product of compound RE58-3 was obtained in the same manner as in step 4 of Reference Example 10 using compound RE58-2 (10 mg, 3.13 μmol).
ESI-MS m / z: 1500 (M + 2 H) 2 ⁺ detected as de-DM Tr body

Reference Example 58 Step 4
The crude product of compound RE58-3 was used to obtain compound C7 (8.4 μmol / g, yield 27%) in the same manner as in step 5 of Reference Example 10.

Reference Example 59

Reference Example 59 step 1
Method similar to step 3 of Reference Example 3 using compound D10 (74.1 mg, 0.029 mmol) synthesized by the method described in Reference Example 46 and compound RE22-2 (15 mg, 0.027 mmol) of Reference Example 22; Alternatively, a crude organism of compound RE59-1 was obtained by the method described in Bioconjugate Chemistry, Vol. 26, pp. 1451-1455 (2015).
ESI-MS m / z: 1392 (M + H) ⁺ , detected as de-DMTr body

Reference Example 59 Step 2
Compound RE59-1 (0.083 g, 0.027 mmol) was used to obtain a crude organism of compound RE59-2 according to the method described in International Publication WO 201505083.
ESI-MS m / z: 2669 (M + H) ⁺ , detected as de-DMTr body

Reference Example 59 Step 3
The compound RE59-2 (0.08 g, 0.027 mmol) was used to obtain a crude product of a compound RE59-3 in the same manner as in step 4 of Reference Example 10.
ESI-MS m / z: 1556 (M + HOOH-2H) ^2-
¹ H-NMR (400 MHz, MeOD): δ 1.45-1.86 (48 H, m), 1. 93 (12 H, s), 1. 94 (12 H, s), 2.02 (12 H, s), 2. 13 (12 H, s), 2.16-2.28 (17H, m), 2.48 (4H, s), 3.10-3.16 (6H, m), 3.36-3.56 (19H, m), 3.77 (6H, s), 3.80-3.89 (6H, m), 3.98-4.35 (30H, m), 4.53-4.59 (4H, m), 4.66-4.72 (1H, m), 5.04-5.10 (4H, m), 5.31-5.36 (4H, m), 6.81-6.88 (4H , m), 7.17-7.23 (1H, m), 7.26-7.32 (6H, m), 7.38-7.44 (2H, m), 7.54 (2H, br s), 7.90 (1 H, br s).

Reference Example 60
Tables P-1 and P-2 in the same manner as in Reference Example 59, Steps 1 and 2, using Compound D1 and Compound RE20-4 of the structure of Reference Example 20 described in Table Z-1 or Reference Example 20 The compound described in was obtained.
The mass spectrometry results of the compound synthesized according to this example are shown in Table P-3.

Reference Example 61: Synthesis of Compound C8

Using compound RE59-3 described in Reference Example 59, compound C8 (10.0 μmol / g) was obtained in the same manner as in step 5 of reference example 10.

Reference Example 62 Synthesis of C9 to C17 Using compounds shown in Tables P-1 and P-2, compounds shown in Tables Q-1 and Q-2 in the same manner as Reference Example 61 I got
The amount of the compound synthesized according to this example is shown in Table Q-3.

Example 1: Synthesis of Compound 1-2

Step 1
Reference Example Compound A1 and a terminal thiolated oligonucleotide synthesized by the method described in Molecules, Vol. 17, p. 13825-13843, 2012 were added and allowed to stand at room temperature for 4 hours. Sodium carbonate was added to the reaction mixture and allowed to stand overnight at 4 ° C. Anion exchange chromatography (GE Healthcare, MonoQ 5/50 GL, 10 μm, 5.0 mm x 50 mm, solution A: 10 mmol / L Tris buffer solution / 30% acetonitrile, solution B: 10 mmol / L Tris buffer solution / 30% Acetonitrile / 1 mol / L NaBr gradient) or reverse phase liquid chromatography (Waters, X Bridge C18, 5 μm, 4.6 mm × 250 mm, 0.1 mol / L triethylammonium acetate buffer, solution B: acetonitrile gradient) Purification gave compound 1-1.

Step 2
Compound 1-1 (3′-b2GPI-ssRNA) synthesized in step 1 was prepared in a mixed buffer (100 mmol / L potassium acetate, 30 mmol / L 2- [4- (2-hydroxyethyl) piperazin-1-yl The concentration was adjusted (50 μmol / L) with ethanesulfonic acid, HEPES) -KOH (pH 7.4), 2 mmol / L magnesium acetate). Equal amounts of the sense strand and the antisense strand (50 μmol / L) were mixed and allowed to stand at 80 ° C. for 10 minutes. Antisense strand sequences are as described in Table R-3. The temperature was gradually lowered and allowed to stand at 37 ° C. for 1 hour to obtain a double-stranded compound 1-2.

Examples 2 to 7 Synthesis of Compounds 2-2 to 7-2 Compounds 2-2 to 7-2 were synthesized in the same manner as in Example 1 except that oligonucleotides different in base sequence from Example 1 were used.

Example 8 Synthesis of Compound 8-2

Step 1
Reference Example Compound D5 is used to add an amino-terminal modified oligonucleotide synthesized by the method described in Molecules, Vol. 17, p. 13825-13843, 2012, to obtain bioconjugate chemistry. 22: 1723-1728 (2011) or Bioconjugate Chemistry, 26: 1451-1455 (2015). Purification by the method described in Example 1 gave compound 8-1.

Step 2
Compound 8-2 was obtained in the same manner as in Step 2 of Example 1.

Example 9 Synthesis of Compound 9-2

In the same manner as in Example 1, Compound 9-2 was obtained.

Example 10 Synthesis of Compound 10-2

Compound 10-2 was synthesized in the same manner as in Example 8 using Reference Example Compound D1.

Example 11 Synthesis of Compound 11-2

Step 1
A 0.2 μmol scale was performed using a nucleic acid synthesizer (Ultra Fast Parallel Synthesizer, manufactured by Sigma, hereinafter UFPS). Compound C16 was used as a solid phase carrier. Dimethoxytrityl dT phosphoramidite (SAFC-PROLIGO) was adjusted to 0.06 mol / L with acetonitrile. The condensation reaction was carried out for 10 minutes each using acetonitrile solution of 5-benzylthio-1H-tetrazole (SAFC-PROLIGO) and 0.06 mol / L dT phosphoramidite as a phosphoroamidite activator. After the reaction, it was immersed in a 28% ammonia solution and left at 55 ° C. for 4 hours. After concentration under reduced pressure, 1-butanol was added to quench the reaction. By purification using reverse phase liquid chromatography (Shiseido, CAPSELL PAK C18, SG300, 6.0 mm x 75 mm, 5% acetonitrile / 0.1% triethylammonium buffer, solution B: gradient with 50% acetonitrile / water) , Compound 11-1 was obtained.

Step 2
Compound 11-2 was obtained in the same manner as in Example 1, step 2.

Example 12 Synthesis of Compound 12-2

The compound 12-2 was obtained in the same manner as in Example 8.

Example 13 Synthesis of Compound 13-2

Compound 13-2 was obtained in the same manner as in Example 8.

Example 14 Synthesis of Compounds 14-2 to 19-2 Compound 14-1 was prepared in the same manner as in Example 12, except that the nucleotide sequence of the nucleic acid of Compound 12-2 described in Example 12 was changed. Compound 14-2 to compound 19-2 were synthesized via .about.19-1.

The ligand-linker structure of the compound synthesized according to this example is shown below in Table R-1.

The sequences (sense strand) of Compounds 1-1 to 19-1 synthesized according to this example and the results of mass analysis are shown in Table R-2. In Table R-2, N (M) represents 2'-O-methyl-modified RNA, N (F) represents 2'-fluorine-modified RNA, and ^ represents phosphorothioate.

The sequences of compounds 1-2 to 19-2 synthesized according to this example are shown in Table R-3 below. In Table R-3, N (M) represents 2'-O-methyl modified RNA, N (F) represents 2'-fluorine modified RNA, p represents phosphorylation at the 5 'end, and ^ represents phosphorothioate. .

Reference Test Example 1: Measurement of the knockdown activity of β2GPI mRNA HepG2 cells (obtained from ATCC, ATCC No .: HB-8065), a cell line derived from human liver cancer, in a 96-well culture plate, 5,000 cells / 80 μL / well It sowed so that it might become. As the medium, MEM medium (Life Technology, catalog number 11095-098) containing 10% fetal bovine serum (FBS) was used. The double-stranded nucleic acid and RNAiMax transfection reagent (Life Technology, catalog number: 1401251) described in Table 1-1 to Table 1-16 are treated with Opti-MEM medium (Life technology, catalog number 11058-021) Dilute and add 20 μl of the siRNA / RNAiMax mixed solution to each 96 well culture plate so that the final concentration of double stranded nucleic acid is 100 pmol / L, and culture for 24 hours at 37 ° C, 5% CO ₂ did. Thereafter, the cells are washed with PBS (Phosphate buffered saline), and each plate is described in the instruction attached to the product using a Cells-to-Ct kit (Applied Biosystems, catalog number: AM1728). CDNA was synthesized according to the method. Add 5 μl of this cDNA to the MicroAmp Optical 96-well plate (Applied Biosystems, catalog number 4326659), and further add 10 μl of TaqMan Gene Expression Master Mix (Applied Biosystems, catalog number 4369016), 3 μl of UltraPure Distilled Water (Life Technologies) Corporation catalog number: 10977-015), 1 μL of human β2GPI probe, 1 μL of human GAPDH probe were added. Real-time PCR of human β2GPI gene and human GAPDH (D-glyceraldehyde-3-phosphate dehydrogenase) was performed using ABI 7900 HT real-time PCR system. GAPDH is a constitutively expressed gene and was measured as an internal control, and the β2GPI expression level was corrected. When HepG2 cells were treated with the transfection reagent alone without addition of siRNA, the amount of β2GPI mRNA was 1.0, and the relative expression amount of β2GPI mRNA was calculated when each siRNA was introduced. This experiment was performed three times, and the average value of the relative expression amount of β2GPI mRNA is shown in Tables 1-1 to 1-16.

Test Example 1 In Vitro Knockdown Test of Nucleic Acid Complex on Human Primary Hepatocytes The in vitro knockdown activity of human primary hepatocytes was measured for each of the nucleic acid complexes obtained in Examples 1-19. Each nucleic acid complex diluted with Optimem (Opti-MEM) (Thermo Fisher Scientific, catalog number 31985) is brought to a final concentration of 100, 30, 10 or 3 nmol / L in 96 wells. Human primary hepatocytes (Bioprediction International, catalog number HEP 187) suspended in plating medium (Bioprediction International, catalog number LV 0304-2) after dispensing 20 μl each into a culture plate The cells were seeded at a cell number of 10000 cells / 80 μL / well and cultured at 37 ° C., 5% CO ₂ for 6 hours, after which the culture supernatant was carefully removed, and the incubation medium (Biopredic International, Inc. , Catalog No. LV 0304-2), and each nucleic acid complex was subjected to human primary hepatocytes. In addition, as a negative control group, cells treated with nothing were seeded. The cells to which each nucleic acid complex has been added are cultured in a 5% CO ₂ incubator at 37 ° C. for 18 hours, washed with ice-cold phosphate buffered saline (DPBS) (manufactured by Nacalai Tesque), and superprep cell Total RNA was recovered and the obtained total RNA was templated according to the method described in the instruction attached to the product, using Lysis and T kit ForQ PCR (Toyobo Co., Ltd., catalog number SCQ-201). The cDNA was prepared by reverse transcription reaction. Using the obtained cDNA as a template and Tachman® Gene Expression Assay Probe (manufactured by Thermo Fisher Scientific Co., Ltd.) as a probe, Quant Studio 12 Caflex Real-Time PC System (manufactured by Thermo Fisher Scientific Inc.) Glyceraldehyde 3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate dehydrogenase, β-GPI gene and constitutively-expressed gene) by PCR reaction according to the method described in the attached instruction manual using The following PCR reaction was performed on the gene (hereinafter referred to as gapdh) to measure the amount of mRNA amplification, and the amount of amplification of gapdh was used as an internal control to calculate a semi-quantitative value of β2GPI mRNA. The expression rate of β2GPI mRNA was determined from the semiquantitative value of β2GPI mRNA, where the semiquantitative value of β2GPI mRNA in the negative control similarly measured was 1 as well. The results of the expression rate of the obtained β2GPI mRNA are shown in Table S1 and Table S2.
As apparent from Tables S1 and S2, each nucleic acid complex suppressed the expression of mRNA of β2 GPI gene after addition to human primary hepatocytes.

Test Example 2 In Vitro Knockdown Test of Nucleic Acid Complexes on Mouse Primary Hepatocytes Among the nucleic acid complexes obtained in Examples 1 to 13, each of the nucleic acid complexes in Tables S3 and S4 was tested in mouse primary hepatocytes in The in vitro knockdown activity was measured. 96 well culture plate of each nucleic acid complex diluted with Optimem (Opti-MEM) (Thermo Fisher Scientific, catalog number 31985) to a final concentration of 10, 3 or 1 nmol / L After dispensing 20 μl each, Williams' E Medium containing Primary Hepatocyte Thawing and Plating Supplements (Thermo Fisher Scientific, Catalog No. CM 3000). (William's E Medium) CD1 (1-CD-1) -derived mouse primary hepatocytes (Thermo Fisher Scientific, catalog number MSCP 10) suspended in (Thermo Fisher Scientific, Catalog No. A12176-01), cell number 10000 cells / 80 [mu] L were seeded so that / well, 37 ° C., the After incubation for 6 hours under 5% CO _2, culture Each nucleic acid complex was added to mouse primary hepatocytes by carefully removing the supernatant and adding Williams Emedium containing Primary Hepatocyte Maintenance Supplements (Thermo Fisher Scientific, catalog number CM4000). Provided for. In addition, as a negative control group, cells treated with nothing were seeded. The cells to which each nucleic acid complex has been added are cultured in a 5% CO ₂ incubator at 37 ° C. for 18 hours, washed with ice-cold phosphate buffered saline (DPBS) (manufactured by Nacalai Tesque) and superprep cell Total RNA was recovered and the obtained total RNA was templated according to the method described in the instruction attached to the product, using Lysis and T kit ForQ PCR (Toyobo Co., Ltd., catalog number SCQ-201). The cDNA was prepared by reverse transcription reaction. Using the obtained cDNA as a template and Tachman® Gene Expression Assays probe (manufactured by Thermo Fisher Scientific Co., Ltd.) as a probe, Quant Studio 12 Caflex Real-Time PC System (manufactured by Thermo Fisher Scientific Co.) Glyceraldehyde 3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate dehydrogenase, β-GPI gene and constitutively-expressed gene) by PCR reaction according to the method described in the attached instruction manual using The following PCR reaction was performed on the gene (hereinafter referred to as gapdh) to measure the amount of mRNA amplification, and the amount of amplification of gapdh was used as an internal control to calculate a semi-quantitative value of β2GPI mRNA. The expression rate of β2GPI mRNA was determined from the semiquantitative value of β2GPI mRNA, where the semiquantitative value of β2GPI mRNA in the negative control similarly measured was 1 as well. The expression rates of the obtained β2GPI mRNA are shown in Table S3 and Table S4.

As apparent from Tables S3 and S4, each nucleic acid complex suppressed the expression of mRNA of β2 GPI gene after addition to mouse primary hepatocytes.

Test Example 3 In Vivo Knockdown Test of Nucleic Acid Complex in Mouse The in vivo knockdown test of each nucleic acid complex in Table S5 was carried out according to the following method. Each nucleic acid complex was diluted with phosphate buffered saline (DPBS) (manufactured by Nacalai Tesque) according to the test. After conditioned breeding of mice (BALB / cA, obtained from CLEA Japan, Inc.), each nucleic acid complex was injected subcutaneously to the mice at 3 mg / kg or 1 mg / kg, respectively. In addition, as a control group, only DPBS was subcutaneously injected to mice. Three days after administration, they were euthanized, and livers were collected and cryopreserved with liquid nitrogen. Frozen liver samples were prepared using Trizol® ARE Isolation Reagents (Thermo Fisher Scientific, Catalog No. 1559 026) and Magna Pure 96 (MagNA Pure 96) (Roche Life Sciences). Total RNA was recovered according to the method described in the attached manual. Furthermore, reverse transcription using the obtained total RNA as a template according to the method described in the instruction attached to the product, using Transcriptor First Strand CDNA Synthesis Kit (Roche Life Sciences, catalog number 04897030001). The cDNA was prepared by the reaction. Using the obtained cDNA as a template and Tachman® Gene Expression Assay Probe (manufactured by Thermo Fisher Scientific Co., Ltd.) as a probe, Quant Studio 12 Caflex Real-time PC System (manufactured by Thermo Fisher Scientific Co.) The PCR reaction of the β2GPI gene and the gapdh gene is performed by PCR according to the method described in the attached instruction manual, and the amount of amplified mRNA is measured respectively, and the amount of amplified mRNA of gapdh is used as an internal control, β2GPI The semiquantitative value of mRNA was calculated. Assuming that the quasi-quantitative value of β2GPI mRNA in the PBS administration group measured in the same manner was 1, the expression rate of β2GPI mRNA was determined. The expression rate of the obtained β2GPI mRNA is shown in Table S5.

As apparent from Table S5, it was revealed that the nucleic acid complex of the present invention is administered to mice to reduce the expression of the β2GPI gene in the liver.

The nucleic acid complex of the present invention can be administered to mammals and used in vivo to treat β2 GPI related diseases.

Claims

A nucleic acid complex represented by the following formula 1.
Formula 1:

(In the formula 1,
X is a double stranded nucleic acid consisting of a sense strand and an antisense strand, comprising a duplex region of at least 11 base pairs,
The double-stranded nucleic acid is an oligonucleotide chain of 17 to 30 nucleotides in length in the antisense strand, and any one of the target β2GPI mRNA sequences described in Tables 1-1 to 1-16. Complementary and
The 3 'or 5' end of the sense strand is linked to S3,
L1 and L2 are each independently a sugar ligand,
S1, S2 and S3 are each independently a linker. )
The nucleic acid complex according to claim 1, which has a structure represented by the following formula 2.
Formula 2:

(In the formula 2,
X, L1, L2 and S3 are as defined above,
P1, P2, P3, P4, P5 and P6, and T1 and T2 are each independently absent, or -CO-, -NH-, -O-, -S-, -O-CO- , -S-CO-, -NH-CO-, -CO-O-, -CO-S- or -CO-NH-,
Q1, Q2, Q3 and Q4 are each independently absent, or substituted or unsubstituted alkylene having 1 to 12 carbon atoms or-(CH 2 CH 2 O) n -CH 2 CH 2- , N is an integer of 0 to 99,
B1 and B2 are each independently a bond or a structure represented by the following formula 2-1, and the terminal black dot in each structure is P2 or P3 or P5, respectively. Or a point of attachment to P6, and m1, m2, m3 and m4 are each independently an integer of 0 to 10,
Formula 2-1:

p1 and p2 are each independently an integer of 1, 2 or 3;
q1, q2, q3 and q4 are each independently an integer of 0 to 10,
However, when p1 and p2 are integers of 2 or 3, respectively, P3 and P6, Q2 and Q4, T1 and T2 and L1 and L2 may be the same or different, and q1 to q4 are 2 to 10 And each of the combinations of-[P2-Q1]-,-[Q2-P3]-,-[P5-Q3]-and-[Q4-P6]-may be the same or different. )
The nucleic acid complex according to claim 2, wherein P1 and P4 are each independently -CO-NH-, -NH-CO- or -O-.
-[P2-Q1] q1 - and- [P5-Q3] q3 -are each independently absent, or have any of the structures represented by the following formulas 3-1 to 3-3 Item 4. The nucleic acid complex according to item 2 or 3.
Formula 3-1:

Formula 3-2:

Formula 3-3:

(In Formula 3-1 to Formula 3-3,
m5 and m6 are each independently an integer of 0 to 10, and the terminal black dot in the structure of Formula 3-1 to Formula 3-3 is a bonding point to B1 or B2 or P1 or P4, respectively is there. )
The nucleic acid complex according to any one of claims 2 to 4, having any one of the structures represented by the following formulas 4-1 to 4-9.
Formula 4-1:

Equation 4-2:

Equation 4-3:

Formula 4-4:

Formula 4-5:

Formula 4-6:

Equation 4-7:

Formula 4-8:

Formula 4-9:

(In the formulas 4-1 to 4-9,
X, L1, L2, S3, P3, P6, T1, T2, Q2, Q4, q2 and q4 are as defined above. )
The nucleic acid complex according to claim 1, having a structure represented by the following formula 5.
Formula 5:

(In the equation 5,
X, S3, P1, P2, P3, Q1, Q2, B1, T1, L1, p1, q1 and q2 are as defined above. )
The nucleic acid complex according to claim 6, wherein P1 is -CO-NH-, -NH-CO- or -O-.
The nucleic acid complex according to claim 6, which has any of the structures represented by the following formulas 6-1 to 6-9.
Formula 6-1:

Formula 6-2:

Equation 6-3:

Equation 6-4:

Formula 6-5:

Formula 6-6:

Formula 6-7:

Formula 6-8:

Formula 6-9:

(In the formulas 6-1 to 6-9,
X, S3, P3, Q2, T1, L1 and q2 are as defined above. )
The nucleic acid complex according to any one of claims 2 to 5, having any one of the structures represented by the following formulas 7-1 to 7-9.
Formula 7-1:

Equation 7-2:

Formula 7-3:

Equation 7-4:

Formula 7-5:

Formula 7-6:

Formula 7-7:

Formula 7-8:

Formula 7-9:

(In the formulas 7-1 to 7-9,
X, S3, L1 and L2 are as defined above. )
The nucleic acid complex according to any one of claims 1 to 9, wherein the sugar ligand is N-acetylgalactosamine.
The nucleic acid complex according to any one of claims 1 to 10, wherein the double stranded nucleic acid comprises a modified nucleotide.
The nucleic acid complex according to any one of claims 1 to 11, wherein the 3 'end of the sense strand and the 5' end of the antisense strand form a blunt end.
The nucleic acid complex according to claim 11, wherein the double stranded nucleic acid comprises a sugar moiety modified nucleotide.
X is a pair of sense strand / antisense strand selected from the group consisting of sense strand / antisense strand described in Table M1-1 to Table M1-18 or Table 1-1 to Table 1-16, The nucleic acid complex according to any one of claims 1 to 13.
The nucleic acid complex according to any one of claims 1 to 14, which has a structure represented by the following formula 7-8-1.
Formula 7-8-1:

(In formula 7-8-1, X is as defined above.)
The nucleic acid complex according to claim 15, wherein X is a double-stranded nucleic acid selected from the group consisting of CHOO 96, CHOO 99, CH0125, CH0126, CH0318, CH0943 and CH1139 in Tables M1-1 to M1-18. .
A pharmaceutical composition comprising the nucleic acid complex according to any one of claims 1 to 16.
The pharmaceutical composition according to claim 17 for introduction into cells.
19. The pharmaceutical composition according to claim 17 or 18, which is administered intravenously or subcutaneously.
A disease comprising administering a nucleic acid complex according to any one of claims 1 to 16 or a pharmaceutical composition according to any one of claims 17 to 19 to a patient in need thereof. Methods of treatment or prevention.
Introducing double stranded nucleic acid into cells using the nucleic acid complex according to any one of claims 1 to 16 or the pharmaceutical composition according to any one of claims 17 to 19 A method for suppressing the expression of β2 GPI gene.
A method of treating a β2GPI related disease, comprising administering the nucleic acid complex according to any one of claims 1 to 16 or the pharmaceutical composition according to any one of claims 17 to 19 to a mammal.
A medicament for use in the treatment of a β2 GPI related disease, comprising the nucleic acid complex according to any one of claims 1 to 16 or the pharmaceutical composition according to any one of claims 17 to 19.
A therapeutic agent for a β2GPI related disease, comprising the nucleic acid complex according to any one of claims 1 to 16 or the pharmaceutical composition according to any one of claims 17 to 19.