TW201920668A - Nucleic acid complex - Google Patents

Abstract

The present invention provides a nucleic acid complex represented by formula 1, the nucleic acid complex being capable of suppressing the expression of [beta]2GPI. 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 [beta]2GPI 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 and a pharmaceutical composition containing the same.

As nucleic acid medicine, aptamer, antisense nucleic acid, Decoy nucleic acid, ribonuclease, small interfering RNA (siRNA), miRNA (micro RNA), and antimiRNA (anti-RNA) are known. micro RNA, anti-microRNA). Nucleic acid medicine is expected to be used clinically for various diseases that have been considered difficult to treat because of its high versatility in controlling all genes in cells. In addition, nucleic acid medicine is expected to become the next generation of medicine after antibodies and low-molecular medicine because of its high target selectivity and activity in cells. However, nucleic acid medicine has a problem that it is difficult to deliver to target tissues.

As one of the delivery methods of nucleic acid medicine effective in the body, a nucleic acid conjugate using a targeting compound and a nucleic acid has been reported. Examples of the targeting compound include ligands capable of binding to a receptor expressed outside the cell. Among them, N-acetyl-D-galactosamine (GalNAc) and the like have been reported several times as ligands capable of binding to asialoglycoprotein receptor (ASGPR), which is highly expressed in liver cells. Nucleic acid complex. In recent years, it has been reported that a nucleic acid complex formed by binding these ligands to siRNAs can be efficiently transmitted to hepatocytes (Non-Patent Document 1).

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

[Chemical 1] (In the formula, Ac represents an ethenyl group; the same applies in the following description)

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

[Chemical 2]

In addition, Patent Document 4 discloses a nucleic acid complex having a structure shown below as a sugar ligand-tethered unit.

[Chemical 3]

On the other hand, β2-glycoprotein 1 (β2GPI) (also known as lipoprotein H, apoH) is a glycoprotein composed of 326 amino acids, and has a higher-order structure formed by connecting five regions. Β2GPI is thought to have a variety of physiological effects, and it has been reported to participate in platelet aggregation, coagulation / fibrinolysis, and the binding of oxidized LDL to macrophages (Non-Patent Document 2).

Regarding the association with diseases, β2GPI is known as the main corresponding antigen of antiphospholipid antibodies that appear in autoimmune diseases such as antiphospholipid antibody syndrome (APS) or systemic lupus erythematosus (SLE). Through the use of animal models and clinical studies, it is known that anti-β2GPI antibodies are also deeply related to disease conditions. The complex formed by β2GPI and anti-β2GPI antibodies will cause endothelial cells, monocytes, platelets, and vegetative bud cells ( Receptors on the membrane of various cells such as trophoblast) generate activation signals, which may cause APS-specific disorders such as thrombosis or abnormal pregnancy (Non-Patent Document 3). By specifically inhibiting the formation of immune complexes containing β2GPI and anti-β2GPI antibodies, it is expected to prevent or treat the above diseases, but β2GPI is present in the blood at a relatively high concentration of 50 to 500 μg / mL, for example, using general antibody medicine It is not easy to continuously suppress all of these β2GPIs (Non-Patent Document 4). Prior Art Literature Patent Literature

Patent Literature 1: International Publication No. 2009/073809 Patent Literature 2: International Publication No. 2013/075035 Patent Literature 3: International Publication No. 2015/105083 Patent Literature 4: International Publication No. 2014/179620 Non-Patent Literature

Non-Patent Literature 1: Journal of American Chemical Society, 2014, Vol. 136, p16958-16961 Non-Patent Literature 2: Annals of the New York Academy of Sciences, 2013, No. Volume 1285, p44-58 Non-Patent Literature 3: The New England Journal of Medicine, 2013, Volume 368, p1033-1044 Non-Patent Literature 4: Journal of Thrombosis and Haemostasis, 2011, Volume 9, p1275-1284

[Problems to be solved by the invention]

An object of the present invention is to provide a nucleic acid complex capable of inhibiting the expression of β2GPI. [Technical means to solve the problem]

The present invention relates to the following. [1] A nucleic acid complex represented by the following formula 1. Formula 1: [化 4] (In Formula 1, X is a double-stranded nucleic acid including a sense strand and an antisense strand and including a double-stranded region of at least 11 base pairs, and the length of the double-stranded nucleic acid in the antisense strand is 17 to 30 The oligonucleotide strand of the nucleotide is complementary to any of the target β2GPI mRNA sequences described in Table 1-1 to Table 1-16, and the 3 'end or 5' end of the sense strand is bonded to S3, L1 And L2 are each independently a sugar ligand, and 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. Equation 2: [化 5] (In Formula 2, X, L1, L2, and S3 have the same meanings as above, and P1, P2, P3, P4, P5, and P6, and T1 and T2 are independent, or 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 independent or absent, or are substituted or unsubstituted alkylene groups having 1 to 12 carbon atoms or-(CH ₂ CH ₂ O) _n -CH ₂ CH _2- , n is 0 to 99 Integer, B1 and B2 are each independently a bonding bond, or any structure represented by the following formula 2-1. The black dot at the end of each structure is the bond with P2 or P3 or P5 or P6 Nodes, m1, m2, m3, and m4 are each independently an integer of 0 to 10, Formula 2-1: [化 6] 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, wherein when p1 and p2 are integers of 2 or 3, each of 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, each of-[P2-Q1]-,-[Q2-P3]-,-[P5-Q3]- And the combination of-[Q4-P6]-may be the same or different) [3] The nucleic acid complex according to [2], wherein P1 and P4 are independently -CO-NH-, -NH-CO- or -O- . [4] The nucleic acid complex according to [2] or [3], in _which- [P2-Q1] _q1 - _and- [P5-Q3] _q3 -are independent, or do not exist, or are represented by the following formula 3-1 ~ Any one of the structures represented by Formula 3-3. Formula 3-1: Equation 3-2: [化 8] Equation 3-3: [化 9] (In Formulas 3-1 to 3-3, m5 and m6 are each independently an integer of 0 to 10, and the black dots at the ends of the structures in Formulas 3-1 to 3-3 are respectively B1 or B2, or Bonding point with P1 or P4) [5] The nucleic acid complex according to any one of [2] to [4], which has any one of the structures represented by the following formulae 4-1 to 4-9. Equation 4-1: Equation 4-2: Equation 4-3: [化 12] Equation 4-4: [Chem. 13] Equation 4-5: [Chem. 14] Equation 4-6: Equation 4-7: [化 16] Equation 4-8: Equation 4-9: (In formulas 4-1 to 4-9, X, L1, L2, S3, P3, P6, T1, T2, Q2, Q4, q2, and q4 have the same meanings as above.) [6] The nucleic acid according to [1] The composite has a structure represented by the following formula 5. Equation 5: [化 19] (In Formula 5, X, S3, P1, P2, P3, Q1, Q2, B1, T1, L1, p1, q1, and q2 have the same meanings as 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 the following formulae 6-1 to 6-9. Equation 6-1: Equation 6-2: Equation 6-3: Equation 6-4: Formula 6-5: [Chem. 24] Formula 6-6: [Chem. 25] Formula 6-7: [Chem. 26] Equation 6-8: Equation 6-9: (In formulas 6-1 to 6-9, X, S3, P3, Q2, T1, L1, and q2 have the same meanings as above.) [9] The nucleic acid complex according to any one of [2] to [5] It has any one of the following formulae 7-1 to 7-9. Equation 7-1: [Chem. 29] Equation 7-2: [Chem 30] Equation 7-3: [Chem. 31] Equation 7-4: Equation 7-5: [Chem. 33] Equation 7-6: Equation 7-7: Equation 7-8: Equation 7-9: (In formulae 7-1 to 7-9, X, S3, L1, and L2 have the same meanings as above.) [10] The nucleic acid complex according to any one of [1] to [9], wherein the sugar ligand N-acetylgalactosyl. [11] The nucleic acid complex according to any one of [1] to [10], wherein the double-stranded nucleic acid includes 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 smooth end. [13] The nucleic acid complex according to any one of [1] to [12], wherein the double-stranded nucleic acid includes a sugar moiety-modified nucleotide. [14] The nucleic acid complex according to any one of [1] to [13], wherein X is a justice selected from Tables M1-1 to M1-18 or Tables 1-1 to 1-16 One pair of justice / antisense stocks in the group consisting of stocks / antisense stocks. [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. Equation 7-8-1: (In the formula 7-8-1, X has the same meaning as above.) [16] The nucleic acid complex according to [15], wherein X is selected from the group consisting of CHOO96, CHOO99, CH0125, and CH0125 in Tables M1-1 to M1-18. Double-stranded nucleic acids in a group consisting of CH0126, CH0318, CH0943 and CH1139. [17] A pharmaceutical composition comprising the nucleic acid complex according to any one of [1] to [16]. [18] The pharmaceutical composition according to [17], which is used for introduction into a cell. [19] The pharmaceutical composition according to [17] or [18], which is for intravenous or subcutaneous administration. [20] A method for treating or preventing a disease, comprising the step of combining a nucleic acid complex according to any one of [1] to [16] or a pharmaceutical composition according to any one of [17] to [19] Patients who need it are administered. [21] A method for inhibiting the expression of the β2GPI gene, which comprises 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] Introduction of double-stranded nucleic acid into cells. [22] A method for treating a β2GPI-related disease, comprising: comparing a nucleic acid complex according to any one of [1] to [16] or a pharmaceutical composition according to any one of [17] to [19] Mammals are administered. [23] A medicine for treating β2GPI-related diseases, 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 β2GPI-related diseases, 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 method according to [22], wherein the β2GPI-related disease is an autoimmune disease or thrombosis. [26] The medicine according to [23], wherein the β2GPI-related disease is an autoimmune disease or thrombosis. [27] The therapeutic agent according to [24], wherein the β2GPI-related disease is an autoimmune disease or thrombosis. [Effect of the invention]

For example, a pharmaceutical composition comprising 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 system of the present invention is a nucleic acid complex represented by the following formula 1. Formula 1:

[Chemical 39]

In Formula 1, X is a double-stranded nucleic acid including a sense strand and an antisense strand and including a double-stranded region of at least 11 base pairs, and the length of the double-stranded nucleic acid in the antisense strand is 17 to 30 The oligonucleotide strand of the nucleotide is complementary to any of the target β2GPI mRNA sequences described in Table 1-1 to Table 1-16, and the 3 'end or 5' end of the sense strand is bonded to S3, L1 And L2 are each independently a sugar ligand, and S1, S2, and S3 are each independently a linker.

In the present invention, relative to the substitution position of S3 on the benzene ring, S1 and S2 may be bonded to the benzene ring in the ortho, meta, and para positions respectively, but it is preferably a nucleic acid complex represented by the following formula 1-1 body. It means that the bond between S1 and S2 on the benzene ring in Formula 1 may be any position other than the substitution position of S3 on the benzene ring. Formula 1-1:

[Chemical 40]

In Formula 1-1, X, L1, L2, S1, S2, and S3 have the same meanings as described above. In this specification, the so-called "same meaning as above" is described by taking Formula 1-1 as an example, which means that each of X, L1, L2, S1, and S2 in Formula 1-1 can be the same as the above The related definitions of X, L1, L2, S1, and S2 in Formula 1 described above are the same.

In the present invention, X is a double-stranded nucleic acid comprising a sense strand and an antisense strand and comprising a double-stranded region of at least 11 base pairs. In addition, the length of the double-stranded nucleic acid in the antisense strand is an oligonucleotide chain of 17 to 30 nucleotides and the target β2GPI mRNA described in Tables 1-1 to 1-16 described below Either of the sequences is complementary. In addition, the 3 'end or 5' end of the justice unit is bonded to S3.

L1 and L2 are each independently a sugar ligand. In the present invention, the sugar ligand means a group derived from a saccharide (monosaccharide, disaccharide, trisaccharide, polysaccharide, etc.) capable of binding to a receptor expressed by a target cell. In the present invention, in the case where the sugar ligand is bonded to the linker of S1 and S2 by O-bonding, the removed portion of the bond-related hydroxyl group of the sugar constituting the sugar ligand means as the source A sugar ligand based on a sugar. In the present invention, it is only necessary to select a sugar ligand targeted to a cell as an oligonucleotide. Examples of the monosaccharide include allose, aldose, arabinose, cladinose, erythrose, erythrulose, fructose, D-fucitol, and L-fucoid Sugar alcohol, fucosamine, fucose, fuculose, galactosyl, D-galactosaminitol, N-acetamido-galactosamine, Galactose, Glutamine, N-Acetyl-Glucosamine, Glucosamine (glucosaminitol), Glucose, Glucose-6-phosphate, Gulose, Glyceraldehyde, L-Glycerol-D-Mannose, Glycerin , Glycerone, gulose, idurose, lyxose, mannose, mannose, mannose-6-phosphate, allosene, isorhamnetose, isorhamnetin, rat Ritol, rhamnose, rhamnose, ribose, ribulose, sedoheptulose, sorbose, tagatose, talose, tartaric acid, threose, xylose, and xylulose. Examples of disaccharides, trisaccharides, and polysaccharides include abicose, acarbose, amicetose, amylopectin, amylose, celery, arcanose, ascarylose, Ascorbic acid, 2-deoxy-6-boivinose, cellobiose, cellotriose, cellulose, chacotriose, chalcose, chitin, colitose , Cyclodextrin, cymarose, dextrin, 2-deoxyribose, 2-deoxyglucose, 2-deoxy digitalis (digitalin), digitalis, digitalis (digitoxose), evalose, evemitrose, fructooligosaccharides, galto-oligosaccharide, gentiotriose, gentiobiose, glycan, glycogen, glycogen, wamamelose ), Heparin, inulin, isolevoglucosenone, isomaltose, isomaltotriose, isopanose, kojibiose, lactose, lactosamine, lactosediamine, Laminarabiose, Levoglucosan, L-glucose (levoglucosenone), β-maltose, maltotriose, mannose-oligosaccharide, manninotriose, melibiose, melibiose, muramic acid, micarose, mycinose, neuraminic acid ( neuraminic acid), sialic acid-containing sugar chains, aspergillus niger, nojirimycin, noviose, oleandrose, panose, paratose, psyllium, cherry Primeverose, raffinose, rhodinose, rutose, sarmentose, sedoheptulose, sedoheptulosan, solantriose, Sophorose, stachyose, streptose, sucrose, α, α-trehalose, trahalosamine, melibiose, tyvelose, xylobiose, umbrella sugar, etc. .

Each of the monosaccharides in the saccharide may be D-form or L-form, or a mixture of D-form and L-form in any ratio. Carbohydrates can also include deoxysugar (made by replacing an alcoholic hydroxyl group with a hydrogen atom), amino sugar (made by replacing an alcoholic hydroxyl group with an amine group), and thiosaccharides (made by replacing an alcoholic hydroxyl group with a thiol group) Or by replacing C = O with C = S or by replacing epoxy with sulfur), seleno sugar, tellurose, and azasugar (replace ring carbon with Nitrogenous), imino sugar (made by replacing epoxy with nitrogen), phosphon sugar (made by replacing epoxy with phosphorus), phosphorus sugar (ring carbon Substituted by phosphorus), C-substituted monosaccharides (substituted by hydrogen atoms on non-terminal carbon atoms by carbon atoms), unsaturated monosaccharides, alditols (substituted by carbonyl groups substituted with CHOH groups) ), Aldonic acid (made by replacing an aldehyde group with a carboxyl group), ketoaldonic acid, uronic acid, aldonic acid, and the like. Regarding amino sugars, examples of the amino monosaccharides in sugars include galactosamine, glucosamine, mannose, fucosamine, isorhamnamine, neuraminic acid, and muramic acid , Lactose diamine, acosamine, bacillosamine, daunosamine, desosamine, forosamine, garosamine, Kanosamine, kansosamine, carbon mold Mycaminose, mycosamine, perosamine, pneumosamine, purpurosamine, rhodosamine, and the like. The amino group of the amino sugar may be substituted with an acetamyl group or the like. Examples of the sialic acid-containing sugar chain include a sugar chain containing NeuAc at a non-reducing end of the sugar chain, and a sugar chain containing NeuAc-Gal-GlcNAc, or a Neu5Acα (2-6) Galβ (1-3) GlcNAc, etc. . As long as each monosaccharide in the saccharide can bind to a receptor expressed by a target cell, it may also be substituted with a substituent. For example, a hydroxyl group may be substituted, and a hydrogen atom in each monosaccharide may be substituted with 1 to plural azides and / Or substituted aryl.

As the sugar ligand, it is preferable to select a sugar ligand that binds to a receptor expressed on the surface of the target cell in accordance with the target organs. For example, when the target cell is a hepatocyte, it is preferable to correspond to the sugar ligand. The sugar ligand of a receptor expressed on the surface of a hepatocyte is more preferably a sugar ligand corresponding to an asialoglycoprotein receptor (ASGPR).

As the sugar ligand corresponding to ASGPR, mannose or N-acetylgalactosyl sugar is preferred, and N-acetylgalactosyl sugar is more preferred. As sugar ligands with higher affinity for ASGPR, for example, Bioorganic Medicinal Chemistry, Vol. 17, p. 7254 (2009) and Journal of the American Chemical Society are known. American Chemical Society), Vol. 134, p. 1978 (2012) and the like, sugar derivatives can be used.

In the present invention, S1, S2, and S3 are linkers. S1 and S2 are not particularly limited as long as they have a structure in which L1 and L2 as saccharide ligands are linked to a benzene ring, and a known structure used in a nucleic acid complex can be used. S1 and S2 may be the same or different. L1 and L2 as sugar ligands are preferably linked through glycosidic bonds and S1 and S2, and S1 and S2 can be respectively -CO-, -NH-, -O-, -S-, -O-CO- , -S-CO-, -NH-CO-, -CO-O-, -CO-S- or -CO-NH- bond and benzene ring are connected.

S3 is not particularly limited as long as it is a structure in which X, which is a double-stranded nucleic acid, is bonded to a benzene ring, and a known structure used in a nucleic acid complex can be used. X as an oligonucleotide is preferably linked to S3 through a phosphodiester bond. S3 can be, for example, -CO-, -NH-, -O-, -S-, -O-CO-, -S- A CO-, -NH-CO-, -CO-O-, -CO-S-, or -CO-NH- bond is connected to the benzene ring.

As the linkers of S1, S2, and S3, for example, International Publication No. 2009/073809, International Publication No. 2013/075035, International Publication No. 2015/105083, International Publication No. 2014/179620, and International Publication No. 2015 / The structure disclosed in No. 006740.

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

[Chemical 41]

In Formula 2, X, L1, L2, and S3 have the same meanings as above, and P1, P2, P3, P4, P5, and P6, and T1 and T2 are independent, or absent, or -CO-, -NH-, -O-, -S-, -O-CO-, -S-CO-, -NH-CO-, -CO-O-, -CO-S- or -CO-NH-, Q1, Q2, Q3 and Q4 is independent or does not exist, or is a substituted or unsubstituted alkylene group having 1 to 12 carbon atoms or-(CH ₂ CH ₂ O) _n -CH ₂ CH _2- , n is an integer from 0 to 99 .

P1 and P4 are independent or absent, or -CO-, -NH-, -O-, -S-, -O-CO-, -S-CO-, -NH-CO-, -CO-O -, -CO-S- or -CO-NH-, preferably -O-, -O-CO-, -NH-CO- or -CO-NH-, more preferably -O-, -NH-CO -Or -CO-NH-, more preferably -NH-CO-. 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 independent or absent, or are substituted or unsubstituted alkylene groups having 1 to 12 carbon atoms or-(CH ₂ CH ₂ O) _n -CH ₂ CH _2- , n An integer of 0 to 99, preferably substituted or unsubstituted alkylene group having 1 to 12 carbon atoms, more preferably unsubstituted alkylene group having 1 to 12 carbon atoms, and further preferably The substituted alkylene group having 1 to 6 carbon atoms is more preferably an unsubstituted alkylene group having 1 to 4 carbon atoms.

P2 and P5 are independent or absent, or -CO-, -NH-, -O-, -S-, -O-CO-, -S-CO-, -NH-CO-, -CO-O -, -CO-S- or -CO-NH-, preferably or absent or -CO-O- or -CO-NH-, more preferably or absent or -CO-NH-. 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 -are independent of each other, and preferably do not exist or have any one of the following formulae 3-1 to 3-3. Equation 3-1:

[Chemical 42] Equation 3-2:

[Chemical 43] Equation 3-3:

[Chemical 44]

In formulas 3-1 to 3-3, m5 and m6 are each independently an integer of 0 to 10, and the black dots at the ends of the structures in formulas 3-1 to 3-3 are respectively B1 or B2, or P1. Or P4 key node.

B1 and B2 are independently bond bonds, or any structure represented by the following formula. The black dots at the ends of each structure are the bond points with P2 or P3, or with P5 or P6, m1, m2, m3 and m4 are each independently an integer of 0-10.

[Chemical 45]

B1 and B2 are preferably derived from amino acids containing unnatural amino acids, such as glutamic acid, aspartic acid, lysine, and imino diacetic acid, or derived from 2-amino-1,3- When B1 and B2 are derived from glutamic acid and aspartic acid, the amino groups of propylene glycol and the like are bonded to the amino groups of glutamic acid and aspartic acid, respectively, as P2 and P5. Preferably, it is a -NH-CO- bond. When B1 and B2 are radicals derived from lysine, the carboxyl groups of lysine are respectively bonded. As P2 and P5, the -CO-NH- bond is preferred. When B1 and B2 are groups derived from iminodiacetic acid, the amine groups of iminodiacetic acid are respectively bonded, and P2 and P5 are preferably -CO- bonds. Specifically, B1 and B2 preferably have the following structures.

[Chemical 46]

When p1 and p2 are integers of 2 or 3, each of 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, each combination of-[P2-Q1]-,-[Q2-P3]-,-[P5-Q3]-,-[Q4-P6]-may be the same or different. The combinations of the so-called-[P2-Q1]-,-[Q2-P3]-,-[P5-Q3]-, and-[Q4-P6]-are the same or different, meaning-[P2-Q1]-,- [Q2-P3]-,-[P5-Q3]-, and-[Q4-P6]-each 2 to 10 units indicated may be the same or different.

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

[Chemical 47] Equation 4-2:

[Chemical 48] Equation 4-3:

[Chemical 49] Equation 4-4:

[Chemical 50] Equation 4-5:

[Chemical 51] Equation 4-6:

[Chemical 52] Equation 4-7:

[Chem 53] Equation 4-8:

[Chemical 54] Equation 4-9:

[Chem 55]

In the formulae 4-1 to 4-9, X, L1, L2, S3, P3, P6, T1, T2, Q2, Q4, q2, and q4 have the same meanings as described above.

P3 and P6 are independent or absent, or -CO-, -NH-, -O-, -S-, -O-CO-, -S-CO-, -NH-CO-, -CO-O -, -CO-S- or -CO-NH-, preferably -O-CO- or -NH-CO-, 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 independent or absent, or are -CO-, -NH-, -O-, -S-, -O-CO-, -S-CO-, -NH-CO-, -CO-O -, -CO-S- or -CO-NH-, 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 Formula 5 below. 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 Same as q4. Equation 5:

[Chemical 56]

In Formula 5, X, S3, P1, P2, P3, Q1, Q2, B1, T1, L1, p1, q1, and q2 have the same meanings as described above. In addition, X, S3, P1, P2, P3, Q1, Q2, B1, T1, L1, p1, q1, and q2 in Formula 5 may be appropriate bases described above, and P1 is preferably -CO-NH -, -NH-CO- or -O-. -(P2-Q1) _q1 -in Formula 5 is preferably absent or has any of the structures represented by 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 formulae 6-1 to 6-9. Equation 6-1:

[Chemical 57] Equation 6-2:

[Chem 58] Equation 6-3:

[Chemical 59] Equation 6-4:

[Chemical 60] Equation 6-5:

[Chem 61] Equation 6-6:

[Chem 62] Equation 6-7:

[Chem 63] Equation 6-8:

[Chemical 64] Equation 6-9:

[Chem 65]

In Formulas 6-1 to 6-9, X, S3, P3, Q2, T1, and L1 have the same meanings as described 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 formulae 7-1 to 7-9. Equation 7-1:

[Chemical 66] Equation 7-2:

[Chemical 67] Equation 7-3:

[Chemical 68] Equation 7-4:

[Chemical 69] Equation 7-5:

[Chem 70] Equation 7-6:

[Chemical 71] Equation 7-7:

[Chemical 72] Equation 7-8:

[Chemical 73] Equation 7-9:

[Chemical 74]

In Formulas 7-1 to 7-9, X, L1, L2, and S3 have the same meanings as described above. L1 and L2 may be the same or different, and preferably the same. In the formulae 7-1 to 7-9, by introducing an alkylene chain having a different chain length for each of the alkylene moieties, and by replacing a amide bond or the like with another bond, it is possible to produce a compound having the formula 7- Nucleic acid derivatives other than the nucleic acid complex of the structure represented by Formula 1 to Formula 9-9.

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

[Chemical 75]

In Formula 11, L1, L2, S1, and S2 have the same meanings as above, and P7 and P8 are independent or absent, or -CO-, -NH-, -O-, -S-, -O-CO- , -S-CO-, -NH-CO-, -CO-O-, -CO-S- or -CO-NH-, Q5, Q6 and Q7 are independent, or not present, or are substituted or unsubstituted Substituted carbon number of 1 to 12 or-(CH ₂ CH ₂ O) _n8 -CH ₂ CH _2- , n8 is an integer of 0 to 99, and B3 is referred to as a brancher unit in this specification, and is Any structure represented by the following formula 11-1, where the curve represents the bond with Q5 and Q6, respectively, Formula 11-1:

[Chemical 76]

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

P7 is either 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 benzene ring -O-.

P8 is either 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 independent, or not present, or are substituted or unsubstituted alkylene groups having 1 to 12 carbon atoms or-(CH ₂ CH ₂ O) _n8 -CH ₂ CH _2- , n8 is 0 An integer from 99 to 99, preferably a substituted or unsubstituted alkylene group having 1 to 12 carbon atoms, more preferably an unsubstituted alkylene group having 1 to 12 carbon atoms, and more preferably an unsubstituted alkylene group The alkylene group having 1 to 6 carbon atoms is more preferably an unsubstituted alkylene group having 1 to 4 carbon atoms.

-(P7-Q5) _q5 -is preferably -O- (CH ₂ ) _m15 -NH- and -NH-CO- (CH ₂ ) _m16 -NH, m15 and m16 are each independently an integer of 1-10.

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

[Chemical 77] Equation 12-2:

[Chem. 78] Equation 12-3:

[Chem. 79] Equation 12-4:

[Chemical 80] Equation 12-5:

[Chemical 81] Equation 12-6:

[Chem 82] Equation 12-7:

[Chemical 83] Equation 12-8:

[Chemical 84] Equation 12-9:

[Chemical 85] Equation 12-10:

[Chem. 86] Equation 12-11:

[Chemical 87] Equation 12-12:

[Chem 88]

In the formulae 12-1 to 12-12, X, L1, L2, S1, and S2 have the same meanings as above, and n1 'to n12' are each independently an integer of 1 to 10.

The nucleic acid complex of the present invention is preferably a nucleic acid complex represented by Formula 1 in which a structure described in Formula 2 corresponding to S1 and S2 and a structure corresponding to Formula 11 in S3 are combined. Formula 2 can be Formula 4-1 to Formula 4-9, Formula 6-1 to Formula 6-9, Formula 7-1 to Formula 7-9, and Formula 2 is Formula 4-1 to Formula In the case of 4-9, Formula 6-1 to Formula 6-9, or Formula 7-1 to Formula 7-9, Formula 11 may be Formula 12-1 to Formula 12-12. The nucleic acid complex of the present invention is more preferably a combination of any one of the formulae 4-1 to 4-9 corresponding to S1 and S2 and the formula 12-1 corresponding to S3 in the nucleic acid complex represented by Formula 1 ~ The nucleic acid complex of any one of the structures described in Formula 12-12, which combines any of the structures described in Formulas 6-1 to 6-9 corresponding to S1 and S2 and Formula 12-1 to Formula 12- corresponding to S3 The nucleic acid complex of any one of the structures described in 12 is a combination of any one of the structures described in formulas 7-1 to 7-9 corresponding to S1 and S2 and any of the structures described in formulas 12-1 to 12-12 corresponding to S3. A structural nucleic acid complex.

The nucleic acid complex of the present invention is preferably represented by the following formula 7-8-1. Equation 7-8-1: (In Formula 7-8-1, X has the same meaning as above.)

X in Formula 1 is a double-stranded nucleic acid that includes a sense strand and an antisense strand and includes a double-stranded region of at least 11 base pairs. The length of the double-stranded nucleic acid in the antisense strand is 17 to 30 cores. The oligonucleotide chain is complementary to any of the target β2GPI mRNA sequences described in Tables 1-1 to 1-16. Since the 3 'end or the 5' end of the sense strand is bonded to S3, in Formula 1, X bonded to S3 is a sense strand constituting a double-stranded nucleic acid, that is, Table 1-1 to Table 1-16 described below Or the justice stock represented by the justice stock sequence in Tables M1-1 to M1-18.

In the present invention, a nucleic acid containing a base sequence complementary to the β2GPI mRNA is referred to as an antisense strand nucleic acid, and a nucleic acid containing a base sequence complementary to the base sequence of the antisense strand nucleic acid is also referred to as a 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 weaken or stop the expression of the β2GPI gene when introduced into mammalian cells. Strand nucleic acid. In addition, the sense strand and the antisense strand have at least 11 base pairs, at least 17 nucleotides and at most 30 in the antisense strand, that is, an oligonucleotide having a chain length of 17 to 30 nucleotides. The nucleotide chain is complementary to the 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 as long as it is a molecule obtained by polymerizing a nucleotide or a molecule having the same function as the nucleotide, and examples thereof include: RNA of a polymer of ribonucleotides, DNA of a polymer of deoxyribonucleotides, a chimeric nucleic acid comprising RNA and DNA, and at least one nucleotide of the nucleic acid are equivalent to the nucleotide Molecularly substituted nucleotide polymers. In addition, the double-stranded nucleic acid used as a drug in the present invention also includes derivatives in which these nucleic acids include at least one molecule having the same function as a nucleotide. In addition, uracil (U) may be replaced with thymine (T).

Examples of molecules having functions equivalent to nucleotides include nucleotide derivatives. As a nucleotide derivative, any molecule can be used as long as it is a molecule obtained by modifying nucleotides, for example, to enhance or stabilize nuclease resistance compared to RNA or DNA, and to improve affinity with complementary strand nucleic acids In order to improve cell permeability or to achieve visualization, it is suitable to use molecules obtained by modifying ribonucleotides or deoxyribonucleotides.

Examples of molecules obtained by modifying nucleotides include sugar-modified nucleotides, phosphodiester bond-modified nucleotides, base-modified nucleotides, and sugar moieties, phosphodiester bonds, and bases. At least one of the modified nucleotides and the like.

As the sugar moiety modified nucleotide, any part of the chemical structure of the sugar structure of the nucleotide that is modified or substituted with any substituent, or substituted with any atom may be any one, and 2 may be preferably used. '-Modified nucleotide.

As the 2'-modified nucleotide, for example, the 2'-OH group of ribose is selected from H, OR, R, R'OR, SH, SR, NH ₂ , NHR, NR ₂ , N ₃ , CN, F, Cl In the group consisting of Br and I (R is an alkyl group or an aryl group, preferably an alkyl group having 1 to 6 carbon atoms, and R 'is an alkylene group, preferably an alkylene group having 1 to 6 carbon atoms) The nucleotide substituted by the substituent is more preferably a nucleotide in which the 2'-OH group is replaced by H, F or methoxy group, and even more preferably a nucleoside in which the 2'-OH group is replaced by F or methoxy group. acid. In addition, the 2'-OH group may be selected from the group consisting of 2- (methoxy) ethoxy (2- (methoxy) ethoxy group), 3-aminopropoxy group (3-aminopropoxy group), and 2- [ (N, N-dimethylamino) oxy] ethoxy (2-[(N, N-dimethylamino) oxy) ethoxy), 3- (N, N-dimethylamino) propoxy (3- (N, N-dimethylamino) propoxy group), 2- [2- (N, N-dimethylamino) ethoxy] ethoxy (2- [2- (N, N-dimethylamino) (ethoxy) ethoxy), 2- (methylamino) -2- pendant ethoxy (2- (methylamino) -2-oxoethoxy), 2- (N-methylamine formamyl) ethoxy (2- (N-methylcarbamoyl) ethoxy group) and 2-cyanoethoxy group (2-cyanoethoxy group) substituted with a substituent substituted nucleotides and the like. Relative to the nucleotides in the double-stranded nucleic acid region, it is preferred to include 50% to 100% of 2'-modified nucleotides, more preferably 70% to 100%, and still more preferably 90% to 100%. . In addition, it is preferable to include 20% to 100% of 2'-modified nucleotides, more preferably 40% to 100%, and even more preferably 60% to 100% of the nucleotides of the sense strand. In addition, it is preferable to contain 20% to 100% of 2'-modified nucleotides with respect to the nucleotides of the antisense strand, more preferably 40% to 100%, and even more preferably 60% to 100%. .

As the phosphodiester bond-modified nucleotide, any part of the chemical structure of the phosphodiester bond of the nucleotide that is partially or wholly modified or substituted with any substituent, or substituted with any atom may be any, such as Examples include nucleotides in which phosphodiester bonds are replaced by phosphorothioate bonds, nucleotides in which phosphodiester bonds are replaced by dithiophosphate bonds, cores in which phosphodiester bonds are replaced by alkylphosphonate bonds Nucleotides, phosphodiester bonds, nucleotides substituted with aminophosphonate bonds, etc.

As the base-modified nucleotide, any part of the chemical structure of the base of the nucleotide that is modified or substituted with any substituent, or substituted with any atom may be any one, and examples include base If the oxygen atom in the group is replaced by a sulfur atom, the hydrogen atom is replaced by an alkyl group having 1 to 6 carbon atoms and the halogen group, the methyl group is replaced by a hydrogen atom, a hydroxymethyl group, and an alkyl group having 2 to 6 carbon atoms, the amine The group is substituted by an alkyl group having 1 to 6 carbons, an alkyl fluorenyl group having 1 to 6 carbons, a pendant oxygen group, a hydroxyl group, and the like.

Examples of the nucleotide derivative include a nucleotide derivative modified with at least one of a nucleotide or a sugar moiety, a phosphodiester bond, or a base, or a peptide, protein, sugar, or Lipids, phospholipids, morphine, folate, phenanthridine, anthraquinone, acridine, fluorescein, rhodamine, coumarin, pigment and other chemical substances, specifically, 5 ' -Polyamine addition nucleotide derivative, cholesterol addition nucleotide derivative, steroid addition nucleotide derivative, bile acid addition nucleotide derivative, vitamin addition nucleotide derivative, Cy5 addition Nucleotide derivatives, Cy3 addition nucleotide derivatives, 6-FAM addition nucleotide derivatives, and biotin addition nucleotide derivatives. A nucleotide derivative may also form an alkylene structure, a peptide structure, a nucleotide structure, an ether structure, an ester structure, and at least one of these in combination with other nucleotides or nucleotide derivatives in a nucleic acid. Bridge structure.

In the present specification, the term "complementary" means a relationship capable of base pairing between two bases. For example, the relationship between adenine and thymine or uracil and the relationship between guanine and cytosine are relaxed through The double-stranded region forms a double-helix structure as a whole.

In the present specification, the term "complementarity" does not mean only the case where two nucleotide sequences are completely complementary, and the nucleotide sequences may also have 0-30%, 0-20%, or 0-10% mismatch bases. The base, for example, means that an antisense strand that is complementary to the β2GPI mRNA may include a substitution of one or a plurality of bases in a base sequence that is completely complementary to a partial base sequence of the mRNA. Specifically, the antisense strand may have 1 to 8, preferably 1 to 6, 1 to 4, 1 to 3, especially 2 or 1 mismatched bases relative to the target sequence of the target gene. . For example, when the base length of the antisense strand is 21, there may be 6, 5, 4, 3, 2 or 1 mismatched bases relative to the target sequence of the target gene. The matching position may be the 5 'end or the 3' end of each sequence. The term "complementarity" includes a case where one nucleotide sequence is a sequence in which one or a plurality of bases are added and / or deleted from a base sequence that is completely complementary to another nucleotide sequence. For example, β2GPI mRNA and the antisense strand nucleic acid of the present invention may have one or two raised bases in the antisense strand and / or the target β2GPI mRNA region due to the addition and / or deletion of bases in the antisense strand.

As long as the double-stranded nucleic acid used as a drug 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 The nucleic acid may be composed of an arbitrary nucleotide or a derivative thereof. The double-stranded nucleic acid of the present invention can form at least 11 base pairs as long as the nucleic acid contains a base sequence complementary to the target β2GPI mRNA sequence and the nucleic acid contains a base sequence complementary to the base sequence of the nucleic acid The length of the strand can be any length. The length of the sequence capable of forming a double strand is usually 11 to 27 bases, preferably 15 to 25 bases, more preferably 17 to 23 bases, and even more preferably 19 ~ 23 bases.

As the antisense strand of the nucleic acid complex of the present invention, a nucleic acid containing a base sequence complementary to the target β2GPI mRNA sequence is used, and 1 to 3 bases of the nucleic acid may also be used, preferably 1 to 2 bases. One base, more preferably one base deletion, substitution or addition.

As a nucleic acid that inhibits the expression of β2GPI, a single-stranded nucleic acid that contains a base sequence that is complementary to the target β2GPI mRNA sequence and that inhibits the performance of β2GPI, or that contains a base sequence that is complementary to the target β2GPI mRNA sequence And a double-stranded nucleic acid comprising a nucleic acid having a base sequence complementary to the base sequence of the nucleic acid and inhibiting the expression of β2GPI.

The single-stranded antisense strand nucleic acid and sense strand nucleic acid constituting the double-stranded nucleic acid are respectively the same or different, and usually contain 11 to 30 bases, preferably the same or different, respectively, containing 17 to 27 bases, and more preferably containing 17 to 25 bases, more preferably 19 to 25 bases, and even more preferably 21 to 23 bases.

In the case where the double-stranded nucleic acid used as a drug in the present invention has an additional nucleotide or a nucleotide derivative that does not form a double-strand on the 3 'side or 5' side connected to the double-stranded region, It is called a protrusion (overhang). In the case where there is a protrusion, the nucleotide constituting the protrusion may be a ribonucleotide, a deoxyribonucleotide, or a derivative thereof.

As a double-stranded nucleic acid having a protruding portion, it is preferably used if it has a protruding portion containing 1 to 6 bases, usually 1 to 3 bases, at least at the 3 'end or 5' end of a single strand. Examples of the protruding portion include those having a protruding portion containing dTdT (dT represents deoxythymidine) or UU (U represents uridine). The antisense stock, the justice stock only, or both the antisense stock and the justice stock may have a protruding portion. In the present invention, it is preferably used for a double-stranded nucleic acid having an protruding portion in the antisense stock. Furthermore, the antisense strand is selected from the oligonucleotides comprising 17 to 30 nucleotides including a double-stranded region and a protruding portion connected thereto, and is selected from those listed in Tables 1-1 to 1-16. The target β2GPI mRNA sequences in this group are fully complementary. Furthermore, as the double-stranded nucleic acid of the present invention, for example, a nucleic acid molecule (WO2005 / 089287) that generates a double-stranded nucleic acid by the action of ribonuclease such as Dicer, or a protrusion without a 3 'end or a 5' end can be used. A double-stranded nucleic acid forming a smooth end, a double-stranded nucleic acid (US2012 / 0040459) with only a positive strand protruding, and the like.

As the double-stranded nucleic acid constituting the nucleic acid complex used in the present invention, a nucleic acid containing the same sequence as the base sequence of the target gene or its complementary strand may be used, and at least a single strand containing the nucleic acid may be used. A double-stranded nucleic acid having a nucleic acid of 1 to 4 bases deleted from its 5 'end or 3' end and a nucleic acid containing a base sequence complementary to the base sequence of the nucleic acid.

The double-stranded nucleic acid constituting the nucleic acid complex used in the present invention may be double-stranded RNA (dsRNA) where RNA forms double strands with each other, double-stranded DNA (dsDNA) where DNA forms double-strands with each other, or double-stranded DNA with RNA and DNAs Hybrid nucleic acid. In addition, one or both of the double strands may be a chimeric nucleic acid of DNA and RNA. Double-stranded RNA (dsRNA) is preferred.

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 more preferably an antisense ribonucleotide. The second to seventh nucleotides from the 5 'end of the sense strand are completely complementary to the second to seventh deoxyribonucleotides from the 3' end of the target β2GPI mRNA sequence, and further preferably from the 5th of the antisense strand. The 2-11th nucleotide from the 'end is completely complementary to the 2-11th deoxyribonucleotide from the 3' end of the target β2GPI mRNA sequence. Furthermore, it is preferred that the eleventh nucleotide from the 5 'end of the antisense strand in the nucleic acid of the present invention is complementary to the eleventh deoxyribonucleotide from the 3' end of the target β2GPI mRNA sequence, and more preferably The 9th to 13th nucleotides from the 5 'end of the antisense strand are completely complementary to the 9th to 13th deoxyribonucleotides from the 3' end of the target β2GPI mRNA sequence. The 7 to 15 nucleotides from the 5 'end are completely complementary to the 7 to 15 deoxyribonucleotides from the 3' end of the target β2GPI mRNA sequence.

The antisense strand and the sense strand of the nucleic acid complex of the present invention can be designed based on the base sequence (sequence number 3541) of the cDNA (sense strand) of the full-length mRNA of human β2GPI registered as Genbank Accession No. NM_000042, for example.

The double-stranded nucleic acid can be designed to interact with a target sequence within the β2GPI gene sequence.

One strand of the double-stranded nucleic acid is complementary to the target site sequence described above. The double-stranded nucleic acid can be chemically synthesized using the method described in this specification.

RNA can be generated by enzymatic or partial / total organic synthesis, or modified ribonucleotides can be introduced in vitro by enzymatic or organic synthesis. In one aspect, each strand is prepared chemically. Methods for chemically synthesizing RNA molecules are well known in the art [see Nucleic Acids Research, 1998, Vol. 32, p. 936-948]. Generally speaking, double-stranded nucleic acids can be synthesized by using a solid-phase oligonucleotide synthesis method (for example, refer to Usman et al., US Patent No. 5,804,683; US Patent No. 5,831,071; US Patent No. 5,998,203; US; Patent No. 6,117,657; US Patent No. 6,353,098; US Patent No. 6,362,323; US Patent No. 6,437,117; US Patent No. 6,469,158; Scaringe et al., US Patent No. 6,111,086; US Patent No. 6,008,400 Specification; US Patent No. 6,111,086).

Single-stranded nucleic acids were synthesized using the solid-phase phosphoramidite method [see Nucleic Acids Research, 1993, Vol. 30, p. 2435-2443], deprotected, and applied to a NAP-5 column (Amersham Pharmacia Biotech, Piscataway, NJ). The oligomers were Amersham Source 15Q columns with a linear gradient of 15 minutes-1.0 cm. Purification was performed using an ion exchange high performance liquid chromatography (IE-HPLC) with a height of 25 cm (Amersham Pharmacia Biotech, Piscataway, NJ). The gradient was changed from 90:10 buffer A: B to 52:48 buffer A: B, buffer A was 100 mmol / L Tris pH 8.5, and buffer B was 100 mmol / L Tris pH 8.5 ( 1 mol / L of NaCl). The samples were monitored at 260 nm, and the peaks corresponding to the full-length oligonucleotide species were collected, pooled, desalted using a NAP-5 column, and freeze-dried.

The purity of each single-stranded nucleic acid was determined by capillary electrophoresis (CE) using Beckman PACE 5000 (Beckman Coulter, Inc., Fullerton, Calif). CE capillary has an inner diameter of 100 μm and contains ssDNA 100R Gel (Beckman-Coulter). Typically, an oligonucleotide of about 0.6 nmole is injected into a capillary tube, electrophoresis is performed under an electric field of 444 V / cm, and detection is performed by UV absorbance at 260 nm. Modified Tris-boric acid-7 mol / L-urea electrophoresis buffer was purchased from Beckman-Coulter. A single-stranded nucleic acid having a purity of at least 90% when evaluated by CE for the experiments described below was obtained. Compound identity is based on the manufacturer's recommended operating instructions by using matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry using Voyager DE. TM. Biospectometry Workstation (Applied Biosystems, Foster City, Calif.) Method for verification. The relative molecular mass of a single-stranded nucleic acid can be obtained within 0.2% of the predicted molecular mass.

The single-stranded nucleic acid was resuspended at a concentration of 100 μmol / L in a buffer solution containing 100 mmol / L potassium acetate and 30 mmol / L HEPES (pH 7.5). The complementary sense and antisense strands were mixed in equal molar amounts to obtain a final solution of 50 μmol / L double-stranded nucleic acid. The sample was heated to 95 ° C for 5 minutes and cooled to room temperature before use. Double-stranded nucleic acids were stored at -20 ° C. Single-stranded nucleic acid lines were freeze-dried or stored at -80 ° C in nuclease-free water.

As the double stranded region including the sense strand and the antisense strand and comprising at least 11 base pairs in the present invention, and the strand length in the antisense strand is 11 to 30 nucleotides Double-stranded nucleic acid partially complementary to the target β2GPI mRNA sequence selected from the group described in Table 1-1 to Table 1-16, which can be used: contains antisense selected from Table 1-1 to Table 1-16 A double-stranded nucleic acid of a sequence in a group consisting of strands, or a group selected from the group consisting of righteous stocks described in Tables M1-1 to M1-18 and Tables 1-1 to 1-16 described below The double-stranded nucleic acid of the above sequence, or one selected from the group consisting of the justice stock / antisense stock described in Tables M1-1 to M1-18 and Tables 1-1 to 1-16 described below Double-stranded nucleic acid to the sequence of the sense / antisense strand. That is, specific examples of the double-stranded nucleic acid constituting the nucleic acid complex used in the present invention include the justice shares and reactions in Tables M1-1 to M1-18 and Tables 1-1 to 1-16 described below. Double stranded nucleic acid. In Tables M1-1 to M1-18, N (M) represents a 2'-O-methyl-modified RNA, N (F) represents a 2'-fluoro-modified RNA, and ＾ represents a phosphorothioate. In addition, the 5 ′ terminal nucleotides 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 or may not be phosphorylated. Is preferably phosphorylated. A double-stranded nucleic acid including the sequences of the sense strand / antisense strand described in Tables 1-1 to 1-16 described below is preferably used in the measurement of knock down activity, and the relative expression of β2GPI is 0.15 The following.

[Table 1] Table M1-1

[Table 2] Table M1-2

[Table 3] Table M1-3

[Table 4] Table M1-4

[Table 5] Table M1-5

[Table 6] Table M1-6

[Table 7] Table M1-7

[Table 8] Table M1-8

[Table 9] Table M1-9

[Table 10] Table M1-10

[Table 11] Table M1-11

[Table 12] Table M1-12

[Table 13] Table M1-13

[Table 14] Table M1-14

[Table 15] Table M1-15

[Table 16] Table M1-16

[Table 17] Table M1-17

[Table 18] Table M1-18

The manufacturing method of the nucleic acid complex of this invention is demonstrated. In addition, in the manufacturing method shown below, when the defined base is changed under the conditions of the manufacturing method or it is not suitable to implement the manufacturing method, it can be introduced by using a protective group commonly used in organic synthetic chemistry. And removal method [for example, "Protective Groups in Organic Synthesis, third edition", method described in TW Greene, John Wiley & Sons Inc. (1999), etc.] . In addition, the order of reaction steps such as introduction of a substituent may be changed as necessary. The nucleic acid polymer represented by Formula 1 can also be synthesized by solid-phase synthesis.

The nucleic acid polymer represented by Formula 1 can be synthesized with reference to a synthetic method of a linker structure known as a nucleic acid complex. Regarding the synthesis of the L1-benzene ring unit with S1 as the linker or the L2-benzene ring unit with S2 as the linker in the nucleic acid complex represented by Formula 1, for example, the nucleic acid complex represented by Formula 2 is used as an example. Instructions. The L1-benzene ring unit or L2-benzene ring unit in the nucleic acid complex represented by Formula 2 is linked by P1, P2, P3, P4, P5 and P6, and T1 and T2. -CO-, -NH-, -O-, -S-, -O-CO-, -S-CO-, -NH-CO- for P1, P2, P3, P4, P5 and P6, and T1 and T2 , -CO-O-, -CO-S-, or -CO-NH- bonds can be referred to, for example, the 4th edition of Experimental Chemistry 19 "Synthesis of Organic Compounds I" Maruzen (1992), 4th Edition of Experimental Chemistry 20 "Synthesis of Organic Compounds II" The method of the bond reaction described in Maruzen (1992) and the like is appropriately synthesized by selecting a raw material suitable for forming the structure represented by Formula 2. Further, a compound having Q1 as a partial structure and a compound having B1 as a partial structure are sequentially bonded to a benzene ring, whereby a partial structure of an L1-benzene ring unit can be produced. Separately synthesize compounds with L1 and Q2 as partial structures, and bond compounds with L1 and Q2 as partial structures to compounds with partial structures with L1-benzene ring units having benzene rings, Q1 and B1 as partial structures, and borrow This can make L1-benzene ring unit structure. The same applies to the L2-benzene ring unit. A compound having Q3 as a partial structure and a compound having B2 as a partial structure are sequentially bonded to the benzene ring, thereby producing a partial structure of the L2-benzene ring unit. Separately synthesize compounds with L2 and Q4 as partial structures, bond compounds with L2 and Q4 as partial structures and compounds with partial structures with L2-benzene ring units as benzene rings, Q3 and B2 as partial structures, and borrow This can produce an L2-benzene ring unit structure.

Examples of the compound having Q1 as a partial structure and the compound having Q3 as a partial structure include an alkylene group having 1 to 10 carbon atoms or-(CH ₂ CH ₂ O) _n -CH ₂ CH ₂ -having hydroxyl groups at both ends. , Carboxyl, amine, thiol-based compounds. Examples of the compound having B1 as a partial structure and the compound having B2 as a partial structure include a structure represented by the following formula 2-1, and a black dot at the end of each structure has a hydroxyl group, a carboxyl group, Amine or thiol-based compounds. Formula 2-1:

[Chemical 90]

Specific examples of the compound having B1 as a partial structure and the compound having B2 as a partial structure include glycols, glutamic acid, aspartic acid, lysine, Tris (Tris (hydroxymethyl) aminomethane, trimethylol) Aminoaminomethane), iminodiacetic acid, 2-amino-1,3-propanediol, and the like, preferably glutamic acid, aspartic acid, lysine, and iminodiacetic acid. Specifically, B1 and B2 preferably have the following structures. [Chemical 91]

The L1-benzene ring unit structure can also be produced by synthesizing a compound having L1, Q2, and B1 as partial structures and bonding it with a compound having Q1 and a benzene ring. The L2-benzene ring unit structure can also be produced by synthesizing a compound having L2, Q4, and B2 as partial structures and bonding it with a compound having Q3 and a benzene ring. In the present invention, as [L1-T1- (Q2-P3 ) q2 -] p1 -B1- (P2-Q1) and a partial structure of _{q1 -P1-} [L2-T2- (Q3-P6 ) q4 -] p2 The partial structures of -B2- (P5-Q3) _q3 -P2- may be the same or different, but are preferably the same.

Examples of the unit corresponding to L1-T1-Q2 of a sugar ligand include L3-T1-Q2-COOH, L3-T1- (Q2-P3) _q2-1 -Q2-NH ₂ and the like. Specific examples include: L3-O-alkylene-COOH having 1 to 12 carbon atoms, or L3-alkylene-CO-NH-alkylene having 2 to 12 carbon atoms- NH ₂ and so on. L3 is not particularly limited as long as it is a sugar ligand derivative that becomes L1 by deprotection. The substituent of the sugar ligand is not particularly limited as long as it is a substituent commonly used in the field of glycochemistry, and an Ac group is preferred.

Regarding the synthesis of the L1-benzene ring unit with S1 as the linker or the L2-benzene ring unit with S2 as the linker, specifically, the method described in the examples is used as a reference, and the carbon of the alkyl chain is appropriately increased or decreased. Number, and replace the terminal amine group or terminal carboxyl group with -CO-, -NH-, -O-, -S-, -O-CO-, -S-CO-, -NH-CO-, -CO -O-, -CO-S-, or -CO-NH-bonded compounds can be synthesized. In addition, as for the sugar ligand of L1, mannose or N-ethylgalactosylamine was exemplified in the examples, but the sugar ligand may be changed to another sugar ligand and implemented.

The synthesis of the X-benzene ring unit using S3 as a linker in the nucleic acid complex represented by Formula 1 will be described by taking, for example, the nucleic acid complex represented by Formula 12. 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 an oligonucleotide bond. -CO-, -NH-, -O-, -S-, -O-CO-, -S-CO-, -NH-CO-, -CO-O-, -CO-S- or P7 and P8 The -CO-NH- bond can be referred to, for example, the fourth edition of Experimental Chemistry 19 "Synthesis of Organic Compounds I" Maruzen (1992), the fourth edition of Experimental Chemistry Lecture 20 "Synthesis of Organic Compounds II" Maruzen (1992) The method of the bonding reaction described in the above is appropriately synthesized by selecting a raw material suitable for forming the structure represented by Formula 12. In addition, a compound having Q5 as a partial structure and a compound having B3 as a partial structure are sequentially bonded to a benzene ring, whereby a partial structure of an X-benzene ring unit can be produced.

Separately synthesize compounds with X and Q7 as partial structures or compounds with X and Q6 as partial structures, so that compounds with X and Q7 as partial structures or compounds with X and Q6 as partial structures and benzene rings and Q5 as sections A compound having a partial structure of an X-benzene ring unit is bonded to form a B3 moiety, whereby an X-benzene ring unit structure can be manufactured. Specifically, in the case where a compound having a benzene ring and Q5 as a partial structure and a partial structure of an X-benzene ring unit has an azide group at the terminal, as an example, the terminal end via The bonded functionalized oligonucleotide reacts to cyclize and add to form a triazole ring, thereby constructing a B3 moiety, thereby producing an X-benzene ring unit structure.

Examples of the compound having Q5 as a partial structure, the compound having Q6 as a partial structure, and the compound having Q7 as a partial structure include an alkylene group having 1 to 10 carbon atoms or-(CH ₂ CH ₂ O) _n8 -CH ₂ A compound having a hydroxyl group, a carboxyl group, an amine group, and a thiol group at both ends of CH _2- .

The L1-benzene ring unit structure, the L2-benzene ring unit structure and the X-benzene ring unit structure can be sequentially manufactured separately. It is preferable to synthesize the L1-benzene ring unit structure and the L2-benzene ring unit structure before bonding the X-benzene. Ring unit structure. In particular, X having an oligonucleotide moiety is preferably introduced into the compound near the final step of the synthesis of the sugar ligand complex.

In the present invention, a compound represented by the following formulae 8 to 10 is obtained as a synthetic intermediate. Equation 8:

[Chemical 92]

(In Formula 8, R1 and R2 are each independently a hydrogen atom, a third butoxycarbonyl group (Boc group), a benzyloxycarbonyl group (Z group), a 9-fluorenylmethoxycarbonyl group (Fmoc group), and -CO- R4, or -CO-B4-[(P9-Q8) _q7 -T3-L3] _p3 , P9 and T3 are independent or absent, or -CO-, -NH-, -O-, -S-, -O-CO-, -S-CO-, -NH-CO-, -CO-O-, -CO-S-, or -CO-NH-, Q8 is either absent or substituted or unsubstituted Alkyl group having 1 to 12 carbon atoms or-(CH ₂ CH ₂ O) _n1 -CH ₂ CH _2- , n1 is an integer of 0 to 99, and B4 is independently a bonding bond, or is the following formula 8-1 For any structure shown, the black dot at the end of each structure is the bond point with carbonyl or P9, m7, m8, m9, and m10 are each independently an integer of 0 to 10, Formula 8-1:

[Chemical 93]

p3 is an integer of 1, 2 or 3, q7 is an integer of 0 to 10, L3 is a sugar ligand, Y is -O- (CH ₂ ) _m11 -NH- and -NH-CO- (CH ₂ ) _m12 -NH M11 and m12 are each independently an integer of 1-10, and R3 is a hydrogen atom, a third butoxycarbonyl group, a benzyloxycarbonyl group, a 9-fluorenylmethoxycarbonyl group, -CO-R4, -CO- (CH ₂ CH ₂ O) _n2 -CH ₂ CH ₂ -N ₃ , or -CO-Q9-B5- (Q10-P10) _q8 -X1, n2 is an integer from 0 to 99, P10 or not exists, or -CO-, -NH-, -O-, -S-, -O-CO-, -S-CO-, -NH-CO-, -CO-O-, -CO-S- or -CO-NH-, Q9 and Q10 is independent, or does not exist, or is a substituted or unsubstituted alkylene group having 1 to 12 carbon atoms or-(CH ₂ CH ₂ O) _n3 -CH ₂ CH _2- , n3 is an integer from 0 to 99 , B5 is any one of the structures represented by the following formula 8-2, and the curve shows the bond with Q9 and Q10, respectively, Formula 8-2:

[Chemical 94]

In Formula 8-2, the substitution in the group having a triazole ring is any of the nitrogen atoms at the 1-position and the 3-position of the triazole ring. q8 is an integer from 0 to 10, X1 is a hydrogen atom or a solid support, and R4 is an amine selected from unsubstituted or substituted by a third butoxycarbonyl group, a benzyloxycarbonyl group, or a 9-fluorenylmethoxycarbonyl group. 1 or 2 substituents in the group consisting of alkyl, carboxyl, maleimide, and aralkyloxycarbonyl groups substituted with 2-10 carbon alkyl groups)

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

[Chem 95]

(In Formula 9, R5 and R6 are each independently a hydrogen atom, a third butoxycarbonyl group, a benzyloxycarbonyl group, a 9-fluorenylmethoxycarbonyl group, -CO-R4 ', or -CO-Q11- (P11- Q11 ') _q9 -T4-L4, P11 and T4 are independent or absent, or -CO-, -NH-, -O-, -S-, -O-CO-, -S-CO-,- NH-CO-, -CO-O-, -CO-S- or -CO-NH-, Q11 and Q11 'are either absent or substituted or unsubstituted alkylene groups having 1 to 12 carbon atoms, or -(CH ₂ CH ₂ O) _n4 -CH ₂ CH _2- , n4 is an integer from 0 to 99, q9 is an integer from 0 to 10, L4 is a sugar ligand, and Y 'is -O- (CH ₂ ) _m11' -NH- and -NH-CO- (CH ₂ ) _{m12 '} -NH, m11' and m12 'are each independently an integer of 1 to 10, and R3' is a hydrogen atom, a third butoxycarbonyl group, a benzyloxycarbonyl group, 9-fluorenylmethoxycarbonyl, -CO-R4 ', -CO- (CH ₂ CH ₂ O) _n2' -CH ₂ CH ₂ -N ₃ , or -CO-Q9'-B5 '-(Q10'- P10 ') _q8' -X1 ', n2' is an integer from 0 to 99, P10 'is either absent or -CO-, -NH-, -O-, -S-, -O-CO-, -S -CO-, -NH-CO-, -CO-O-, -CO-S- or -CO-NH-, Q9 'and Q10' are independent, or not present, or are substituted or unsubstituted carbons Alkyl number 1 to 12 or-(CH ₂ CH ₂ O) _{n3 '} -CH ₂ CH _2- , n3' is an integer from 0 to 99, B5 'is any structure represented by the following formula 9-1, and the curve represents a bond with Q9' and Q10 ', respectively. -1:

[Chem 96]

In the formula 9-1, the substitution in the group having a triazole ring is any of nitrogen atoms at the 1-position and the 3-position of the triazole ring. q8 'is an integer from 0 to 10, X1' is a hydrogen atom or a solid phase support, and R4 'is selected from a group substituted with a third butoxycarbonyl group, a benzyloxycarbonyl group, a 9-fluorenylmethoxycarbonyl group, or not (Substituted amine group, carboxyl group, maleimide group, and aralkyloxycarbonyl group with 1 or 2 substituents substituted with 2 to 10 carbon alkyl groups)

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

[Chem 97]

In Formula 10, R7 and R8 are each independently a hydroxyl group, a third butoxy group, a benzyloxy group, -NH-R10, or -NH-Q12- (P12-Q12 ') _q10 -T4-L4, and P12 and T4 are independent , Or not present, or -CO-, -NH-, -O-, -S-, -O-CO-, -S-CO-, -NH-CO-, -CO-O-, -CO- S- or -CO-NH-, Q12 and Q12 'are either absent, or substituted or unsubstituted alkylene having 1 to 12 carbon atoms or-(CH ₂ CH ₂ O) _n2 -CH ₂ CH ₂ -, N2 is an integer from 0 to 99, L4 is a sugar ligand, Y2 is -O- (CH ₂ ) _m9 -NH- and -NH-CO- (CH ₂ ) _m10 -NH, m9 and m10 are independently 1 An integer of -10, q10 is an integer of 0-10, R9 is a hydrogen atom, a third butoxycarbonyl group, a benzyloxycarbonyl group, a 9-fluorenylmethoxycarbonyl group, -CO-R10, -CO- (CH ₂ CH ₂ O) _n6 -CH ₂ CH ₂ -N ₃ or -CO-Q13-B6- (Q14-P13) _q11 -X2, n6 is an integer from 0 to 99, P13 is either absent or -CO-, -NH-, -O-, -S-, -O-CO-, -S-CO-, -NH-CO-, -CO-O-, -CO-S- or -CO-NH-, Q13 and Q14 is independent or does not exist, or is a substituted or unsubstituted alkylene group having 1 to 12 carbon atoms _or- (CH ₂ CH ₂ O) _n7 -CH ₂ CH _2- , n7 is an integer from 0 to 99 , B6 is the following formula For any structure represented by 10-1, the curve shows the bond with Q13 and Q14, respectively. Formula 10-1:

[Chemical 98]

In Formula 10-1, the substitution in the group having a triazole ring is any of nitrogen atoms at the 1-position and the 3-position of the triazole ring. q11 is an integer from 0 to 10, X2 is a hydrogen atom or a solid support, and R10 is an amine selected from the group consisting of substituted or unsubstituted by a third butoxycarbonyl group, a benzyloxycarbonyl group, and a 9-fluorenylmethoxycarbonyl group. 1 or 2 substituents in the group consisting of alkyl, carboxyl, maleimide, and aralkyloxycarbonyl groups substituted with 2-10 carbon alkyl groups)

Hereinafter, an example of the manufacturing method is illustrated about this invention. In addition, in the following descriptions of Production Method 1 to Production Method 17, among the compounds represented by Formula 1 to Formula 12 in the nucleic acid derivatives and the like of the present invention, the symbols representing the groups have the same symbols, but It should be understood that the two are separate, and the present invention is not limited to the explanation of the bases described in Manufacturing Method 1 to Manufacturing Method 12. In addition, regarding the nucleic acid derivative in the present invention, X representing an oligonucleotide is described as -O-X in Production Method 1 to Production Method 17.

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

[Chemical 99]

(In the formula, P1 is a protecting group such as Fmoc which can be deprotected by a base, DMTr represents p, p'-dimethoxytrityl, R represents a sugar ligand-tethered unit, and R 'represents R in Each hydroxyl group of a sugar ligand is protected by a protecting group capable of being deprotected by a base such as acetamyl. The polymer represents a solid phase support, and Q 'is -CO-)

Step 1 Compound (IB) can be obtained by dissolving compound (IA) and p, p'-dimethoxytriphenylchloromethane in a solvent such as pyridine and optionally in the presence of a co-solvent at 0 ° C and 100 ° C. It is produced by reacting at an intermediate temperature for 5 minutes to 100 hours. Examples of the co-solvent include methanol, ethanol, dichloromethane, chloroform, 1,2-dichloroethane, toluene, ethyl acetate, acetonitrile, diethyl ether, tetrahydrofuran, and 1,2-dimethoxyethane. , Dioxane, N, N-dimethylformamide (DMF), N, N-dimethylacetamide, N-methylpyrrolidone, pyridine, water, etc. These can be used alone or in combination .

Step 2 Compound (IC) can be reacted in a solvent-free condition or in a solvent in the presence of 1 to 1,000 equivalents of a secondary amine at a temperature between room temperature and 200 ° C. 5 minutes to 100 hours. Examples of the solvent include methanol, ethanol, dichloromethane, chloroform, 1,2-dichloroethane, toluene, ethyl acetate, acetonitrile, diethyl ether, tetrahydrofuran, 1,2-dimethoxyethane, Dioxane, N, N-dimethylformamide (DMF), N, N-dimethylacetamide, N-methylpyrrolidone, pyridine, water, etc. These can be used alone or in combination. Examples of the secondary amine include diethylamine and piperidine.

Step 3 Compound (1-E) can be obtained by leaving compound (IC) and compound (ID) in a solvent-free condition or in a solvent at 1-30 equivalents of a base, a condensing agent, and 0.01-30 equivalents as needed. It is produced by reacting in the presence of additives at a temperature between room temperature and 200 ° C for 5 minutes to 100 hours. Examples of the solvent include those exemplified in Step 2. Examples of the base include cesium carbonate, potassium carbonate, potassium hydroxide, sodium hydroxide, sodium methoxide, potassium tert-butoxide, triethylamine, diisopropylethylamine, N-methylline, pyridine, 1,8-diazabicyclo [5.4.0] -7-undecene (DBU), N, N-dimethyl-4-aminopyridine (DMAP), etc. Examples of the condensing agent include 1,3-dicyclohexanecarbodiimide (DCC), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, and salts Acid salt (EDC), carbonyldiimidazole, benzotriazol-1-yloxytris (dimethylamino) fluorene, hexafluorophosphate (benzotriazol-1-yloxy) tripyrrole Pyridylfluorene, hexafluorophosphate O- (7-azabenzotriazol-1-yl) -N, N, N ', N'-tetramethylurenium (HATU), hexafluorophosphate O- (benzene Benzotriazol-1-yl) -N, N, N ', N'-tetramethylurenium (HBTU), 2-chloro-1-methylpyridinium iodide, and the like. Examples of the additive include 1-hydroxybenzotriazole (HOBt), 4-dimethylaminopyridine (DMAP), and the like. Compound (I-D) can be obtained by a known method (for example, with reference to the Journal of the American Chemical Society, vol. 136, p. 16958, (2014)) or a method based thereon.

Step 4 Compound (IF) can be obtained 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. Manufacturing.

Examples of the solvent include those exemplified in Step 2.

Examples of the base include those exemplified in Step 3.

Step 5 The compound (IG) can be obtained by subjecting the compound (IF) to a solid-phase support having an aminated end in a solvent-free condition or in a solvent at a base of 1 to 30 equivalents, a condensing agent, and 0.01 to as needed In the presence of 30 equivalents of additives, react at a temperature between room temperature and 200 ° C for 5 minutes to 100 hours, and then react in an acetic anhydride / pyridine solution at a temperature between room temperature and 200 ° C for 5 minutes to Made in 100 hours. Examples of the solvent include those exemplified in Step 2. Examples of the base, the condensing agent, and the additives include those exemplified in Step 3. Examples of the aminated solid phase support include long-chain alkylamine fine-porous glass (LCAA-CPG). These are available as commercially available products.

Step 6 A nucleic acid complex having a sugar ligand-tethered-branched unit represented by the formula (I ') can be produced by using a compound (IG) and correspondingly by a known oligonucleotide chemical synthesis method. After the nucleotide chain is extended, it is detached from the solid phase, and the protective group is deprotected and refined.

Examples of well-known methods for chemical synthesis of oligonucleotides include phosphoramidite method, phosphorothioate method, phosphate triester method, and CEM (Cation Exchange Membrane) method (see Nucleic Acids Research, 35, 3287 (2007)) and the like can be synthesized by, for example, an ABI3900 high-throughput nucleic acid synthesizer (manufactured by Applied Biosystems).

Detachment and deprotection from the solid phase can be achieved by using alkali treatment for 10 seconds to 72 hours in a solvent or without solvent at a temperature between -80 ° C and 200 ° C after the oligonucleotide chemical synthesis. get on.

Examples of the base include ammonia, methylamine, dimethylamine, ethylamine, diethylamine, isopropylamine, diisopropylamine, piperidine, triethylamine, ethylenediamine, and 1,8-diazepine. Cyclobis [5.4.0] -7-undecene (DBU), potassium carbonate, etc.

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

The purification of the oligonucleotide can be performed by a method using a C18 reverse phase column or an anion exchange column, preferably by combining the above two methods. The purity of the purified nucleic acid complex is preferably 90% or more, and more preferably 95% or more.

Further, in the above step 3, if necessary, the compound (ID) may be divided into two units, and the compound (IC) may be condensed in two stages. Specifically, for example, when R-Q 'is R-NH-CO-Q4'-CO- (Q4' is a substituted or unsubstituted alkylene group having 1 to 12 carbon atoms), in step 3 In the same manner as in Step 3, the compound (IC) is condensed with CH ₃ CH ₂ -O-CO-Q4'-CO-OH (Q4 'has the same meaning as above), and the ethyl ester of the obtained compound is condensed in The target compound can be obtained by hydrolysis with a base such as lithium hydroxide in a solvent such as ethanol or water, and condensation with R'-NH ₂ (R 'has the same meaning as above). In addition, CH ₃ CH ₂ -O-CO-Q4'-CO-OH (Q4 'has the same meaning as above) and R'-NH ₂ (R' has the same meaning as above) can be obtained by a known method (for example, refer to the US Journal of the Chemical Society, Vol. 136, p. 16958 (2014)) or according to its method. Here, in Q4 ′, the substituent and the alkylene moiety in the substituted or unsubstituted alkylene group having 1 to 12 carbon atoms are the same as described above.

The case where Q is -CO- is exemplified, but regarding the case where Q is not -CO-, the structure of Q can be changed appropriately by the same method as above, a known method or a combination of these methods, It is prepared by appropriately changing the reaction conditions.

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

[Chemical 100]

(In the formula, DMTr, R, R ', X, Q' and polymer have the same meanings as above. TBDMS represents the third butyldimethylsilyl group, and Fmoc represents the 9-fluorenylmethoxycarbonyl group.)

Step 7 The compound (II-A) can be obtained by dissolving the compound (IA), the third butyl chlorochlorosilane and dimethylaminopyridine in a solvent such as N, N-dimethylformamide (DMF), It is preferably produced by reacting for 5 minutes to 100 hours at a temperature between 0 ° C and 100 ° C in the presence of 2 equivalents of bases. Examples of the base include those exemplified in Step 3 of Production Method 1.

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) can be obtained 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. Manufacturing. Examples of the solvent include those exemplified in Step 2.

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 (I-D) under the same conditions as in step 3 of production method 1.

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

In addition, in the above step 11, if necessary, the compound (ID) may be divided into two units and condensed with the compound (II-C) in two stages. Specifically, for example, when R-Q 'is -NH-CO-Q4'-CO- (Q4' is a substituted or unsubstituted alkylene group having 1 to 12 carbon atoms), in step 11 The compound (II-C) is condensed with CH ₃ CH ₂ -O-CO-Q4'-CO-OH (Q4 'has the same meaning as above) by the same method as in Step 11, and the ethyl ester of the obtained compound is condensed. The target compound can be obtained by hydrolysis with a base such as lithium hydroxide in a solvent such as ethanol or water, and condensation with R'-NH ₂ (R 'has the same meaning as above). In addition, CH ₃ CH ₂ -O-CO-Q4'-CO-OH (Q4 'has the same meaning as above) and R'-NH ₂ (R' has the same meaning as above) can be obtained by a known method (for example, refer to the US Journal of the Chemical Society, Vol. 136, p. 16958 (2014)) or according to its method.

The case where Q is -CO- is exemplified, but regarding the case where Q is not -CO-, the structure of Q can be changed appropriately by the same method as above, a known method or a combination of these methods, It is prepared by appropriately changing the reaction conditions.

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

[Chemical 101]

(In the formula, DMTr, Fmoc, R, R ', Q', X and polymer have the same meanings as above.)

Compound (III ') can be produced using compound (III-A) under the same conditions as in steps 1 to 6 of production method 1. The compound (III-A) can be obtained as a commercially available 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 The compound (III ') can be produced using the compound (III-E) under the same conditions as in the steps 4 to 6 of the production method 1.

In Step 17 described above, if necessary, the compound (ID) may be divided into two units and condensed with the compound (III-C) in two stages. Specifically, for example, when R-Q 'is -NH-CO-Q4'-CO- (Q4' is a substituted or unsubstituted alkylene group having 1 to 12 carbon atoms), in step 17 The compound (III-C) is condensed with CH ₃ CH ₂ -O-CO-Q4'-CO-OH (Q4 'has the same meaning as above) by the same method as in Step 17, and the ethyl ester of the obtained compound is condensed. The target compound can be obtained by hydrolysis with a base such as lithium hydroxide in a solvent such as ethanol or water, and condensation with R'-NH ₂ (R 'has the same meaning as above). In addition, CH ₃ CH ₂ -O-CO-Q4'-CO-OH (Q4 'has the same meaning as above) and R'-NH ₂ (R' has the same meaning as above) can be obtained by a known method (for example, refer to the US Journal of the Chemical Society, Vol. 136, p. 16958 (2014)) or according to its method.

The case where Q is -CO- is exemplified, but regarding the case where Q is not -CO-, the structure of Q can be changed appropriately by the same method as above, a known method or a combination of these methods, It is prepared by appropriately changing the reaction conditions.

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

[Chemical 102]

(In the formula, DMTr, Fmoc, R, R ', Q', X and polymer have the same meanings as above.)

Compound (IV ') can be produced using compound (IV-A) under the same conditions as in steps 1 to 6 of production method 1. The compound (IV-A) can be obtained as a commercially available product.

Further, in the above step 23, if necessary, the compound (ID) may be divided into two units, and the compound (IV-C) may be condensed in two stages. Specifically, for example, when R'-Q 'is -NH-CO-Q4'-CO- (Q4' is a substituted or unsubstituted alkylene group having 1 to 12 carbon atoms), then step 23 In the same manner as in Step 23, the compound (IV-C) is condensed with CH ₃ CH ₂ -O-CO-Q4'-CO-OH (Q4 'has the same meaning as above), and the ethyl group of the obtained compound is condensed. After the ester is hydrolyzed with a base such as lithium hydroxide in a solvent such as ethanol or water, and then condensed with R'-NH ₂ (R 'has the same meaning as above), the target compound can be obtained. In addition, CH ₃ CH ₂ -O-CO-Q4'-CO-OH (Q4 'has the same meaning as above) and R'-NH ₂ (R' has the same meaning as above) can be obtained by a known method (for example, refer to the US Journal of the Chemical Society, Vol. 136, p. 16958 (2014)) or according to its method.

The case where Q is -CO- is exemplified, but regarding the case where Q is not -CO-, the structure of Q can be changed appropriately by the same method as above, a known method or a combination of these methods, It is prepared by appropriately changing the reaction conditions.

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

[Chemical 103]

(In the formula, DMTr, R, R ', X, Q', TBDMS, Fmoc and polymer have the same meaning as above.)

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

Further, in the above step 31, if necessary, the compound (ID) may be divided into two units and condensed with the compound (VD) in two stages. Specifically, for example, when R'-Q 'is -NH-CO-Q4'-CO- (Q4' is a substituted or unsubstituted alkylene group having 1 to 12 carbon atoms), in step 31 In the same manner as in Step 31, the compound (VD) is condensed with CH ₃ CH ₂ -O-CO-Q4'-CO-OH (Q4 'has the same meaning as above), and the ethyl ester of the obtained compound is condensed in The target compound can be obtained by hydrolysis with a base such as lithium hydroxide in a solvent such as ethanol or water, and condensation with R'-NH ₂ (R 'has the same meaning as above). In addition, CH ₃ CH ₂ -O-CO-Q4'-CO-OH (Q4 'has the same meaning as above) and R'-NH ₂ (R' has the same meaning as above) can be obtained by a known method (for example, refer to the US Journal of the Chemical Society, Vol. 136, p. 16958 (2014)) or according to its method.

The case where Q is -CO- is exemplified, but regarding the case where Q is not -CO-, the structure of Q-OH can also be changed by appropriately changing the structure of Q-OH by the same method, a known method, or the method It is prepared by combining and appropriately changing reaction conditions.

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

[Chemical 104] (Wherein R, R ', Q', DMTr and X have the same meanings as above)

Step 35 Compound (IH) can be obtained by using compound (II-E) and 2-cyanoethyl-N, N, N ', N'-tetraisopropylphosphoramidene It is produced by reacting in a solvent with a base and a reaction accelerator at a temperature between room temperature and 200 ° C for 5 minutes to 100 hours. Examples of the solvent include those exemplified in Step 2 of Production Method 1. Examples of the base include those exemplified in Step 3 of Production Method 1. Examples of the reaction accelerator include 1H-tetrazole, 4,5-dicyanoimidazole, 5-ethylthiotetrazole, and 5-benzylthiotetrazole, and they are commercially available.

Step 36 Extend the oligonucleotide chain, and finally use compound (IH) to modify the 5 'end of the oligonucleotide with a sugar ligand-tethering-branching unit, and then detach from the solid phase and deprotect the protecting group. And purification, whereby compound (I '') can be produced. Here, the detachment from the solid phase, the deprotection of the protecting group, and the purification can be performed in the same manner as in Step 7 of Production Method 1, respectively.

Production method 7 The method for producing a nucleic acid complex of the present invention in which a sugar ligand-tethered-branched unit is bonded to the 5 'end of the oligonucleotide is exemplified below.

It can be manufactured under the same conditions as steps 35 and 36 of manufacturing method 6.

[Chem. 105]

(Wherein R, R ', Q', DMTr and X have the same meanings as above)

Production method 8 The method for producing a nucleic acid complex of the present invention in which a sugar ligand-tethered-branched unit is bonded to the 5 'end of the oligonucleotide is exemplified below.

It can be manufactured under the same conditions as steps 35 and 36 of manufacturing method 6.

[Chem. 106]

(Wherein R, R ', Q', DMTr and X have the same meanings as above)

Production method 9 The method for producing a nucleic acid complex of the present invention in which a sugar ligand-tethered-branched unit is bonded to the 5 'end of the oligonucleotide is exemplified below.

It can be manufactured under the same conditions as steps 35 and 36 of manufacturing method 6.

[Chemical 107]

(Wherein R, R ', Q', DMTr and X have the same meanings as above)

Production method 10 The method for producing a nucleic acid complex of the present invention in which a sugar ligand-tethered-branched unit is bonded to the 5 'end of the oligonucleotide is exemplified below.

It can be manufactured under the same conditions as steps 35 and 36 of manufacturing method 6.

[Chemical 108]

(Wherein R, R ', Q', DMTr and X have the same meanings as above)

Production method 11 Dissolve the right strand with a sugar ligand-tethering-branching unit at the 3 'or 5' end of the sense strand constituting the double-stranded nucleic acid and the antisense strand constituting the double-stranded nucleic acid, respectively, in water or an appropriate one. A nucleic acid complex having a double-stranded nucleic acid can be obtained by mixing in a buffer solution. Examples of the buffer solution include an acetate buffer solution, a Tris buffer solution, a citric acid buffer solution, a phosphate buffer solution, and water. These can be used alone or in combination.

As the mixing ratio of the justice stock and the antisense stock, the antisense stock is preferably 0.5 to 2 equivalents, more preferably 0.9 to 1.1 equivalents, and still more preferably 0.95 to 1.05 equivalents relative to 1 equivalent of the justice shares.

Further, after the justice stock and the antisense stock are mixed, an annealing treatment may be performed as appropriate. The annealing treatment can be performed by heating the mixture of the sense stock and the antisense stock to preferably 50 to 100 ° C, more preferably 60 to 100 ° C, and still more preferably 80 to 100 ° C, and then slowly cooling to room temperature.

Antisense strands can be obtained according to the known oligonucleotide synthesis methods described above.

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

[Chem. 109]

(In the formula, DMTr, R, R ', X, Q', polymer, and Fmoc have the same meanings as above, TBS represents the third butyldimethylsilyl group, and R0 and Rx are the same or different and represent a hydrogen atom, C1 to C10 alkylene or C3-C8 cycloalkylene, W is C1-C10 alkylene, C3-C8 cycloalkylene, or nitrogen-containing heterocyclic ring that can form C4-C8 with R0)

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 commercial product, or by a known method (for example, Bioorganic & Medicinal Chemistry Letters, Vol. 11, pp. 383-386) or based thereon Way to obtain.

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 using compound (VI-E) and compound (VI-F) under the same conditions as in step 3 of production method 1.

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

Steps 45 to 53 can also be performed by a known method (for example, the method described in International Publication No. 2015/105083) or a method based thereon. The compound (VI-F) can be obtained by a known method (for example, Journal of the American Chemical Society, Vol. 136, p. 16958, method described in 2014) or a method based thereon.

Production method 13 The sugar ligand-tethered unit in which P1 and P4 in Formula 2 are -NH-CO-, -O-CO-, or -S-CO- can be produced by the following method.

[Chemical 110]

(In the formula, Q1, Q2, Q3, Q4, Q5, P2, P3, P5, P6, P7, T1, T2, L1, L2, q1, q2, q3, and q4 have the same meanings as above, and q2 'represents a ratio q2 An integer smaller than 1, q4 'represents an integer smaller than q4, P1' and P4 'independently represent -NH-CO-, -O-CO-, or -S-CO-, and Z represents H, OH, NH ₂ , SH, a chlorine atom, a bromine atom, an iodine atom, a methanesulfonyloxy group, a p-toluenesulfonyloxy group, or a carboxylic acid, B1 ′ and B2 ′ represent any one of the structures of the following formula, PG1, PG2, PG3, PG4, PG5, PG6 and PG7 respectively represent suitable protecting groups) Formula:

[Chemical 111]

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

Step 54 The compound (VII-C) can be obtained by adding the compound (VII-A) and the compound (VII-B) to a solvent such as tetrahydrofuran, adding a triphenylphosphine polymer carrier, and cooling with an azobisene in an ice bath. It is produced by reacting a diisopropyl formate toluene solution. Examples of the solvent include those exemplified in Step 2 of Production Step 1. Compound (VII-A) is available as a commercial product.

Step 55 The compound (VII-D) can be produced by reacting the compound (VII-C) in a solvent such as methanol under ice-cooling in the presence of a base. Examples of the solvent include those exemplified in Step 2 of Production Step 1. Examples of the base include those exemplified in Step 3 of Production Step 1.

Step 56 The compound (VII-F) can be produced using the compound (VII-D) and the compound (VII-E) under the same conditions as in the step 3 of the production step 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 step 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 step 1. Further, by repeatedly performing the DP step and step 58, a compound (VII-J) having an ideal q1 value 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 step 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 step 1.

Steps 61 to 63 The compound (VII ′) can be produced using the compound (VII-O), the compound (VII-P), and the compound (VII-Q) under the same conditions as in the step 3 of the production step 1. In addition, by repeatedly performing the DP step and step 61, a compound (VII ') having an ideal q3 value can be produced.

Step DP can be produced by appropriately adopting a method commonly used in organic synthetic chemistry [for example, a method described in "Protective Groups in Organic Synthesis", 3rd Edition, by T.W. Greene, John Wiley & Sons Inc. (1999), etc.]. 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 compound (VII-Q) are available as commercially available products, or they can be prepared by "Experimental Chemistry Lecture 4th Edition Organic Synthesis, p.258, Maruzen (1992)", "March Advanced Organic Chemistry: Reactions 2. Mechanisms and Structures (March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure) 7th Edition "or a combination of the methods described in the method.

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

[Chemical 112]

(In the formula, Q5, P7, and q5 have the same meanings as above, q5 '' represents an integer less than q5, q5 'represents an integer less than q5, and Z2 represents H, OH, NH ₂ or SH, PG8 and PG9 (Respectively suitable protecting groups, LC represents a saccharide ligand-tethered unit, and E represents a carboxylic acid or maleimide)

Step 64 The compound (VIII-C) can be produced using the compound (VIII-A) and the compound (VIII-B) under the same conditions as in the step 3 of the production step 1. Further, by repeatedly performing the DP step and step 64, a compound (VIII-C) having an ideal q5 '' value can be produced. Compound (VIII-B) can be obtained as a commercial product, or it can be obtained through "Organic Synthesis of Experimental Chemistry 4th Edition, p.258, Maruzen (1992)", "March Advanced Organic Chemistry: Reactions, Mechanisms and Structures 7th Edition "or a combination of the methods described in" Version ".

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 step 1. Compound (VIII-D) can be obtained as a commercial product, or it can be obtained by "Organic Synthesis of Experimental Chemistry 4th Edition, p.258, Maruzen (1992)", "March Advanced Organic Chemistry: Reactions, Mechanisms and Structures 7th Edition "or a combination of the methods described in" Version ".

Step DP can be produced by appropriately adopting a method commonly used in organic synthetic chemistry [for example, a method described in "Protective Groups in Organic Synthesis", 3rd Edition, by T.W. Greene, John Wiley & Sons Inc. (1999), etc.].

Production method 15 The sugar ligand-tethered unit in which P1 and P4 are -O- in formula 2 can be produced by the following method.

[Chem 113]

(Where Q1, Q2, Q3, Q4, P2, P3, P5, P6, T1, T2, L1, L2, q1, q2, q3, q4, q2 ', q4', Z, B1 ', B2' are respectively Same meaning as above, PG10, PG11, PG12, PG13, PG14 and PG15 respectively represent suitable protecting groups) Formula:

[Chemical 114]

m1, m2, m3, and m4 have the same meanings as described above.

Step 66 Compound (IX-C) can be prepared by dissolving compound (IX-A) and compound (IX-B) in a solvent such as N, N'-dimethylformamide, and adding a base such as potassium bicarbonate to It is produced by reacting at room temperature to 200 ° C for 5 minutes to 100 hours. Examples of the solvent include those exemplified in Step 2 of Production Step 1. Examples of the base include those exemplified in Step 3 of Production Step 1.

Step 67 Compound (IX-E) can be prepared by dissolving compound (IX-C) and compound (IX-D) in a solvent such as N, N'-dimethylformamide, and adding a base such as potassium bicarbonate to It is produced by reacting at room temperature to 200 ° C for 5 minutes to 100 hours. Examples of the solvent include those exemplified in Step 2 of Production Step 1. Examples of the base include those exemplified in Step 3 of Production Step 1. Compound (IX-A) is available 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 step 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 step 1. Further, by repeatedly performing the DP step and step 69, a compound (VII-J) having an ideal q1 value can be produced.

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 step 1.

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

Step 72-74 Compound (IX ') can use compound (IX-M), compound (IX-N), compound (IX-O), and compound (IX-P) under the same conditions as in step 3 of production step 1 Under Manufacturing. In addition, by repeatedly performing the DP step and step 72, a compound (IX ') having an ideal q3 value can be produced.

Step DP can be produced by appropriately adopting a method commonly used in organic synthetic chemistry [for example, a method described in "Protective Groups in Organic Synthesis", 3rd Edition, by T.W. Greene, John Wiley & Sons Inc. (1999), etc.].

Compound (IX'-B), Compound (IX'-D), Compound (IX'-F), Compound (IX'-H), Compound (IX'-J), Compound (IX'-L), Compound ( IX'-N), compound (IX'-O), and compound (IX'-P) are available as commercially available products, or by "Organic Synthesis, Experimental Chemistry 4th Edition, p.258, Maruzen (1992) ", Or" March Advanced Organic Chemistry: Reactions, Mechanisms, and Structures, 7th Edition ".

Production method 16 As a method for producing a nucleic acid complex of Formula 1 to Formula 8, the following method can also be used.

[Chem 115]

(In the formula, LC, Q5, Q6, Q7, P7, P8, q5 ', q6 and X have the same meanings as above)

Step 75 The compound (X-B) can be produced by reacting the compound (XIII '') and the compound (X-A) in a solvent at 0 ° C to 100 ° C for 10 seconds to 100 hours. Examples of the solvent include water, a phosphate buffer solution, a sodium acetate buffer solution, and dimethylsulfinium. These can be used alone or in combination. Compound (VIII ′) can be obtained by using Production Method 14.

The compound (XA) can be obtained by a known method (for example, Bioconjugate Chemistry, Vol. 21, pages 187-202, 2010 or Current Protocols in Nucleic Acid Chemistry, 2010, September; CHAPTER: Unit 4.41) or according to its method.

Step 76 The compound (X ') can be produced by using the compound (XB) and reacting it at a temperature between room temperature and 200 ° C for 5 minutes to 100 hours under the conditions of pH 8 or higher, such as sodium carbonate aqueous solution or ammonia water. .

Production method 17 As a method for producing a nucleic acid complex of Formula 1 to Formula 8, the following method can also be adopted.

[Chem 116]

(In the formula, LC, Q5, Q6, Q7, P7, P8, q5 ', q6 and X have the same meanings as above)

Step 77 Compound (XI-A) can use compound (VIII '' ') by a known method (for example, "Bioconjugate Conjugation", vol. 26, pp. 1451-1455, method described in 2015) or based on it Way to obtain. Compound (VIII "') is obtained by using Production Method 14.

Step 78 The compound (XI ') can use the compound (XI-A) and the compound (XI-B) by a known method (for example, "Bioconjugate Conjugation", Vol. 26, pp. 1451-1455, 2015) Method) or according to its method. The compound (XI-B) can be obtained by a method described in or based on Bioconjugated Chemistry, Vol. 26, pp. 1451-1455, 2015.

Step 79 As another method, the compound (XI ′) can be prepared by a known method (for example, refer to “Bioconjugate Conjugation”, Vol. 22, pp. 1723-1728, 2011) or a method based thereon. XI-A) obtained directly.

The nucleic acid complex in the present specification can also be obtained, for example, in the form of a salt such as an acid addition salt, a metal salt, an ammonium salt, an organic amine addition salt, or an amino acid addition salt.

Examples of the acid addition salt include inorganic acid salts such as hydrochloride, sulfate, and phosphate; acetate, maleate, fumarate, citrate, and mesylate. Organic acid salts; Examples of metal salts include alkali metal salts such as sodium and potassium salts; alkaline earth metal salts such as magnesium and calcium salts; aluminum salts and zinc salts; and examples of ammonium salts include: Salts such as methyl ammonium; examples of the addition salts of organic amines include addition salts such as phospholine and piperidine; examples of the addition salts of amino acids include additions of lysine, glycine, and phenylalanine A salt.

In the case of preparing a salt of a nucleic acid complex of the present specification, it may be directly purified when the complex system is obtained in the form of a desired salt, and the complex is dissolved or suspended in the form of a free form when obtained. Appropriate solvent, add corresponding acid or base, and separate and refine. In addition, when the counter ion forming the complex salt is replaced with a different counter ion, the complex salt is dissolved or suspended in a suitable solvent, and a number of equivalents to a large excess of an acid, base, and / or salt (chloride) are added. Inorganic salts such as sodium and ammonium chloride, etc.) can be isolated and purified.

There may be geometric isomers, stereoisomers such as optical isomers, tautomers, etc. in the nucleic acid complexes of this specification, and all possible isomers and mixtures thereof are included in the present invention.

In addition, the nucleic acid complex of the present specification may exist in the form of adducts with water or various solvents, and such adducts are also included in the present invention.

Furthermore, the nucleic acid complex of the present invention also includes those in which some or all of the atoms in the molecule are replaced by atoms (isotopes) (such as deuterium atoms) having different mass numbers.

The pharmaceutical composition of the present invention includes a nucleic acid complex represented by Formula 1. The nucleic acid complex of the present invention is recognized by a target cell and introduced into a cell by a sugar ligand having L1 and L2. The nucleic acid complex of the present invention reduces or stops the expression of a target gene in an organism by administering it to a mammal, thereby suppressing the expression of the target gene, and thus can be used for treating a target gene-related disease. In the case where the nucleic acid complex of the present invention is used as a therapeutic agent or a preventive agent, it is preferable to use the administration route that is most effective for the treatment as the administration route, and there is no particular limitation. For example, intravenous administration, Subcutaneous administration and intramuscular administration are preferred, and subcutaneous administration is preferred. The amount to be administered varies depending on the condition, age, administration route, etc. of the object to be administered. For example, it can be administered by converting the amount of double-stranded oligonucleotides per day to 0.1 μg to 1000 mg. Preferably, it is administered in such a manner that the daily dose becomes 1 to 100 mg.

Examples of preparations suitable for intravenous or intramuscular administration include injections. The prepared liquids can be used directly as, for example, injections, or they can be removed from the liquids by, for example, filtration or centrifugation. Use the solvent after use, freeze-dry the solution before using it, and / or freeze-dry the solution with excipients such as mannitol, lactose, trehalose, maltose, or glycine. . In the case of an injection, it is preferred to prepare a solution by mixing a liquid or a solvent-free or freeze-dried composition with, for example, water, an acid, an alkali, various buffers, physiological saline or an amino acid infusion. In addition, an injection such as citric acid, ascorbic acid, cysteine, or EDTA (ethylenediamine tetraacetic acid) or an isotonicity agent such as glycerin, glucose, or sodium chloride can be added to prepare an injection. . In addition, a cryopreservation agent such as glycerin may be added and frozen.

By administering the composition of the present invention to a mammalian cell, the double-stranded nucleic acid in the composition of the present invention can be introduced into the cell.

The method for introducing the nucleic acid complex of the present invention into mammalian cells in vivo may be performed according to a known transfection procedure that can be performed in vivo. By intravenously administering the composition of the present invention to mammals including humans, the composition can be transmitted to the liver, and the double-stranded nucleic acid in the composition of the present invention can be introduced into the liver or hepatocytes.

If the double-stranded nucleic acid in the composition of the present invention is introduced into the liver or liver cells, the expression of the β2GPI gene in the liver cells can be reduced, and β2GPI-related diseases can be treated or prevented. Examples of β2GPI-related diseases include autoimmune diseases or thrombosis, and more specifically, systemic lupus erythematosus (SLE), antiphospholipid antibody syndrome, hemodialysis complications and arteriosclerosis in patients with end stage renal failure . The subject to be administered 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 effectiveness of β2GPI gene inhibition in an in vivo pharmacodynamic evaluation model of a therapeutic or preventive agent for the above-mentioned diseases. Examples of the in vivo efficacy evaluation model include a lupus anticoagulant (LA) test and the like. Here, LA and the anti-β2GPI antibody are both types of antiphospholipid antibodies, which exhibit the activity of inhibiting the phospholipid-dependent coagulation reaction of the collected blood in vitro. LA occurs mainly in diseases such as SLE or APS, and multiple reports have shown that it is associated with the onset or condition of thrombosis or infertility with anti-β2GPI antibodies (blood, 2003, volume 101, p. No. 5, p1827-1832). Furthermore, it has been reported that anti-β2GPI antibodies acquire phospholipid-binding activity via β2GPI, and thus exert LA activity dependent on β2GPI ("Thrombosis and Haemostasis", 1998, Vol. 79, No. 1, p79- 86). Furthermore, it has been reported that this β2GPI-dependent LA is deeply related to the incidence of the disease (blood, 2004, Vol. 104, No. 12, p3598-3602), and it is thought that if the expression of β2GPI in blood is suppressed The release of LA activity may provide a novel and effective treatment for β2GPI-related diseases. In clinical examination, LA can be detected by measuring the activated partial clotting hormone time, kaolin clotting time and / or diluted Russell snake venom time (dRVVT).

The amount to be administered varies depending on the condition, age, administration route, etc. of the object to be administered. For example, it can be administered by converting the amount of double-stranded nucleic acid to a daily dose of 0.1 μg to 1000 mg. Dosage is 1 to 100 mg per day.

In addition, the present invention provides: a nucleic acid complex for the treatment of a disease; a pharmaceutical composition for the treatment of a disease; the use of a nucleic acid complex for the treatment of a disease; the use of a nucleic acid complex in the manufacture of a medicine for the treatment of a disease; Nucleic acid complexes for the manufacture of medicines for the treatment of diseases; including methods for treating or preventing diseases in which an effective amount of the nucleic acid complexes is administered to a subject in need thereof. [Example]

Next, the present invention will be specifically described with reference examples, examples, and test examples. However, the present invention is not limited to these examples and test examples. Furthermore, the proton nuclear magnetic resonance spectra ( ¹ H NMR) shown in the examples and reference examples are those measured at 270 MHz, 300 MHz, or 400 MHz. Depending on the compound and the measurement conditions, there are no clearly observed exchange protons. Situation. In addition, the multiplicity of the signal is used in common use, br is broad, which means a wide signal in appearance. UPLC analysis uses the following conditions. Mobile phase A: 0.1% formic acid in water, B: acetonitrile solution. Gradient: mobile phase B is a linear gradient of 10% to 90% (3 minutes). Column: ACQUITY UPLC BEH C18 (1.7 μm, inner diameter) manufactured by Waters. 2.1 × 50 mm) Flow rate: 0.8 mL / min PDA detection wavelength: 254 nm (detection range 190 ～ 800 nm)

Reference examples are shown in Tables X-1 to X-4.

Table X-1: Compounds A1 to A7

[Table 20] Continued from table X-1

[Table 21] Table X-2: Compounds B1 to B3

Table X-3: Compounds C1 to C17

[Table 23] Continued from Table X-3

[Table 24] Continue to table X-3

[Table 25] Continued from Table X-3

[Table 26] Continued from Table X-3

Table X-4: Compounds D1 to D11

[Table 28] Continue to table X-4

Reference example 1

[Chem. 117]

Synthesis of compound RE1-2 The compound RE1-1 (0.8755 g, 1.9567 mmol) synthesized by the method described in the Journal of the American Chemical Society, Vol. 136, pages 16958-16961, 2014 was dissolved in tetrahydrofuran (10 mL), and 1,3-dicyclohexanecarbodiimide (DCC, 0.4247 g, 2.0584 mmol) and N-hydroxysuximide (0.2412 g, 2.0958 mmol) were stirred at room temperature for a while. . The solid precipitated from the reaction mixture was removed, and the solvent was distilled off under reduced pressure. The obtained mixture was dissolved in N, N'-dimethylformamide (DMF), and 2-aminoethylcis-butenediamidoimine hydrobromide (0.6479 g, 2.5491 mmol) and diiso After propylethylamine (1.7 mL, 9.7835 mmol), stir at room temperature overnight. The solvent of the reaction solution was distilled off under reduced pressure, and dissolved by reverse-phase column chromatography (water / methanol = 80/20) to obtain 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 (1H, d, J = 3.5 Hz), 6.99 (2H, s), 7.81-7.87 (2H, m).

Synthesis of compound RE1-4 Step 1 Compound RE1-1 (0.9602 g, 2.1460 mmol) was dissolved in N, N'-dimethylformamide (10 mL), and N-Boc-ethylenediamine (Sigma-Aldrich) was added. Manufactured by the company, 0.6877 g, 4.292 mmol), diisopropylethylamine (1.90 mL, 10.87 mmol), and 2- (1H-benzotriazol-1-yl) -1,1,3, hexafluorophosphate, 3-Tetramethylurenium (manufactured by Wako Pure Chemical Industries, Ltd., 1.6437 g, 4.3229 mmol) was stirred overnight at room temperature. Water was added to the reaction solution, and extraction was performed twice with chloroform, 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 RE1-3. ESI-MS m / z: 590 (M + H) ⁺

Step 2 Synthesis of compound RE1-4 The 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 at room temperature. A moment. Water was added to the reaction 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 distilled off under reduced pressure, and the residue was eluted by reversed-phase column chromatography (water / methanol = 80/20), whereby Compound RE1-4 (0.3879 g, yield 37%) was obtained. ESI-MS m / z: 490 (M + H) ⁺ ; ¹ H-NMR (DMSO-D ₆ ) δ: 1.46-1.52 (4H, m), 1.78 (3H, s), 1.90 (3H, s), 2.00 (3H, s), 2.08 (2H, t, J = 7.4 Hz), 2.11 (3H, s), 2.85 (2H, t, J = 6.3 Hz), 3.27 (2H, dd, J = 12.3, 6.2 Hz ), 3.67-3.69 (1H, m), 3.68-3.73 (1H, m), 3.86-3.90 (1H, m), 4.01-4.04 (3H, m), 4.49 (1H, d, J = 8.4 Hz), 4.97 (1H, dd, J = 11.3, 3.4 Hz), 5.22 (1H, d, J = 3.5 Hz), 7.86 (1H, d, J = 9.1 Hz), 7.95-8.02 (1H, m).

Reference example 2

[Chemical 118]

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

Step 2 of Reference Example 2 The compound RE2-2 (1.07 g, 1.699 mmol) synthesized in Step 1 of Reference Example 2 was dissolved in N, N-dimethylformamide (10 mL), and piperine was added at room temperature. Pyridine (0.336 mL, 3.40 mmol) and stirred for 3 hours. Water was added to the reaction 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 distilled off under reduced pressure, and the residue was purified by amine silica gel column chromatography (chloroform / methanol = 90/10) to obtain compound RE2-3 (0.59 g, yield 85%). ESI-MS m / z: 408 (M + H) ⁺

Reference example 3

[Chemical 119]

Reference Example 3 Step 1 Dissolve dimethyl 5-hydroxyisophthalate (compound RE3-1, manufactured by Wako Pure Chemical Industries, Ltd., 5.0443 g, 24 mmol) in tetrahydrofuran (manufactured by Wako Pure Chemical Industries, Ltd., 25 mL), Added 2- (third butoxycarbonylamino) -1-ethanol (manufactured by Tokyo Chemical Industry Co., Ltd., 4.0343 g, 25.03 mmol) and triphenylphosphine polymer carrier (manufactured by Sigma-Aldrich Co., Ltd., 6.61 g, 25.2) Then, a 40% diisopropyl azodicarboxylate (DIAD) toluene solution (manufactured by Tokyo Chemical Industry Co., Ltd., 13.26 mL, 25.2 mmol) was added under ice-cooling, and the mixture was stirred at room temperature for one night. The reaction solution was filtered, and the filtrate was distilled off under reduced pressure, and then the residue was purified by amine silica gel column chromatography (hexane / ethyl acetate = 95/5 to 80/20) to obtain compound RE3. -2 (5.3071 g, 63% yield). ESI-MS m / z: 254 (M + H) ⁺ , detected as de-Boc

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

Step 3 of Reference Example 3 The compound RE3-3 (1.9296 g, 5.93 mmol) synthesized in Step 2 of Reference Example 3 was dissolved in N, N'-dimethylformamide (70 mL), and N-1- ( 9H-fluoren-9-ylmethoxycarbonyl) -ethylenediamine hydrochloride (3.3493 g, 11.86 mmol), diisopropylethylamine (5.18 mL, 29.7 mmol), and hexafluorophosphate 2- (1H -Benzotriazol-1-yl) -1,1,3,3-tetramethylurenium (4.5168 g, 11.88 mmol), and stirred at room temperature for 4 hours. The reaction solution was cooled in an ice bath, and a 10% citric acid aqueous solution was added. After extraction with chloroform, the organic layer was washed with a saturated sodium bicarbonate aqueous solution and a saturated saline solution, and dried over anhydrous magnesium sulfate. The solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (chloroform / methanol) to obtain compound RE3-4 (3.4407 g, yield 68%). ESI-MS m / z: 898 (M + HCOO) ^-

Step 4 of Reference Example 3 The compound RE3-4 (1.6087 g, 1.884 mmol) synthesized in Step 3 of Reference Example 3 was dissolved in dichloromethane (20 mL), and under ice-cooling, trifluoroacetic acid (5 mL, 64.9 mmol), and stirred at room temperature for 4 hours. The reaction solution was evaporated under reduced pressure to remove the solvent, thereby quantitatively obtaining a compound RE3-5 (2.4079 g). ESI-MS m / z: 798 (M + HCOO) ^-

Step 5 of Reference Example 3 The compound RE3-5 (386 mg, 0.512 mmol) synthesized in Step 4 of Reference Example 3 was dissolved in tetrahydrofuran (10 mL). Under cooling in an ice bath, benzamidine chloride (175 mg, 1.024) was added. mmol) and stirred at room temperature for 1 hour. The reaction solution was evaporated under reduced pressure to remove the solvent, and the residue was purified by silica gel column chromatography (chloroform / methanol) to obtain a compound RE3-6 (373 mg, yield 82%). ESI-MS m / z: 888 (M + H) ⁺

Step 6 of Reference Example 3 The compound RE3-6 (108 mg, 0.122 mmol) synthesized in Step 5 of Reference Example 3 was dissolved in dichloromethane (5 mL). Diethylamine (0.5 mL, 4.8 mmol) was added at room temperature ) And stirred for 1 hour. The solvent was distilled off under reduced pressure, whereby Compound RE3-7 (54.9 mg) was quantitatively obtained. ESI-MS m / z: 444 (M + H) ⁺

Reference Example 3, Step 7 Add the compound RE3-7 (180 mg, 0.406 mmol), Nα, Nε-bis (third butoxycarbonyl) -L-lysine (Nova Biochem) synthesized in Step 6 of Reference Example 3. Manufacturing, 295 mg, 0.852 mmol), diisopropylethylamine (0.354 mL, 2.029 mmol), and 2- (1H-benzotriazol-1-yl) -1,1,3,3 hexafluorophosphate -Tetramethylurenium (324 mg, 0.852 mmol), stirred overnight at room temperature. The reaction solution was cooled in an ice bath, a 10% citric acid aqueous solution was added, and extraction was performed with chloroform. Then, the organic layer was washed with saturated brine, and dried over anhydrous magnesium sulfate. The solvent was distilled off under reduced pressure, and the residue was purified by amine silica gel column chromatography (chloroform / methanol) to thereby quantitatively obtain compound RE3-8 (450 mg). ESI-MS m / z: 1101 (M + H) ⁺

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

Reference example 4

[Chem 120] (Bn in the figure represents benzyl)

Reference Example 4 Step 1 The compound RE4-1 synthesized by the method described in Reference Example 3 (the compound RE3-5 in Reference Example 3, 0.5716 g, 0.7582 mmol) was used. Volumes, pp. 690-699, dodecanoic acid monobenzyl ester (0.4859 g, 1.5164 mmol), diisopropylethylamine (0.662 mL, 3.79 mmol), and hexafluorophosphate 2 synthesized by the method described in 2011 -(1H-benzotriazol-1-yl) -1,1,3,3-tetramethylurenium (0.5766 g, 1.516 mmol) dissolved in N, N dimethylformamide (12 mL), Stir at room temperature for 1 hour. The reaction solution was cooled in an ice bath, a saturated aqueous citric acid solution was added, and extraction was performed with chloroform. Then, the organic layer was washed with a saturated saline solution, and dried over anhydrous magnesium sulfate. The solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (chloroform / methanol) to obtain compound RE4-2 (0.88 g, yield 84%). ESI-MS m / z: 1057 (M + H) ⁺

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

Reference Example 4 Step 3 Add the compound synthesized in Step 2 of Reference Example RE4-3 (0.437 g, 0.7143 mmol), Nα, Nε-bis (third butoxycarbonyl) -L-lysine (Nova Biochem) Manufacturing, 0.5483 g, 1.583 mmol), diisopropylethylamine (0.624 mL, 3.57 mmol), 2- (1H-benzotriazol-1-yl) -1,1,3,3-hexafluorophosphate Tetramethylurenium (0.5703 g, 1.5 mmol) was stirred at room temperature for 2 hours. A 10% citric acid aqueous solution was added to the reaction solution, and extraction was performed 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 RE4-4. ESI-MS m / z: 1269 (M + H) ⁺

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

Reference example 5

[Chemical 121]

Reference Example 5 Step 1 The compound RE5-1 (RE3-8, 100 mg, 0.091 mmol in Reference Example 3) synthesized by the method described in Reference Example 3 was dissolved in methanol (3 mL), and acetic acid (2 μL) was added. ), Catalytic hydrogen reduction using palladium / carbon is performed. The obtained solution fraction was distilled to remove the solvent under reduced pressure, thereby quantitatively obtaining a 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-cis-butenediamidohexyloxy) succinamine (48 mg, 0.155 mmol) were dissolved in tetrahydrofuran (2 mL ), Diisopropylethylamine (0.045 mL, 0.259 mmol) was added and stirred overnight at room temperature. The solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (chloroform / methanol) to obtain compound RE5-3 (18 mg, yield 30%). ESI-MS m / z: 1160 (M + H) ⁺

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

Reference example 6

[Chem 122]

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

Step 2 of Reference Example 6 The compound RE6-2 (0.9622 g, 1.952 mmol) synthesized in Step 1 of Reference Example 6 was used to quantitatively obtain the compound RE6-3 by the same method as Step 4 of Reference Example 3. ESI-MS m / z: 396 (M + H) ⁺

Reference Example 6 Step 3 The compound synthesized in Step 2 of Reference Example RE6-3 (0.1146 g, 0.290 mmol) and N-butanesulfinoimino 15-azido-4,7,10,13-tetra Hexadecanadecanoic acid (N3-PEG4-NHS, manufactured by Tokyo Chemical Industry Co., Ltd., 0.0750 g, 0.1931 mmol) was used to quantitatively obtain compound RE6-4 by the same method as in step 2 of Reference Example 5. ESI-MS m / z: 669 (M + H) ⁺

Step 4 of Reference Example 6 Using compound RE6-4 (0.1291 g, 0.193 mmol) synthesized in Step 3 of Reference Example 6, compound RE6-5 was quantitatively obtained by the same method as that of Step 2 of Reference Example 3. ESI-MS m / z: 641 (M + H) ⁺

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

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

Reference example 7

[化 123]

Reference Example 7 Step 1 The compound RE7-1 (RE3-9, 0.2586 g, 0.3695 mmol in Reference Example 3) synthesized by the method described in Reference Example 3 was used and the Journal of the American Chemical Society, Vol. 136 , Pages 16958 to 16961, the compound RE-1 (0.8559 g, 1.7927 mmol) described in Reference Example 1 synthesized by the method described in 2014. Compound RE7-2 was obtained by the same method as in Step 3 of Reference Example 3. (0.5272 g, 61% yield). ESI-MS m / z: 2418 (M + H) ⁺

Step 2 of Reference Example 7 Using compound RE7-2 (0.2653 g, 0.1097 mmol) synthesized in Step 1 of Reference Example 7, compound RE7-3 (0.1524 g, yield) was obtained in the same manner as in Step 1 of Reference Example 5. 61%). ESI-MS m / z: 2284 (M + H) ⁺

Reference Example 8: Synthesis of compounds A1 and B1

[Chem 124]

The compound A1 was synthesized by using the compound RE8-1 synthesized by the method described in Reference Example 7 (the compound RE7-3 in Reference Example 7, 0.0152 g, 0.006657 mmol). Compound A1 was obtained (0.0077 g, 47% yield). ESI-MS m / z: 1239 (M + 2H) ^{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 (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, brs), 8.01-8.08 (3H, brm), 8.54-8.60 (2H, brm).

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 obtained by the same method as in step 3 of Reference Example 3. ). ESI-MS m / z: 1279 (M + 2H) ^{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), 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), 7.52 (2H, s), 7.66-7.75 (2H, m), 7.78-7.87 ( 6H, m), 7.91 (1H, brs), 8.01-8.08 (3H, brm), 8.54-8.60 (2H, brm).

Reference Example 9: Synthesis of compounds B2 and B3

[Chemical 125]

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

The compound B3 was synthesized by using the compound RE9-2 synthesized by the method described in Reference Example 6 (the compound RE6-7 in Reference Example 6, 0.0100 g, 0.01112 mmol) by the same method as in Step 3 of Reference Example 3. Compound B3 was obtained (0.0223 g, 72% yield). ESI-MS m / z: 1393 (M + 2H) ²⁺

Reference Example 10: Synthesis of compounds C1 and D1

[Chem. 126]

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

Step 2 of Reference Example 10 Using compound RE10-2 (0.1459 g, 0.056 mmol) synthesized in Step 1 of Reference Example 10, compound D1 (112 mg, yield 80%) was obtained in the same manner as in Step 1 of Reference Example 5. ). ESI-MS m / z: 1249 (M + 2H) ^{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, brs), 8.01-8.08 (3H, brm), 8.54-8.60 (2H, brm).

Step 3 of Reference Example 10 The compound D1 (0.1091 g, 0.044 mmol) synthesized in Step 2 of Reference Example 10 and the compound RE2-3 (0.0748 g, 0.184 mmol) of Reference Example 2 were used. The same method was used to obtain a compound RE10-3 as a crude product. ESI-MS m / z: 1292 (M + 2H) ²⁺ , detected as a de-DMTr

Step 4 of Reference Example 10 The compound RE10-3 (0.161 g, 0.05586 mmol) synthesized in Step 3 of Reference Example 10 was dissolved in dichloromethane (5 mL), and succinic anhydride (manufactured by Tokyo Chemical Industry Co., Ltd., 0.1182 g, 1.181) was added. mmol), N, N-dimethylaminopyridine (0.0224 g, 0.183 mmol), and triethylamine (0.55 mL, 3.95 mmol), and stirred at room temperature overnight. The reaction solution was cooled in an ice bath, water was added, and extraction was performed 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 deDMr

Step 5 of Reference Example 10 The compound RE10-4 (0.0816 g, 0.02734 mmol) synthesized in Step 4 of Reference Example 10, O- (benzotriazol-1-yl) -N, N, N ', N'-Tetramethylurenium (0.0221 g, 0.05827 mmol) and diisopropylethylamine (0.02 mL, 0.1094 mmol) were dissolved in N, N-dimethylformamidine (4 mL), and LCAΑ was added -CPG (manufactured by Chem Gene, 0.4882 g), stirred overnight at room temperature. The mixture was separated by filtration, washed with dichloromethane, 10% methanol in dichloromethane, and diethyl ether in order, and then reacted with an acetic anhydride / pyridine solution to obtain compound C1 (49.5 μmol / g, yield 89%). In addition, the yield is calculated by adding a 1% trifluoroacetic acid / dichloromethane solution to the solid phase support, and calculating based on the introduction rate into the solid phase support that can be calculated from the absorption derived from DMTr groups.

Reference example 11

[Chemical 127]

Compound RE11-2 (1.050 g) was synthesized from compound RE11-1 (1.200 g, 3.640 mmol) by the method described in Journal of Medicinal Chemistry, Vol. 59, pages 2718-2733, 2016. , Yield 50%). ESI-MS m / z: 582 (M + H) ⁺

Reference example 12

[Chem 128]

Compound RE12-1 (4.000 g, 10.27 mmol) was dissolved in dichloromethane (60 mL), and benzyl 2- (2-hydroxyethoxy) ethylaminoformate (2.700 g, 11.30 mmol) and trifluoro were added Methanesulfonic acid (0.1360 mL, 1.541 mmol) was stirred overnight under reflux. A 10 wt% potassium carbonate aqueous solution was added to the reaction solution, and the solution was separated with dichloromethane. The organic phase was washed with water and dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and 2-methyltetrahydrofuran was substituted and concentrated. The residue was dropped into heptane, and the obtained crystal was filtered to obtain compound RE12-2 (5.130 g, yield 88%). ESI-MS m / z: 570 (M + H) ⁺

Reference example 13

[Chem. 129]

Compound RE13-1 (Compound RE1-1 in Reference Example 1, 898.0 mg, 2.007 mmol) was dissolved in dichloromethane (15 mL), and 1-hydroxybenzotriazole monohydrate (338.0 mg, 2.208 mmol) was added. , 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (343 mg, 2.208 mmol), and N-1-Z-1,3-diaminopropane The hydrochloride (0.4910 mL, 2.208 mmol) was stirred at room temperature for 3 hours. Water was added to the reaction solution, and the mixture was separated with dichloromethane. The organic layer was washed with a saturated sodium bicarbonate aqueous solution and dried over anhydrous sodium sulfate. The solvent was distilled off 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

[Chem 130]

Reference Example 14 Step 1 The compound RE14-1 (the compound RE1-1 in Reference Example 1, 3.00 g, 6.70 mmol) was dissolved in dichloromethane (60 mL), and benzyl L-lysine diamine was added at room temperature. P-Toluenesulfonate (1.75 g, 3.02 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) and stirred for 2.5 hours. The reaction solution was washed with water and a saturated aqueous sodium hydrogen carbonate solution, and dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (chloroform / methanol = 90/10) to thereby quantitatively obtain compound RE14-2. ESI-MS m / z: 1096 (M + H) ⁺

Reference Example 14 Step 2 Dissolve compound RE14-2 (2.30 g, 2.10 mmol) in tetrahydrofuran (46 mL), add 10% palladium-carbon powder (aqueous product, 54.29%, 424 mg) at room temperature, and place in a hydrogen atmosphere Stir overnight. The reaction solution was filtered, and the solvent was distilled off under reduced pressure, whereby the compound was quantitatively obtained as RE14-3. ESI-MS m / z: 1006 (M + H) ⁺

Reference example 15

[Chemical 131]

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

Step 2 of Reference Example 15 Using compound RE15-2 (1.5 g, 1.37 mmol), Compound RE15-3 was quantitatively obtained by the same method as Step 2 of Synthesis of Compound RE1-4 of Reference Example 1. ESI-MS m / z: 990 (M + H) ⁺

Reference Example 16

[Chem 132]

Reference Example 16 Step 1 Using N- (third butoxycarbonyl) -L-glutamic acid (manufactured by Tokyo Chemical Industry Co., Ltd.), a compound RE16-1 (1.855 g, 3.19 mmol), and the crude product of compound RE16-2 was obtained in the same manner as in Step 1 of Reference Example 15. ESI-MS m / z: 1105 (M + H) ⁺

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

Reference Example 17

[Chemical 133]

The compound RE17-1 (90 mg, 0.173 mmol) synthesized by the method described in Journal of Organic Chemistry, Volume 74, pages 6837-6842, 2009 was dissolved in tetrahydrofuran (1 mL) Then, a triphenylphosphine polymer carrier (manufactured by Sigma-Aldrich, 63 mg, 0.189 mmol) was added, and the mixture was stirred under a heating loop for 3 hours. The reaction solution was filtered, and the solvent was distilled off under reduced pressure to obtain 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, brs), 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 (1H, m), 7.27-7.33 (6H, m), 7.41-7.43 (2H, m).

Reference Example 18

[Chemical 134]

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

Reference Example 18 Step 2 The crude product of compound RE18-2 (1.5 g) and 1,4-diaminobutane (1,4-Diaminobutane) (manufactured by Tokyo Chemical Industry Co., Ltd., 3.29 g, 37.3 mmol) were used. Compound RE18-3 (0.18 g, two-stage yield 10%) was obtained by the same method as Step 3 of Reference Example 3. ESI-MS m / z: 551 (M + HCOO) - 1 H-NMR (400 MHz, MeOD) δ: 1.09 (3H, s), 1.45-1.52 (4H, m), 2.80 (2H, t, J = 7.2 Hz), 2.91 (2H, 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

[Chem. 135]

The compound RE19-1 (110 mg, 0.212 mmol) synthesized by the method described in Organic Chemistry, Vol. 74, pp. 6837-6842, 2009 was dissolved in tetrahydrofuran (2 mL), and N- (1R , 8S, 9S) -Bicyclo [6.1.0] non-4-yne-9-ylmethoxycarbonyl-1,8-diamino-3,6-dioxoctane (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), and stirred at room temperature 1 hour. Water was added to the reaction solution, and extraction was performed with chloroform. Then, the organic layer was washed with saturated brine, and dried over anhydrous magnesium sulfate. The solvent was distilled off under reduced pressure, and the residue was purified by amine 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, 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).

Reference example 20

[Chemical 136]

Reference Example 20 Step 1 Compound RE20-1 (manufactured by AstaTech, 100 mg, 1.148 mmol) and Fmoc-Ser (tBuMe2Si) -OH (manufactured by Watanabe Chemical Industry Co., Ltd., 532 mg, 1.205 mmol) were used. Compound RE20-2 (410 mg, yield 70%) was obtained in the same manner as in Step 3. ESI-MS m / z: 511 (M + H) ^{+ 1} H-NMR (400 MHz, CDCl ₃ ) δ: 0.06 (6H, s), 0.90 (9H, s), 2.76-2.85 (1H, m), 3.65-3.86 (5H, m), 4.02-4.23 (3H, 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).

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

Step 3 of Reference Example 20 Using a crude product of compound RE20-3 (680 mg), compound RE20-4 (330 mg, 70% in two stages) was obtained by the same method as 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 (6H, m), 0.89 (9H, d, J = 28.8 Hz), 2.84- 2.94 (1H, m), 3.24-3.30 (2H, 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

[Chemical 137]

Reference Example 21 Step 1 N- (third butoxycarbonyl) -1,3-diaminopropane (produced by Tokyo Chemical Industry Co., Ltd., 1.788 g, 10.26 mmol) was dissolved in dichloromethane (22.8 mL), and three Ethylamine (1.907 mL, 13.68 mmol) was stirred at room temperature for 15 minutes. To the reaction solution was added dropwise a dichloromethane solution (5 mL) of compound RE21-1 (1.126 g, 6.84 mmol) synthesized by the method described in Organic Letter, Volume 16, pages 6318-6321, 2014, Stir at room temperature for 2 hours. Water was added to the reaction solution, and extraction was performed with chloroform. Then, the organic layer was washed with saturated brine, and dried over anhydrous magnesium sulfate. The solvent was distilled off 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) ⁺

Step 2 of Reference Example 21 Using compound RE21-2 (1.65 g, 5.46 mmol), compound RE21-3 (1.10 g, yield 100%) was obtained by the same method as step 2 of the synthesis of compound RE1-4 of Reference Example 1. . ESI-MS m / z: 203 (M + H) ^{+ 1} H-NMR (400 MHz, CDCl ₃ ) δ: 1.74 (2H, dt, J = 12.0, 6.0 Hz), 2.95 (2H, t, J = 6.0 Hz), 3.18 (1H, s), 3.60 (2H, 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 (1H, brs).

Reference example 22

[Chemical 138]

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

Step 2 of Reference Example 22 Using the crude product of compound RE22-2, compound R22-3 (1.34 g, 52% yield in two stages) was obtained 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 (2H, t, J = 6.4 Hz), 3.47 (2H, t, J = 6.4 Hz) , 3.79 (6H, s), 6.78-6.84 (4H, m), 7.17-7.21 (1H, m), 7.27-7.35 (6H, m), 7.42-7.46 (2H, m).

Reference Example 22 Step 3 The compound RE22-3 (1.15 g, 3.16 mmol) and Fmoc-Ser (tBuMe2Si) -OH (manufactured by Watanabe Chemical Industry Co., Ltd., 1.677 g, 3.8 mmol) were used in the same manner as in Step 3 of Reference Example 3. This method gave compound RE22-4 (560 mg, yield 31%). ¹ H-NMR (400 MHz, CDCl ₃ ) δ: 0.00-0.07 (6H, m), 0.83-0.89 (9H, m), 3.18-3.26 (2H, m), 3.39-3.46 (2H, m), 3.61 -3.68 (1H, m), 3.76 (6H, s), 3.89 (1H, dd, J = 10.0, 4.0 Hz), 4.03 (1H, dd, J = 10.0, 4.0 Hz), 4.15-4.20 (1H, 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 and Fmoc-Ser (tBuMe2Si) -OH described in Table Y-1 were used to obtain the compound RE23 described in Table Y-2 by the same method as in Reference Example 22. -6 to RE23-10. Using the compound RE22-3 and Fmoc-Thr (tBuMe ₂ Si) -OH of Reference Example 22, the compound RE23-11 described in Table Y-2 was 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 compound RE23-12 described in Table Y-2 was obtained in the same manner as in Reference Example 22. The NMR analysis data of the compounds synthesized in this example are shown in Table Y-3.

[Table 29] Table Y-1

[Table 30] Table Y-2

[表 31] Table Y-3

Reference example 24

[Chemical 139]

Using the compound RE24-1 synthesized by the method described in Reference Example 22 (the compound RE22-4, 2.487 g, 3.16 mmol in Reference Example 22), compound RE24-2 was obtained in the same manner as in Step 2 of Reference Example 2. (1.2 g, yield 67%). 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, m), 7.27-7.33 (6H, m), 7.40-7.44 (2H, m), 7.72-7.75 (1H, brm).

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

[Table 32] Table Z-1

[TABLE 33]

Reference example 26

[Chem 140]

Reference Example 26 Step 1 The compound RE26-1 (2.00 g, 9.47 mmol) was dissolved in N, N'-dimethylformamide (40 mL), and iminodiacetic acid di-tert-butyl ester was added at room temperature. (5.11 g, 20.84 mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (4.00 g, 20.84 mmol), 1-hydroxybenzotriazole-1 Hydrate (145 mg, 0.947 mmol) and stirred for 2 hours. Water was added to the reaction solution, and extraction was performed with ethyl acetate, and then the organic layer was washed with a saturated sodium bicarbonate aqueous solution and a saturated saline solution, and dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (heptane / ethyl acetate = 50/50). Further, the slurry was purified by using methanol to obtain compound RE26-2 (4.07 g, yield 65%). ESI-MS m / z: 664 (M-H) ^-

Reference Example 26 Step 2 The compound RE26-2 (2.66 g, 4.00 mmol) was dissolved in tetrahydrofuran (53 mL), and 10% palladium-carbon powder (aqueous product, 54.29%, 490 mg) was added at room temperature. Stir for 3 hours. The reaction solution was filtered, and the solvent was distilled off under reduced pressure to obtain 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), and hexafluorophosphate O- (7-azabenzotriazole was added. -1-yne) -N, N, N ', N'-tetramethylurenium (625.0 mg, 1.644 mmol), diisopropylethylamine (0.5730 mL, 3.290 mmol), and dodecanedioic acid Monobenzyl ester (527.0 mg, 1.644 mmol) was stirred overnight at room temperature. Water was added to the reaction solution, and extraction was performed with ethyl acetate. The organic layer was washed with a saturated sodium bicarbonate aqueous solution and a saturated saline solution, and dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (heptane / ethyl acetate = 60/40) to obtain compound RE26-4 (1.030 g, yield 80%). ESI-MS m / z: 939 (M + H) ⁺

Reference Example 26 Step 4 The 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 distilled off under reduced pressure to obtain a crude product of compound RE26-5. ESI-MS m / z: 713 (M-H) ^-

Reference example 27

[Chem. 141]

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

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

Reference Example 27 Step 3 The 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 distilled off under reduced pressure to obtain 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) was dissolved in N, N'-dimethylformamide (7 mL), and 1-hydroxybenzotriazole monohydrate (327.0 mg, 2.137 mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (410.0 mg, 2.137 mmol), and di-t-butyl iminodiacetate ( 524.0 mg, 2.137 mmol), and stirred at room temperature for 5 hours. Water was added to the reaction solution, and extraction was performed with ethyl acetate. The organic layer was washed with a saturated sodium bicarbonate aqueous solution and a saturated saline solution, and dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (heptane / ethyl acetate = 60/40) to obtain compound RE27-5 (617.0 mg, yield 78%). ESI-MS m / z: 814 (M-H) ^-

Reference Example 27 Step 5 The 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

[Chem. 142]

Reference Example 28 Step 1 The compound RE28-1 (the compound RE26-3 in Reference Example 26, 474 mg, 0.744 mmol) synthesized by the method described in Reference Example 26 was dissolved in N, N'-dimethylformamidine Amine (10 mL) was added at room temperature to trans-cyclohexane-1,4-dicarboxylate synthesized by the method described in the Journal of Medical Chemistry, Vol. 54, p. 2433-2446, 2011 Acid monobenzyl ester (0.234 mg, 0.893 mmol), diisopropylethylamine (0.312 mL, 1.79 mmol), hexafluorophosphate O- (7-azabenzotriazol-1-yl) -N, N , N ', N'-tetramethylurenium (339 mg, 0.893 mmol) and stirred for 6 hours. Water was added to the reaction solution, and extraction was performed with ethyl acetate, and then the organic layer was washed with a saturated sodium bicarbonate aqueous solution and a saturated saline solution, and dried over anhydrous sodium sulfate. The solvent was distilled off 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 The compound RE28-2 (341 mg, 0.387 mmol) was dissolved in dichloromethane (3.4 mL), and trifluoroacetic acid (3.4 mL) was added at room temperature, followed by stirring overnight. The reaction solution was concentrated under reduced pressure, azeotroped with ethyl acetate, and slurry purification with heptane, to thereby obtain compound RE28-3 (254 mg, yield 100%). ESI-MS m / z: 656 (M + H) ⁺

Reference Example 29

[Chemical 143]

Reference Example 29 Step 1 The compound RE29-1 (500 mg, 2.75 mmol) was dissolved in N, N'-dimethylformamidine (10 mL), and ditrimethylene diacetate was added at room temperature. (1.48 g, 6.04 mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (1.16 g, 6.04 mmol), 1-hydroxybenzotriazole-1 Hydrate (42.0 mg, 0.275 mmol) and stirred for 4 hours. Water was added to the reaction solution, and the mixture was extracted with ethyl acetate. The organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. The solvent was distilled off 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 The compound RE29-2 (323 mg, 0.507 mmol) was dissolved in N, N'-dimethylformamide (6v5 mL), and potassium carbonate (84.0 mg, 0.609 mmol) was added at room temperature. Benzyl bromoacetate (139 mg, 0.609 mmol) and stirred for 3 hours. Water was added to the reaction solution, and the mixture was extracted with ethyl acetate. The organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. The solvent was distilled off 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 The compound RE29-3 (312 mg, 0.398 mmol) was dissolved in dichloromethane (3.1 mL), and trifluoroacetic acid (3.1 mL) was added at room temperature, followed by stirring overnight. The reaction solution was concentrated under reduced pressure, and azeotroped with ethyl acetate to quantitatively obtain compound RE29-4 (252 mg). ESI-MS m / z: 561 (M + H) ⁺

Reference example 30

[Chemical 144]

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

Reference Example 30 Step 2 The compound RE30-2 (500 mg, 1.40 mmol) was suspended in acetonitrile (10 mL), and tert-butyl acrylate (3.59 g, 28.0 mmol) and benzyltrimethyl hydroxide were added at room temperature. Ammonium (40% aqueous solution, 1.76 mL, 702 mmol) and stirred overnight. The solvent was distilled off under reduced pressure, water was added, and extraction was performed with ethyl acetate. The organic layer was washed with saturated brine, and dried over anhydrous sodium sulfate. The solvent was distilled off 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 The compound RE30-3 (340 mg, 0391 mmol) was dissolved in tetrahydrofuran (6.8 mL), and 10% palladium-carbon powder (aqueous product, 54.29%, 31.3 mg) was added at room temperature in a hydrogen atmosphere. Stir for 6 hours. The reaction solution was filtered, the solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (heptane / ethyl acetate = 30/70) to obtain compound RE30-4 (235 mg, product Rate 72%). ESI-MS m / z: 841 (M + H) ⁺

Reference Example 30 Step 4 The compound RE30-4 (232 mg, 0.276 mmol) was dissolved in N, N'-dimethylformamide (4.6 mL), and monobenzyl dodecanoate (0.133 mg, 0.414 mmol), diisopropylethylamine (0.145 mL, 0.829 mmol), hexafluorophosphate O- (7-azabenzotriazol-1-yl) -N, N, N ', N'-tetrakis Methylurenium (158 mg, 0.414 mmol) and stirred overnight. Water was added to the reaction solution, and extraction was performed with ethyl acetate, and then the organic layer was washed with a saturated sodium bicarbonate aqueous solution and a saturated saline solution, and dried over anhydrous sodium sulfate. The solvent was distilled off 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), and trifluoroacetic acid (2.7 mL) was added at room temperature, followed by stirring overnight. The reaction solution was concentrated under reduced pressure and azeotroped with ethyl acetate to quantitatively obtain compound RE30-6 (231 mg). ESI-MS m / z: 919 (M + H) ⁺

Reference Example 31

[Chem. 145]

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 Amidine (10 mL), add triethylamine (0.90 mL, 7.11 mmol), 1-hydroxybenzotriazole monohydrate (703 mg, 5.21 mmol), and 1- (3-dimethylamine at room temperature. Propyl) -3-ethylcarbodiimide hydrochloride (1.36 g, 7.11 mmol) and stirred for 16 hours. The reaction solution 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 The compound RE31-2 (500 mg, 1.01 mmol) and zinc powder (330 mg, 5.05 mmol) were suspended in methanol (3.5 mL) and tetrahydrofuran (3.5 mL), and ammonium chloride was added dropwise at 0 ° C. (378 mg, 7.07 mmol) in water, and stirred at room temperature for 24 hours. The reaction solution was worked up, and the crude product was purified by silica gel column chromatography to obtain compound RE31-3 (160 mg, yield 34%).

Reference Example 31 Step 3 The compound RE31-3 (200 mg, 0.430 mmol) and N-benzyloxycarbonylglycine (sometimes, the benzyloxycarbonyl group is also referred to as Cbz) (90.0 mg, 0.430 mmol) were dissolved in N , N'-dimethylformamide (2.0 mL), and diisopropylethylamine (0.220 mL, 1.29 mmol) and hexafluorophosphate O- (7-azabenzotriazole- 1-yl) -N, N, N ', N'-tetramethylurenium (245 mg, 0.645 mmol) and stirred for 16 hours. The reaction solution 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

[Chemical 146]

Reference Example 32 Step 1 3,5-Dinitrobenzoic acid RE32-1 (500 mg, 2.36 mmol) and N-Cbz-ethylenediamine (588 mg, 2.83 mmol) were dissolved in N, N'-dimethyl Formamidine (5.0 mL), add triethylamine (0.65 mL, 4.72 mmol), 1-hydroxybenzotriazole monohydrate (380 mg, 2.83 mmol), and 1- (3-dimethyl) at room temperature. Aminopropyl) -3-ethylcarbodiimide hydrochloride (675 mg, 3.54 mmol) and stirred for 16 hours. The reaction solution 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 Step 2 Dissolve compound RE32-2 (200 mg, 0.515 mmol) in ethanol (5.0 mL), and add tin (II) chloride (584 mg, 3.09 mmol) and concentrated hydrochloric acid (0.2 mL) at room temperature. And stirred at 80 ° C for 16 hours. The reaction solution was worked up to quantitatively obtain compound RE32-3 (180 mg). ESI-MS m / z: 329 (M + H) ⁺

Reference example 33

[Chemical 147]

Step 1 of Reference Example 33 The compound RE33-1 (the compound RE3-2, 8.17 g, 23.12 mmol in Reference Example 3) synthesized by the method described in Reference Example 3 was used, and was the same as that in Step 4 of Reference Example 3. Method RE33-2 (3.7 g, yield 63%) was obtained. ESI-MS m / z: 254 (M + H) ⁺

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

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

Reference example 34

Reference Example 34 Step 1 The compound RE34-1 (2 g, 9.53 mmol) and a third butoxycarbonylamino-1-pentanol (manufactured by Tokyo Chemical Industry Co., Ltd., 2 g, 10 mmol) were used. The same method as in step 1 was used to obtain compound RE34-2 (2.40 g, yield 63%). ESI-MS m / z: 296 (M + H) ⁺ , detected as de-Boc

Step 2 of Reference Example 34 Using compound RE34-2, compound RE34-3 (1.579 g, yield 21%) was obtained by the same method as steps 2 to 4 of Reference Example 3 and steps 1 to 4 of Reference Example 4. ESI-MS m / z: 910 (M + H) ⁺

Reference Example 35: Synthesis of compound D2

[Chem. 149]

Reference Example 35 Step 1 The compound RE35-1 synthesized by the method described in Reference Example 11 (the compound RE11-2 in Reference Example 11, 1.015 g, 1.748 mmol) was dissolved in N, N'-dimethylformamidine Amine (12 mL), 10% palladium-carbon powder (aqueous product, 54.29%, 187 mg) was added at room temperature, and stirred under a hydrogen atmosphere for 6 hours. The reaction solution was filtered. To the filtrate were added the compound RE26-5 (250.0 mg, 0.350 mmol), 1-hydroxybenzotriazole monohydrate (26.80 mg, 0.1750 mmol), and 1- (3-dimethyl) synthesized in Reference Example 26. Aminopropyl) -3-ethylcarbodiimide hydrochloride (402.0 mg, 2.099 mmol) was stirred overnight at room temperature. Water was added to the reaction solution, and extraction was performed with ethyl acetate. The organic layer was washed with a saturated sodium bicarbonate aqueous solution and a saturated saline solution, and dried over anhydrous sodium sulfate. The solvent was distilled off 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 + 2H) ²⁺

Reference Example 35 Step 2 The compound RE35-2 (0.7380 g, 0.3040 mmol) was dissolved in tetrahydrofuran (7 mL), and 10% palladium-carbon powder (aqueous product, 54.29%, 135.90 mg) was added at room temperature in a hydrogen atmosphere. Stir overnight. The reaction solution was filtered, the solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (chloroform / methanol = 87/13) to obtain compound D2 (581 mg, yield 82%). ESI-MS m / z: 1170 (M + 2H) ^{2+ 1} H-NMR (400 MHz, DMSO-d ₆ ) 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

[Chemical 150]

Reference Example 36 Step 1 The compound RE36-1 synthesized by the method described in Reference Example 12 (the compound RE12-2 in Reference Example 12, 500 mg, 0.879 mmol) was dissolved in N, N'-dimethylformamidine Amine (6.5 mL), 10% palladium-carbon powder (aqueous product, 54.29%, 94 mg) was added at room temperature, and stirred under a hydrogen atmosphere for 4 hours. The reaction solution was filtered. To the filtrate were added the compound RE26-5 (126.0 mg, 0.176 mmol), 1-hydroxybenzotriazole monohydrate (13.47 mg, 0.088 mmol), and 1- (3-dimethylamino group) in Reference Example 26. (Propyl) -3-ethylcarbodiimide hydrochloride (202.0 mg, 1.055 mmol), and stirred overnight at room temperature. The reaction solution was evaporated under reduced pressure to remove the solvent, 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 + 2H) ²⁺

Reference Example 36 Step 2 The compound RE36-2 (0.242 g, 0.102 mmol) was dissolved in tetrahydrofuran (3.6 mL) and water (1.2 mL), and 10% palladium-carbon powder (aqueous product, 54.29%, 45%) was added at room temperature. mg), and stirred under a hydrogen atmosphere for 4 hours. The reaction solution was filtered, and the solvent was distilled off under reduced pressure to obtain compound D3 (216 mg, yield 93%). ESI-MS m / z: 1146 (M + 2H) ^{2+ 1} H-NMR (400 MHz, DMSO-d ₆ ) δ: 1.15-1.65 (20H, m), 1.68-2.15 (52H, m), 3.13- 3.29 (6H, m), 3.40-3.67 (16H, 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 (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

[Chemical 151]

Reference Example 37 Step 1 The compound RE37-1 synthesized by the method described in Reference Example 13 (the compound RE13-2 in Reference Example 13, 430 mg, 0.674 mmol) was dissolved in N, N'-dimethylformamidine Amine (6 mL), 10% palladium-carbon powder (aqueous product, 54.29%, 79 mg) was added at room temperature, and stirred under a hydrogen atmosphere for 4 hours. The reaction solution was filtered. To the filtrate were added the compound RE26-5 (105.0 mg, 0.148 mmol), 1-hydroxybenzotriazole monohydrate (11.31 mg, 0.074 mmol), and 1- (3-dimethylamino group) in Reference Example 26. Propyl) -3-ethylcarbodiimide hydrochloride (170.0.0 mg, 0.887 mmol) was stirred overnight at room temperature. The reaction solution was evaporated under reduced pressure to remove the solvent, and the residue was purified by reverse-phase column chromatography (water / acetonitrile) to obtain compound RE37-2 (218.1 mg, yield 56%). ESI-MS m / z: 1329 (M + 2H) ²⁺

Reference Example 37 Step 2 The compound RE37-2 (0.210 g, 0.079 mmol) was dissolved in tetrahydrofuran (3.1 mL) and water (1.0 mL), and 10% palladium-carbon powder (aqueous product, 54.29%, 39) was added at room temperature. mg), and stirred under a hydrogen atmosphere for 4 hours. The reaction solution was filtered, and the solvent was distilled off under reduced pressure to obtain compound D4 (192.7 mg, yield 95%). ESI-MS m / z: 1284 (M + 2H) ^{2+ 1} H-NMR (400 MHz, DMSO-d ₆ ) δ: 1.17-1.65 (42H, m), 1.69-2.13 (61H, m), 2.95- 3.17 (16H, m), 3.65-3.77 (3H, 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 (1H, s), 7.64-7.92 (11H, m), 8.26-8.48 (4H, m), 10.14 (1H , s)

Reference Example 38: Synthesis of Compound D5

[Chemical 152]

Reference Example 38 Step 1 The compound RE38-1 synthesized by the method described in Reference Example 12 (the compound RE12-2 in Reference Example 12, 450 mg, 0.791 mmol) was dissolved in N, N'-dimethylformamidine Amine (6 mL), 10% palladium-carbon powder (aqueous product, 54.29%, 85 mg) was added at room temperature, and stirred under a hydrogen atmosphere for 5 hours. The reaction solution was filtered. To the filtrate were added the compound RE27-6 (94 mg, 0.158 mmol), 1-hydroxybenzotriazole monohydrate (133.0 mg, 0.871 mmol), and 1- (3-dimethylamino group) in Reference Example 27. Propyl) -3-ethylcarbodiimide hydrochloride (182.0 mg, 0.950 mmol) was stirred overnight at room temperature. The reaction solution was evaporated under reduced pressure to remove the solvent, 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 + 2H) ²⁺

Reference Example 38 Step 2 The compound RE38-2 (80 mg, 0.035 mmol) was dissolved in tetrahydrofuran (1.7 mL) and water (0.85 mL), and 10% palladium-carbon powder (aqueous product, 54.29%, 26) was added at room temperature. mg), and stirred under a hydrogen atmosphere for 2 hours. The reaction solution was filtered, and the solvent was distilled off under reduced pressure to obtain compound D5 (57.5 mg, yield 75%). ESI-MS m / z: 1084 (M + 2H) ^{2+ 1} H-NMR (400 MHz, DMSO-d ₆ ) δ: 1.69-2.21 (46H, m), 3.14-3.65 (28H, m), 3.67- 4.22 (27H, m), 4.43-4.66 (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

[Chemical 153]

Reference Example 39 Step 1 The compound RE39-1 synthesized by the method described in Reference Example 13 (the compound RE13-2 in Reference Example 13, 418 mg, 0.655 mmol) was dissolved in N, N'-dimethylformamidine Amine (6 mL), 10% palladium-carbon powder (aqueous product, 54.29%, 77 mg) was added at room temperature, and stirred under a hydrogen atmosphere for 5 hours. The reaction solution was filtered. To the filtrate were added the compound RE27-6 (85 mg, 0.144 mmol), 1-hydroxybenzotriazole monohydrate (121.0 mg, 0.791 mmol), and 1- (3-dimethyl) synthesized in Reference Example 27. Aminopropyl) -3-ethylcarbodiimide hydrochloride (165.0 mg, 0.863 mmol) was stirred overnight at room temperature. The reaction solution was evaporated under reduced pressure to remove the solvent, and the residue was purified by reverse-phase column chromatography (water / acetonitrile) to obtain compound RE39-2 (99 mg, yield 28%). ESI-MS m / z: 1268 (M + 2H) ²⁺

Reference Example 39 Step 2 The compound RE39-2 (186 mg, 0.073 mmol) was dissolved in tetrahydrofuran (2.8 mL) and water (0.93 mL), and 10% palladium-carbon powder (aqueous product, 54.29%, 40%) was added at room temperature. mg), and stirred under a hydrogen atmosphere for 2 hours. The reaction solution was filtered, and the solvent was distilled off under reduced pressure to obtain compound D6 (156.7 mg, yield 87%). ESI-MS m / z: 1222 (M + 2H) ^{2+ 1} H-NMR (400 MHz, DMSO-d ₆ ) δ: 1.36-1.62 (27H, m), 1.67-2.17 (64H, m), 2.92- 3.21 (15H, m), 3.58-3.77 (2H, 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

[Chemical 154]

Reference Example 40 Step 1 The compound D5 (171 mg, 0.079 mmol) synthesized by the method described in Reference Example 38 was dissolved in N, N'-dimethylformamide (3.4 mL), and benzyl glycine was added. P-Toluenesulfonate (32.0 mg, 0.095 mmol), 1-hydroxybenzotriazole monohydrate (12.09 mg, 0.079 mmol), and 1- (3-dimethylaminopropyl) -3-ethyl Carbodiimide hydrochloride (18.16 mg, 0.095 mmol) was stirred overnight at room temperature. The reaction solution was evaporated under reduced pressure to remove the solvent, 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 + 2H) ²⁺

Reference Example 40 Step 2 The compound RE40-2 (54 mg, 0.023 mmol) was dissolved in tetrahydrofuran (0.83 mL) and water (0.28 mL), and 10% palladium-carbon powder (aqueous product, 54.29%, 18) was added at room temperature. mg), and stirred under a hydrogen atmosphere for 2 hours. The reaction solution was filtered, and the solvent was distilled off under reduced pressure to obtain compound RE40-3 (50.1 mg, yield 97%). ESI-MS m / z: 1112 (M + 2H) ^{2+ 1} H-NMR (400 MHz, DMSO-d ₆ ) 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, 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

[Chem. 155]

Reference Example 41 Step 1 The compound RE41-1 (500 mg, 0.861 mmol) was dissolved in N, N'-dimethylformamide (10 mL), and 10% palladium carbon powder (aqueous product, 54.29%, 92.1 mg), and stirred under a hydrogen atmosphere for 2 hours. Under argon atmosphere, add the compound RE27-3 (113 mg, 0.172 mmol) and 1- (3-dimethylaminopropyl) -3-ethylcarbodicarbonate synthesized in Reference Example 27 at room temperature. Amidine imine hydrochloride (198 mg, 1.03 mmol), 1-hydroxybenzotriazole monohydrate (13.2 mg, 0.086 mmol) and stirred overnight. The reaction solution was filtered through diatomaceous earth, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (chloroform / methanol = 90/10), and then HPLC (acetonitrile / water) was obtained by reverse phase separation. Purification was carried out to obtain compound RE41-2 (195 mg, yield 48%). ESI-MS m / z: 1186 (M + 2H) ²⁺

Reference Example 41 Step 2 The compound RE41-2 (194 mg, 0.082 mmol) was dissolved in tetrahydrofuran (2.9 mg) and water (1.0 mL), and 10% palladium-carbon powder (aqueous product, 54.29%, 35.7) was added at room temperature. mg), and stirred under a hydrogen atmosphere for 8 hours. The reaction solution was filtered, and the solvent was distilled off under reduced pressure to obtain compound D7 (183 mg, yield 98%). ESI-MS m / z: 1141 (M + 2H) ^{2+ 1} H-NMR (400 MHz, DMSO-d ₆ ) 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, 4H), 4.96 (dd, J = 3.1, 11.2 Hz, 4H), 5.21 (d, J = 3.1 Hz , 4H), 6.98 (s, 1H), 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

[Chemical 156]

Reference Example 42 Step 1 The compound RE42-1 synthesized by the method described in Reference Example 11 (the compound RE11-2 in Reference Example 11, 500 mg, 0.861 mmol) was dissolved in N, N'-dimethylformamidine Amine (10 mL), 10% palladium-carbon powder (aqueous product, 54.29%, 92.1 mg) was added at room temperature, and stirred under a hydrogen atmosphere for 2 hours. Under argon atmosphere, add the compound RE29-4 (97.0 mg, 0.172 mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodicarbonate synthesized in Reference Example 29 at room temperature. Amidine imine hydrochloride (198 mg, 1.03 mmol), 1-hydroxybenzotriazole monohydrate (13.2 mg, 0.086 mmol) and stirred overnight. The reaction solution was filtered through celite, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (chloroform / methanol = 85/15), and then HPLC (acetonitrile / water) was obtained by reverse phase separation. Purification was performed to obtain compound RE42-2 (179 mg, yield 46%). ESI-MS m / z: 1138 (M + 2H) ²⁺

Reference Example 42 Step 2 The compound RE42-2 (175 mg, 0.077 mmol) was dissolved in tetrahydrofuran (2.6 mg) and water (0.9 mL), and 10% palladium-carbon powder (aqueous product, 54.29%, 32.4) was added at room temperature. mg), and stirred for 1 hour under a hydrogen atmosphere. The reaction solution was filtered, and the solvent was distilled off under reduced pressure to obtain compound D8 (160 mg, yield 95%). ESI-MS m / z: 1093 (M + 2H) ^{2+ 1} H-NMR (400 MHz, DMSO-d ₆ ) 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, 1H), 6.94 (s, 2H), 7.87 (d, J = 9.4 Hz, 2H), 7.92 (d, J = 8.5 Hz, 2H), 8.33 (brs, 2H), 8.50 (brs, 2H).

Reference Example 43: Synthesis of Compound D9

[Chemical 157]

Reference Example 43 Step 1 The compound RE43-1 synthesized by the method described in Reference Example 13 (the compound RE13-2 in Reference Example 13, 500 mg, 0.784 mmol) was dissolved in N, N'-dimethylformamidine Amine (10 mL), 10% palladium-carbon powder (aqueous product, 54.29%, 92.1 mg) was added at room temperature, and stirred under a hydrogen atmosphere for 2 hours. Under argon atmosphere, add the compound RE30-6 (144 mg, 0.157 mmol) and 1- (3-dimethylaminopropyl) -3-ethylcarbodicarbonate synthesized in Reference Example 30 at room temperature. Amidine imine hydrochloride (180 mg, 0.941 mmol), 1-hydroxybenzotriazole monohydrate (12.0 mg, 0.078 mmol) and stirred overnight. The reaction solution was filtered through diatomaceous earth, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (chloroform / methanol = 80/20), and then HPLC (acetonitrile / water) was obtained by reverse phase separation. Purification was performed to obtain compound RE43-2 (191 mg, yield 43%). ESI-MS m / z: 1431 (M + 2H) ²⁺

Reference Example 43 Step 2 The compound RE43-2 (186 mg, 0.065 mmol) was dissolved in tetrahydrofuran (2.8 mg) and water (0.9 mL), and 10% palladium-carbon powder (aqueous product, 54.29%, 34.3) was added at room temperature. mg), and stirred for 1 hour under a hydrogen atmosphere. The reaction solution was filtered, and the solvent was distilled off under reduced pressure to obtain compound D9 (174 mg, yield 97%). ESI-MS m / z: 1386 (M + 2H) ^{2+ 1} H-NMR (400 MHz, DMSO-d ₆ ) δ: 1.25-4.02 (m, 164H), 4.18-4.26 (m, 2H), 4.47 ( d, J = 8.1 Hz, 4H), 4.96 (dd, J = 3.1, 11.2 Hz, 4H), 5.21 (d, J = 3.6 Hz, 4H), 7.74-7.91 (m, 13H), 8.18 (s, 2H ), 8.36 (d, J = 7.2 Hz, 2H), 10.27 (brs, 1H).

Reference Example 44: Synthesis of compound A2

[化 158]

Reference Example 44 Step 1 The compound RE44-1 (the compound RE31-4 in Reference Example 31, 150 mg, 0.228 mmol) 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 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 the compound of Reference Example 14 RE14-3 (574 mg, 0.571 mmol) and 1- (3-dimethylamine were added at room temperature. Propyl) -3-ethylcarbodiimide hydrochloride (109 mg, 0.571 mmol), triethylamine (0.159 mL, 1.14 mmol), 1-hydroxybenzotriazole monohydrate (3.50 mg, 0.023 mmol) and stirred overnight. The reaction solution was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (chloroform / methanol = 90/10), and further purified by reverse-phase fractionation HPLC (acetonitrile / water) to obtain compound RE44-2. (242 mg, 44% yield). ESI-MS m / z: 1216 (M + 2H) ²⁺

Reference Example 44 Step 2 The compound RE44-2 (242 mg, 0.100 mmol) was dissolved in tetrahydrofuran / water (4/1; 12 mL), and 10% palladium-carbon powder (aqueous product, 54.29%, 44.6) was added at room temperature. mg), and stirred under a hydrogen atmosphere for 2 hours. Under argon atmosphere, add 6-butenediamidohexanoic acid (23.2 mg, 0.110 mmol), 4- (4,6-dimethoxy-1,3,5-tri- 2-yl) -4-methylmorpholine hydrochloride (55.2 mg, 0.199 mmol) and stirred overnight. The reaction solution was filtered through celite, and the solvent was distilled off under reduced pressure. The residue was purified with HP20 resin (acetone / water) to obtain compound A2 (97.4 mg, yield 39%). ESI-MS m / z: 1245 (M + 2H) ^{2+ 1} H-NMR (400 MHz, DMSO-d ₆ ) 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

[Chemical 159]

Reference Example 45 Step 1 The compound RE45-1 synthesized by the method described in Reference Example 32 (the compound RE32-3, 75.0 mg, 0.228 mmol in Reference Example 32) was dissolved in N, N'-dimethylformamidine Amine (3.0 mL), the compound of Reference Example 14 RE14-3 (574 mg, 0.571 mmol), diisopropylethylamine (0.199 mL, 1.14 mmol), and hexafluorophosphate O- (7- Azabenzotriazol-1-yl) -N, N, N ', N'-tetramethylurenium (217 mg, 0.571 mmol) and stirred overnight. The solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (chloroform / methanol = 90/10), and further purified by reverse-phase fractionation HPLC (acetonitrile / water) to obtain compound RE45. -2 (202 mg, yield 38%). ESI-MS m / z: 1152 (M + 2H) ²⁺

Reference Example 45 Step 2 The compound RE45-2 (196 mg, 0.085 mmol) was dissolved in tetrahydrofuran / water (4/1; 10 mL), and 10% palladium-carbon powder (aqueous product, 54.29%, 36.1) was added at room temperature. mg), and stirred under a hydrogen atmosphere for 2 hours. Under argon atmosphere, add 6-butenediamidohexanoic acid (19.8 mg, 0.094 mmol), 4- (4,6-dimethoxy-1,3,5-tri- 2-yl) -4-methylmorpholine hydrochloride (47.0 mg, 0.170 mmol) and stirred overnight. The reaction solution was filtered through celite, and the solvent was distilled off under reduced pressure. The residue was purified with HP20 resin (acetone / water) to obtain compound A3 (42.4 mg, yield 21%). ESI-MS m / z: 1182 (M + 2H) ²⁺

Reference Example 46: Compound D10

[Chemical 160]

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, product Rate 58%). ESI-MS m / z: 2437 (M + H) ⁺

Reference Example 47: Synthesis of Compound A4

[Chemical 161]

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

Step 2 of Reference Example 47 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 Step 2 of Reference Example 3. ESI-MS m / z: 2170 (M + H) ⁺

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

Reference Example 48: Synthesis of Compound A5

[Chemical 162]

Step 1 of Reference Example 48 The compound RE48-1 synthesized by the method described in Reference Example 33 (the compound RE33-4, 0.099 g, 0.277 mmol in Reference Example 33) was used, and the compound synthesized in Step 2 of Reference Example 16 was used. Compound RE16-3 (0.618 g, 0.615 mmol) was obtained by the same method as step 3 of Reference Example 3 to obtain compound RE48-2 (0.343 g, yield 53%). ESI-MS m / z: 2333 (M + H) ⁺

Step 2 of Reference Example 48 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

[Chemical 163]

Using compound RE49-1 (0.048 g, 0.021 mmol) and 3-cis-butene diimide propionate N-butane diimide (manufactured by Tokyo Chemical Industry Co., Ltd., 0.017 g, 0.064 mmol), with reference examples Compound A1 was synthesized in the same manner as in Compound 8 to obtain compound A6 (0.040 g, yield 78%). ESI-MS m / z: 2480 (M + HCOO) ^-

Reference Example 50: Synthesis of Compound A7

[Chemical 164]

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

Reference Example 51

[Chemical 165]

Reference Example 51 Step 1 Using the compound RE51-1 (0.122 g, 0.054 mmol) synthesized by the method described in Reference Example 7 and by using the compound with "Bioconjugate Conjugation", Volume 22, pages 690? 699, 2011 The monobenzyl hexanoate synthesized by the same method as described in 2010 was obtained by the same method as that in step 1 of Reference Example 4 to obtain compound RE51-2 (0.076 g, yield 56%). ESI-MS m / z: 2503 (M + H) ⁺

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

Reference Example 52

[Chemical 166]

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

Reference Example 53: Synthesis of compound C2

[Chemical 167]

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

Step 2 of Reference Example 53 Using compound RE53-1 (0.129 g, 0.044 mmol) and step 4 of Reference Example 10, a crude product of compound RE53-2 was obtained. ESI-MS m / z: 1535 (M + HCOOH-2H) ^2-

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

Reference Example 54: Synthesis of compound C3

[Chemical 168]

Reference Example 54 Step 1 The compound D1 (100 mg, 0.040 mmol) synthesized by the method described in Reference Example 10 and the compound RE17-2 of Reference Example 17 were used. The compound was obtained in the same manner as in Step 3 of Reference Example 10. RE54-1 (90 mg, yield 76%). ESI-MS m / z: 1335 (M-DMTr + 2H) ²⁺

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

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

Reference Example 55: Synthesis of compound C4

[Chemical 169]

Reference Example 55 Step 1 The compound D10 (100 mg, 0.039 mmol) synthesized by the method described in Reference Example 46 and the compound RE17-2 synthesized in Reference Example 17 were used. The same procedures as in Step 3 of Reference Example 10 were used. Method: Compound RE55-1 (90 mg, yield 76%) was obtained. ESI-MS m / z: 1356 (M + 2H) ²⁺ , detected as deDMr body

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

Step 3 of Reference Example 55 Using the crude product of compound RE55-2, compound C4 (17.0 μmol / g, two-stage yield of 26%) was obtained by the same method as step 5 of Reference Example 10.

Reference Example 56: Synthesis of compound C5

[Chem 170]

Reference Example 56 Step 1 The compound D10 (100 mg, 0.039 mmol) synthesized by the method described in Reference Example 46 and the compound RE18-3 synthesized in Step 2 of Reference Example 18 were used. 3 The same method was used to obtain compound RE56-1 (50 mg, yield 42%). ESI-MS m / z: 1363 (M + 2H) ²⁺ , detected as deDMr

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

Step 3 of Reference Example 56 Using the crude product of compound RE56-2, compound C5 (0.5 μmol / g, two-stage yield of 1%) was obtained by the same method as step 5 of Reference Example 10.

Reference Example 57: Synthesis of compound C6

[Chemical 171]

Step 1 of Reference Example 57 The compound D10 (100 mg, 0.039 mmol) synthesized by the method described in Reference Example 46 and the compound RE19-2 of Reference Example 19 were used, and the compound was obtained by the same method as in Step 3 of Reference Example 10. RE57-1 (40 mg, 30% yield). ESI-MS m / z: 1532 (M + 2H) ²⁺ , detected as deDMr

Step 2 of Reference Example 57 Using compound RE57-1 (40 mg, 0.012 mmol), the crude product of compound RE57-2 was obtained by the same method as Step 4 of Reference Example 10. ESI-MS m / z: 1582 (M + 2H) ²⁺ , detected as deDMr

Step 3 of Reference Example 57 Using the crude product of compound RE57-2, Compound C6 (0.2 μmol / g, 1% yield in two stages) was obtained by the same method as Step 5 of Reference Example 10.

Reference Example 58: Synthesis of compound C7

[Chemical 172]

Step 1 of Reference Example 58 Using compound D1 (70 mg, 0.028 mmol) synthesized by the method described in Reference Example 10 and the compound RE21-3 of Reference Example 21, compound RE58 was obtained in the same manner as in Step 3 of Reference Example 10. -1 (58 mg, 77% yield). ESI-MS m / z: 1341 (M + 2H) ²⁺

Reference Example 58 Step 2 The compound RE58-1 (58 mg, 0.022 mmol) was dissolved in methanol (0.15 mL), and the compound was synthesized by the method described in Organic Chemistry, Volume 74, pages 6837-6842, 2009. Compound RE17-1 (16.9 mg, 0.032 mmol) of Reference Example 17, 1 mol / L SODIUM L-ASCORBATE aqueous solution (0.022 mL, 0.022 mmol), 20 mmol / L copper (II) sulfate aqueous solution (0.011 mL, 0.22 μmol) and 10 mmol / L tris (2-benzimidazolylmethyl) amine / DMSO solution (0.022 mL, 0.22 μmol), and 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: 1450 (M + 2H) ²⁺ , detected as deDMr body

Step 3 of Reference Example 58 Using compound RE58-2 (10 mg, 3.13 μmol), the crude product of compound RE58-3 was obtained by the same method as Step 4 of Reference Example 10. ESI-MS m / z: 1500 (M + 2H) ²⁺ , detected as a deDMR body

Step 4 of Reference Example 58 Using the crude product of compound RE58-3, Compound C7 (8.4 μmol / g, yield 27%) was obtained by the same method as that of Step 5 of Reference Example 10.

Reference Example 59

[Chemical 173]

Reference Example 59 Step 1 The compound D10 (74.1 mg, 0.029 mmol) synthesized by the method described in Reference Example 46 and the compound RE22-2 (15 mg, 0.027 mmol) of Reference Example 22 were used. The crude product of compound RE59-1 was obtained by the same method as in step 3 or the method described in "Bioconjugate Conjugation", Vol. 26, pp. 1451-1455, 2015. ESI-MS m / z: 1392 (M + H) ⁺ , detected as de-DMTr

Step 2 of Reference Example 59 Using compound RE59-1 (0.083 g, 0.027 mmol), a crude product of compound RE59-2 was obtained by a method described in International Publication No. 2015105083. ESI-MS m / z: 2669 (M + H) ⁺ , detected as de-DMTr

Step 3 of Reference Example 59 Using compound RE59-2 (0.08 g, 0.027 mmol), the crude product of compound RE59-3 was obtained 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 (48H, m), 1.93 (12H, s), 1.94 (12H, s ), 2.02 (12H, s), 2.13 (12H, 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 , brs), 7.90 (1H, brs).

Reference Example 60 Using the structure described in Compound D1 and Table Z-1 or compound RE20-4 in Reference Example 20, Table P-1 and Table P-1 were obtained in the same manner as in Step 1 and Step 2 of Reference Example 59. Compounds listed in Table 2. The results of mass spectrometry analysis of the compounds synthesized in this example are shown in Table P-3.

[Table 34] Table P-1

[Table 35] Continued from Table P-1

[Table 36] Table P-2

[TABLE 37]

Reference Example 61: Synthesis of compound C8

[Chemical 174]

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 the compounds described in Tables P-1 and P-2, the compounds described in Tables Q-1 and Q-2 were obtained in the same manner as in Reference Example 61. The loading amount of the compound synthesized by this example is shown in Table Q-3.

[Table 38] Table Q-1

[Table 39] Continued from Table Q-1

[Table 40] Table Q-2

[TABLE 41]

Example 1: Synthesis of Compound 1-2

[Chemical 175]

Step 1 Add the reference compound A1 and the terminal thiolated oligonucleotide synthesized by the method described in "Molecules", Vol. 17, pp. 13825-13843, 2012, and let stand at room temperature. Set for 4 hours. Sodium carbonate was added to the reaction mixture, and the mixture was left standing at 4 ° C for a while. By anion exchange chromatography (GE Healthcare, MonoQ 5 / 50GL, 10 μm, 5.0 mm × 50 mm; solution A: 10 mmol / L Tris buffer / 30% acetonitrile and solution B: 10 mmol / L Tris buffer Solution / 30% acetonitrile / 1 mol / L NaBr gradient) or reverse phase liquid chromatography (Waters, X BridgeC18, 5 μm, 4.6 mm × 250 mm, 0.1 mol / L triethylammonium acetate buffer Purification was performed by either method with a solution B: gradient formed by acetonitrile, whereby Compound 1-1 was obtained.

Step 2 Use a mixed buffer (100 mmol / L potassium acetate, 30 mmol / L 2- [4- (2-hydroxyethyl) pipe-1-yl] ethanesulfonic acid (HEPES) -KOH (pH 7.4), 2 mmol / L magnesium acetate) The concentration of the compound 1-1 (3'-b2GPI-ssRNA) synthesized in step 1 was adjusted (50 μmol / L). Mix equal amounts of the above-mentioned sense stock and anti-sense stock (50 μmol / L), respectively, and leave them at 80 ° C for 10 minutes. The sequence of antisense shares is as described in Table R-3. Slowly lower the temperature and leave it at 37 ° C for 1 hour to obtain 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 using an oligonucleotide having a base sequence different from that of Example 1.

Example 8: Synthesis of Compound 8-2

[Chemical 176]

Step 1 Using Reference Example Compound D5, add an oligonucleotide modified at the end with an amine group synthesized by the method described in Molecular Science, Vol. 17, pp. 13825-13843, 2012. "Yoke Chemistry", Vol. 22, pp. 1723-1728, 2011 or "Bioconjugate Chemistry", Vol. 26, pp. 1451-1455, 2015. The compound 8-1 was obtained by purification by the method described in Example 1.

Step 2 Compound 8-2 was obtained by the same method as step 2 of Example 1.

Example 9: Synthesis of Compound 9-2

[Chemical 177]

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

Example 10: Synthesis of Compound 10-2

[Chemical 178]

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

Example 11: Synthesis of Compound 11-2

[Chemical 179]

Step 1 Use a nucleic acid synthesizer (Ultra Fast Parallel Syntheizer, manufactured by Sigma, hereinafter referred to as UFPS), and perform it at 0.2 μmol. Compound C16 was used as the solid support. Dimethoxytrityl dT phosphoramidite (SAFC-PROLIGO) was adjusted to 0.06 mol / L by acetonitrile. As the activator of phosphoramidite, 5-benzylthio-1H-tetrazole (SAFC-PROLIGO) and 0.06 mol / L dT phosphoramidite in acetonitrile were used for 10 minutes each. Condensation reaction. After the reaction, it was immersed in a 28% ammonia solution and left at 55 ° C for 4 hours. The reaction mixture was concentrated under reduced pressure, and 1-butanol was added to stop the reaction. Reverse phase liquid chromatography (Shiseido, CAPSELL PAK C18, SG300, 6.0 mm × 75 mm, gradient of 5% acetonitrile / 0.1% triethylammonium acetate buffer and B solution: 50% acetonitrile / water) Purification was carried out to obtain compound 11-1.

Step 2 Compound 11-2 was obtained by the same method as step 2 of Example 1.

Example 12: Synthesis of Compound 12-2

[Chemical 180]

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

Example 13: Synthesis of Compound 13-2

[Chemical 181]

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

Example 14: Synthesis of Compounds 14-2 to 19-2 The base sequence of the nucleic acid of Compound 12-2 described in Example 12 was changed, except that Compound 14- 1 to 19-1 to synthesize compound 14-2 to compound 19-2, respectively.

[Chemical 182]

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

[Table 42] Table R-1

[Table 43] Continued from Table R-1

The sequences (sense strands) and mass spectrometric analysis results of compounds 1-1 to 19-1 synthesized in this example are shown in Table R-2 below. In Table R-2, N (M) represents 2'-O-methyl-modified RNA, N (F) represents 2'-fluoro-modified RNA, and ＾ represents phosphorothioate.

[Table 44] Table R-2

The sequences of the compounds 1-2 to 19-2 synthesized in 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'-fluoro-modified RNA, p represents phosphorylation at the 5 'end, and ＾ represents thio. Phosphate.

[Table 45] Table R-3

[Table 46] Continued from Table R-3

Reference Test Example 1: Measurement of β2GPI mRNA attenuated activity HepG2 cells (obtained from ATCC, ATCC number: HB- 8065). As the culture medium, a MEM medium (manufactured by Life Technologies, catalog number 11095-098) containing 10% fetal bovine serum (FBS) was used. The double-stranded nucleic acid and RNAiMax transfection reagents (manufactured by Life Technologies, catalog number 1401251) described in Tables 1-1 to 1-16 were performed using Opti-MEM medium (manufactured by Life Technologies, catalog number 11058-021). Dilute and add 20 μL of siRNA / RNAiMax mixture to each well of a 96-well culture plate such that the final concentration of double-stranded nucleic acid becomes 100 pmol / L, and incubate at 37 ° C and 5% CO ₂ for 24 hours. Thereafter, the cells were washed with PBS (Phosphate buffered saline), and a Cells-to-Ct kit (manufactured by Applied Biosystems, catalog number AM1728) was used. CDNA synthesis. 5 μL of this cDNA was added to a MicroAmp optical 96-well plate (manufactured by Applied Biosystems, catalog number 4326659), and 10 μL of TaqMan gene expression high-performance premix (Gene Expression Master Mix) (manufactured by Applied Biosystems, catalog number 4369016) was added. , 3 μL of UltraPure Distilled Water (manufactured by Life Technologies, catalog number 10977-015), 1 μL of human β2GPI probe, 1 μL of human GAPDH probe. ABI7900 HT real-time PCR system was used for real-time PCR of human β2GPI gene and human GAPDH (D-glyceraldehyde-3-phosphate dehydrogenase, D-glyceraldehyde-3-phosphate dehydrogenase). GAPDH is a constitutive expression gene, and it is measured as an internal control to correct β2GPI expression. The amount of β2GPI mRNA when HepG2 cells were treated with only transfection reagent without adding siRNA was set to 1.0, and the relative expression amount of β2GPI mRNA when each siRNA was introduced was calculated. This experiment was performed three times, and the average values of the relative expression amounts of β2GPI mRNA are shown in Tables 1-1 to 1-16.

[TABLE 47]

[TABLE 48]

[TABLE 49]

[TABLE 50]

[TABLE 51]

[TABLE 52]

[TABLE 53]

[TABLE 54]

[TABLE 55]

[TABLE 56]

[TABLE 57]

[TABLE 58]

[TABLE 59]

[TABLE 60]

[TABLE 61]

[TABLE 62]

Test Example 1 In Vitro Attenuation Test of Human Primary Hepatocytes by Nucleic Acid Complex For each of the nucleic acid complexes obtained in Examples 1 to 19, the in vitro attenuation activity in human primary hepatocytes was measured. 20 μL of each well of each nucleic acid complex diluted with Opti-MEM (manufactured by Thermo Fisher Scientific, catalog number 31985) so that the final concentration becomes 100, 30, 10, or 3 nmol / L is dispensed into a 96-well culture plate Thereafter, human primary hepatocytes (manufactured by Biopredic International, catalog number HEP187) were suspended in a plating medium (manufactured by Biopredic International, catalog number LV0304-2) such that the number of cells became 10,000 cells / 80 μL / well. ), After incubating at 37 ° C and 5% CO ₂ for 6 hours, carefully remove the culture supernatant and add incubation medium (manufactured by Biopredic International, catalog number LV0304-2) to each nucleic acid complex For human primary hepatocytes. As a negative control group, cells without any treatment were inoculated. The cells to which each nucleic acid complex was added were cultured in a 5% CO ₂ incubator at 37 ° C for 18 hours, washed with phosphate-buffered saline (DPBS) (manufactured by Nacalai Tesque) cooled with ice bath, and SuperPrep cells Cell Lysis & RT Kit for qPCR (manufactured by Toyobo Co., Ltd., catalog number SCQ-201), total RNA was recovered in accordance with the method described in the manual accompanying the product, and the total RNA obtained was used CDNA was prepared as a template by a reverse transcription reaction. Using the obtained cDNA as a template, a Taqman (registered trademark) Gene Expression Assays probe (Thermo Fisher Scientific) was used as a probe, and a QuantStudio 12K Flex real-time PCR system (Thermo Fisher Scientific) was used. According to the method described in the attached instruction manual, a PCR reaction is performed to thereby make the β2GPI gene and the glyceraldehyde 3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate dehydrogenase, hereinafter referred to as gapdh) as a constitutive expression gene The gene was subjected to a PCR reaction, and the amount of mRNA amplification was measured, and the amount of mRNA of gapdh was used as an internal control to calculate the quasi-quantitative value of mRNA of β2GPI. The quasi-quantitative value of β2GPI mRNA in the negative control similarly measured was set to 1, and the expression rate of β2GPI mRNA was obtained based on the quasi-quantitative value of β2GPI mRNA. The results of the expression rate of the obtained β2GPI mRNA are shown in Tables S1 and S2. According to Tables S1 and S2, it can be known that each nucleic acid complex inhibits the expression of the mRNA of the β2GPI gene after being added to human primary hepatocytes.

[Table 63]

[TABLE 64]

Test Example 2 In vitro attenuation test of nucleic acid complexes on mouse primary hepatocytes The respective nucleic acid complexes of Tables S3 and S4 among the nucleic acid complexes obtained in Examples 1 to 13 were measured in the mouse first generation Liver cells attenuate activity in vitro. 20 μL of each well of each nucleic acid complex diluted with Opti-MEM (manufactured by Thermo Fisher Scientific, catalog number 31985) so as to have a final concentration of 10, 3, or 1 nmol / L was dispensed into a 96-well culture plate, William's E medium (William's) containing Primary Hepatocyte Thawing and Plating Supplements (manufactured by Thermo Fisher Scientific, catalog number CM3000) was inoculated such that the number of cells became 10,000 cells / 80 μL / well. E Medium) (manufactured by Thermo Fisher Scientific, catalog number A12176-01) suspended mouse primary hepatocytes derived from CD-1 (manufactured by Thermo Fisher Scientific, catalog number MSCP10) at 37 ° C, 5% CO ₂ After 6 hours of incubation under conditions, the culture supernatant was carefully removed, and Williams E medium containing Primary Hepatocyte Maintenance Supplements (manufactured by Thermo Fisher Scientific, catalog number CM4000) was added. The nucleic acid complex is provided to mouse primary hepatocytes. As a negative control group, cells without any treatment were inoculated. The cells to which each nucleic acid complex was added were cultured in a 5% CO ₂ incubator at 37 ° C for 18 hours, washed with phosphate-buffered saline (DPBS) (manufactured by Nacalai Tesque) cooled with ice bath, and SuperPrep cells were used. Cell Lysis & RT Kit for qPCR (manufactured by Toyobo Co., Ltd., catalog number SCQ-201), total RNA was recovered in accordance with the method described in the manual accompanying the product, and the total RNA obtained was used CDNA was prepared as a template by a reverse transcription reaction. Using the obtained cDNA as a template, using a Taqman (registered trademark) gene expression analysis probe (manufactured by Thermo Fisher Scientific) as a probe, and using a QuantStudio 12K Flex real-time PCR system (manufactured by Thermo Fisher Scientific) according to the attached instruction manual Perform the PCR reaction according to the method described in the above, whereby the β2GPI gene and the glyceraldehyde 3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate dehydrogenase (hereinafter referred to as gapp)) gene constituting the expression gene are subjected to PCR reaction, respectively Measure the amount of mRNA amplification, and use the amount of gapdh mRNA as an internal control to calculate the quasi-quantitative value of mRNA for β2GPI. The quasi-quantitative value of β2GPI mRNA in the negative control similarly measured was set to 1, and the expression rate of β2GPI mRNA was obtained based on the quasi-quantitative value of β2GPI mRNA. The expression rates of the obtained β2GPI mRNA are shown in Tables S3 and S4.

[TABLE 65]

[TABLE 66]

According to Table S3 and Table S4, it can be known that each nucleic acid complex inhibits the expression of the mRNA of the β2GPI gene after being added to the mouse primary liver cells.

Test Example 3 In vivo attenuation test of nucleic acid complexes in mice For each nucleic acid complex of Table S5, the in vivo attenuation test of mice was performed by the following method. In addition, in accordance with the test, each nucleic acid complex was diluted with phosphate-buffered saline (DPBS) (manufactured by Nacalai Tesque) and used. After mice (BALB / cA, purchased from CLEA Japan) were domesticated and bred, each nucleic acid complex was administered by subcutaneous injection at 3 mg / kg or 1 mg / kg in each mouse, respectively. As a control group, DPBS was administered to mice by subcutaneous injection only. Euthanasia was performed 3 days after administration, and livers were harvested and frozen in liquid nitrogen. Using Trizol (registered trademark) RNA Isolation Reagent (manufactured by Thermo Fisher Scientific, catalog number 15596026) and MagNA Pure 96 (manufactured by Roche Life Science), the liver was frozen according to the method described in the manual accompanying the product. Total RNA was recovered from the sample. Furthermore, a Transcriptor First Strand cDNA synthesis kit (manufactured by Roche Life Science, catalog number 04897030001) was used, and the total RNA obtained was used as the method described in the manual accompanying the product. CDNA was prepared by reverse transcription reaction of the template. Using the obtained cDNA as a template, using a Taqman (registered trademark) gene expression analysis probe (manufactured by Thermo Fisher Scientific) as a probe, and using a QuantStudio 12K Flex real-time PCR system (manufactured by Thermo Fisher Scientific) according to the attached instruction manual Perform the PCR reaction according to the method described in the above, whereby the β2GPI gene and the gapdh gene are subjected to the PCR reaction, and the amount of mRNA amplification is measured, and the amount of mRNA amplification of gapdh is used as an internal control to calculate the quantified value of the mRNA of β2GPI. The quasi-quantitative value of the β2GPI mRNA in the PBS administration group measured in the same manner was set to 1 to obtain the expression rate of the β2GPI mRNA. The expression ratio of the obtained β2GPI mRNA is shown in Table S5.

[TABLE 67]

According to Table S5, it was found that administration of the nucleic acid complex of the present invention to mice reduced the expression of the β2GPI gene in the liver. [Industrial availability]

The nucleic acid complex of the present invention can be used to treat β2GPI-related diseases in vivo by administering to a mammal.

Claims (24)

A nucleic acid complex, which is represented by the following Formula 1, Formula 1: [化 1] (In Formula 1, X is a double-stranded nucleic acid including a sense strand and an antisense strand and including a double-stranded region of at least 11 base pairs, and the length of the double-stranded nucleic acid in the antisense strand is 17 to 30 The oligonucleotide strand of the nucleotide is complementary to any of the target β2GPI mRNA sequences described in Table 1-1 to Table 1-16, and the 3 'end or 5' end of the sense strand is bonded to S3, L1 And L2 are each independently a sugar ligand, and S1, S2, and S3 are each independently a linker). For example, the nucleic acid complex of claim 1 has a structure represented by the following formula 2, where: [化 2] (In Formula 2, X, L1, L2, and S3 have the same meanings as above, and P1, P2, P3, P4, P5, and P6, and T1 and T2 are independent, or 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 independent or absent, or are substituted or unsubstituted alkylene groups having 1 to 12 carbon atoms or-(CH ₂ CH ₂ O) _n -CH ₂ CH _2- , n is 0 to 99 Integer, B1 and B2 are each independently a bonding bond, or any structure represented by the following formula 2-1. The black dot at the end of each structure is the bond with P2 or P3 or P5 or P6 Nodes, m1, m2, m3, and m4 are each independently an integer of 0 to 10, Formula 2-1: [化 3] 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, wherein when p1 and p2 are integers of 2 or 3, each of 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, each-[P2-Q1]-,-[Q2-P3]-,-[P5-Q3]- , The combination of-[Q4-P6]-can be the same or different). For example, the nucleic acid complex of claim 2, wherein P1 and P4 are independently -CO-NH-, -NH-CO- or -O-. For example, the nucleic acid complex of claim 2 or 3, in which-[P2-Q1] _q1 - _and- [P5-Q3] _q3 -are independent, or do not exist, or are represented by the following formulas 3-1 to 3-3 Any one of the structures shown, Formula 3-1: [化 4] Equation 3-2: [化 5] Equation 3-3: [化 6] (In Formulas 3-1 to 3-3, m5 and m6 are each independently an integer of 0 to 10, and the black dots at the ends of the structures in Formulas 3-1 to 3-3 are respectively B1 or B2, or Bonding point with P1 or P4). The nucleic acid complex according to any one of claims 2 to 4, which has any one of the following formulae 4-1 to 4-9, formula 4-1: [化 7] Equation 4-2: [化 8] Equation 4-3: [化 9] Equation 4-4: Equation 4-5: Equation 4-6: Equation 4-7: [Chem. 13] Equation 4-8: Equation 4-9: (In formulas 4-1 to 4-9, X, L1, L2, S3, P3, P6, T1, T2, Q2, Q4, q2, and q4 have the same meanings as described above). For example, the nucleic acid complex of claim 1 has a structure represented by the following formula 5, which is: [化 16] (In Formula 5, X, S3, P1, P2, P3, Q1, Q2, B1, T1, L1, p1, q1, and q2 have the same meanings as described above). The nucleic acid complex of claim 6, wherein P1 is -CO-NH-, -NH-CO-, or -O-. The nucleic acid complex according to claim 6 or 7, which has any one of the following formulae 6-1 to 6-9, formula 6-1: [化 17] Equation 6-2: Equation 6-3: Equation 6-4: Formula 6-5: [Chem. 21] Equation 6-6: Equation 6-7: Equation 6-8: Equation 6-9: (In formulas 6-1 to 6-9, X, S3, P3, Q2, T1, L1, and q2 have the same meanings as described above). The nucleic acid complex according to any one of claims 2 to 5, which has any one of the structures represented by the following formulae 7-1 to 7-9, formula 7-1: [化 26] Equation 7-2: Equation 7-3: Equation 7-4: [Chem. 29] Equation 7-5: [Chem 30] Equation 7-6: Equation 7-7: Equation 7-8: [Chem. 33] Equation 7-9: (In formulas 7-1 to 7-9, X, S3, L1, and L2 have the same meanings as described above). The nucleic acid complex according to any one of claims 1 to 9, wherein the above-mentioned sugar ligand is N-acetamylgalactosyl sugar. 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 smooth end. The nucleic acid complex according to claim 11, wherein the double-stranded nucleic acid comprises a sugar moiety-modified nucleotide. The nucleic acid complex according to any one of claims 1 to 13, wherein X is selected from the justice stocks / antisense stocks listed in Tables M1-1 to M1-18 or Tables 1-1 to 1-16 1 pair of justice stocks / antisense stocks in the group. 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: [化 35] (In Formula 7-8-1, X has the same meaning as described above). For example, the nucleic acid complex of claim 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. A pharmaceutical composition comprising a nucleic acid complex according to any one of claims 1 to 16. The pharmaceutical composition according to claim 17, which is for introduction into a cell. The pharmaceutical composition of claim 17 or 18 is for intravenous or subcutaneous administration. A method for treating or preventing 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. A method for inhibiting the expression of a β2GPI gene, comprising introducing a double-stranded nucleic acid into a cell using 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. A method for treating a β2GPI-related 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 mammal. A medicine for treating β2GPI-related diseases, 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 β2GPI-related diseases, 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.