TW201909925A - 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 complement factor B (CFB). 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 CFB mRNA sequences of interest described in Tables 1-1 to 1-3 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 bonds to S3; L1 and L2 each independently represent a sugar ligand; S1, S2, and S3 each independently represent a linker.).

Description

Nucleic acid complex

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

As nucleic acid medicines, aptamers, antisense nucleic acids, decoy nucleic acids, ribonuclease, siRNA (small interfering RNA), miRNA (micro RNA, microRNA), and antimiRNA (anti-) are known. Micro RNA, anti-microRNA, etc. Nucleic acid medicine is expected to be clinically applied to various diseases that have hitherto been considered to be difficult to treat because it can control the high versatility of all genes in cells. Moreover, nucleic acid medicine is expected to become a next-generation medicine after antibodies and low-molecular medicine because of its high target selectivity and activity in cells. However, nucleic acid medicines have problems in that they are difficult to deliver to target tissues.

As one of the methods for the delivery of nucleic acid medicines effective in vivo, a nucleic acid conjugate using a targeting compound and a nucleic acid has been reported. As the targeting compound, a ligand capable of binding to a receptor expressed outside the cell can be mentioned. Among them, in particular, it has been reported that N-acetyl-D-galactosamine (GalNAc) or the like is used as a ligand capable of binding to the asialoglycoprotein receptor (ASGPR) which is highly expressed in hepatocytes. Nucleic acid complex. In recent years, it has been reported that a nucleic acid complex in which these ligands and siRNAs are combined can be efficiently delivered to liver cells (Non-Patent Document 1).

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

[Chemical 1] (wherein Ac represents an ethyl hydrazide; the same applies to the present specification)

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

[Chemical 2]

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

[Chemical 3]

A group of proteins in the blood that mediate an immune response, as a complement, may be exemplified by promoting the phagocytosis of the phagocytic cells by the phagocytic cells or damaging the virus having the outer membrane to lose the infectivity. The complement having the most amount in the serum is C3, and is activated by the complement factor B (CFB) or the like in the second pathway of complement, thereby controlling its action (Non-Patent Document 2).

It is known that if C3 is continuously activated due to an abnormality of a control factor related to the second pathway of complement, or stabilization of C3 converting enzyme due to its own antibody, it may cause atypical hemolytic uremic syndrome (aHUS), array Nocturnal hemoglobinuria (PNH), age-related macular degeneration (AMD), membranous proliferative glomerulonephritis (MPGN), C3 nephritis, membranous nephropathy, acute spheroid nephritis (RPGN), acute kidney injury ( AKI), ANCA-associated vasculitis, lupus nephritis, asthma, autoimmune diseases (such as systemic lupus erythematosus (SLE), psoriasis, optic neuromyelitis, myasthenia gravis, etc.), etc. (Non-Patent Document 3, 4). It is expected that the above-mentioned diseases can be prevented or treated by specifically inhibiting the action of the complement factor B, but the complement factor B is present in the blood at a relatively high concentration of 300 μg/mL (Non-Patent Document 5), for example, using a general antibody medicine. It is not easy to continuously suppress all of the complement B factors. Prior Technical Literature Patent Literature

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

Non-Patent Document 1: Journal of American Chemical Society, 2014, Vol. 136, p16958-16961 Non-Patent Document 2: Proceedings of the National Academy of Sciences of the United States of America ), 1997, Vol. 94, p8720-8725 Non-Patent Document 3: Immunological Reviews, 2008, Vol. 223, p300-316 Non-Patent Document 4: Journal of the American Society of Nephrology, 2015 , vol. 26, p2917-2929 Non-Patent Document 5: Introduction to Complement, 2011, MEDICAL VIEW, p18

[The problem that the invention wants to solve]

An object of the present invention is to provide a nucleic acid complex capable of inhibiting the expression of complement B factor (CFB). [Technical means to solve the problem]

The present invention relates to the following. [1] A nucleic acid complex represented by the following formula 1. Equation 1: [Chemical 4] (In Formula 1, 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, and the strand 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 CFB mRNA sequences described in Tables 1-1 to 1-3, and the 3' end or the 5' end of the sense strand is bonded to S3, L1. And L2 are each a sugar ligand, and each of S1, S2 and S3 is a linker. [2] The nucleic acid complex according to [1], which has a structure represented by the following formula 2. Equation 2: [Chemical 5] (In Formula 2, X, L1, L2, and S3 are respectively the same as defined above, and P1, P2, P3, P4, P5, and P6, and T1 and T2 are independent or absent, respectively, or -CO-, -NH- , -O-, -S-, -O-CO-, -S-CO-, -NH-CO-, -CO-O-, -CO-S- or -CO-NH-, Q1, Q2, Q3 And Q4 are independently 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 The integers, B1 and B2 are each independently a bond key, or any structure represented by the following formula 2-1, and the black dots at the ends in each structure are respectively a bond with P2 or P3 or with P5 or P6. The nodes, m1, m2, m3, and m4 are each independently an integer of 0 to 10, and Equation 2-1: [Chem. 6] P1 and p2 are each independently an integer of 1, 2 or 3, and q1, q2, q3 and q4 are each independently an integer of 0 to 10, wherein each of P3 and P6 is when p1 and p2 are integers of 2 or 3, respectively. 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]- 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-, respectively. . [4] The nucleic acid complex according to [2] or [3], wherein -[P2-Q1] _q1 - and -[P5-Q3] _q3 - are each independently or absent, or are represented by the following formula 3-1 Any of the structures represented by the formula 3-3. Equation 3-1: [Chem. 7] Equation 3-2: [Chem. 8] Equation 3-3: [Chem. 9] (In the formula 3-1 to the formula 3-3, m5 and m6 are each independently an integer of 0 to 10, and the black dots at the ends of the structures of the formulas 3-1 to 3-3 are respectively B1 or B2, or [5] The nucleic acid complex according to any one of [2] to [4], which has any one of the following formulas 4-1 to 4-9. Equation 4-1: [Chem. 10] Equation 4-2: [Chem. 11] Equation 4-3: [Chem. 12] Equation 4-4: [Chem. 13] Equation 4-5: [Chem. 14] Equation 4-6: [Chem. 15] Equation 4-7: [Chem. 16] Equation 4-8: [Chem. 17] Equation 4-9: [Chem. 18] (In the 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) [6] The nucleic acid as described in [1] A composite having a structure represented by the following formula 5. Equation 5: [Chem. 19] (In the formula 5, X, S3, P1, P2, P3, Q1, Q2, B1, T1, L1, p1, q1, and q2 are respectively the same as the above-mentioned meanings.) [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 formulas 6-1 to 6-9. Equation 6-1: [Chem. 20] Equation 6-2: [Chem. 21] Equation 6-3: [Chem. 22] Equation 6-4: [Chem. 23] Equation 6-5: [Chem. 24] Equation 6-6: [Chem. 25] Equation 6-7: [Chem. 26] Equation 6-8: [Chem. 27] Equation 6-9: [Chem. 28] (In the formulas 6-1 to 6-9, X, S3, P3, Q2, T1, L1, and q2 are respectively the same as the above-mentioned meanings.) [9] The nucleic acid complex according to any one of [2] to [5] It has any structure represented by the following formula 7-1 to formula 7-9. Equation 7-1: [Chem. 29] Equation 7-2: [Chem. 30] Equation 7-3: [Chem. 31] Equation 7-4: [Chem. 32] Equation 7-5: [Chem. 33] Equation 7-6: [Chem. 34] Equation 7-7: [Chem. 35] Equation 7-8: [Chem. 36] Equation 7-9: [Chem. 37] In the formulas 7-1 to 7-9, the nucleic acid complex according to any one of the above aspects, wherein the above-mentioned sugar ligand is the same as the above-mentioned one. It is N-acetyl galactosamine. [11] The nucleic acid complex according to any one of [1] to [10] wherein the double-stranded nucleic acid comprises a modified nucleotide. [12] The nucleic acid complex according to any one of [1] to [11] wherein the 3' end of the sense strand and the 5' end of the antisense strand form a smooth end. [13] The nucleic acid complex according to [11], wherein the double-stranded nucleic acid comprises a sugar moiety-modified nucleotide. [14] The nucleic acid complex according to any one of [1] to [13] which has a structure represented by the following formula 7-8-1. Equation 7-8-1: [Chem. 38] The nucleic acid complex according to any one of the above aspects, wherein X is selected from Table 1-1 to Table 1-3. One of the justice stocks/antisense stocks in the group of justice stocks/anti-sense stocks recorded in the stock. [16] The nucleic acid complex according to any one of [1] to [14] wherein X is selected from the group consisting of the justice stocks/antisense stocks listed in Tables M1-1 to M1-3. 1 pair of justice stocks / anti-sense stocks. [17] The nucleic acid complex according to any one of [1] to [14] wherein X is a pair of justice shares selected from the group consisting of the justice stocks/antisense stocks listed in Table M1-4. / Anti-sense stocks. [18] The nucleic acid complex according to [17], wherein X is a pair of sense strands/antisense strands represented by EB0125 described in Table M1-4. [19] A pharmaceutical composition comprising the nucleic acid complex according to any one of [1] to [18]. [20] The pharmaceutical composition according to [19], which is for introduction into a cell. [21] The pharmaceutical composition according to [19] or [20], which is administered intravenously or subcutaneously. [22] A method for treating or preventing a disease, comprising the nucleic acid composition according to any one of [1] to [18], or the pharmaceutical composition according to any one of [19] to [21] Patients who need them are given a dose. [23] A method of inhibiting the expression of a CFB gene, which comprises the use of the nucleic acid composition according to any one of [1] to [18], or the pharmaceutical composition according to any one of [19] to [21] The double-stranded nucleic acid is introduced into the cells. [24] A pharmaceutical composition according to any one of [1] to [18] Mammals are administered. [25] A pharmaceutical composition for treating a CFB-related disease, comprising the nucleic acid composition according to any one of [1] to [18], or the pharmaceutical composition according to any one of [19] to [21] . [26] A pharmaceutical composition according to any one of [19] to [21], wherein the nucleic acid composition according to any one of [19] to [21], or a pharmaceutical composition according to any one of [19] to [21]. [27] The treatment method according to [24], wherein the CFB-related disease is atypical hemolytic uremic syndrome, paroxysmal nocturnal hemoglobinuria, age-related macular degeneration, membranous proliferative spheroid nephritis, C3 nephritis Membranous nephropathy, acute glomerulonephritis (RPGN), acute kidney injury (AKI), ANCA-associated vasculitis, lupus nephritis, asthma, systemic lupus erythematosus (SLE), psoriasis, optic neuromyelitis or myasthenia gravis. [28] The medicine described in [25], wherein the CFB-related diseases are atypical hemolytic uremic syndrome, paroxysmal nocturnal hemoglobinuria, age-related macular degeneration, membranous proliferative spheroid nephritis, C3 nephritis, Membranous nephropathy, acute glomerulonephritis (RPGN), acute kidney injury (AKI), ANCA-associated vasculitis, lupus nephritis, asthma, systemic lupus erythematosus (SLE), psoriasis, optic neuromyelitis, or myasthenia gravis. [29] The therapeutic agent according to [26], wherein the CFB-related disease is atypical hemolytic uremic syndrome, paroxysmal nocturnal hemoglobinuria, age-related macular degeneration, membranous proliferative spheroid nephritis, C3 nephritis Membranous nephropathy, acute glomerulonephritis (RPGN), acute kidney injury (AKI), ANCA-associated vasculitis, lupus nephritis, asthma, systemic lupus erythematosus (SLE), psoriasis, optic neuromyelitis or myasthenia gravis. [Effects 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 of the present invention is a nucleic acid complex represented by the following formula 1. Formula 1:

[39]

In Formula 1, 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, wherein the double-stranded nucleic acid has a chain length of 17 to 30 in the antisense strand. The oligonucleotide strand of the nucleotide is complementary to any of the target CFB mRNA sequences described in Tables 1-1 to 1-3, and the 3' end or the 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, S1 and S2 may be bonded to the benzene ring at the ortho, meta or para position, respectively, with respect to the substitution position of S3 on the benzene ring, but it is preferably a nucleic acid complex represented by the following formula 1-1. body. This means that the bonding bonds of S1 and S2 in the formula 1 on the benzene ring may be any position other than the substitution position of S3 on the benzene ring. Equation 1-1:

[化40]

In Formula 1-1, X, L1, L2, S1, S2, and S3 have the same meanings as described above, respectively. In the present specification, the phrase "is identical to the above meaning" is exemplified by the formula 1-1, and means that each of X, L1, L2, S1, and S2 in the formula 1-1 may be the same as above. In the formula 1, the definitions of X, L1, L2, S1, and S2 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. Further, the double-stranded nucleic acid has an oligonucleotide chain of 17 to 30 nucleotides in the antisense strand and a target CFB mRNA as described in Tables 1-1 to 1-3 described below. Any of the sequences are complementary. In addition, the 3' end or the 5' end of the sense strand is bonded to S3.

L1 and L2 are each independently a sugar ligand. In the present invention, the term "sugar ligand" means a group derived from a saccharide (monosaccharide, disaccharide, trisaccharide, polysaccharide, or the like) 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 portion of the saccharide group constituting the sugar ligand to be bonded is referred to as a source. A sugar ligand based on a sugar group. In the present invention, it is only necessary to select a glycoside ligand which is a target cell of an oligonucleotide. As the monosaccharide, for example, allose, aldose, arabinose, cladinose, erythroside, erythrulose, fructose, D-fucitol, L-fucoid Sugar alcohol, fucosamine, fucose, fuculose, galactosamine, galactosaminitol, N-ethinyl-galactosamine, Galactose, glucosamine, N-ethinyl-glucosamine, glucosaminitol, glucose, glucose-6-phosphate, gulose, glyceraldehyde, L-glycerol-D-mannose heptose, glycerol , glycerone, gulose, idose, lyxose, mannosamine, mannose, mannose-6-phosphate, psicose, isorhamnose, isorhamnetamine, rat Limeitol, rhamnose, rhamnose, ribose, ribulose, sedoheptulose, sorbose, tagatose, talose, tartaric acid, threose, xylose, xylulose, and the like. Examples of the disaccharide, trisaccharide, and polysaccharide include abequose, acarbose, amicetose, amylopectin, amylose, celery sugar, arcanose, and ascarylose. Ascorbic acid, 2-deoxy-6-rhobinose, cellobiose, cellotriose, cellulose, chacotriose, chalcose, chitin, colitose , cyclodextrin, cymarose, dextrin, 2-deoxyribose, 2-deoxyglucose, 2-deoxydone, diginose, digitalose, digitalis (digitoxose), evalose, evemitrose, fructooligosaccharide, galto-oligosaccharide, gentian trisaccharide, gentian disaccharide, glycan, glycogen, glycogen, hammeme ), heparin, inulin, isolevoglucosenone, isomaltose, isomaltotriose, isopanose, kojibiose, lactose, lactosamine, lactosediamine, Laminarabiose, levoglucosan, levoglucose (levoglucosenone), β-maltose, maltotriose, manno-oligosaccharide, manninotriose, raffinose, melibiose, muramic acid, micarose, mycinose, ceramide ( Neuraminic acid), sialic acid sugar chain, aflatoxin, nojirimycin, noviose, oleandrose, panose, paratose, psyllium, cherry Primose, raffinose, rhodinose, rutose, sarmentose, sedoheptulose, sedoheptulosan, solatriose, Sophorose, stachyose, streptose, sucrose, alpha, alpha-trehalose, trahalosamine, pine disaccharide, tyvelose, xylobiose, umbelliferous, etc. .

Each monosaccharide in the saccharide may be a D body or an L body, or may be a mixture of any of the D body and the L body. The sugar may also include deoxy sugar (which is obtained by substituting an alcoholic hydroxyl group into a hydrogen atom), an amino sugar (which is obtained by substituting an alcoholic hydroxyl group into an amine group), and a thio sugar (a substitution of an alcoholic hydroxyl group into a mercaptan group) Or substitution of C=O for C=S or substitution of epoxy for sulfur), seleno sugar, telluro sugar, aza sugar (replacement of ring carbon with Nitrogen), imino sugar (formed by replacing epoxy with nitrogen), phosphano sugar (substituted with epoxy instead of phosphorus), and phospha sugar (cyclocarbon) Substituted as phosphorus), C-substituted monosaccharide (which is obtained by substituting a hydrogen atom on a non-terminal carbon atom for a carbon atom), unsaturated monosaccharide, or alditol (substituting a carbonyl group for a CHOH group) And aldonic acid (which is obtained by substituting an aldehyde group into a carboxyl group), ketoaldonic acid, uronic acid, aldonic acid, and the like. As the amino sugar, examples of the amino monosaccharide in the saccharide include galactosamine, glucosamine, mannosamine, fucose amine, isorhamidosamine, ceramide, and muramic acid. , lactose diamine, acosamine, bacillosamine, daunosamine, desosamine, forosamine, garosamine, kanosamine, kansosamine, carbonaceous Mycaminose, mycosamine, perosamine, pneumosamine, purpurosamine, rhodosamine, and the like. Further, the amine group of the amino sugar may be substituted with an ethyl 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 examples thereof include a sugar chain containing NeuAc-Gal-GlcNAc or a Neu5Acα(2-6)Galβ(1-3)GlcNAc. . Each monosaccharide in the saccharide may be substituted with a substituent as long as it can bind to a receptor expressed by the target cell, for example, a hydroxyl group may be substituted, and a hydrogen atom in each monosaccharide may be substituted with 1 to a plurality of azide groups and / or an aryl group which may be substituted.

The sugar ligand is preferably a sugar ligand which is selected to bind to a receptor which is expressed on the surface of the target cell in accordance with each organ to be targeted, for example, when the target cell is a hepatocyte, preferably corresponds to The sugar ligand of the receptor expressed on the surface of the liver cell is more preferably a sugar ligand corresponding to the asialoglycoprotein receptor (ASGPR).

As the sugar ligand corresponding to ASGPR, mannose or N-ethinylgalactosamine is preferred, and N-ethylmercapto galactosamine is more preferred. As a sugar ligand having higher affinity for ASGPR, for example, "Bioorganic Medicinal Chemistry", Vol. 17, p. 7254 (2009) and "Journal of the American Chemical Society" (Journal of Biochemistry) The sugar derivative described in American Chemical Society, Vol. 134, p. 1978 (2012), etc., can be used.

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

S3 is not particularly limited as long as it is a structure in which X as a double-stranded nucleic acid is linked 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 by a phosphodiester bond, and S3 can be, for example, by -CO-, -NH-, -O-, -S-, -O-CO-, -S- The CO-, -NH-CO-, -CO-O-, -CO-S- or -CO-NH- linkages are linked to a benzene ring.

As the linker 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, International Publication No. 2015/ Structure disclosed in 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:

[化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, respectively, or are -CO-, -NH-, -O-, -S-, -O-CO-, -S-CO-, -NH-CO-, -CO-O-, -CO-S- or -CO-NH-, Q1, Q2, Q3 and Q4 is independently or absent, 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 of 0 to 99 .

P1 and P4 are independent or absent, respectively, or are -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-, and further preferably -NH-CO-. In the case where P1 or P4 is, for example, -NH-CO-, it has a partial structure of -NH-CO-benzene ring.

Q1, Q2, Q3 and Q4 are each independently 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 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 further preferably not The alkylene group having 1 to 6 carbon atoms is substituted, and more preferably an unsubstituted alkylene group having 1 to 4 carbon atoms.

P2 and P5 are independent or absent, respectively, or are -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 a partial structure of B1-CO-NH-Q1 and B2-CO-NH-Q3.

-[P2-Q1] _q1 - and -[P5-Q3] _q3 - are independently independent, and preferably do not exist or are represented by any of the following formulas 3-1 to 3-3. Equation 3-1:

[化42] Equation 3-2:

[化43] Equation 3-3:

[化44]

In the formulae 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 of the formulae 3-1 to 3-3 are respectively B1 or B2 or P1 Or P4 key node.

B1 and B2 are each independently a bonding bond, or any structure represented by the following formula, and the black dots at the ends in each structure are respectively a bond with P2 or P3 or with P5 or P6, m1. M2, m3 and m4 are each independently an integer of 0-10.

[化45]

B1 and B2 are preferably derived from an amino acid containing an unnatural amino acid such as glutamic acid, aspartic acid, lysine or imidodiacetic acid, or derived from 2-amino-1,3- The base of an amino alcohol such as propylene glycol, when B1 and B2 are derived from the base of glutamic acid and aspartic acid, the amine groups of glutamic acid and aspartic acid are respectively bonded to each other as P2 and P5. Preferably, the NH-CO- bond is bonded to the carboxyl group of the amine acid when B1 and B2 are derived from the group of the amine acid, and is preferably a -CO-NH- bond, as P2 and P5. When B1 and B2 are groups derived from iminodiacetic acid, the amine groups of the imidodiacetic acid are bonded to each other, and as P2 and P5, a -CO- bond is preferred. Specifically, B1 and B2 preferably have the following structure.

[Chem. 46]

When p1 and p2 are each an integer 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, the combination of each of -[P2-Q1]-, -[Q2-P3]-, -[P5-Q3]-, and -[Q4-P6]- may be the same or different. The combination of each -[P2-Q1]-, -[Q2-P3]-, -[P5-Q3]-, and -[Q4-P6]- is the same or different, meaning -[P2-Q1]-,- [2 - P3] -, - [P5 - Q3] - and - [Q4-P6] - Each of the 2 to 10 units is 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 formulas 4-1 to 4-9. Equation 4-1:

[化47] Equation 4-2:

[48] Equation 4-3:

[化49] Equation 4-4:

[化50] Equation 4-5:

[化51] Equation 4-6:

[化52] Equation 4-7:

[化53] Equation 4-8:

[54] Equation 4-9:

[化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, respectively, or are -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 a partial structure of B1-NH-CO-Q2 and B2-NH-CO-Q4, respectively.

T1 and T2 are independent or absent, respectively, 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 the following formula 5. In Formula 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 in Formula 2 Same as q4. Equation 5:

[化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, respectively. Further, X, S3, P1, P2, P3, Q1, Q2, B1, T1, L1, p1, q1 and q2 in the formula 5 may each be a suitable base as described above, and P1 is preferably -CO-NH. -, -NH-CO- or -O-. -(P2-Q1) _q1 - in the formula 5 is preferably absent or is any of the structures represented by the above formulas 3-1 to 3-3.

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

[化57] Equation 6-2:

[化58] Equation 6-3:

[化59] Equation 6-4:

[60] Equation 6-5:

[化61] Equation 6-6:

[化62] Equation 6-7:

[化63] Equation 6-8:

[化64] Equation 6-9:

[化65]

In the formulae 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 formulas 7-1 to 7-9. Equation 7-1:

[化66] Equation 7-2:

[67] Equation 7-3:

[化68] Equation 7-4:

[化69] Equation 7-5:

[化70] Equation 7-6:

[71] Equation 7-7:

[化72] Equation 7-8:

[化73] Equation 7-9:

[化74]

In the formula 7-1 to the formula 7-9, X, L1, L2 and S3 have the same meanings as described above. L1 and L2 may be the same or different and are preferably the same. In the formula 7-1 to the formula 7-9, since the alkyl group is introduced into the alkyl chain having a different chain length, and the amide bond or the like is substituted with another bond, the formula 7- can be produced. A nucleic acid derivative other than the nucleic acid complex of the structure represented by the formula 1-9.

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

[化75]

In Formula 11, L1, L2, S1 and S2 have the same meanings as above, and P7 and P8 are independent or absent, respectively, or are -CO-, -NH-, -O-, -S-, -O-CO- , -S-CO-, -NH-CO-, -CO-O-, -CO-S- or -CO-NH-, Q5, Q6 and Q7 are independent, or absent, or substituted or not a substituted alkyl group having 1 to 12 carbon atoms 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 the present specification. In any of the structures represented by the following formula 11-1, the curve indicates a bonding bond with Q5 and Q6, respectively, and Equation 11-1:

[化76]

In the formula 11-1, the substituent in the group having a triazole ring is any one of the nitrogen atom at the 1-position and the 3-position of the triazole ring. Q5 and q6 are each independently an integer from 0 to 10.

P7 or 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-. In the case where P7 is, for example, -O-, it has a partial structure of a benzene ring-O-.

P8 or absent, or -CO-, -NH-, -O-, -S-, -O-CO-, -S-CO-, -NH-CO-, -CO-O-, -CO- S- or -CO-NH-, in the case of presence, is preferably -CO-O- or -CO-NH-, more preferably -CO-NH-. In the case where P8 is, for example, -CO-NH-, it has a partial structure of Q6-CO-NH-.

Q5, Q6 and Q7 are each independently or absent, 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 of from ～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 further preferably unsubstituted. 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, and m15 and m16 are each independently an integer of from 1 to 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 formulas 12-1 to 12-12. Equation 12-1:

[化77] Equation 12-2:

[化78] Equation 12-3:

[化79] Equation 12-4:

[化80] Equation 12-5:

[化81] Equation 12-6:

[化82] Equation 12-7:

[化83] Equation 12-8:

[化84] Equation 12-9:

[化85] Equation 12-10:

[化86] Equation 12-11:

[化87] Equation 12-12:

[化88]

In the formulae 12-1 to 12-12, X, L1, L2, S1 and S2 have the same meanings as described 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 in which the structure described in Formula 2 corresponding to S1 and S2 and the structure described in Formula 11 corresponding to S3 are combined in the nucleic acid complex represented by Formula 1. Formula 2 may be Formula 4-1 to Formula 4-9, and may be Formula 6-1 to Formula 6-9, or 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 preferably one in which any one of the formulas 4-1 to 4-9 corresponding to S1 and S2 and the formula 12-1 corresponding to S3 are combined in the nucleic acid complex represented by Formula 1. The nucleic acid complex of any one of the structures of the formula 12-12, which has any one of the formulas 6-1 to 6-9 corresponding to S1 and S2, and the formula 12-1 to formula 12 corresponding to S3. The nucleic acid complex of any of the structures described in the above, wherein any one of the structures described in the formulas 7-1 to 7-9 corresponding to S1 and S2 and the formula 12-1 to formula 12-12 corresponding to S3 are combined. A structural nucleic acid complex.

X in Formula 1 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, wherein the stranded nucleic acid has a chain length of 17 to 30 cores in the antisense strand. The oligonucleotide chain of the nucleotide is complementary to any of the target CFB mRNA sequences described in Tables 1-1 to 1-3. Since the 3' end or the 5' end of the justice strand is bonded to S3, in Formula 1, X bonded to S3 is a justice strand constituting a double-stranded nucleic acid, that is, Table 1-1 to Table 1-3 described below. Or the justice shares represented by the sequence of the justice shares in Tables M1-1 to M1-3.

In the present invention, a nucleic acid comprising a base sequence complementary to CFB mRNA is referred to as an antisense strand nucleic acid, and a nucleic acid comprising 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 attenuate or stop the performance of the CFB gene when introduced into a mammalian cell, and is a pair having a sense strand and an antisense strand. Nucleic acid. Further, the sense strand has at least 11 base pairs with the antisense strand, and at least 17 nucleotides of the antisense strand and at most 30, ie, an oligonucleotide having a chain length of 17 to 30 nucleotides The nucleotide chain is complementary to the target CFB mRNA sequence selected from the group described in Tables 1-1 to 1-3.

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, for example, RNA of a polymer of ribonucleotides, DNA of a polymer as a deoxyribonucleotide, a chimeric nucleic acid comprising RNA and DNA, and at least one nucleotide of the nucleic acid are equivalent to the nucleotide A molecularly substituted nucleotide polymer. Further, the double-stranded nucleic acid as a drug used in the present invention also includes a derivative in which at least one molecule having the same function as a nucleotide is contained in the nucleic acid. Also, uracil (U) can be replaced with thymine (T).

Examples of the molecule having the same function as the nucleotide include a nucleotide derivative and the like. The nucleotide derivative may be any molecule as long as it is a molecule obtained by modifying a nucleotide, for example, to enhance or stabilize nuclease resistance compared to RNA or DNA, in order to improve affinity with the complementary strand nucleic acid. In order to improve cell permeability or to visualize, it is preferable to use a molecule obtained by modifying a ribonucleotide or a deoxyribonucleotide.

Examples of the molecule obtained by modifying the nucleotide include a sugar moiety modified nucleotide, a phosphodiester bond modified nucleotide, a base modified nucleotide, and a sugar moiety, a phosphodiester bond, and a base. At least one of the modified nucleotides and the like.

The sugar-modified nucleotide may be any one as long as it is a part or the whole of a chemical structure of a sugar of a nucleotide modified or substituted with an arbitrary substituent or substituted by an arbitrary atom, and may be preferably used. '-Modified nucleotides.

As a 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 a 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). a substituent-substituted nucleotide, more preferably a 2'-OH group substituted with H, F or a methoxy group, and further preferably a 2'-OH group substituted with F or a methoxy group acid. Further, it is also exemplified that the 2'-OH group is selected from 2-(methoxy)ethoxy (2-(methoxy)ethoxy), 3-aminopropoxy (3-aminopropoxy), 2-[ (N,N-dimethylamino)oxy]ethoxy (2-[(N,N-dimethylamino)oxy]ethoxy), 3-(N,N-dimethylamino)propoxy (3-(N,N-dimethylamino)propoxy), 2-[2-(N,N-dimethylamino)ethoxy]ethoxy (2-[2-(N,N-Dimethylamino)) Ethoxy]ethoxy), 2-(methylamino)-2-oxoethoxyethoxy (2-(methylamino)-2-oxoethoxy), 2-(N-methylaminecarbamyl)ethoxylate Nucleotides substituted with substituents in the group consisting of 2-(N-methylcarbamoyl)ethoxy group and 2-cyanoethoxy group (2-cyanoethoxy group). Preferably, the nucleotides in the region of the double-stranded nucleic acid comprise 50% to 100% of the 2'-modified nucleotide, more preferably 70% to 100%, and even more preferably 90% to 100%. . Further, the nucleotide of the sense strand preferably contains 20% to 100% of the 2'-modified nucleotide, more preferably 40% to 100%, and still more preferably 60% to 100%. Further, the nucleotide of the antisense strand preferably contains 20% to 100% of the 2'-modified nucleotide, more preferably 40% to 100%, and even more preferably 60% to 100%. .

The phosphodiester bond-modified nucleotide may be any one as long as it is a part or the whole of a chemical structure of a phosphodiester bond of a nucleotide modified or substituted with an arbitrary substituent or substituted by an arbitrary atom, for example, There may be mentioned a nucleotide in which a phosphodiester bond is substituted by a phosphorothioate bond, a nucleotide in which a phosphodiester bond is substituted by a phosphorodithioate bond, and a core in which a phosphodiester bond is substituted by an alkylphosphonate bond. A nucleotide in which a nucleotide or a phosphodiester bond is substituted with an amino phosphate bond or the like.

The base-modified nucleotide may be any one as long as it is a part or the whole of a chemical structure of a base of a nucleotide, which is modified or substituted with an arbitrary substituent or substituted by an arbitrary atom, and may be, for example, a base. The oxygen atom in the group is substituted by a sulfur atom, the hydrogen atom is substituted by an alkyl group having 1 to 6 carbon atoms, a halogen group, and the methyl group is substituted by a hydrogen atom, a hydroxymethyl group or an alkyl group having 2 to 6 carbon atoms. The group is substituted by an alkyl group having 1 to 6 carbon atoms, an alkylene group having 1 to 6 carbon atoms, a pendant oxy group, a hydroxyl group or the like.

The nucleotide derivative may also be a nucleotide derivative modified with at least one of a nucleotide or a sugar moiety, a phosphodiester bond or a base, or a peptide, a protein, a sugar, or a linker. Other chemical substances such as lipids, phospholipids, morphine, folate, phenanthridine, anthraquinone, acridine, luciferin, rhodamine, coumarin, pigment, etc., specifically, 5 '-Polyamine addition nucleotide derivative, cholesterol addition nucleotide derivative, steroid addition nucleotide derivative, bile acid addition nucleotide derivative, vitamin addition nucleotide derivative, Cy5 Nucleotide derivatives such as addition nucleotide derivatives, Cy3 addition nucleotide derivatives, 6-FAM addition nucleotide derivatives, and biotin addition nucleotide derivatives can also be used in nucleic acids Other nucleotides or nucleotide derivatives form a bridging structure such as an alkylene structure, a peptide structure, a nucleotide structure, an ether structure, an ester structure, and a structure in which at least one of them is combined.

In the present specification, "complementary" means a relationship in which base pairing can be performed between two bases, for example, a relationship between adenine and thymine or uracil, and a relationship between guanine and cytosine, and relaxation. The hydrogen bond and the double-stranded region form a double helix structure as a whole.

In the present specification, the term "complementarity" does not mean that the two nucleotide sequences are completely complementary, and the nucleotide sequence may have 0 to 30%, 0 to 20% or 0 to 10% of mismatch base. The base, for example, means that the antisense strand complementary to the CFB mRNA may comprise one or a plurality of base substitutions in the base sequence which is completely complementary to the 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 position may be the 5' end or the 3' end of each sequence. Further, the term "complementarity" includes a case where a nucleotide sequence is a sequence in which one or a plurality of bases are added to and/or deleted from a nucleotide sequence which is completely complementary to another nucleotide sequence. For example, CFB mRNA and the antisense strand nucleic acid of the present invention may have one or two raised bases in the antisense strand and/or target CFB mRNA region due to addition and/or deletion of bases in the antisense strand.

The double-stranded nucleic acid used as a drug in the present invention is a nucleic acid comprising a base sequence complementary to a partial base sequence of CFB mRNA and/or a base sequence complementary to a base sequence of the nucleic acid. The nucleic acid may be composed of any nucleotide or a derivative thereof. The double-stranded nucleic acid of the present invention can form a double of at least 11 base pairs with a nucleic acid comprising a base sequence complementary to the target CFB mRNA sequence and a nucleic acid comprising a base sequence complementary to the base sequence of the nucleic acid. The strands may be of any length, and the length of the sequence capable of forming a double strand is usually 11 to 27 bases, preferably 15 to 25 bases, more preferably 15 to 23 bases, still more preferably 17 ~ 21 bases.

As the antisense strand of the nucleic acid complex of the present invention, a nucleic acid comprising a base sequence complementary to the target CFB mRNA sequence may be used, and 1 to 3 bases, preferably 1 to 2, of the nucleic acid may be used. The base, more preferably one base deleted, substituted or added.

As the nucleic acid which inhibits the expression of CFB, it is preferred to use a single-stranded nucleic acid comprising a base sequence complementary to the target CFB mRNA sequence and inhibiting the expression of CFB, or a base sequence comprising a complementarity to the target CFB mRNA sequence. A nucleic acid 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 CFB.

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

The double-stranded nucleic acid used as a drug in the present invention has a double nucleotide-added nucleotide or nucleotide derivative on the 3' side or the 5' side of the double-stranded region, and is It is called a protrusion (overhang). In the case of a protrusion, the nucleotide constituting the protrusion may be a ribonucleotide, a deoxyribonucleotide or a derivative thereof.

The double-stranded nucleic acid having a protruding portion is used for at least a single strand of the 3' end or the 5' end, and has a protrusion of 1 to 6 bases, usually 1 to 3 bases, preferably having 2 bases. Examples of the protrusions include those having a protrusion including dTdT (dT represents deoxythymidine) or UU (U represents uridine). It may have a protruding portion in only the antisense stock, only the justice stock, or the antisense stock and the justice stock. In the present invention, it is preferably used in the double stranded nucleic acid having the protruding portion of the antisense strand. Further, the antisense strand is selected from the group consisting of the ligated strands comprising 17 to 30 nucleotides in the double-stranded region and the ligated portion thereof, and is selected from the group consisting of Tables 1-1 to 1-3. The sequence of either of the target CFB mRNAs is sufficiently complementary. Further, as the double-stranded nucleic acid of the present invention, for example, a nucleic acid molecule which generates a double-stranded nucleic acid by the action of a ribonuclease such as Dicer (WO2005/089287) or a protrusion having no 3' end or 5' end may be used. A double-stranded nucleic acid with a smooth end, a double-stranded nucleic acid with only a prominent strand (US2012/0040459), and the like are formed.

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

The double-stranded nucleic acid constituting the nucleic acid complex used in the present invention may be a double-stranded RNA (dsRNA) in which RNA forms a double strand, a double-stranded DNA (dsDNA) in which DNA forms a double strand, or a double strand of RNA and DNA. Hybrid nucleic acid. Further, one or both of the double strands may be a chimeric nucleic acid of DNA and RNA. Preferred is double stranded RNA (dsRNA).

Preferably, the second nucleotide of the antisense strand of the nucleic acid complex of the present invention from the 5' end is preferably complementary to the second deoxyribonucleotide from the 3' end of the target CFB mRNA sequence, more preferably The 2nd to 7th nucleotides of the sense strand from the 5' end are completely complementary to the 2nd to 7th deoxyribonucleotides from the 3' end of the target CFB mRNA sequence, and thus preferably the antisense strand from 5 The 2nd to 11th nucleotides from the end are completely complementary to the 2nd to 11th deoxyribonucleotides from the 3' end of the target CFB mRNA sequence. Further, it is preferred that the 11th nucleotide from the 5' end of the antisense strand in the nucleic acid of the present invention is complementary to the 11th deoxyribonucleotide from the 3' end of the target CFB mRNA sequence, more preferably The 9th to 13th nucleotides of the antisense strand from the 5' end are completely complementary to the 9th to 13th deoxyribonucleotides from the 3' end of the target CFB mRNA sequence, and thus preferably the antisense strand. The 7th to 15th nucleotides from the 5' end are completely complementary to the 7th to 15th deoxyribonucleotides from the 3' end of the target CFB mRNA sequence.

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

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

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

RNA may be produced by enzymatic or partial/total organic synthesis, or may be introduced into a modified ribonucleotide by enzymatic or organic synthesis in vitro. In one aspect, each strand is prepared by chemical means. Methods for the chemical synthesis of RNA molecules are well known in the art [see Nucleic Acids Research, 1998, Vol. 32, p. 936-948]. In general, the double-stranded nucleic acid can be synthesized by using a solid phase oligonucleotide synthesis method (for example, see U.S. Patent No. 5,804,683; U.S. Patent No. 5,831,071; U.S. Patent No. 5,998,203; Patent No. 6, 117, 657; U.S. Patent No. 6, 353, 098; U.S. Patent No. 6,362, 323; U.S. Patent No. 6, 437, 117; U.S. Patent No. 6, 469, 158; Scaringe et al., U.S. Patent No. 6,111,086; U.S. Patent No. 6,008,400 No. specification; US Patent No. 6,111,086).

Regarding the single-stranded nucleic acid, it is synthesized by a solid phase phosphite method [Ref. Nucleic Acids Research, 1993, Vol. 30, p. 2435-2443], and deprotected, and applied to a NAP-5 column (Amersham Pharmacia Biotech, Desalting was carried out on Piscataway, NJ). The oligomer was a Amersham Source 15Q column with a linear gradient of 15 minutes - 1.0 cm. Purification was carried out using an ion exchange high performance liquid chromatography (IE-HPLC) using 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, buffer B was 100 mmol/L Tris pH 8.5 ( 1 mol/L NaCl). The samples were monitored at 260 nm, peaks corresponding to the full length oligonucleotide species were collected, pooled, desalted using a NAP-5 column and lyophilized.

The purity of each single-stranded nucleic acid was determined by capillary electrophoresis (CE) using a Beckman PACE 5000 (Beckman Coulter, Inc., Fullerton, Calif). The CE capillary has an inner diameter of 100 μm and contains ssDNA 100R Gel (Beckman-Coulter). Typically, about 0.6 nmole of oligonucleotide is injected into a capillary and electrophoresed at an electric field of 444 V/cm, which is detected by UV absorbance at 260 nm. Modified Tris-boric acid-7 mol/L-urea running buffer was purchased from Beckman-Coulter. A single-stranded nucleic acid having a purity of at least 90% when evaluated by CE was obtained for the experiment described below. Compound identity is based on the manufacturer's recommended operating instructions, using matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry using Voyager DE.TM. Biospectometry Workstation (Applied Biosystems, Foster City, Calif.) The method is verified. The relative molecular mass of the 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 containing 100 mmol/L potassium acetate and 30 mmol/L HEPES (pH 7.5). The complementary sense shares were mixed with the antisense strands in the same molar amount 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. The double-stranded nucleic acid was stored at -20 °C. The single-stranded nucleic acid was freeze-dried or stored at -80 ° C in water without nuclease.

As an oligonucleotide chain comprising a sense strand and an antisense strand in the present invention and comprising a double-stranded region of at least 11 base pairs, and having a strand length of 11 to 30 nucleotides in the antisense strand The double-stranded nucleic acid partially complementary to the target CFB mRNA sequence selected from the group described in Tables 1-1 to 1-3, may be used, comprising: an antisense selected from the group consisting of Tables 1-1 to 1-3. a double-stranded nucleic acid of a sequence in a group consisting of strands, or a group consisting of a group of justices selected from Tables M1-1 to M1-3 and Tables 1-1 to 1-3 described below a double-stranded nucleic acid of the sequence or a group consisting of a group of justices/antisense strands selected from Tables M1-1 to M1-3 and Tables 1-1 to 1-3 described below. A double-stranded nucleic acid for the sequence of the justice strand/antisense strand. That is, the specific examples of the double-stranded nucleic acid constituting the nucleic acid complex used in the present invention include the positive stocks and the counters in Tables M1-1 to M1-3 and Tables 1-1 to 1-3 described below. The double-stranded nucleic acid of the stock. Further, in Tables M1-1 to M1-3, N(M) represents 2'-O-methyl modified RNA, N(F) represents 2'-fluoro modified RNA, and ^ represents phosphorothioate. Further, the 5' terminal nucleotides of the antisense strand sequences described in Tables 1-1 to 1-3 and Tables M1-1 to M1-3 may be phosphorylated at the 5' end or may be unphosphorylated. Preferably, it is phosphorylated. The double-stranded nucleic acid comprising the sequence of the sense strand/antisense strands described in Tables 1-1 to 1-3 described below is preferably in the measurement of the knockdown activity, and the relative expression of CFB is 0.40. The following.

[Table 1] Table M1-1

[Table 2] Table M1-2

[Table 3] Table M1-3

[Table 4]

A method of producing the nucleic acid complex of the present invention will be described. Further, in the manufacturing method shown below, when the defined basis is changed under the conditions of the manufacturing method or is not suitable for the implementation of the manufacturing method, the introduction of a protecting group commonly used in organic synthetic chemistry can be used. And a removal method [for example, "Protective Groups in Organic Synthesis, third edition", TW Greene, John Wiley & Sons Inc. (1999), etc., to produce a target compound . Further, the order of the reaction steps such as introduction of a substituent may be changed as needed. The nucleic acid polymer represented by Formula 1 can also be synthesized by solid phase synthesis.

The nucleic acid polymer represented by Formula 1 can be synthesized by referring to a synthesis method which is a linker structure known as a nucleic acid complex. For the synthesis of the L1-benzene ring unit having S1 as a linker or the L2-benzene ring unit having S2 as a 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. Description. The L1-benzene ring unit or the 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. P1, P2, P3, P4, P5 and P6, and -CO-, -NH-, -O-, -S-, -O-CO-, -S-CO-, -NH-CO- of T1 and T2 The -CO-O-, -CO-S- or -CO-NH- linkage can be referred to, for example, in the fourth edition of Experimental Chemistry Lecture 19 "Synthesis of Organic Compounds I" Maruzen (1992), 4th Edition Experimental Chemistry Lecture 20 "Synthesis of Organic Compounds II" A method of bonding reaction described in Maruzen (1992) and the like, and a material suitable for forming the structure represented by Formula 2 is selected and appropriately synthesized. Further, a compound having a partial structure of Q1 and a compound having a partial structure of B1 are sequentially bonded to the benzene ring, whereby a partial structure of the L1-benzene ring unit can be produced. Further, a compound having L1 and Q2 as a partial structure is separately synthesized, and a compound having L1 and Q2 as a partial structure is bonded to a compound having a benzene ring, Q1 and B1 as partial structures having a partial structure of an L1-benzene ring unit, This makes it possible to produce an L1-benzene ring unit structure. Similarly to the L2-benzene ring unit, a compound having a partial structure of Q3 and a compound having a partial structure of B2 are sequentially bonded to the benzene ring, whereby a partial structure of the L2-benzene ring unit can be produced. Further, a compound having L2 and Q4 as a partial structure is separately synthesized, and a compound having L2 and Q4 as a partial structure is bonded to a compound having a partial structure of a L2-benzene ring unit having a benzene ring, Q3 and B2 as a partial structure, This makes it possible to produce an L2-benzene ring unit structure.

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

[化89]

Specific examples of the compound having a partial structure of B1 and a compound having a partial structure of B2 include diol, glutamic acid, aspartic acid, lysine, Tris (hydroxymethyl) aminomethane, and trishydroxyl Aminoaminomethane, iminodiacetic acid, 2-amino-1,3-propanediol, etc., are preferably glutamic acid, aspartic acid, lysine, and iminodiacetic acid. Specifically, B1 and B2 are preferably the following structures.

[化90]

Further, by synthesizing a compound having L1, Q2 and B1 as a partial structure, and bonding with a compound having Q1 and a benzene ring, an L1-benzene ring unit structure can be produced. The L2-benzene ring unit structure can also be produced by synthesizing a compound having L2, Q4 and B2 as a partial structure and bonding it with a compound having Q3 and a benzene ring. In the present invention, as a partial structure of [L1-T1-(Q2-P3) _q2 -] _p1 -B1-(P2-Q1) _q1 -P1- and as [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 the sugar ligand include L3-T1-Q2-COOH, L3-T1-(Q2-P3) _{q2 - 1} -Q2-NH ₂ and the like. Specific examples thereof include L3-O-alkylene-COOH having a carbon number of 1 to 12, or L3-alkyl-CO-NH having a carbon number of 1 to 12 and an alkylene group 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 which is L1 by deprotection. The substituent of the sugar ligand is not particularly limited as long as it is a substituent generally used in the field of saccharide chemistry, and is preferably an Ac group.

Regarding the synthesis of the L1-benzene ring unit having S1 as a linker or the L2-benzene ring unit having S2 as a 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. And replace the terminal amine group or terminal carboxyl group to form -CO-, -NH-, -O-, -S-, -O-CO-, -S-CO-, -NH-CO-, -CO A compound based on a -O-, -CO-S- or -CO-NH- bond, whereby it can be synthesized. Further, the sugar ligand of L1 is exemplified by mannose or N-ethinyl galactosamine, but may be changed to another sugar ligand.

For the synthesis of the X-benzene ring unit having S3 as a linker in the nucleic acid complex represented by Formula 1, for example, the nucleic acid complex represented by Formula 12 will be described as an example. The X-benzene ring unit in the nucleic acid complex represented by Formula 12 has a bond represented by P7 and P8 in addition to the bond of the oligonucleotide. -CO-, -NH-, -O-, -S-, -O-CO-, -S-CO-, -NH-CO-, -CO-O-, -CO-S- or P7 and P8 The -CO-NH- linkage can be referred to, for example, in the fourth edition of Experimental Chemistry Lecture 19 "Synthesis of Organic Compounds I" Maruzen (1992), 4th Edition Experimental Chemistry Lecture 20 "Synthesis of Organic Compounds II" Maruzen (1992) The method of the bonding reaction described in the above is appropriately selected by selecting a material suitable for forming the structure represented by Formula 12. Further, a compound having a partial structure of Q5 and a compound having a partial structure of B3 are sequentially bonded to the benzene ring, whereby a partial structure of the X-benzene ring unit can be produced.

Further synthesizing a compound having X and Q7 as a partial structure or a compound having X and Q6 as a partial structure, a compound having X and Q7 as a partial structure or a compound having X and Q6 as a partial structure and having a benzene ring and Q5 as a part The structure of the compound having a partial structure of the X-benzene ring unit is bonded, and the B3 moiety is constructed, whereby the X-benzene ring unit structure can be produced. Specifically, the case where the terminal having the benzene ring and Q5 as a partial structure of the compound having a partial structure of the X-benzene ring unit has an azide group is exemplified by the terminal end as disclosed in the examples. The bonded functionalized oligonucleotide is reacted to form a triazole ring to form a B3 moiety, whereby an X-benzene ring unit structure can be produced.

The compound having Q5 as a partial structure, a compound having Q6 as a partial structure, and a compound having Q7 as a partial structure may be exemplified by 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, or 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 produced separately, preferably after synthesizing the L1-benzene ring unit structure and the L2-benzene ring unit structure, and then 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 Formula 8 to Formula 10 which is a synthetic intermediate is obtained. Equation 8:

[化91]

(In the 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), -CO- R4, or -CO-B4-[(P9-Q8) _q7 -T3-L3] _p3 , P9 and T3 are independent or absent, respectively, or are -CO-, -NH-, -O-, -S-, -O-CO-, -S-CO-, -NH-CO-, -CO-O-, -CO-S- or -CO-NH-, Q8 either absent or substituted or unsubstituted The 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 a formula 8-1 In any of the structures shown, the black dots at the ends in each structure are respectively a bond with a carbonyl group or a P9, and m7, m8, m9, and m10 are each independently an integer of 0 to 10, and Equation 8-1:

[化92]

P3 is an integer of 1, 2 or 3, q7 is an integer from 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 from 1 to 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 is either absent or -CO-, -NH-, -O-, -S-, -O-CO-, -S-CO-, -NH-CO-, -CO-O-, -CO-S- or -CO-NH-, Q9 and Q10 is independently or absent, 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 of 0 to 99 B5 is any structure represented by the following formula 8-2, and the curve indicates a bonding bond with Q9 and Q10, respectively, and Equation 8-2:

[化93]

In the formula 8-2, the substituent in the group having a triazole ring is any one of the nitrogen atom at the 1-position and the 3-position of the triazole ring. Q8 is an integer of 0 to 10, X1 is a hydrogen atom or a solid phase carrier, and R4 is an amine selected from the group consisting of a third butoxycarbonyl group, a benzyloxycarbonyl group, a 9-fluorenylmethoxycarbonyl group or an unsubstituted amine. a C 2-10 alkyl group substituted with one or two substituents in the group consisting of a carboxyl group, a carboxyl group, a maleimide group, and an aralkoxycarbonyl group)

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

[化94]

(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, respectively, or are -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 from 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-(CH2) _m11' - NH- and -NH-CO-(CH ₂ ) _m12' -NH, m11' and m12' are each independently an integer of from 1 to 10, and R3' is a hydrogen atom, a third butoxycarbonyl group, a benzyloxycarbonyl group, and 9 - mercaptomethoxycarbonyl, -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 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 absent, or are substituted or unsubstituted carbon number 1 to 12 alkyl or -(CH ₂ CH ₂ O) _n3' -CH ₂ CH ₂ -, n3' is an integer from 0 to 99, and B5' is any structure represented by the following formula 9-1, and the curve indicates a bonding bond with Q9' and Q10', respectively. 1:

[化95]

In the formula 9-1, the substituent in the group having a triazole ring is any one of the nitrogen atom 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 carrier, and R4' is selected from a third butoxycarbonyl group, a benzyloxycarbonyl group, a 9-fluorenylmethoxycarbonyl group or not a C 2-10 alkyl group substituted with one or two substituents in the group consisting of a substituted amine group, a carboxyl group, a maleimide group, and an aralkoxycarbonyl group)

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

[化96]

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 each independently. , or absent, 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 alkyl 1 to 12 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, and m9 and m10 are each independently 1 An integer of ～10, q10 is an integer of 0 to 10, and 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 independently or absent, 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 of 0 to 99 , B6 is the following formula For any structure represented by 10-1, the curve indicates the bond with Q13 and Q14, respectively, and Equation 10-1:

[化97]

In the formula 10-1, the substituent in the group having a triazole ring is any one of the nitrogen atom at the 1-position and the 3-position of the triazole ring. Q11 is an integer of 0 to 10, X2 is a hydrogen atom or a solid phase carrier, and R10 is an amine selected from the group consisting of a third butoxycarbonyl group, a benzyloxycarbonyl group, a 9-fluorenylmethoxycarbonyl group or an unsubstituted amine. a C 2-10 alkyl group substituted with one or two substituents in the group consisting of a carboxyl group, a carboxyl group, a maleimide group, and an aralkoxycarbonyl group)

Hereinafter, an example of a manufacturing method will be exemplified in the present invention. In the description of the production method 1 to the production method 17 in the following, in the compounds represented by the formulas 1 to 12 in the nucleic acid derivative of the present invention, the symbols indicating the radicals have the same symbols, but The two should be understood separately, and the present invention is not limited to the description of the basis of the methods described in the manufacturing method 1 to the manufacturing method 12. Further, in the nucleic acid derivative of the present invention, X representing the oligonucleotide is described as -O-X in Production Method 1 to Production Method 17.

Production Method 1 Nucleic acid derivative in the present invention As a method for producing a compound having a partial structure represented by the formula (I'), a production method thereof can be exemplified.

[化98]

(wherein 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-lanthanide unit, and R' represents R Each of the hydroxyl groups of the sugar ligand is protected by a protecting group such as an acetamyl group which can be deprotected by a base, and the polymer represents a solid phase carrier, and Q' is -CO-)

Step 1 Compound (IB) can be obtained by reacting compound (IA) with p,p'-dimethoxytriphenylchloromethane in a solvent such as pyridine, if necessary in the presence of a cosolvent at 0 ° C and 100 ° C. Manufactured by reacting at a temperature of between 5 minutes and 100 hours. Examples of the cosolvent 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., which may be used singly or in combination. .

Step 2 The compound (IC) can be reacted at a temperature between room temperature and 200 ° C in the presence of from 1 to 1000 equivalents of a secondary amine in the absence of a solvent or in a solvent. Manufactured from 5 minutes to 100 hours. Examples of the 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., may be used singly or in combination. Examples of the secondary amine include diethylamine, piperidine, and the like.

Step 3 The compound (1-E) can be used in an amount of from 1 to 30 equivalents of a base, a condensing agent, and optionally 0.01 to 30 equivalents, in a solvent-free condition or in a solvent. In the presence of an additive, the reaction is carried out at a temperature between room temperature and 200 ° C for 5 minutes to 100 hours. As the solvent, those exemplified in the step 2 can be mentioned. Examples of the base include cesium carbonate, potassium carbonate, potassium hydroxide, sodium hydroxide, sodium methoxide, potassium t-butoxide, triethylamine, diisopropylethylamine, N-methylporphyrin, and pyridine. 1,8-diazabicyclo[5.4.0]-7-undecene (DBU), N,N-dimethyl-4-aminopyridine (DMAP), and the like. Examples of the condensing agent include 1,3-dicyclohexanecarbodiimide (DCC) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide salt. Acid salt (EDC), carbonyl diimidazole, benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate, hexafluorophosphoric acid (benzotriazol-1-yloxy)tripyrrole Iridinyl hydrazine, O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hydride (HATU), hexafluorophosphate O-(benzene) And triazol-1-yl)-N,N,N',N'-tetramethyluronium (HBTU), 2-chloro-1-methylpyridinium iodide and the like. Examples of the additive include 1-hydroxybenzotriazole (HOBt) and 4-dimethylaminopyridine (DMAP). The compound (I-D) can be obtained by a known method (for example, refer to "American Chemical Society", Vol. 136, p. 16958, (2014)) or a method therefor.

Step 4 Compound (IF) can be reacted in the presence of 1 to 30 equivalents of a base in the presence of 1 to 30 equivalents of a base in a solvent at a temperature between room temperature and 200 ° C for 5 minutes to 100 hours. Manufacturing.

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

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

Step 5 Compound (IG) can be obtained by using a solid phase carrier which is aminated with a terminal (IF) and a terminal in a solvent-free condition or in a solvent, in an amount of from 1 to 30 equivalents of a base, a condensing agent, and optionally 0.01 to In the presence of 30 equivalents of the additive, the reaction is carried out at a temperature between room temperature and 200 ° C for 5 minutes to 100 hours, and then reacted in an acetic anhydride/pyridine solution at a temperature between room temperature and 200 ° C for 5 minutes. Manufactured in 100 hours. As the solvent, those exemplified in the step 2 can be mentioned. Examples of the base, the condensing agent, and the additive include those exemplified in the step 3. Examples of the aminated organic carrier include long-chain alkylamine fine glass (LCAA-CPG), and the like, which can be obtained as a commercial product.

Step 6 A nucleic acid complex having a glycoside-lanthanide-branched unit represented by the formula (I') can be produced by using a compound (IG) by a known oligonucleotide chemical synthesis method. After the nucleotide chain is elongated, the self-solid phase is detached, and the protective group is deprotected and purified.

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

The detachment and deprotection from the solid phase can be carried out by alkali treatment for 10 seconds to 72 hours at a temperature between -80 ° C and 200 ° C in a solvent or solvent-free condition after chemical synthesis of the oligonucleotide. get on.

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

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

The purification of the oligonucleotide can be carried out 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, preferably 95% or more.

Further, in the above step 3, if necessary, the compound (ID) may be divided into two units and condensed with the compound (IC) 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 The compound (IC) is condensed with CH ₃ CH ₂ -O-CO-Q4'-CO-OH (Q4' has the same meaning as described above) by the same method as in the step 3, and the ethyl ester of the obtained compound is A solvent such as ethanol or water is hydrolyzed with a base such as lithium hydroxide, and further condensed with R'-NH ₂ (R' has the same meaning as described above) to obtain a target compound. Further, CH ₃ CH ₂ -O-CO-Q4'-CO-OH (Q4' has the same meaning as described above) and R'-NH ₂ (R' has the same meaning as defined above) can be known by a known method (for example, refer to "USA" Chemical Society, Vol. 136, p. 16958 (2014)) or obtained 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 the compound in the case where Q is not -CO- may be modified by appropriately changing the structure of Q, by the same method as described above, a known method, or a combination of the methods, Prepared by appropriately changing the reaction conditions.

Production Method 2 Nucleic Acid Derivative of the Present Invention As a method for producing a compound having a partial structure represented by the formula (II'), a production method thereof can be exemplified.

[化99]

(wherein, DMTr, R, R', X, Q' and the polymer have the same meanings as above. TBDMS means a third butyl dimethyl fluorenyl group, and Fmoc means a 9-fluorenyl methoxycarbonyl group)

Step 7 Compound (II-A) can be obtained by using compound (IA), tert-butyldimethylchloromethane and dimethylaminopyridine in a solvent such as N,N-dimethylformamide (DMF). It is preferably produced by reacting in the presence of 2 equivalents of base at a temperature between 0 ° C and 100 ° C for 5 minutes to 100 hours. Examples of the base include those exemplified in the third step of the production method 1.

Step 8 Compound (II-B) can be produced under the same conditions as in Step 1 of Production Method 1, using Compound (II-A).

Step 9 Compound (II-C) can be reacted 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. As the solvent, those exemplified in the step 2 can be mentioned.

Step 10 Compound (II-D) can be produced under the same conditions as in Step 2 of Production Method 1, using Compound (II-C).

Step 11 Compound (II-E) can be produced under the same conditions as in Step 3 of Production Method 1, using Compound (II-D) and Compound (I-D).

Steps 12 to 14 Compound (II') can be produced under the same conditions as in Step 4 to Step 6 of Production Method 1, using Compound (II-E).

Further, 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 Ethyl ester of the obtained compound is obtained by condensing the compound (II-C) with CH ₃ CH ₂ -O-CO-Q4'-CO-OH (Q4' has the same meaning as described above) by the same method as in the step 11 After hydrolysis by a base such as lithium hydroxide or the like in a solvent such as ethanol or water, and further condensing with R'-NH ₂ (R' has the same meaning as described above), the target compound can be obtained. Further, CH ₃ CH ₂ -O-CO-Q4'-CO-OH (Q4' has the same meaning as described above) and R'-NH ₂ (R' has the same meaning as defined above) can be known by a known method (for example, refer to "USA" Chemical Society, Vol. 136, p. 16958 (2014)) or obtained according to its method.

The case where Q is -CO- is exemplified, but the compound in the case where Q is not -CO- may be modified by appropriately changing the structure of Q, by the same method as described above, a known method, or a combination of the methods, Prepared by appropriately changing the reaction conditions.

Production Method 3 Nucleic acid derivative in the present invention As a method for producing a compound having a partial structure represented by the formula (III'), a production method thereof can be exemplified.

[化100]

(wherein, DMTr, Fmoc, R, R', Q', X and the polymer have the same meaning as above)

Compound (III') can be produced under the same conditions as in Step 1 to Step 6 of Production Method 1, using Compound (III-A). Further, the compound (III-A) can be obtained as a commercial product.

Step 15 Compound (III-B) can be produced under the same conditions as in Step 1 of Production Method 1, using Compound (III-A). Compound (III-A) can be purchased as a commercial product.

Step 16 Compound (III-C) can be produced under the same conditions as in Step 2 of Production Method 1, using Compound (III-B).

Step 17 Compound (III-E) can be produced under the same conditions as in Step 3 of Production Method 1, using Compound (III-C).

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

Further, in the above step 17, 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 Ethyl ester of the obtained compound is obtained by condensing the compound (III-C) with CH ₃ CH ₂ -O-CO-Q4'-CO-OH (Q4' has the same meaning as described above) by the same method as in the step After hydrolysis by a base such as lithium hydroxide or the like in a solvent such as ethanol or water, and further condensing with R'-NH ₂ (R' has the same meaning as described above), the target compound can be obtained. Further, CH ₃ CH ₂ -O-CO-Q4'-CO-OH (Q4' has the same meaning as described above) and R'-NH ₂ (R' has the same meaning as defined above) can be known by a known method (for example, refer to "USA" Chemical Society, Vol. 136, p. 16958 (2014)) or obtained according to its method.

The case where Q is -CO- is exemplified, but the compound in the case where Q is not -CO- may be modified by appropriately changing the structure of Q, by the same method as described above, a known method, or a combination of the methods, Prepared by appropriately changing the reaction conditions.

Production Method 4 Nucleic Acid Derivative of the Present Invention As a method for producing a compound having a partial structure represented by the formula (IV'), a production method thereof can be exemplified.

[化101]

(wherein, DMTr, Fmoc, R, R', Q', X and the polymer have the same meaning as above)

Compound (IV') can be produced under the same conditions as in Step 1 to Step 6 of Production Method 1, using Compound (IV-A). Further, the compound (IV-A) can be obtained as a commercial product.

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

The case where Q is -CO- is exemplified, but the compound in the case where Q is not -CO- may be modified by appropriately changing the structure of Q, by the same method as described above, a known method, or a combination of the methods, Prepared by appropriately changing the reaction conditions.

Production Method 5 Nucleic Acid Derivative of the Present Invention As a method for producing a compound having a partial structure represented by the formula (V'), a production method thereof can be exemplified.

[化102]

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

The compound (V') can be produced under the same conditions as in the steps 1 to 7 of the production method 2, using the compound (IV-A). Further, the compound (IV-A) can be obtained as a commercial 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 The compound (VD) is condensed with CH ₃ CH ₂ -O-CO-Q4'-CO-OH (Q4' has the same meaning as described above) by the same method as in the step 31, and the ethyl ester of the obtained compound is A solvent such as ethanol or water is hydrolyzed with a base such as lithium hydroxide, and further condensed with R'-NH ₂ (R' has the same meaning as described above) to obtain a target compound. Further, CH ₃ CH ₂ -O-CO-Q4'-CO-OH (Q4' has the same meaning as described above) and R'-NH ₂ (R' has the same meaning as defined above) can be known by a known method (for example, refer to "USA" Chemical Society, Vol. 136, p. 16958 (2014)) or obtained according to its method.

The case where Q is -CO- is exemplified, but in the case where Q is not -CO-, the structure of Q-OH may be appropriately changed by the same method, known method or methods as described above. Prepared by combining and appropriately changing the reaction conditions.

Production Method 6 Hereinafter, a method for producing the nucleic acid complex of the present invention in which a sugar ligand-lanthanide-branched unit is bonded to the 5' end of the oligonucleotide is exemplified.

[化103] (wherein R, R', Q', DMTr and X have the same meaning as above)

Step 35 Compound (IH) can be obtained by reacting compound (II-E) with 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphonium amide in a solvent-free condition or The solvent is produced by reacting at room temperature with a temperature of 200 ° C for 5 minutes to 100 hours in the presence of a base and a reaction accelerator. The solvent is exemplified in the step 2 of the production method 1. Examples of the base include those exemplified in the third step of the production method 1. Examples of the reaction accelerator include 1H-tetrazole, 4,5-dicyanoimidazole, 5-ethylthiotetrazole, and 5-benzylthiotetrazole, which are commercially available.

Step 36: The oligonucleotide chain is elongated, and finally, the compound (IH) is used, and after the 5' end of the oligonucleotide is modified by the glycoside-lanthanide-branched unit, the self-solid phase is detached and the protective group is deprotected. And refining, whereby the compound (I'') can be produced. Here, the separation from the solid phase, the deprotection of the protecting group, and the purification can be carried out in the same manner as in the seventh step of the production method 1.

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

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

[化104]

(wherein R, R', Q', DMTr and X have the same meaning as above)

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

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

[化105]

(wherein R, R', Q', DMTr and X have the same meaning as above)

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

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

[化106]

(wherein R, R', Q', DMTr and X have the same meaning as above)

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

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

[107]

(wherein R, R', Q', DMTr and X have the same meaning as above)

The manufacturing method 11 is to dissolve the water in the water or the appropriate one of the 3' terminal ' or 5' terminus of the double stranded nucleic acid having the glycoside-lanthanide-branched unit and the antisense strand constituting the double-stranded nucleic acid. The buffer solution is mixed, whereby a nucleic acid complex having a double-stranded nucleic acid can be obtained. Examples of the buffer solution include an acetate buffer solution, a Tris buffer solution, a citrate buffer solution, a phosphate buffer solution, and water, and these may be used singly or in combination.

As a mixture ratio of the justice stock and the antisense stock, the antisense strand is preferably 0.5 to 2 equivalents, more preferably 0.9 to 1.1 equivalents, still more preferably 0.95 equivalents to 1.05 equivalents, per equivalent of the justice strands.

Further, after the justice strand is mixed with the antisense strand, the annealing treatment can be appropriately performed. The annealing treatment can be carried out by heating the mixture of the sense strand and the antisense strand to preferably 50 to 100 ° C, more preferably 60 to 100 ° C, still more preferably 80 to 100 ° C, and then slowly cooling to room temperature.

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

Production Method 12 Nucleic Acid Derivative of the Present Invention As a method for producing a compound having a partial structure represented by the formula (VI'), a production method thereof can be exemplified.

[化108]

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

Step 45 Compound (VI-B) can be produced under the same conditions as in Step 1 of Production Method 1, using Compound (VI-A). The 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 The method is obtained.

Step 46 Compound (VI-C) can be produced under the same conditions as in Step 2 of Production Method 1, using Compound (VI-B).

Step 47 Compound (VI-D) can be produced under the same conditions as in Step 3 of Production Method 1, using Compound (VI-C).

Step 48 Compound (VI-E) can be produced under the same conditions as in Step 2 of Production Method 1, using Compound (VI-D).

Step 49 Compound (VI-G) can be produced under the same conditions as in Step 3 of Production Method 1, using Compound (VI-E) and Compound (VI-F).

Step 50 Compound (VI-H) can be produced under the same conditions as in Step 9 of Production Method 2, using Compound (VI-G).

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

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

Production Method 13 A sugar ligand-lanthanide unit of the formula 2 in which P1 and P4 are -NH-CO-, -O-CO- or -S-CO- can be produced by the following method.

[化109]

(wherein 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 the ratio q2 An integer of 1, q4' represents an integer less than q4, and P1' and P4' independently represent -NH-CO-, -O-CO- or -S-CO-, and Z represents H, OH, NH ₂ And SH, a chlorine atom, a bromine atom, an iodine atom, a methanesulfonyloxy group, a p-toluenesulfonyloxy group or a carboxylic acid, and 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)

[110]

M1, m2, m3 or m4 each independently represent an integer from 0 to 10.

Step 54 Compound (VII-C) can be obtained by adding compound (VII-A) and compound (VII-B) to a solvent such as tetrahydrofuran, adding a triphenylphosphine polymer carrier, cooling under ice bath, and azo It is produced by reacting a solution of diisopropyl formate in toluene. As the solvent, those exemplified in the step 2 of the production step 1 can be mentioned. The compound (VII-A) can be obtained as a commercial product.

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

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

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

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

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

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

Steps 61 to 63 The compound (VII') can be produced under the same conditions as in the step 3 of the production step 1 using the compound (VII-O), the compound (VII-P) and the compound (VII-Q). Further, by repeating the DP step and the step 61, the compound (VII') having a desired q3 value can be produced.

The step DP can be produced by appropriately adopting a method commonly used in organic synthetic chemistry [for example, "Protective Groups in Organic Synthesis", 3rd edition, 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 the compound (VII-Q) can be obtained as a commercial product, or by "Experimental Chemistry Lecture 4th Edition Organic Synthesis, p.258, Maruzen (1992)", "March Advanced Organic Chemistry: Reaction The combination of the methods described in the "March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure", 7th Edition, or the method according thereto.

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

[111]

(wherein, 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 indicate a suitable protecting group, LC represents a sugar ligand-lanthanide unit, and E represents a carboxylic acid or a maleimide.

Step 64 Compound (VIII-C) can be produced under the same conditions as in Step 3 of Production Step 1, using Compound (VIII-A) and Compound (VIII-B). Further, by repeating the DP step and the step 64, the compound (VIII-C) having a desired q5'' value can be produced. Compound (VIII-B) can be obtained as a commercial product, or by "Experimental Chemistry Lecture 4th Edition Organic Synthesis, p.258, Maruzen (1992)", "March Advanced Organic Chemistry: Reaction, Mechanism and Structure No. 7 A combination of methods described in the "Brand" or obtained according to the method thereof.

Step 65 Compound (VIII') can be produced under the same conditions as in Step 3 of Production Step 1, using Compound (VIII-C) and Compound (VIII-D). Compound (VIII-D) can be obtained as a commercial product, or by "Experimental Chemistry Lecture 4th Edition Organic Synthesis, p.258, Maruzen (1992)", "March Advanced Organic Chemistry: Reaction, Mechanism and Structure No. 7 A combination of methods described in the "Brand" or obtained according to the method thereof.

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

Production Method 15 A sugar ligand-lanthanide unit in which P1 and P4 are -O- in Formula 2 can be produced by the following method.

[化112]

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

[化113]

M1, m2, m3 and m4 have the same meanings as described above.

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

Step 67 Compound (IX-E) can be obtained by dissolving compound (IX-C) and compound (IX-D) in a solvent such as N,N'-dimethylformamide, and adding a base such as potassium hydrogencarbonate. It is produced by reacting at room temperature to 200 ° C for 5 minutes to 100 hours. As the solvent, those exemplified in the step 2 of the production step 1 can be mentioned. As the base, those exemplified in the step 3 of the production step 1 can be mentioned. Compound (IX-A) is available as a commercial product.

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

Step 69 Compound (IX-I) can be produced under the same conditions as in Step 3 of Production Step 1, using Compound (IX-G) and Compound (IX-H). Further, by repeating the DP step and the step 69, the compound (VII-J) having a desired q1 value can be produced.

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

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

Step 72-74 Compound (IX') can be used in the same conditions as in the step 3 of the production step 1 using the compound (IX-M), the compound (IX-N), the compound (IX-O) and the compound (IX-P). Made under. Further, by repeating the DP step and the step 72, the compound (IX') having a desired q3 value can be produced.

The step DP can be produced by appropriately adopting a method commonly used in organic synthetic chemistry [for example, "Protective Groups in Organic Synthesis", 3rd edition, 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) can be obtained as commercially available products, or by "Experimental Chemistry Lecture 4th Edition Organic Synthesis, p.258, Maruzen (1992) The combination of the methods described in "March Advanced Organic Chemistry: Reaction, Mechanism and Structure, 7th Edition" or according to the method thereof.

Production Method 16 As a method for producing the nucleic acid complex of Formulas 1 to 8, the following methods can also be employed.

[化114]

(wherein LC, Q5, Q6, Q7, P7, P8, q5', q6 and X have the same meaning as above)

Step 75 Compound (X-B) can be produced by reacting compound (XIII'') with 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 dimethyl hydrazine. These may be used singly or in combination. Compound (VIII') can be obtained by employing Production Method 14.

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

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

Production Method 17 As a method for producing the nucleic acid complex of Formulas 1 to 8, the following methods can also be employed.

[化115]

(wherein LC, Q5, Q6, Q7, P7, P8, q5', q6 and X have the same meaning as above)

Step 77 Compound (XI-') can be used as a compound (VIII'') by a known method (for example, "Bioconjugated Chemistry", Vol. 26, pp. 1451-1455, 2015) or according to the same. The method is obtained. The compound (VIII''') is obtained by employing the production method 14.

Step 78 Compound (XI') can be used as the compound (XI-A) and the compound (XI-B) by a known method (for example, "Bioconjugate Chemistry", Vol. 26, pp. 1451-1455, 2015. Method) or obtained according to the method thereof. The compound (XI-B) can be obtained by the method described in "Bioconjugate Chemistry", Vol. 26, pp. 1451-1455, 2015, or the method according thereto.

Step 79 Further, as another method, the compound (XI') can be synthesized from a compound by a known method (for example, refer to "Bioconjugate Chemistry", Vol. 22, pp. 1723-1728, 2011) or a method according thereto. XI-A) is 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, and acetate, maleate, fumarate, citrate, methanesulfonate, and the like. The organic salt; for example, an alkali metal salt such as a sodium salt or a potassium salt; an alkaline earth metal salt such as a magnesium salt or a calcium salt; an aluminum salt or a zinc salt; and an ammonium salt, for example, ammonium or tetra Examples of the organic amine addition salt include an addition salt of a porphyrin or a piperidine, and examples of the amino acid addition salt include, for example, an amino acid, a glycine, a phenylalanine, and the like. Addition salt.

In the case of preparing a salt of the nucleic acid complex of the present specification, the composite system may be directly purified when it is obtained in the form of a desired salt, and the complex may be dissolved or suspended when obtained in a free form. A suitable solvent may be added to the corresponding acid or base to carry out separation and purification. Further, 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 few equivalents to a large excess of acid, base and/or salt (chlorination) is added. For example, an inorganic salt such as sodium or ammonium chloride may be isolated or purified.

Stereoisomers, tautomers and the like of geometric isomers, optical isomers and the like may also be present in the nucleic acid complex of the present specification, and all possible isomers and mixtures thereof are included in the present invention.

Further, the nucleic acid complex of the present specification may be present in the form of an adduct with water or various solvents, and such adducts are also included in the present invention.

Further, the nucleic acid complex of the present invention also includes a part or all of atoms in the molecule which are substituted with atoms (isotopes) having different mass numbers (for example, a ruthenium atom or the like).

The pharmaceutical composition of the present invention comprises the nucleic acid complex represented by Formula 1. The nucleic acid complex of the present invention is recognized by a target cell and introduced into a cell by a glycoside having L1 and L2. The nucleic acid complex of the present invention can be used for the treatment of a target gene-related disease by administering a mammal to a target to reduce or stop the expression of a target gene in an organism and inhibit the expression of a target gene. In the case where the nucleic acid complex of the present invention is used as a therapeutic or prophylactic agent, it is preferred to use a route of administration which is most effective for the treatment, and is not particularly limited, and examples thereof include intravenous administration, Subcutaneous administration, intramuscular administration, etc., preferably subcutaneous administration. The amount of administration may vary depending on the condition, age, administration route, and the like of the administration target, and for example, the administration amount of the double-stranded oligonucleotide may be 0.1 μg to 1000 mg per day. Jia is administered in such a manner that the amount is 1 to 100 mg per day.

Examples of the preparation suitable for intravenous administration or intramuscular administration include an injection, and the prepared preparation can be directly used in the form of, for example, an injection, or can be removed from the solution by, for example, filtration, centrifugation or the like. After the solvent is used, the solution is freeze-dried and then used, and/or a liquid agent to which an excipient such as mannitol, lactose, trehalose, maltose or glycine is added is freeze-dried and then used. . In the case of an injection, it is preferred to prepare an injection by mixing a liquid or a solvent or a lyophilized composition such as water, an acid, a base, various buffers, physiological saline or an amino acid infusion. Further, an injection such as citric acid, ascorbic acid, cysteine or EDTA (ethylenediamine tetraacetic acid) or an isotonic agent such as glycerin, glucose or sodium chloride may be added to prepare an injection. . Further, a cryopreservation agent such as glycerin may be added and stored frozen.

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

The method of introducing the nucleic acid complex of the present invention into a mammalian cell in vivo may be carried out according to a known transfection procedure which can be carried out in vivo. By administering the composition of the present invention intravenously to a mammal including a human, it can be delivered to the liver, and the double-stranded nucleic acid in the composition of the present invention can be introduced into the liver or liver cells.

When the double-stranded nucleic acid in the composition of the present invention is introduced into the liver or liver cells, the expression of the CFB gene in the hepatocytes can be lowered, and the damage mediated by the abnormality of the second pathway of complement can be treated or prevented. Examples of CFB-related diseases include atypical hemolytic uremic syndrome (aHUS), paroxysmal nocturnal hemoglobinuria (PNH), age-related macular degeneration (AMD), and membranous proliferative glomerulonephritis (MPGN). , C3 nephritis, membranous nephropathy, asthma, autoimmune diseases (for example, systemic lupus erythematosus (SLE), psoriasis, optic neuromyelitis, myasthenia gravis, etc.). The subject to be administered is a mammal, preferably a human.

The amount of administration may vary depending on the condition, age, administration route, and the like of the administration target, and may be administered, for example, in such a manner that the dose of the double-stranded nucleic acid is 0.1 μg to 1000 mg per day. The dosage is 1 to 100 mg on the 1st.

Further, the present invention provides a nucleic acid complex for use in the treatment of a disease; a pharmaceutical composition for treating a disease; a use of a nucleic acid complex for treating a disease; and a use of a nucleic acid complex in the manufacture of a medicine for treating a disease; A nucleic acid complex for the manufacture of a medicament for the treatment of a disease; and a method for treating or preventing a disease in which an effective amount of the nucleic acid complex is administered to a subject in need thereof. [Examples]

Next, the present invention will be specifically described by way of Reference Examples, Examples and Test Examples. However, the present invention is not limited to the examples and test examples. Further, the proton nuclear magnetic resonance spectrum ( ¹ H NMR) shown in the examples and the reference examples was measured at 270 MHz, 300 MHz or 400 MHz, and the exchange protons were not clearly observed depending on the compound and the measurement conditions. The situation. Moreover, the multiplicity of signals is expressed by the usual use, and the br is broad, which means that the appearance is a wide signal. UPLC analysis uses the following conditions. Mobile phase A: aqueous solution containing 0.1% formic acid, B: gradient of acetonitrile solution: linear gradient of mobile phase B from 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 example compounds are disclosed in Tables X-1 to X-4.

[Table 5] Table X-1: Compounds A1 to A7

[Table 6] Table X-1

[Table 7] Table X-2: Compounds B1 to B3

[Table 8] Table X-3: Compounds C1 to C17

[Table 9] Table X-3

[Table 10] Table X-3

[Table 11] Table X-3

[Table 12] Table X-3

[Table 13] Table X-4: Compounds D1 to D11

[Table 14] Table X-4

Reference example 1

[116]

Synthesis of Compound RE1-2 Compound RE1-1 (0.8755 g, 1.9567 mmol) synthesized by the method described in American Chemical Society, Vol. 136, No. 16958-16961, 2014, was dissolved in tetrahydrofuran (10). (mL), 1,3-dicyclohexanecarbodiimide (DCC, 0.4247 g, 2.0584 mmol) and N-hydroxysulphonide (0.2412 g, 2.0958 mmol) were stirred at room temperature. . 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-aminoethyl maleimide hydrobromide (0.6479 g, 2.5491 mmol) and diiso was added. After propylethylamine (1.7 mL, 9.7835 mmol), it was stirred at room temperature. The solvent of the reaction liquid was distilled off under reduced pressure, and it was dissolved by reverse phase column chromatography (water/methanol = 80/20), whereby compound RE1-2 (0.8502 g, yield 76%) was obtained. ). 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. Made 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, 3-Tetramethyluronium (manufactured by Wako Pure Chemical Industries, Ltd., 1.6437 g, 4.3229 mmol), stirred at room temperature overnight. After adding water to the reaction mixture and extracting twice with chloroform, 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 The compound RE1-3 (1.2654 g, 2.1460 mmol) synthesized in the synthesis of the compound RE1-4 was dissolved in dichloromethane (15 mL), trifluoroacetic acid (4 mL) was added, and stirred at room temperature. A trip. After adding water to the reaction mixture and extracting 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 mixture was dissolved by reverse-column 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

[化117]

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

Reference Example 2 Step 2 The compound RE2-2 (1.07 g, 1.699 mmol) synthesized in Step 1 of Reference Example 2 was dissolved in N,N-dimethylformamide (10 mL), and the mixture was added at room temperature. Pyridine (0.336 mL, 3.40 mmol) and stirred for 3 hours. After adding water to the reaction mixture and extracting 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 ethylamine hexane 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

[化118]

Reference Example 3, step 1 dimethyl 5-hydroxyisophthalate (compound RE3-1, manufactured by Wako Pure Chemical Industries, Ltd., 5.0443 g, 24 mmol) was dissolved in tetrahydrofuran (manufactured by Wako Pure Chemical Industries, Ltd., 25 mL). 2-(Tertibutoxycarbonylamino)-1-ethanol (manufactured by Tokyo Chemical Industry Co., Ltd., 4.0343 g, 25.03 mmol), and a triphenylphosphine polymer carrier (manufactured by Sigma-Aldrich Co., Ltd., 6.61 g, 25.2) After mmol), a 40% toluene solution of diisopropyl azodicarboxylate (DIAD) (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. The reaction liquid was filtered, and the filtrate was distilled off under reduced pressure, and then the residue was purified by an amine-purified column chromatography (hexane/ethyl acetate = 95/5 to 80/20) to obtain a compound RE3. -2 (5.3071 g, yield 63%). ESI-MS m/z: 254 (M + H) ⁺ , detected as de Boc

Reference Example 3 Step 2 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 a 2 mol/L sodium hydroxide aqueous solution was added under cooling in an ice bath ( Manufactured by Wako Pure Chemical Industries, Inc., 13 mL), and stirred at room temperature for 4 hours. The reaction solution was cooled in an ice bath, and a 10% aqueous citric acid solution was added thereto, and the mixture was extracted with ethyl acetate. The organic layer was washed with 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) ^-

Reference Example 3 Step 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 2-(1H) - benzotriazol-1-yl)-1,1,3,3-tetramethyluronium (4.5168 g, 11.88 mmol), stirred at room temperature for 4 hours. The reaction mixture was cooled in an ice-bath, and a 10% aqueous citric acid solution was added, and the mixture was extracted with chloroform, and the organic layer was washed with saturated aqueous sodium hydrogen carbonate and 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 (chloroform/methanol) to give compound RE3-4 (3.4407 g, yield 68%). ESI-MS m/z: 898 (M + HCOO) ^-

Reference Example 3 Step 4 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 then trifluoroacetic acid (5 mL, 64.9 mmol), stirred at room temperature for 4 hours. The solvent was distilled off under reduced pressure to give a compound RE3-5 (2.4079 g). ESI-MS m/z: 798 (M + HCOO) ^-

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

Reference Example 3 Step 6 The compound RE3-6 (108 mg, 0.122 mmol) synthesized in Step 3 of Reference Example 3 was dissolved in dichloromethane (5 mL), and diethylamine (0.5 mL, 4.8 mmol) was added at room temperature. ) and stir 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) synthesized in Step 6 of Reference Example 3, Nα, Nε-bis(t-butoxycarbonyl)-L-isoamine (Nova Biochem) Manufactured, 295 mg, 0.852 mmol), diisopropylethylamine (0.354 mL, 2.029 mmol), and 2-(1H-benzotriazol-1-yl)-1,1,3,3 Tetramethyluronium (324 mg, 0.852 mmol) was stirred overnight at room temperature. The reaction solution was cooled in an ice bath, and a 10% aqueous citric acid solution was added thereto, and the mixture was extracted with chloroform, and the organic layer was washed with brine and dried over anhydrous magnesium sulfate. The solvent was distilled off under reduced pressure, and the residue was purified by ethylamine hexane column chromatography (chloroform/methanol), whereby compound RE3-8 (450 mg) was quantitatively obtained. ESI-MS m/z: 1101 (M + H) ⁺

Reference Example 3 Step 8 Compound RE3-8 (2.1558 g, 1.9593 mmol) synthesized in Step 3 of Reference Example 3 was dissolved in dichloromethane (20 mL) Stir at room temperature for 4 hours. The solvent was distilled off under reduced pressure, and the compound RE3-9 was quantitatively obtained. ESI-MS m/z: 700(M + H) ⁺

Reference example 4

[化119] (In the figure, Bn represents a benzyl group)

Reference Example 4 Step 1 Compound RE4-1 synthesized by the method described in Reference Example 3 (Compound RE3-5 in Reference Example 3, 0.5716 g, 0.7582 mmol), by Bioconjugated Chemistry, No. 22 Vol., pp. 690-699, the method described in 2011, the synthesis of monobenzyl dodecanoate (0.4859 g, 1.5164 mmol), diisopropylethylamine (0.662 mL, 3.79 mmol), and hexafluorophosphate 2 -(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium (0.5766 g, 1.516 mmol) was dissolved in N,N-dimethylformamide (12 mL). Stir at room temperature for 1 hour. The reaction solution was cooled in an ice bath, and a saturated aqueous solution of citric acid was added, and the mixture was extracted with chloroform, and 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 (chloroform/methanol) to give compound RE4-2 (0.88 g, yield 84%). ESI-MS m/z: 1057 (M + H) ⁺

Reference Example 4 Step 2 The compound RE4-2 (0.7545 g, 0.714 mmol) synthesized in Step 1 of Reference Example 4 was dissolved in dichloromethane (20 mL), and diethylamine (5 mL, 47.9 Mmmol) and stirred 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 RE4-3 (0.437 g, 0.7143 mmol) synthesized in Step 2 of Reference Example 4, Nα, Nε-bis(t-butoxycarbonyl)-L-isoamine (Nova Biochem) Manufactured, 0.5483 g, 1.583 mmol), diisopropylethylamine (0.624 mL, 3.57 mmol), 2-(1H-benzotriazol-1-yl)-hexafluorophosphate-1,1,3,3- Tetramethyluronium (0.5703 g, 1.5 mmol) was stirred at room temperature for 2 hours. After adding a 10% aqueous citric acid solution to the reaction mixture and extracting 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 a crude product of compound RE4-4 was obtained. ESI-MS m/z: 1269 (M + H) ⁺

Reference Example 4 Step 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 then trifluoroacetic acid (3 mL, 38.9 mmol), stirred at room temperature for 4 hours. The solvent was distilled off under reduced pressure to give a crude product of compound RE4-5. ESI-MS m/z: 869(M + H) ⁺

Reference example 5

[化120]

Reference Example 5 Step 1 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 thereto. After that, catalytic hydrogen reduction using palladium/carbon is performed. The obtained solution fraction was distilled off under reduced pressure to remove the solvent, whereby the compound RE5-2 was quantitatively obtained. ESI-MS m/z: 967 (M + H) ⁺

Reference Example 5, Step 2 Compound RE5-2 (50 mg, 0.052 mmol) and N-(6-methyleneimide hexamethyleneoxy) succinamine (48 mg, 0.155 mmol) were dissolved in tetrahydrofuran (2 mL) Diisopropylethylamine (0.045 mL, 0.259 mmol) was added and stirred at room temperature overnight. The solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (chloroform/methanol) to give compound RE5-3 (18 mg, yield 30%). ESI-MS m/z: 1160 (M + H) ⁺

Reference Example 5 Step 3 The compound RE5-3 (18 mg, 0.016 mmol) synthesized in Step 2 of Reference Example 5 was dissolved in dichloromethane (2 mL), and then trifluoroacetic acid (0.2 mL, 2.6 mmol), stir overnight at room temperature. The solvent was evaporated under reduced pressure, and the crystals were crystallised from diethyl ether to afford compound RE5-4 (7.5 mg, yield: 64%) as white solid. ESI-MS m/z: 759 (M + H) ⁺

Reference example 6

[化121]

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

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

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

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

Reference Example 6 Step 6 Using the 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 procedure as in Step 4 of Reference Example 3. 86%). ESI-MS m/z: 899 (M + H) ⁺

Reference example 7

[化122]

Reference Example 7 Step 1 The compound RE7-1 synthesized by the method described in Reference Example 3 (RE3-9, 0.2586 g, 0.3695 mmol in Reference Example 3) and the American Chemical Society, Vol. 136, were used. , Compound No. RE-1 (0.8559 g, 1.7927 mmol) described in Reference Example 1 synthesized by the method described in 2014, the compound RE7-2 was obtained by the same method as Step 3 of Reference Example 3. (0.5272 g, yield 61%). ESI-MS m/z: 2418 (M + H) ⁺

Reference Example 7 Step 2 Using the 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 by the same procedure 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

[化123]

For the synthesis of the compound A1, the compound RE8-1 (the compound RE7-3 in Reference Example 7, 0.0152 g, 0.006657 mmol) synthesized by the method described in Reference Example 7 was used in the same manner as in the step 2 of Reference Example 5. Compound A1 (0.0077 g, yield 47%) was obtained. 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 procedure 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

[化124]

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

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

Reference Example 10: Synthesis of Compounds C1 and D1

[化125]

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 American Chemical Society, 136 Vol., No. 16958-16961, the compound RE1-1 (0.4162 g, 0.93 mmol) of Reference Example 1 synthesized by the method described in 2014, was obtained by the same method as Step 3 of Reference Example 3 to obtain the compound RE10-2 ( 334.8 mg, yield 58%). ESI-MS m/z: 1294 (M + 2H) ²⁺

Reference Example 10, Step 2 Using the compound RE10-2 (0.1459 g, 0.056 mmol) synthesized in the step 1 of the title of Example 10, Compound D1 (112 mg, yield 80%) was obtained by the same procedure ). 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).

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

Reference Example 10 Step 4 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. Methyl), N,N-dimethylaminopyridine (0.0224 g, 0.183 mmol), and triethylamine (0.55 mL, 3.95 mmol) were stirred at room temperature. The reaction solution was cooled in an ice bath, water was added, and the mixture was extracted twice 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 a crude product of compound RE10-4 was obtained. ESI-MS m/z: 1342 (M + 2H) ²⁺ , detected as de-DMTr

Reference Example 10, Step 5 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'-tetramethyluronium oxime (0.0221 g, 0.05827 mmol) and diisopropylethylamine (0.02 mL, 0.1094 mmol) were dissolved in N,N-dimethylformamide (4 mL). -CPG (manufactured by Chem Gene, 0.4882 g), stirred at room temperature overnight. The mixture was separated by filtration, washed successively with dichloromethane, 10% methanol dichloromethane and diethyl ether, and then reacted with acetic anhydride/pyridine solution to obtain compound C1 (49.5 μmol/g, yield 89%). Further, the yield was calculated by adding a 1% trifluoroacetic acid/dichloromethane solution to the solid phase carrier, and calculating the rate of introduction into the solid phase carrier based on the absorption derived from the DMTr group.

Reference example 11

[化126]

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, pp. 2718-2733, 2016. , yield 50%). ESI-MS m/z: 582 (M + H) ⁺

Reference example 12

[化127]

Compound RE12-1 (4.000 g, 10.27 mmol) was dissolved in dichloromethane (60 mL). EtOAc (EtOAc (EtOAc) 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 mixture, and the mixture was separated from dichloromethane. The organic phase was washed with water and dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and the residue was concentrated with 2-methyltetrahydrofuran. The residue was dropped into heptane, and the obtained crystal was filtered to give compound RE12-2 (5.130 g, yield 88%). ESI-MS m/z: 570 (M + H) ⁺

Reference example 13

[化128]

Compound RE13-1 (Compound RE1-1, 898.0 mg, 2.007 mmol) in Reference Example 1 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 Hydrochloride (0.4910 mL, 2.208 mmol) was stirred at room temperature for 3 h. Water was added to the reaction mixture, and the organic layer was washed with a saturated aqueous solution of sodium hydrogencarbonate 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), whereby Compound RE13-2 (873.0 mg, yield 68%) was obtained. ESI-MS m/z: 639 (M + H) ⁺

Reference example 14

[化129]

Reference Example 14 Step 1 Compound RE14-1 (Compound RE1-1 in Reference Example 1, 3.00 g, 6.70 mmol) was dissolved in dichloromethane (60 mL), and L-isobutylate 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 h. The reaction solution was washed with water and a saturated aqueous sodium hydrogen sulfate 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), whereby compound RE14-2 was quantitatively obtained. ESI-MS m/z: 1096 (M + H) ⁺

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

Reference example 15

[化130]

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

Reference Example 15 Step 2 Using the compound RE15-2 (1.5 g, 1.37 mmol), the compound RE15-3 was quantitatively obtained by the same procedure as the the the ESI-MS m/z: 990 (M + H) ⁺

Reference example 16

[化131]

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

Reference Example 16 Step 2 Using the compound RE16-2 (1.421 g, 1.2866 mmol), the compound RE16-3 was quantitatively obtained by the same procedure as the the the ESI-MS m/z: 1004(M + H) ⁺

Reference example 17

[化132]

The compound RE17-1 (90 mg, 0.173 mmol) synthesized by the method described in Journal of Organic Chemistry, Vol. 74, pp. 6837-6842, 2009 was dissolved in tetrahydrofuran (1 mL). A triphenylphosphine polymer carrier (manufactured by Sigma-Aldrich, 63 mg, 0.189 mmol) was added, and stirred under a heating loop for 3 hours. The reaction liquid was filtered, and the solvent was evaporated under reduced pressure, whereby Compound RE17-2 (70 mg, yield 82%) was obtained. 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

[化133]

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

Reference Example 18, Step 2 Using the crude product of Compound RE18-2 (1.5 g) and 1,4-diaminobutane (manufactured by Tokyo Chemical Industry Co., Ltd., 3.29 g, 37.3 mmol), Compound RE18-3 (0.18 g, two-stage yield 10%) was obtained by the same procedure as in the step 3 of the title compound. 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

[化134]

Compound RE19-1 (110 mg, 0.212 mmol) synthesized by the method described in Organic Chemistry, Vol. 74, No. 6837-6842, 2009, was dissolved in tetrahydrofuran (2 mL), and N-(1R was added. ,8S,9S)-bicyclo[6.1.0]dec-4-yne-9-ylmethoxycarbonyl-1,8-diamino-3,6-dioxooctane (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), stirred at room temperature 1 hour. After adding water to the reaction mixture and extracting with chloroform, 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 ethylamine hexane 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

[化135]

Reference Example 20, Step 1 Using Compound RE20-1 (manufactured by AstaTech Co., Ltd., 100 mg, 1.148 mmol) and Fmoc-Ser(tBuMe2Si)-OH (manufactured by Watanabe Chemical Industries, Ltd., 532 mg, 1.205 mmol), with Reference Example 3 The same procedure as in Step 3 gave Compound RE20-2 (410 mg, yield 70%). 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).

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

Reference Example 20, Step 3 Using the crude product of Compound RE20-3 (680 mg), Compound RE20-4 (330 mg, yield of a two-stage yield of 70%) was obtained by the same procedure as in Step 5 of Reference Example 2. ESI-MS m/z: 519 (M + H) ^{+ 1} H-NMR (400 MHz, CDCl ₃ ) δ: 0.02-0.09 (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

[化136]

Reference Example 21 Step 1 N-(Tertilybutoxycarbonyl)-1,3-diaminopropane (manufactured by Tokyo Chemical Industry Co., Ltd., 1.788 g, 10.26 mmol) was dissolved in dichloromethane (22.8 mL), and three were added. Ethylamine (1.907 mL, 13.68 mmol) was stirred at room temperature for 15 min. A solution of the compound RE21-1 (1.126 g, 6.84 mmol) in methylene chloride (5 mL) synthesized by the method described in Organic Letter, Vol. 16, No. 6318-6321, 2014, was added dropwise to the reaction solution. Stir at room temperature for 2 hours. After adding water to the reaction mixture and extracting with chloroform, 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), whereby Compound RE21-2 (1.65 g, yield 80%) was obtained. ESI-MS m/z: 303 (M + H) ⁺

Reference Example 21, Step 2 Using Compound RE21-2 (1.65 g, 5.46 mmol), Compound RE21-3 (1.10 g, yield 100%) was obtained by the same procedure as in the step 2 . 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

[化137]

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

Reference Example 22 Step 2 Using the crude product of Compound RE22-2, Compound R22-3 (1.34 g, a two-stage yield of 52%) was obtained by the procedure described in Step 5 of 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, using the compound RE22-3 (1.15 g, 3.16 mmol) and Fmoc-Ser(tBuMe2Si)-OH (manufactured by Watanabe Chemical Industries, Ltd., 1.677 g, 3.8 mmol) by the same procedure as in Step 3 of Reference Example 3. The compound obtained 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 Using the compounds RE23-1 to RE23-5 and Fmoc-Ser(tBuMe ₂ Si)-OH described in the Table Y-1, the same as in Reference Example 22 was used to obtain the results described in Table Y-2. Compounds RE23-6 to RE23-10. Using the compound RE22-3 of Reference Example 22 and Fmoc-Thr(tBuMe ₂ Si)-OH, the compound RE23-11 described in the Table Y-2 was obtained by the same method as Reference Example 22. The compound RE23-12 described in the Table Y-2 was obtained by the same method as Reference Example 22 using the compound RE23-1 and the Fmoc-Thr(tBuMe ₂ Si)-OH described in the Table Y-1. The NMR analysis data of the compound synthesized in the present example is shown in Table Y-3.

[Table 15] Table Y-1

[Table 16] Table Y-2

[Table 17] Table Y-3

Reference example 24

[化138]

The compound RE24-1 (Compound RE22-4 in Reference Example 22, 2.487 g, 3.16 mmol) synthesized by the method described in Referential Example 22 was used to obtain Compound RE24-2 by the same procedure 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 the Table Y-2, the compounds RE25-1 to RE25-7 described in Table Z-1 were obtained by the same method as Reference Example 24. The results of mass spectrometry analysis of the compounds synthesized in this example are shown in Table Z-2.

[Table 18] Table Z-1

[Table 19]

Reference example 26

[化139]

Reference Example 26 Step 1 Compound RE26-1 (2.00 g, 9.47 mmol) was dissolved in N,N'-dimethylformamide (40 mL), and dibutyl succinimide diacetate 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- The hydrate (145 mg, 0.947 mmol) was stirred for 2 hours. After adding water to the reaction mixture and extracting with ethyl acetate, the organic layer was washed with a saturated aqueous sodium hydrogen carbonate solution and 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). Further, the slurry was purified by methanol, whereby Compound RE26-2 (4.07 g, yield: 65%) was obtained. ESI-MS m/z: 664 (M - H) ^-

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

Reference Example 26 Step 3 Compound RE26-3 (871.0 mg, 1.370 mmol) was dissolved in N,N'-dimethylformamide (17 mL), and O-(7-azabenzotriazole) hexafluorophosphate was added. -1- alkyne)-N,N,N',N'-tetramethyluronium (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 at room temperature overnight. After adding water to the reaction mixture and extracting with ethyl acetate, the organic layer was washed with a saturated aqueous sodium hydrogen carbonate solution and 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 = 60 / 40) to obtain compound RE26-4 (1.030 g, yield: 80%). ESI-MS m/z: 939 (M + H) ⁺

Reference Example 26 Step 4 Compound RE26-4 (1.030 g, 1.98 mmol) was dissolved in dichloromethane (10 mL), and trifluoroacetic acid (10.00 mL, 130.0 mmol) was added and 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

[化140]

Reference Example 27 Step 1 Compound RE27-1 (2.000 g, 12.98 mmol) was dissolved in N,N'-dimethylformamide (30 mL), and potassium hydrogencarbonate (1.559 g, 15.57 mmol) and benzyl chloride were added. (2.328 mL, 19.47 mmol), stirred at room temperature for 4 h. Saturated ammonium chloride was added to the reaction mixture, 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), whereby Compound RE27-2 (2.850 g, yield: 90%) was obtained. ESI-MS m/z: 243 (M - H) ^-

Reference Example 27 Step 2 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 mixture, and the mixture was extracted with dichloromethane. The organic layer was washed with 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), whereby Compound RE27-3 (4.300 g, yield 89%) was obtained. ESI-MS m/z: 472 (M - H) ^-

Reference Example 27 Step 3 Compound RE27-3 (1.000 g, 2.16 mmol) was dissolved in dichloromethane (10 mL), trifluoroacetic acid (10.00 mL, 130.0 mmol) was added and 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 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-tert-butyl iminodiacetic acid ( 524.0 mg, 2.137 mmol), stirred at room temperature for 5 hours. After adding water to the reaction mixture and extracting with ethyl acetate, the organic layer was washed with a saturated aqueous sodium hydrogen carbonate solution and 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 = 60 / 40), whereby Compound RE27-5 (617.0 mg, yield 78%) was obtained. ESI-MS m/z: 814 (M - H) ^-

Reference Example 27 Step 5 Compound RE27-5 (610.0 mg, 0.7490 mmol) was dissolved in dichloromethane (6 mL), and trifluoroacetic acid (6 mL, 78.00 mmol) was added and 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

[化141]

Reference Example 28 Step 1 Compound RE28-1 (Compound RE26-3, 474 mg, 0.744 mmol in Reference Example 26) synthesized by the method described in Referential Example 26 was dissolved in N,N'-dimethylformamidine. Amine (10 mL), trans-cyclohexane-1,4-dicarboxyl synthesized by the method described in Journal of Pharmaceutical Chemistry, Vol. 54, pp. 2433-2446, 2011, at room temperature 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'-tetramethyluronium (339 mg, 0.893 mmol) and stirred for 6 hours. After adding water to the reaction mixture and extracting with ethyl acetate, the organic layer was washed with a saturated aqueous sodium hydrogen carbonate solution and 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), whereby Compound RE28-2 (448 mg, yield 68%) was obtained. ESI-MS m/z: 879 (M - H) ^-

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

Reference example 29

[化142]

Reference Example 29 Step 1 Compound RE29-1 (500 mg, 2.75 mmol) was dissolved in N,N'-dimethylformamide (10 mL), and dibutyl succinimide 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- The hydrate (42.0 mg, 0.275 mmol) was stirred for 4 hours. After adding water to the reaction mixture and extracting 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 give compound RE29-2 (329 mg, yield 19%). ESI-MS m/z: 635 (M - H) ^-

Reference Example 29 Step 2 Compound RE29-2 (323 mg, 0.507 mmol) was dissolved in N,N'-dimethylformamide (6 v 5 mL), and potassium carbonate (84.0 mg, 0.609 mmol) was added at room temperature. Benzyl bromide (139 mg, 0.609 mmol) was stirred for 3 hours. After adding water to the reaction mixture and extracting 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 Compound RE29-3 (312 mg, 0.398 mmol) was dissolved in dichloromethane (3.1 mL), trifluoroacetic acid (3.1 mL) was added at room temperature and stirred overnight. The reaction solution was concentrated under reduced pressure and aq. ethyl acetate was used to afford compound RE29-4 (252 mg). ESI-MS m/z: 561 (M + H) ⁺

Reference example 30

[化143]

Reference Example 30 Step 1 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- The hydrate (145 mg, 0.947 mmol) was 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). Further, the slurry was purified by ethyl acetate, whereby Compound RE30-2 (2.68 g, yield: 79%) was obtained. ESI-MS m/z: 356 (M - H) ^-

Reference Example 30 Step 2 Compound RE30-2 (500 mg, 1.40 mmol) was suspended in acetonitrile (10 mL), and butyl butyl acrylate (3.59 g, 28.0 mmol), benzyl trimethyl hydroxyacetate was added at room temperature. Ammonium (40% in water, 1.76 mL, 702 mmol) was stirred overnight. The solvent was distilled off under reduced pressure, and water was added, 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 give compound RE30-3 (300 mg, yield 24%). ESI-MS m/z: 871 (M + H) ⁺

Reference Example 30 Step 3 Compound RE30-3 (340 mg, 0391 mmol) was dissolved in tetrahydrofuran (6.8 mL), and 10% palladium carbon powder (aqueous product, 54.29%, 31.3 mg) was added at room temperature under a hydrogen atmosphere. Stir under 6 hours. The reaction solution was filtered, and the solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography (heptane / ethyl acetate = 30 / 70) to obtain compound RE30-4 (235 mg. Rate 72%). ESI-MS m/z: 841 (M + H) ⁺

Reference Example 30, Step 4 Compound RE30-4 (232 mg, 0.276 mmol) was dissolved in N,N'-dimethylformamide (4.6 mL), and monobutyl laurate (0.133 mg, 0.414 mmol), diisopropylethylamine (0.145 mL, 0.829 mmol), O-(7-azabenzotriazol-1-yl)-N,N,N',N'-four Methyl urea guanidine (158 mg, 0.414 mmol) was stirred overnight. After adding water to the reaction mixture and extracting with ethyl acetate, the organic layer was washed with a saturated aqueous sodium hydrogen carbonate solution and 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 = 30 / 70) to yield compound RE30-5 (274 mg, yield 87%). ESI-MS m/z: 1141 (M - H) ^-

Reference Example 30 Step 5 Compound RE30-5 (273 mg, 0.239 mmol) was dissolved in dichloromethane (2.7 mL), trifluoroacetic acid (2.7 mL) The reaction solution was concentrated under reduced pressure, and then aq. ethyl acetate was used to obtain a compound RE30-6 (231 mg). ESI-MS m/z: 919 (M + H) ⁺

Reference example 31

[化144]

Reference Example 31 Step 1 4-Nitroisophthalic acid RE31-1 (500 mg, 2.37 mmol) and N-Boc-ethylenediamine (808 mg, 5.21 mmol) were dissolved in N,N'-dimethylmethyl Indoleamine (10 mL), 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 h. The reaction liquid was worked up, and the crude product was purified by silica gel column chromatography, whereby Compound RE31-2 (650 mg, yield 55%) was obtained.

Reference Example 31 Step 2 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. An aqueous solution of (378 mg, 7.07 mmol) was stirred at room temperature for 24 hours. The reaction liquid was worked up, and the crude product was purified by silica gel column chromatography, whereby compound RE31-3 (160 mg, yield 34%) was obtained.

Reference Example 31, Step 3 Compound #31-3 (200 mg, 0.430 mmol) and N-benzyloxycarbonylglycine (sometimes also benzyloxycarbonyl as Cbz) (90.0 mg, 0.430 mmol) were dissolved in N , N'-dimethylformamide (2.0 mL), adding diisopropylethylamine (0.220 mL, 1.29 mmol), hexafluorophosphate O-(7-azabenzotriazole) at room temperature 1-Base)-N,N,N',N'-tetramethyluronium (245 mg, 0.645 mmol) and stirred for 16 h. The reaction liquid was worked up, and the crude product was purified by silica gel column chromatography, whereby Compound RE31-4 (180 mg, yield: 64%) was obtained. ESI-MS m/z: 657 (M + H) ⁺

Reference example 32

[化145]

Reference Example 32, Step 1 Dissolve 3,5-dinitrobenzoic acid RE32-1 (500 mg, 2.36 mmol) and N-Cbz-ethylenediamine (588 mg, 2.83 mmol) in N,N'-dimethyl Methionamine (5.0 mL), adding 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) was stirred for 16 h. The reaction solution was worked up, and the crude product was purified by silica gel column chromatography, whereby Compound RE32-2 (445 mg, yield: 48%) was obtained.

Reference Example 32 Step 2 Compound RE32-2 (200 mg, 0.515 mmol) was dissolved in ethanol (5.0 mL), and tin (II) chloride ( 584 mg, 3.09 mmol) and concentrated hydrochloric acid (0.2 mL) were added at room temperature. Stir at 80 ° C for 16 hours. The reaction solution was worked up to quantitatively obtain the compound RE32-3 (180 mg). ESI-MS m/z: 329 (M + H) ⁺

Reference example 33

[化146]

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

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

Reference Example 33 Step 3 Compound RE33-4 (3.08 g, yield: 87%) was obtained by the same procedure as in the procedure of the the ESI-MS m/z: 360(M + H) ⁺

Reference example 34 [Chem. 147]

Reference Example 34 Step 1 Compound RE34-1 (2 g, 9.53 mmol) and tributyloxycarbonylamino-1-pentanol (manufactured by Tokyo Chemical Industry Co., Ltd., 2 g, 10 mmol) were used, with reference example 3 The same procedure as in Step 1 gave Compound RE34-2 (2.40 g, yield 63%). ESI-MS m/z: 296 (M + H) ⁺ , detected as de Boc

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

Reference Example 35: Synthesis of Compound D2

[化148]

Reference Example 35 Step 1 Compound RE35-1 (Compound RE11-2 in Reference Example 11, 1.015 g, 1.748 mmol) synthesized by the method described in Referential Example 11 was dissolved in N,N'-dimethylformamidine. Amine (12 mL) was added with a 10% palladium carbon powder (aqueous product, 54.29%, 187 mg) at room temperature, and stirred under a hydrogen atmosphere for 6 hours. The reaction solution was filtered. The compound RE26-5 (250.0 mg, 0.350 mmol) synthesized in Reference Example 26, 1-hydroxybenzotriazole monohydrate (26.80 mg, 0.1750 mmol), and 1-(3-dimethyl group) were added to the filtrate. Aminopropyl)-3-ethylcarbodiimide hydrochloride (402.0 mg, 2.099 mmol) was stirred at room temperature overnight. After adding water to the reaction mixture and extracting with ethyl acetate, the organic layer was washed with a saturated aqueous sodium hydrogen carbonate solution and 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 (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 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 under a hydrogen atmosphere. Stir under night. The reaction solution was filtered, and the solvent was evaporated 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

[化149]

Reference Example 36 Step 1 Compound RE36-1 (Compound RE12-2, 500 mg, 0.879 mmol in Reference Example 12) synthesized by the method described in Reference Example 12 was dissolved in N,N'-dimethylformamidine. Amine (6.5 mL) was added with a 10% palladium carbon powder (aqueous product, 54.29%, 94 mg) at room temperature, and stirred under a hydrogen atmosphere for 4 hours. The reaction solution was filtered. 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 were added to the filtrate. Propyl)-3-ethylcarbodiimide hydrochloride (202.0 mg, 1.055 mmol) was stirred at room temperature overnight. The solvent was distilled off under reduced pressure, and the residue was purified by reversed column chromatography (water/acetonitrile) to give compound RE36-2 (249.7 mg, yield 60%). ESI-MS m/z: 1191 (M + 2H) ²⁺

Reference Example 36 Step 2 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), stirred under a hydrogen atmosphere for 4 hours. The reaction solution was filtered, and the solvent was evaporated under reduced pressure to give 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

[化150]

Reference Example 37 Step 1 Compound RE37-1 (Compound RE13-2, 430 mg, 0.674 mmol in Reference Example 13) synthesized by the method described in Referential Example 13 was dissolved in N,N'-dimethylformamidine. Amine (6 mL) was added with a 10% palladium carbon powder (aqueous product, 54.29%, 79 mg) at room temperature, and stirred under a hydrogen atmosphere for 4 hours. The reaction solution was filtered. 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 were added to the filtrate. Propyl)-3-ethylcarbodiimide hydrochloride (170.0.0 mg, 0.887 mmol) was stirred at room temperature overnight. The solvent was distilled off under reduced pressure, and the residue was purified by reversed column chromatography (water / acetonitrile) to give compound RE37-2 (218.1 mg, yield 56%). ESI-MS m/z: 1329 (M + 2H) ²⁺

Reference Example 37 Step 2 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), stirred under a hydrogen atmosphere for 4 hours. The reaction solution was filtered, and the solvent was evaporated under reduced pressure to give Compound D4 (192.7 mg, yield 95%). ESI-MS m/z: 1284 (M + 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

[化151]

Reference Example 38 Step 1 Compound RE38-1 (Compound RE12-2, 450 mg, 0.791 mmol in Reference Example 12) synthesized by the method described in Reference Example 12 was dissolved in N,N'-dimethylformamidine. Amine (6 mL) was added with a 10% palladium carbon powder (aqueous product, 54.29%, 85 mg) at room temperature, and stirred under a hydrogen atmosphere for 5 hours. The reaction solution was filtered. 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 were added to the filtrate. Propyl)-3-ethylcarbodiimide hydrochloride (182.0 mg, 0.950 mmol) was stirred at room temperature overnight. The solvent was distilled off under reduced pressure, and the residue was purified by reversed column chromatography (water / acetonitrile) to give compound RE38-2 (99 mg, yield 28%). ESI-MS m/z: 1129 (M + 2H) ²⁺

Reference Example 38 Step 2 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 was added at room temperature (aqueous product, 54.29%, 26) Mg), stirred under a hydrogen atmosphere for 2 hours. The reaction liquid was filtered, and the solvent was evaporated under reduced pressure to give Compound D5 (57.5 mg, yield 75%). ESI-MS m/z: 1084 (M + 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

[化152]

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

Reference Example 39 Step 2 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), stirred under a hydrogen atmosphere for 2 hours. The reaction solution was filtered, and the solvent was evaporated under reduced pressure to give Compound D6 (156.7 mg, yield 87%). ESI-MS m/z: 1222 (M + 2H) ^{2+ 1} H-NMR (400 MHz, DMSO-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

[Chem. 153]

Reference Example 40 Step 1 Compound D5 (171 mg, 0.079 mmol) synthesized by the method of Reference 38 was dissolved in N,N'-dimethylformamide (3.4 mL), and benzyl glycinate 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 The carbodiimide hydrochloride (18.16 mg, 0.095 mmol) was stirred at room temperature overnight. The solvent was evaporated under reduced pressure, and the residue was purified by reversed column chromatography (water / acetonitrile) to give compound RE40-2 (55.7 mg, yield 31%). ESI-MS m/z: 1158 (M + 2H) ²⁺

Reference Example 40 Step 2 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 was added at room temperature (aqueous product, 54.29%, 18) Mg), stirred under a hydrogen atmosphere for 2 hours. The reaction liquid was filtered, and the solvent was evaporated under reduced pressure to give compound RE40-3 (50.1 mg, yield 97%). ESI-MS m/z: 1112 (M + 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

[化154]

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

Reference Example 41 Step 2 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), stirred under a hydrogen atmosphere for 8 hours. The reaction solution was filtered, and the solvent was evaporated under reduced pressure to give 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

[化155]

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

Reference Example 42 Step 2 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 was added at room temperature (aqueous product, 54.29%, 32.4) Mg), stirred under a hydrogen atmosphere for 1 hour. The reaction liquid was filtered, and the solvent was evaporated under reduced pressure to give 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

[化156]

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

Reference Example 43 Step 2 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 was added at room temperature (aqueous product, 54.29%, 34.3) Mg), stirred under a hydrogen atmosphere for 1 hour. The reaction solution was filtered, and the solvent was evaporated to dryness to give 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157]

Reference Example 44 Step 1 Compound RE44-1 (Compound RE31-4, 150 mg, 0.228 mmol in Reference Example 31) synthesized by the method described in Referential Example 31 was dissolved in dichloromethane (1.5 mL). Trifluoroacetic acid (1.5 mL) was added at room temperature and stirred for 4 hours. The reaction solution was concentrated under reduced pressure and then evaporated with ethyl acetate. The residue was dissolved in N,N'-dimethylformamide (3 mL), and the compound RE14-3 (574 mg, 0.571 mmol) and 1-(3-dimethylamine) of Reference Example 14 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 liquid was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (chloroform/methanol=90/10), and purified by reverse phase separation HPLC (acetonitrile/water) to obtain compound RE44-2. (242 mg, yield 44%). ESI-MS m/z: 1216 (M + 2H) ²⁺

Reference Example 44 Step 2 Compound RE44-2 (242 mg, 0.100 mmol) was dissolved in tetrahydrofuran/water (4/1; 12 mL), and 10% palladium carbon powder was added at room temperature (aqueous product, 54.29%, 44.6) Mg), stirred under a hydrogen atmosphere for 2 hours. Add 6-methyleneimide caproic acid (23.2 mg, 0.110 mmol), 4-(4,6-dimethoxy-1,3,5-triterpene) at room temperature under argon atmosphere. 2-yl)-4-methylmorpholine hydrochloride (55.2 mg, 0.199 mmol) was stirred overnight. The reaction solution was filtered through celite, and the solvent was evaporated under reduced pressure, and the residue was purified from ethylbenzene 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

[化158]

Reference Example 45 Step 1 Compound RE45-1 (Compound RE32-3, Reference Example 32, 75.0 mg, 0.228 mmol) synthesized by the method described in Referential Example 32 was dissolved in N,N'-dimethylformamidine. The amine (3.0 mL) was added at room temperature under the compound of Example 14 RE14-3 (574 mg, 0.571 mmol), diisopropylethylamine (0.199 mL, 1.14 mmol), hexafluorophosphate O-(7- Azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium (217 mg, 0.571 mmol) was 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 purified by reverse phase separation 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 Compound RE45-2 (196 mg, 0.085 mmol) was dissolved in tetrahydrofuran/water (4/1; 10 mL), and 10% palladium carbon powder was added at room temperature (aqueous product, 54.29%, 36.1) Mg), stirred under a hydrogen atmosphere for 2 hours. Add 6-methyleneimine hexanoic acid (19.8 mg, 0.094 mmol), 4-(4,6-dimethoxy-1,3,5-triterpene) at room temperature under argon atmosphere. 2-yl)-4-methylmorpholine hydrochloride (47.0 mg, 0.170 mmol) was stirred overnight. The reaction solution was filtered through celite, and the solvent was evaporated under reduced pressure, and the residue was purified by ethylbenzene 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

[化159]

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

Reference Example 47: Synthesis of Compound A4

[化160]

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

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

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

Reference Example 48: Synthesis of Compound A5

[化161]

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

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

Reference Example 49: Synthesis of Compound A6

[化162]

Compound RE49-1 (0.048 g, 0.021 mmol) and 3-butyleneimine propionic acid N-butylimide (manufactured by Tokyo Chemical Industry Co., Ltd., 0.017 g, 0.064 mmol) were used, with reference examples. The synthesis of the compound A1 of 8 was carried out in the same manner to obtain the compound A6 (0.040 g, yield 78%). ESI-MS m/z: 2480 (M + HCOO) ^-

Reference Example 50: Synthesis of Compound A7

[化163]

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

Reference example 51

[化164]

Reference Example 51 Step 1 The compound RE51-1 (0.122 g, 0.054 mmol) synthesized by the method described in Reference Example 7 was used and by Bioconjugated Chemistry, Vol. 22, pp. 690-699, 2011. The compound RE51-2 (0.076 g, yield 56%) was obtained by the same procedure as in the step 1 of the titled compound. ESI-MS m/z: 2503 (M + H) ⁺

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

Reference example 52

[Chem. 165]

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

Reference Example 53: Synthesis of Compound C2

[化166]

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

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

Reference Example 53 Step 3 Compound C53 (19.4 μmol/g, yield 35%) was obtained using the compound RE53-2 (0.0467 g, 0.013 mmol).

Reference Example 54: Synthesis of Compound C3

[化167]

Reference Example 54 Step 1 Using 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, the compound was obtained by the same procedure as in Step 3 of Reference Example 10. RE54-1 (90 mg, yield 76%). ESI-MS m/z: 1335 (M - DMTr + 2H) ²⁺

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

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

Reference Example 55: Synthesis of Compound C4

[化168]

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 in the same manner as in Step 3 of Reference Example 10. Method Compound RE55-1 (90 mg, yield 76%) was obtained. ESI-MS m/z: 1356 (M + 2H) ²⁺ , detected as de-DMTr

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

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

Reference Example 56: Synthesis of Compound C5

[化169]

Reference Example 56 Step 1 The compound D10 (100 mg, 0.039 mmol) synthesized by the method described in Referential Example 46 and the compound RE18-3 synthesized in Step 2 of Reference Example 18 were used, and the procedure of Reference Example 10 was used. 3 The same procedure gave compound RE56-1 (50 mg, yield 42%). ESI-MS m/z: 1363 (M + 2H) ²⁺ , detected as de-DMTr

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

Reference Example 56, Step 3 Compound C5 (0.5 μmol/g, a two-stage yield of 1%) was obtained by the same procedure as the procedure

Reference Example 57: Synthesis of Compound C6

[化170]

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

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

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

Reference Example 58: Synthesis of Compound C7

[化171]

Reference Example 58 Step 1 Using the 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 by the same procedure as Step 3 of Reference Example 10. -1 (58 mg, yield 77%). ESI-MS m/z: 1341 (M + 2H) ²⁺

Reference Example 58 Step 2 Compound RE58-1 (58 mg, 0.022 mmol) was dissolved in methanol (0.15 mL) and added by the method described in Organic Chemistry, Vol. 74, pp. 6837-6842, 2009. Reference Example 17 Compound RE17-1 (16.9 mg, 0.032 mmol), 1 mol/L SODI® M L-ASCORBATE aqueous solution (0.022 mL, 0.022 mmol), 20 mmol/L aqueous copper (II) sulfate solution (0.011 mL, 0.22 μmol), and 10 mmol/L of tris(2-benzimidazolylmethyl)amine/DMSO solution (0.022 mL, 0.22 μmol), and stirred at room temperature for 3 hours. The reaction liquid was purified by a hydrazine 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 de-DMTr

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

Reference Example 58 Step 4 Compound C7 (8.4 μmol/g, yield: 27%) was obtained by the same procedure as the procedure

Reference example 59

[化172]

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

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

Reference Example 59 Step 3 Using a compound RE59-2 (0.08 g, 0.027 mmol), a crude product of compound RE59-3 was obtained by the same procedure as in the step 4 of the referenced 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 structures described in the compound D1 and the table of the Z-1 or the compound RE20-4 of the reference example 20, the P-1 and P- were obtained by the same method as the steps 1 and 2 of Reference Example 59. 2 The compounds described in the table. The results of mass spectrometry analysis of the compounds synthesized in this example are shown in Table P-3.

[Table 20] Table P-1

[Table 21] Pick up the P-1 form

[Table 22] Table P-2

[Table 23]

Reference Example 61: Synthesis of Compound C8

[化173]

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

Reference Example 62: Synthesis of C9 to C17 The compound described in Tables Q-1 and Q-2 was obtained by the same method as Reference Example 61 using the compounds described in Tables P-1 and P-2. The supported amount of the compound synthesized by the present example is shown in Table Q-3.

[Table 24] Table Q-1

[Table 25] Pick up the Q-1 form

[Table 26] Table Q-2

[Table 27]

Example 1: Synthesis of Compound 1-1 and Compound 1-2

[174]

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, which was allowed to stand at 4 ° C. By anion exchange chromatography (GE Healthcare, MonoQ 5/50GL, 10 μm, 5.0 mm × 50 mm; from liquid A: 10 mmol/L Tris buffer/30% acetonitrile and B solution: 10 mmol/L Tris buffer Liquid / 30% acetonitrile / 1 mol / L NaBr gradient) or reverse phase liquid chromatography (Waters, X Bridge C18, 5 μm, 4.6 mm × 250 mm, from 0.1 mol / L triethylammonium acetate buffer Purification is carried out by any of the methods of the liquid B: a gradient of acetonitrile formation, whereby Compound 1-1 is obtained.

Step 2 Using a mixed buffer (100 mmol/L potassium acetate, 30 mmol/L 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES)-KOH (pH 7.4) , 2 mmol/L magnesium acetate) The concentration of compound 1-1 (3'-CFB-ssRNA) synthesized in step 1 was adjusted (50 μmol/L). An equal amount of the above-mentioned justice and antisense strands (50 μmol/L) were separately mixed and allowed to stand at 80 ° C for 10 minutes. The antisense strand sequence is as described in Table R-2. The mixture was slowly cooled, and allowed to stand at 37 ° C for 1 hour to obtain a double-stranded compound 1-2.

Examples 2 to 11: Synthesis of Compounds 2-1 to 11-1 and Compounds 2-2 to 11-2 were synthesized in the same manner as in Example 1 using an oligonucleotide different from the nucleotide sequence of Example 1. Compounds 2-1 to 11-1 and compounds 2-2 to 11-2.

Example 12: Synthesis of Compound 12-1 and Compound 12-2

[化175]

Step 1 Using the reference compound D5, add an amine group-modified oligonucleotide synthesized by the method described in Molecular Science, Vol. 17, pp. 13825-13843, 2012, by Yoke Chemistry, Vol. 22, pp. 1723-1728, 2011 or Bioconjugated Chemistry, Vol. 26, pp. 1451-1455, 2015, describes the method of reaction. Purification was carried out by the method described in Example 1, whereby Compound 12-1 was obtained.

Step 2 Compound 12-2 was obtained by the same method as Step 2 of Example 1.

Examples 13 to 15: Synthesis of Compound 13-1 to 15-1 and Compound 13-2 to 15-2 An oligonucleotide different from the base sequence of Example 1 was synthesized in the same manner as in Example 1. Compounds 13-1 to 15-1 and compounds 13-2 to 15-2.

The ligand-linker structure of the compound synthesized by this example is shown below.

[化176]

The sequence of the nucleic acid complex synthesized by the present example (JP) and the results of mass spectrometry are shown in Table R-1. Further, in the table of the R-1, N(M) represents a 2'-O-methyl modified RNA, N(F) represents a 2'-fluoro modified RNA, and ^ represents a phosphorothioate.

[Table 28] Table R-1

The sequence of the nucleic acid complex synthesized by the present example is shown in the R-2 table. Further, in the table of R-2, 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 ester.

[Table 29] Table R-2

[Table 30] Table R-2

Reference test Example 1: Measurement of attenuating activity of CFB mRNA in human cells Huh7 cells which were obtained as human liver cancer-derived cell lines were seeded at 10,000 cells/80 μL/well in a 96-well culture plate (obtained from National Research and Development) JCRB Cell Bank of the Institute of Pharmaceutical and Pharmaceutical Sciences, Health and Nutrition. The medium was DMEM medium (manufactured by Life Technologies, catalog number 08458-16) containing 10% fetal calf serum (FBS). The double-stranded nucleic acid is used by synthesizing those described in Tables 1-1 to 1-3 by gene design. Specifically, the annealed double-stranded nucleic acid comprises a sense strand comprising the ribonucleotides of SEQ ID NOs: 2 to 124, and an antisense strand comprising the ribonucleotides of SEQ ID NOs: 125 to 247 (SEQ ID NO: The justice shares shown in (n=2 to 124) form a pair with the antisense strands shown in the sequence number [n+123]. The double-stranded nucleic acid described in Tables 1-1 to 1-3 and RNAiMax transfection reagent (manufactured by Life Technologies, catalog number 1401251) were diluted with Opti-MEM medium (manufactured by Life Technologies, catalog number 11058-021). 20 μL of the siRNA/RNAiMax mixture was added to each well of a 96-well culture plate at a final concentration of the double-stranded nucleic acid of 1 nM or 0.1 nM, and cultured at 37 ° C under 5% CO ₂ for 24 hours. Thereafter, the cells were washed with PBS (Phosphate buffered saline, phosphate buffered saline), and a SuperPrep cell decomposition & qPCR reverse transcription kit (Cell Lysis & RT Kit for qPCR) (manufactured by Toyobo Co., Ltd., catalog number SCQ-101), cDNA was synthesized from each disk according to the method described in the accompanying instructions of the product. 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 performance high-performance premix (Gene Expression Master Mix) (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 CFB probe, and 1 μL of human β-actin probe. QuantStudio ^TM 12K Flex using real time PCR system for CFB human and human β-actin gene of real time PCR. --actin is a constitutive expression gene, which is measured as an internal control to correct the amount of CFB expression. The amount of CFB mRNA when Huh7 cells were treated with only the transfection reagent without using siRNA was 1.0, and the relative expression amount of CFB mRNA when each double-stranded nucleic acid was introduced was calculated. The experiment was carried out a plurality of times, and the average value of the relative amount of CFB mRNA was shown in Table 1-1 to Table 1-3.

[Table 31]

[Table 32]

[Table 33]

The in vitro weakening activity in human primary hepatocytes was measured for each of the nucleic acid complexes obtained in Examples 1 to 10 and 12 to 15. Each nucleic acid complex diluted with Opti-MEM (manufactured by Thermo Fisher Scientific Co., Ltd., catalog No. 31985) at a final concentration of 10 or 30 nmol/L was dispensed into a 96-well culture plate at a volume of 20 μL per well to the cells. The primary hepatocytes (manufactured by Biopredic International, catalog number HEP187) suspended in a plating medium (manufactured by Biopredic International, catalog number LV0304-2) in a number of 10000 cells/80 μL/well, at 37 After incubation for 6 hours at ° C and 5% CO ₂ , the culture supernatant was carefully removed, and an incubation medium (manufactured by Biopredic International, catalog number LV0304-2) was added, and each nucleic acid complex was supplied to the first generation. Hepatocyte. Further, as a negative control group, cells which were not treated were inoculated. The cells to which the respective nucleic acid complexes were added were cultured in a 5% CO ₂ incubator at 37 ° C for 18 hours, and washed with an ice bath-cooled phosphate buffered saline (DPBS) (manufactured by Nacalai Tesque Co., Ltd.) to use SuperPrep cells. The decomposition & qPCR reverse transcription kit (manufactured by Toyobo Co., Ltd., catalog number SCQ-201) was used to recover total RNA according to the method described in the accompanying instructions, and cDNA was produced by reverse transcription reaction using the obtained total RNA as a template. Using the obtained cDNA as a template, a Taqman (registered trademark) Gene Expression Assays probe (manufactured by Thermo Fisher Scientific Co., Ltd.) was used as a probe, and a QuantStudio 12K Flex real-time PCR system (manufactured by Thermo Fisher Scientific Co., Ltd.) was used. The PCR reaction is carried out according to the method described in the instruction manual, whereby the CFB gene and the glyceraldehyde 3-phosphate dehydrogenase (hereinafter referred to as gapdh) which is a constitutive gene are used. The gene was subjected to a PCR reaction, and the amount of mRNA amplification was measured, and the amount of mRNA amplification of gapdh was used as an internal control to calculate a quasi-quantitative value of CFB mRNA. The quasi-quantitative value of the CFB mRNA in the negative control which was similarly measured was set to 1, and the expression rate of CFB mRNA was determined from the quasi-quantitative value of the CFB mRNA. The results of the expression rate of the obtained CFB mRNA are shown in Table S1. According to Table S1, each nucleic acid complex inhibits the expression of the mRNA of the CFB gene after being added to human primary hepatocytes.

[Table 34]

Test Example 2 In vitro attenuation test of nucleic acid complexes on mouse primary hepatocytes The respective nucleic acid complexes obtained in Examples 10, 11, 14, and 15 were assayed for in vitro attenuating activity in primary hepatocytes of mice. . Each nucleic acid complex diluted with Opti-MEM (manufactured by Thermo Fisher Scientific, catalog No. 31985) at a final concentration of 30, 10, 3 or 1 nmol/L was dispensed to a 96-well culture plate at 20 μL per well. Thereafter, Williams E medium containing Primary Hepatocyte Thawing and Plating Supplements (manufactured by Thermo Fisher Scientific, catalog number CM3000) was inoculated in such a manner that the number of cells became 10000 cells/80 μL/well. CD-1-derived mouse primary hepatocytes (manufactured by Thermo Fisher Scientific, catalog number MSCP10) suspended in (William's E Medium) (manufactured by Thermo Fisher Scientific, catalog number A12176-01) at 37 ° C, 5% After 6 hours of incubation under CO ₂ 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. Each nucleic acid complex was administered to mouse primary hepatocytes. Further, as a negative control group, cells which were not treated were inoculated. The cells to which the respective nucleic acid complexes were added were cultured in a 5% CO ₂ incubator at 37 ° C for 18 hours, and washed with an ice bath-cooled phosphate buffered saline (DPBS) (manufactured by Nacalai Tesque Co., Ltd.) to use SuperPrep cells. The decomposition & qPCR reverse transcription kit (manufactured by Toyobo Co., Ltd., catalog number SCQ-201) was used to recover total RNA according to the method described in the accompanying instructions, and cDNA was produced by reverse transcription reaction using the obtained total RNA as a template. Using the obtained cDNA as a template, a Taqman (registered trademark) gene expression analysis probe (manufactured by Thermo Fisher Scientific Co., Ltd.) was used as a probe, and a QuantStudio 12K Flex real-time PCR system (manufactured by Thermo Fisher Scientific Co., Ltd.) was used, according to the accompanying instruction manual. The PCR reaction is carried out by the method described in the above, whereby the CFB gene and the glyceraldehyde-3-phosphate dehydrogenase (hereinafter referred to as the gapdh) gene which is a constitutive gene are subjected to a PCR reaction, respectively. The amount of mRNA amplification was measured, and the amount of mRNA amplification of gapdh was used as an internal control, and the quasi-quantitative value of the mRNA of CFB was calculated. The quasi-quantitative value of the CFB mRNA in the negative control which was similarly measured was set to 1, and the expression rate of CFB mRNA was determined from the quasi-quantitative value of the CFB mRNA. The expression rate of the obtained CFB mRNA is shown in Table S2.

[Table 35]

According to Table S2, each nucleic acid complex inhibits the expression of mRNA of the CFB gene after being added to primary mouse hepatocytes.

Test Example 3 In Vivo Attenuation Test of Nucleic Acid Complexes in Mice Each of the nucleic acid complexes of Table S3 was subjected to a mouse in vivo attenuation test by the following method. In addition, each nucleic acid complex was diluted with phosphate buffered physiological saline (DPBS) (manufactured by Nacalai Tesque Co., Ltd.) and used in the test. After domestication and breeding of mice (BALB/cA, purchased from CLEA Japan), 30 mg/kg, 10 mg/kg, 3 mg/kg or 0.3 mg/kg were administered subcutaneously to each mouse. Nucleic acid complex. Further, as a control group, DPBS was administered only by subcutaneous injection into mice. Three days after the administration, euthanasia was performed, and the liver was removed and stored frozen in liquid nitrogen. The liver was frozen using Trizol (registered trademark) RNA isolation reagent (RNA Isolation Reagent) (manufactured by Thermo Fisher Scientific, catalog No. 15596026) and MagNA Pure 96 (manufactured by Roche Life Science) according to the method described in the accompanying instructions of the product. The sample recovers total RNA. Further, using the Transcriptor First Strand cDNA synthesis kit (Roche Life Science, catalog number 04897030001), the total RNA obtained was used as the method described in the accompanying instructions of the product. cDNA was produced by reverse transcription of the template. Using the obtained cDNA as a template, a Taqman (registered trademark) gene expression analysis probe (manufactured by Thermo Fisher Scientific Co., Ltd.) was used as a probe, and a QuantStudio 12K Flex real-time PCR system (manufactured by Thermo Fisher Scientific Co., Ltd.) was used, according to the accompanying instruction manual. The PCR reaction was carried out by the method described above, whereby the CFB gene and the gapdh gene were subjected to a PCR reaction, and the amount of mRNA amplification was measured, and the amount of mRNA amplification of gapdh was used as an internal control to calculate a quasi-quantitative value of CFB mRNA. The quasi-quantitative value of the CFB mRNA in the PBS administration group measured in the same manner was set to 1, and the expression rate of CFB mRNA was determined. The expression rate of the obtained CFB mRNA is shown in Table S3.

[Table 36]

In the table, N.T. indicates that no evaluation has been made.

According to Table S3, the nucleic acid complex of the present invention was administered to mice to reduce the expression of the CFB gene in the liver.

Test Example 4 In vitro attenuation test of nucleic acid complexes on rat primary hepatocytes The in vitro attenuating activity in rat primary hepatocytes was measured for each nucleic acid complex shown in Table S4. Each nucleic acid complex diluted with Opti-MEM (Thermo Fisher Scientific, Cat. No. 31985) at a final concentration of 30, 10, 3 or 1 nmol/L was dispensed into a 96-well culture plate after 20 μL per well. Williams E medium containing primary Hepatocyte Thawing and Plating Supplements (manufactured by Thermo Fisher Scientific, catalog number CM3000) was inoculated with a cell number of 10,000 cells/80 μL/well ( Rat primary hepatocytes (manufactured by Thermo Fisher Scientific, catalog number RTCP10) suspended in Thermo Fisher Scientific, catalog number A12176-01), carefully removed at 37 ° C, 5% CO ₂ for 6 hours, carefully removed The supernatant was cultured, and Williams E medium containing Primary Hepatocyte Maintenance Supplements (manufactured by Thermo Fisher Scientific, catalog number CM4000) was added, and each nucleic acid complex was administered to rat primary hepatocytes. . Further, as a negative control group, cells which were not treated were inoculated. The cells to which the respective nucleic acid complexes were added were cultured in a 5% CO ₂ incubator at 37 ° C for 18 hours, and washed with an ice bath-cooled phosphate buffered saline (DPBS) (manufactured by Nacalai Tesque Co., Ltd.) to use SuperPrep cells. The Cell Lysis & RT Kit for qPCR (manufactured by Toyobo Co., Ltd., catalog number SCQ-201), the total RNA was recovered according to the method described in the accompanying instructions of the product, and the total RNA obtained was used as the cDNA was produced by reverse transcription of the template. Using the obtained cDNA as a template, a Taqman (registered trademark) gene expression analysis probe (manufactured by Thermo Fisher Scientific Co., Ltd.) was used as a probe, and a QuantStudio 12K Flex real-time PCR system (manufactured by Thermo Fisher Scientific Co., Ltd.) was used, according to the accompanying instruction manual. The PCR reaction is carried out by the method described in the above, whereby the CFB gene and the glyceraldehyde-3-phosphate dehydrogenase (hereinafter referred to as the gapdh) gene which is a constitutive gene are subjected to a PCR reaction, respectively. The amount of mRNA amplification was measured, and the amount of mRNA amplification of gapdh was used as an internal control, and the quasi-quantitative value of the mRNA of CFB was calculated. The quasi-quantitative value of the CFB mRNA in the negative control which was similarly measured was set to 1, and the expression rate of CFB mRNA was determined. The results of the expression rate of the obtained CFB mRNA are shown in Table S4. As can be seen from Table S4, each nucleic acid complex inhibits the expression of the mRNA of the CFB gene after addition to rat primary hepatocytes.

[Table 37]

Test Example 5 In vivo activity of nucleic acid complexes in rats The rat in vivo attenuation test was carried out by the following methods for each of the nucleic acid complexes of Table S5. In addition, each nucleic acid complex was diluted with phosphate buffered physiological saline (DPBS) (manufactured by Nacalai Tesque Co., Ltd.) and used in the test. After domestication and breeding of rats (WKY, purchased from Charles River Japan Co., Ltd.), each nucleic acid complex was administered subcutaneously at a dose of 10 mg/kg in each rat. Further, as a control group, DPBS was administered only by subcutaneous injection into rats. Three days after the administration, euthanasia was performed, and the liver was removed and stored frozen in liquid nitrogen. Total RNA was collected from frozen samples of liver using Trizol (registered trademark) RNA isolation reagent (manufactured by Thermo Fisher Scientific, catalog No. 15596026) and MagNA Pure 96 (manufactured by Roche Life Science) according to the method described in the accompanying instructions. Further, using the transcript first cDNA synthesis kit (manufactured by Roche Life Science, catalog number 04897030001), cDNA was produced by reverse transcription reaction using the obtained total RNA as a template according to the method described in the accompanying instructions of the product. . Using the obtained cDNA as a template, a Taqman (registered trademark) gene expression analysis probe (manufactured by Thermo Fisher Scientific Co., Ltd.) was used as a probe, and a QuantStudio 12K Flex real-time PCR system (manufactured by Thermo Fisher Scientific Co., Ltd.) was used, according to the accompanying instruction manual. The PCR reaction was carried out by the method described above, whereby the CFB gene and the gapdh gene were subjected to a PCR reaction, and the amount of mRNA amplification was measured, and the amount of mRNA amplification of CFB was used as an internal control to calculate a quasi-quantitative value of CFB mRNA. The quasi-quantitative value of the CFB mRNA in the PBS-administered group was determined to be 1, and the expression rate of the CFB mRNA was determined from the quasi-quantitative value of the CFB mRNA. The expression rate of the obtained CFB mRNA is shown in Table S5.

[Table 38]

As can be seen from Table S5, the nucleic acid complex of the present invention was administered to rats to reduce the expression of the CFB gene in the liver. [Industrial availability]

The nucleic acid complex of the present invention can be used for administering a mammal to treat a CFB-related disease in vivo.

Claims (26)

A nucleic acid complex represented by the following formula 1, Formula 1: [Chemical Formula 1] (In Formula 1, 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, and the strand 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 CFB mRNA sequences described in Tables 1-1 to 1-3, and the 3' end or the 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). The nucleic acid complex of claim 1, which has a structure represented by the following formula 2, and formula 2: [Chemical 2] (In Formula 2, X, L1, L2, and S3 are respectively the same as defined above, and P1, P2, P3, P4, P5, and P6, and T1 and T2 are independent or absent, respectively, or -CO-, -NH- , -O-, -S-, -O-CO-, -S-CO-, -NH-CO-, -CO-O-, -CO-S- or -CO-NH-, Q1, Q2, Q3 And Q4 are independently 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 The integers, B1 and B2 are each independently a bond key, or any structure represented by the following formula 2-1, and the black dots at the ends in each structure are respectively a bond with P2 or P3 or with P5 or P6. The nodes, m1, m2, m3, and m4 are each independently an integer of 0 to 10, and Equation 2-1: [Chem. 3] P1 and p2 are each independently an integer of 1, 2 or 3, and q1, q2, q3 and q4 are each independently an integer of 0 to 10, wherein each of P3 and P6 is when p1 and p2 are integers of 2 or 3, respectively. 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]- may be the same or different). The nucleic acid complex of claim 2, wherein P1 and P4 are independently -CO-NH-, -NH-CO- or -O-, respectively. The nucleic acid complex of claim 2 or 3, wherein -[P2-Q1] _q1 - and -[P5-Q3] _q3 - are each independently or absent, or are represented by the following formula 3-1 to formula 3-3 Any structure expressed, Equation 3-1: [Chem. 4] Equation 3-2: [Chemical 5] Equation 3-3: [Chem. 6] (In the formula 3-1 to the formula 3-3, m5 and m6 are each independently an integer of 0 to 10, and the black dots at the ends of the structures of the formulas 3-1 to 3-3 are respectively B1 or B2, or Bond node with P1 or P4). The nucleic acid complex according to any one of claims 2 to 4, which has any one of the following formulas 4-1 to 4-9, and formula 4-1: [Chem. 7] Equation 4-2: [Chem. 8] Equation 4-3: [Chem. 9] Equation 4-4: [10] Equation 4-5: [Chem. 11] Equation 4-6: [Chem. 12] Equation 4-7: [Chem. 13] Equation 4-8: [Chem. 14] Equation 4-9: [Chem. 15] (In the 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). A nucleic acid complex according to claim 1, which has a structure represented by the following formula 5, and a formula 5: (In the formula 5, X, S3, P1, P2, P3, Q1, Q2, B1, T1, L1, p1, q1, and q2 are respectively the same as defined above). The nucleic acid complex of claim 6, wherein P1 is -CO-NH-, -NH-CO- or -O-. The nucleic acid complex of claim 6 or 7, which has any of the structures represented by the following formulas 6-1 to 6-9, and formula 6-1: [Chem. 17] Equation 6-2: [Chem. 18] Equation 6-3: [Chem. 19] Equation 6-4: [Chem. 20] Equation 6-5: [Chem. 21] Equation 6-6: [Chem. 22] Equation 6-7: [Chem. 23] Equation 6-8: [Chem. 24] Equation 6-9: [Chem. 25] (In the formulae 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 following formulas 7-1 to 7-9, and formula 7-1: [Chem. 26] Equation 7-2: [Chem. 27] Equation 7-3: [Chem. 28] Equation 7-4: [Chem. 29] Equation 7-5: [Chem. 30] Equation 7-6: [Chem. 31] Equation 7-7: [Chem. 32] Equation 7-8: [Chem. 33] Equation 7-9: [Chem. 34] (In the 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 sugar ligand is N-ethylmercapto galactosamine. The nucleic acid complex of any one of claims 1 to 10, wherein the double-stranded nucleic acid comprises a modified nucleotide. The nucleic acid complex of 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 of 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, which has the structure represented by the following formula 7-8-1, and the formula 7-8-1: [Chem. 35] (In the formula 7-8-1, X has the same meaning as described above). The nucleic acid complex according to any one of claims 1 to 14, wherein X is a pair of justice shares selected from the group consisting of the justice stocks/antisense stocks listed in Tables 1-1 to 1-3. Anti-sense stocks. The nucleic acid complex according to any one of claims 1 to 14, wherein X is a pair of justice shares selected from the group consisting of the justice stocks/antisense stocks listed in Tables M1-1 to M1-3/ Anti-sense stocks. The nucleic acid complex according to any one of claims 1 to 14, wherein X is a pair of justice shares/antisense shares selected from the group consisting of the justice shares/antisense stocks listed in Table M1-4. The nucleic acid complex of claim 17, wherein X is a pair of sense strands/antisense strands represented by EB0125 as described in Table M1-4. A pharmaceutical composition comprising the nucleic acid complex of any one of claims 1 to 18. The pharmaceutical composition of claim 19, which is for introduction into a cell. A pharmaceutical composition according to claim 19 or 20 which is administered intravenously or subcutaneously. A method of treating or preventing a disease, which comprises administering a nucleic acid complex according to any one of claims 1 to 18 or a pharmaceutical composition according to any one of claims 19 to 21 to a patient in need thereof. A method of inhibiting the expression of a CFB gene, which comprises introducing a double-stranded nucleic acid into a cell using the nucleic acid complex according to any one of claims 1 to 18 or the pharmaceutical composition according to any one of claims 19 to 21. A method of treating a CFB-associated disease, which comprises administering a nucleic acid complex according to any one of claims 1 to 18 or a pharmaceutical composition according to any one of claims 19 to 21 to a mammal. A pharmaceutical composition for the treatment of a CFB-related disease, comprising the nucleic acid complex of any one of claims 1 to 18, or the pharmaceutical composition according to any one of claims 19 to 21. A therapeutic agent for a CFB-associated disease, comprising the nucleic acid complex of any one of claims 1 to 18, or the pharmaceutical composition according to any one of claims 19 to 21.