Nucleic acid complex and pharmaceutical composition containing the same

Abstract

Disclosed is a nucleic acid complex represented by formula (I) or a pharmaceutically acceptable salt thereof (X is CH2 or O; Y is a sugar ligand having mannose or GalNac; n is an integer of 1 to 8; and Z is a group containing an oligonucleotide).

Description

[Technical field]

[0001] The present invention relates to a nucleic acid complex and a pharmaceutical composition containing the nucleic acid complex. [Background technology]

[0002] Known nucleic acid drugs include antisense, decoy nucleic acid, ribozyme, aptamer, siRNA, miRNA, and messenger RNA (mRNA). Among them, nucleic acid drugs acting on mRNA are expected to be clinically applied to diseases that have been difficult to treat so far because of their high versatility in controlling any gene in the cell. In other words, nucleic acid drugs are expected to be the next generation of drugs after small molecules and antibody drugs. However, problems with nucleic acid drugs include the difficulty of delivering them into cells, which are the site of action of nucleic acid drugs, due to the relatively large size of the nucleic acid drug, the extremely low cell membrane permeability due to the negative charge of the phosphate backbone, and the degradation of siRNA by nucleases in the blood (Non-Patent Document 1).

[0003] In order to solve such problems, a method of applying a delivery means to a nucleic acid drug has been adopted. As one of the delivery means, a nucleic acid complex (conjugated nucleic acid) of a targeting ligand and a nucleic acid has been reported. The targeting ligand can be in the form of binding to a receptor expressed on the cell surface. For example, several nucleic acid complexes using N-acetyl-D-galactosamine (GalNAc) or the like have been reported as a ligand for the asialoglycoprotein receptor (ASGPR) highly expressed in hepatic parenchymal cells (Patent Document 1, Patent Document 2, Patent Document 3, Non-Patent Document 2). Furthermore, mannose conjugates and mannosylated nucleic acid complexes have been reported as drug carriers for delivery to the mannose receptor (CD206) highly expressed in immune cells such as macrophages and dendritic cells (Patent Document 4). It has also been reported that lipophilic compounds such as cholesterol and fatty acids can be modified into nucleic acid drugs to increase their affinity with lipoproteins in plasma, thereby achieving delivery to cells expressing the corresponding lipoprotein receptors (LDL receptor, HDL receptor, scavenger receptor SRB1) (Non-Patent Document 3). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2009 / 073809 [Patent Document 2] International Publication No. 2014 / 179620 [Patent Document 3] International Publication No. 2016 / 100401 [Patent Document 4] International Publication No. 2018 / 004004 [Non-patent literature]

[0005] [Non-Patent Document 1] Nature Reviews Drug Discovery. 8, 129-138 (2009). [Non-Patent Document 2] J. Am. Chem. Soc. 2014,136, 16958-16961. [Non-Patent Document 3] Nucleic Acids Research, 2019, Vol.47, No.3,1082-1096. Summary of the Invention [Problem to be solved by the invention]

[0006] Many targeting ligands and nucleic acid complexes containing ligands have been proposed to date, but at present, no fully satisfactory ligands have been found that are capable of improving specific binding to receptors or enhancing the cellular uptake of nucleic acid drugs. [Means for solving the problem]

[0007] The present inventors have conducted extensive research to achieve the above object, and as a result have discovered a nucleic acid complex that is selectively taken up by cells. That is, the present invention relates to the following [1] to

[27] . [1] Formula (I): [ka] [X is CH 2 or O; Y is a sugar ligand having mannose or GalNac; n represents an integer from 1 to 8; Z is a group that comprises an oligonucleotide. or a pharma- ceutically acceptable salt thereof. [2] Formula (II): [ka] [Y is a sugar ligand having mannose or GalNac; Z is a group that comprises an oligonucleotide. or a pharma- ceutically acceptable salt thereof. [3] Formula (III): [ka] [Z is a group that includes an oligonucleotide.] or a pharma- ceutically acceptable salt thereof. [4] Formula (IV): [ka] [Z is a group that includes an oligonucleotide.] or a pharma- ceutically acceptable salt thereof. [5] Formula (V): [ka] [Z is a group that includes an oligonucleotide.] or a pharma- ceutically acceptable salt thereof. [6] Formula (VI): [ka] [Z is a group that includes an oligonucleotide.] or a pharma- ceutically acceptable salt thereof. [7] A nucleic acid complex represented by the following formula (VII): or a pharma- ceutically acceptable salt thereof. [ka] [Z is a group that includes an oligonucleotide.] or a pharma- ceutically acceptable salt thereof. [8] Formula (VIII): [ka] [Z is a group that includes an oligonucleotide.] or a pharma- ceutically acceptable salt thereof. [9] Formula (IX): [ka] [Z is a group that includes an oligonucleotide.] or a pharma- ceutically acceptable salt thereof.

[10] Formula (X): [ka] [Z is a group that includes an oligonucleotide.] or a pharma- ceutically acceptable salt thereof.

[11] Formula (XI): [ka] [Z is a group that includes an oligonucleotide.] or a pharma- ceutically acceptable salt thereof.

[12] Formula (XII): [ka] [Z is a group that includes an oligonucleotide.] or a pharma- ceutically acceptable salt thereof.

[13] Formula (XIII): [ka] [Z is a group that includes an oligonucleotide.] or a pharma- ceutically acceptable salt thereof.

[14] Formula (XIV): [ka] [Z is a group that includes an oligonucleotide.] or a pharma- ceutically acceptable salt thereof.

[15] The nucleic acid complex according to any one of [1] to

[14] above, wherein the oligonucleotide is single-stranded.

[16] The nucleic acid complex of

[15] above, wherein the oligonucleotide is bound via the 3' end.

[17] The nucleic acid complex of

[15] above, wherein the oligonucleotide is bound via the 5' end.

[18] The nucleic acid complex according to any one of [1] to

[14] above, wherein the oligonucleotide is double-stranded.

[19] The nucleic acid complex of

[18] above, wherein the oligonucleotide is bound via the 3' end of one strand.

[20] The nucleic acid complex of

[18] above, wherein the oligonucleotide is bound via the 5' end of one strand.

[21] A pharmaceutical composition comprising the nucleic acid complex according to any one of [1] to

[20] above.

[22] The pharmaceutical composition described in

[21] above, which regulates the expression of a target gene in a cell.

[23] The pharmaceutical composition according to

[22] above, wherein the cell is a dendritic cell, a macrophage or a hepatic parenchymal cell.

[24] A nucleic acid complex according to any one of [1] to

[20] above, for use in a method for regulating expression of a target gene in a cell.

[25] The nucleic acid complex according to

[24] above, wherein the cell is a dendritic cell, a macrophage or a hepatic parenchymal cell.

[26] A method for regulating expression of a target gene in a cell of a subject, comprising administering to the subject the nucleic acid complex described in any one of [1] to

[20] above or the pharmaceutical composition described in

[21] above.

[27] The method according to

[26] above, wherein the cell is a dendritic cell, a macrophage or a hepatic parenchymal cell. Effect of the Invention

[0008] As shown in the pharmacological test described below, the nucleic acid complex according to the present invention is selectively taken up by cells expressing mannose receptor or ASGPR. By administering a pharmaceutical composition containing the nucleic acid complex according to the present invention to a mammal (including a human), various related diseases may be cured. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] The present invention will be described in detail below.

[0010] The structural formula of the compound in this specification may conveniently represent a certain isomer, but is not limited to the description of the formula for convenience, and includes all isomers and isomer mixtures such as geometric isomers, optical isomers, rotational isomers, stereoisomers, tautomers, etc. that arise from the structure of the compound, and may be either one isomer or a mixture containing each isomer in any ratio. Thus, for example, the compound in this specification may have optical isomers and racemates, but is not limited to either in this specification, and may be a racemate, any of the optically active substances, or a mixture containing each optically active substance in any ratio.

[0011] In addition, the compounds in the present specification may exist in the form of crystalline polymorphs, but are not limited to any of them, and may be a single crystal form or a mixture of any of them. In addition, the compounds in the present specification also include amorphous forms, and further, the compounds in the present specification include anhydrates and solvates (particularly hydrates).

[0012] The compounds herein also include isotopically labeled compounds of the compounds. Isotopically labeled compounds are identical to the compounds except that one or more atoms are replaced with an atom having an atomic mass or mass number different from the atomic mass or mass number normally found in nature. Isotopes that can be incorporated into the compounds herein include, for example, isotopes of hydrogen, carbon, nitrogen, oxygen, fluorine, phosphorus, sulfur, iodine, and chlorine. 2 H, 3 H, 11 C. 14 C. 15 N, 18 O. 18 F and 35 Includes S.

[0013] In this specification, the term "pharmaceutical acceptable salt" is not particularly limited as long as it is a salt that forms a salt with a compound, and specific examples thereof include acid addition salts, metal salts, ammonium salts, organic amine addition salts, amino acid addition salts, etc.

[0014] Preferred examples of the acid addition salts include inorganic acid salts such as hydrochloride, hydrobromide, sulfate, nitrate, and phosphate, and organic acid salts such as acetate, succinate, fumarate, maleate, tartrate, citrate, lactate, stearate, benzoate, methanesulfonate, p-toluenesulfonate, and benzenesulfonate. Preferred examples of the metal salt include alkali metal salts such as sodium salts and potassium salts, alkaline earth metal salts such as magnesium salts and calcium salts, aluminum salts, and zinc salts. Preferred examples of the ammonium salt include ammonium and tetramethylammonium salts. Preferred examples of the organic amine addition salts include addition salts of morpholine, piperidine, and the like. Preferred examples of amino acid addition salts include addition salts of lysine, glycine, phenylalanine, aspartic acid, glutamic acid, and the like.

[0015] When the compounds in the present specification are obtained in a free form, they can be converted into a salt which the compounds may form or into a hydrate thereof in a conventional manner.

[0016] When the compounds herein are obtained as salts or hydrates, they can be converted to the free forms in a conventional manner.

[0017] Furthermore, various isomers (e.g., geometric isomers, optical isomers, rotamers, stereoisomers, tautomers, etc.) obtained with respect to the compounds in the present specification can be purified and isolated by using conventional separation means, for example, recrystallization, a diastereomeric salt method, an enzymatic resolution method, various types of chromatography (e.g., thin layer chromatography, column chromatography, gas chromatography, high performance liquid chromatography, etc.).

[0018] The pharmaceutical composition of the present invention can be produced by mixing a pharma- ceutical acceptable additive with the nucleic acid complex or a pharma- ceutical acceptable salt thereof. The pharmaceutical composition of the present invention can be produced according to a known method, for example, the method described in the General Provisions for Preparations of the Japanese Pharmacopoeia, 17th Edition.

[0019] The pharmaceutical composition according to the present invention can be administered to a patient appropriately depending on its dosage form.

[0020] The dosage of the nucleic acid complex of the present invention varies depending on the severity of symptoms, age, sex, body weight, dosage form, type of salt, specific type of disease, etc., but typically, for adults, the daily oral dosage converted into oligonucleotide is about 30 μg to 10 g, preferably 100 μg to 1 g, and by injection about 1 μg to 1 g, preferably 100 μg to 300 mg, administered once or in divided doses.

[0021] The nucleic acid complex of the present invention is a nucleic acid complex in which a sugar ligand is bound to an oligonucleotide via a linker, and the sugar ligand has an O-, N-, or C-linkage, preferably an O-linked mannose or GalNac. That is, the nucleic acid complex of the present invention has a structure of (sugar ligand)-(linker)-(oligonucleotide).

[0022] In one embodiment, the nucleic acid complex contains two or more sugars, preferably three or four sugars, hi one embodiment, the nucleic acid complex contains at least three Mannoses or at least three GalNacs, which can be targeted and delivered to macrophages or hepatocytes, respectively.

[0023] <About sugar ligands> Mannose receptors are highly expressed on certain cells, such as macrophages and dendritic cells, where CD206 is highly expressed. Mannose conjugates and mannosylated drug carriers exhibit high binding affinity to CD206 and have been successfully used to deliver drug molecules, such as oligonucleotides, to cells, such as macrophages and dendritic cells.

[0024] ASGPR is highly expressed in certain cells, such as hepatocytes. GalNac conjugates and GalNacylated drug carriers exhibit high binding affinity to ASGPR and have been successfully used to deliver drug molecules, such as oligonucleotides, to cells, such as hepatocytes.

[0025] The sugar ligand refers to a group derived from a sugar capable of binding to a receptor expressed in a target cell, and the following structure can be exemplified as one preferred embodiment of the sugar ligand in the nucleic acid complex of the present invention. The wavy line represents a bond to the linker. [ka] [ka]

[0026] <About Oligonucleotides> As the oligonucleotide in the nucleic acid complex according to the present invention, an oligonucleotide known to be used as a nucleic acid drug can be used. In this specification, the nucleic acid drug refers to a nucleotide used as an antisense, a decoy nucleic acid, a ribozyme, an siRNA, an miRNA, an antimiRNA, an mRNA, etc.

[0027] Additionally, the oligonucleotide may be a single-stranded or double-stranded oligonucleotide.

[0028] The linker and oligonucleotide in the nucleic acid complex according to the present invention may be bound to a nucleotide such as an oligonucleotide, for example, at the 3' or 5' end of the oligonucleotide. When the oligonucleotide is double-stranded, the linker is preferably bound to the 3' or 5' end of the sense strand constituting the double-stranded nucleic acid, but is not limited to this bond.

[0029] The number of linkers to which the oligonucleotide is bound in the nucleic acid complex is not limited to one, and may be two or more.

[0030] In some embodiments, it is possible to include particular bases in the overhangs or to include modified nucleotides or nucleotide surrogates in the single-stranded overhangs (e.g., the 5' overhang or the 3' overhang, or both), for example to enhance stability.

[0031] The oligonucleotide constituting the nucleic acid complex of the present invention may be in any form so long as it has the ability to control the expression of a target gene when introduced into a mammalian cell, and a single-stranded oligonucleotide or a double-stranded oligonucleotide is preferably used.

[0032] The oligonucleotide may be any molecule that is a polymer of nucleotides or molecules having a function equivalent to that of nucleotides, such as DNA, which is a polymer of deoxyribonucleotides, RNA, which is a polymer of ribonucleotides, and chimeric nucleic acids, which are polymers of DNA and RNA. In addition, in DNA, RNA, and chimeric nucleic acids, it may be a nucleotide polymer in which at least one nucleotide such as a deoxyribonucleotide or ribonucleotide is replaced with a molecule having a function equivalent to that of a nucleotide. Note that uracil (U) in RNA is unambiguously read as thymine (T) in DNA.

[0033] Molecules having the same functions as nucleotides include, for example, nucleotide derivatives in which nucleotides have been modified. For example, compared to DNA or RNA, molecules in which deoxyribonucleotides or ribonucleotides have been modified are preferably used in order to improve or stabilize nuclease resistance, increase affinity with complementary nucleic acid, increase cell permeability, or enable visualization.

[0034] Examples of nucleotide derivatives include sugar-modified nucleotides, phosphodiester bond-modified nucleotides, and base-modified nucleotides, ie, nucleotides in which at least one of the sugar moiety, the phosphodiester bond, and the base is modified.

[0035] The sugar-modified nucleotide may be any nucleotide in which part or all of the chemical structure of the sugar of the nucleotide is modified or substituted with any substituent, or substituted with any atom, but 2'-modified nucleotides are preferably used.

[0036] Examples of 2'-modified nucleotides include those in which the 2'-OH group of ribose is OR, R, R'OR, SH, SR, or NH 2 , N.H.R., N.R. 2 , N 3 , CN, F, Cl, Br and I (wherein R is alkyl or aryl, preferably alkyl having 1 to 6 carbon atoms, and R' is alkylene, preferably alkylene having 1 to 6 carbon atoms), and preferred examples of the 2'-modification include substitution with F, a methoxy group and an ethoxy group. Alternatively, the 2'-modified nucleotide may be a locked nucleic acid, in which the oxygen atom at the 2' position of ribose and the carbon at the 4' position are bridged with methylene.

[0037] The phosphodiester bond-modified nucleotide may be any nucleotide in which a part or all of the chemical structure of the phosphodiester bond of a nucleotide has been modified or substituted with any substituent, or substituted with any atom, and examples thereof include a nucleotide in which a phosphodiester bond has been substituted with a phosphorothioate bond, a nucleotide in which a phosphodiester bond has been substituted with a phosphorodithioate bond, a nucleotide in which a phosphodiester bond has been substituted with an alkylphosphonate bond, and a nucleotide in which a phosphodiester bond has been substituted with a phosphoroamidate bond, and preferably a nucleotide in which a phosphodiester bond has been substituted with a phosphorothioate bond.

[0038] Oligonucleotides also include those in which some or all of the atoms in the molecule are substituted with atoms having different mass numbers (isotopes).

[0039] In one embodiment, the oligonucleotide in the nucleic acid complex of the invention modulates the expression of a target gene in a cell.

[0040] In one embodiment, the oligonucleotide in the nucleic acid complex of the invention is linked to the ligand (also referred to as linker-ligand) via a linker.

[0041] <About the linker> As the "linker" in the present invention, any linker that can be used in a nucleic acid complex can be used.

[0042] Examples of linker structures that can be adopted include the structures disclosed in WO 2009 / 073809, WO 2013 / 075035, and WO 2015 / 105083.

[0043] In one embodiment, the linker comprises at least one cleavable linking group.

[0044] A cleavable linking group is one that is sufficiently stable outside a cell but which is cleaved once inside a target cell to release the moiety to which the linker is attached.

[0045] For example, a phosphate-based cleavable linking group is cleaved by an agent that degrades or hydrolyzes the phosphate group. An example of an agent that cleaves a phosphate group within a cell is an enzyme such as a phosphatase. A preferred embodiment of a phosphate-based linking group is -OP(O)(OH)-O- or -OP(S)(OH)-O-.

[0046] An embodiment of the linker is any of the following structures, where X and Y are each independently at each occurrence; X is CH 2 or O, Y represents a sugar ligand, Z represents a group comprising an oligonucleotide, and n represents an integer from 1 to 8. [ka] EXAMPLES

[0047] The nucleic acid complex according to the present invention can be produced, for example, by the method described in the Production Examples below, and the effect of the compound can be confirmed by the method described in the Test Examples below. However, these are merely illustrative, and the present invention is not limited to the following specific examples in any case, and may be modified without departing from the scope of the present invention. The examples of the present invention can be produced using synthetic chemistry techniques known to those skilled in the art.

[0048] The abbreviations used in this specification are conventional abbreviations well known to those skilled in the art. Cbz: benzyloxycarbonyl CTC: 2-chlorotrityl chloride DCE: 1,2-dichloroethane DCM: dichloromethane DIPEA: N,N-diisopropylethylamine DMF: N,N-dimethylformamide DMSO: Dimethyl sulfoxide EDCI: 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride EtOAc: ethyl acetate ESI: electrospray ionization Fmoc: 9-fluorenylmethyloxycarbonyl HBTU: 1-[bis(dimethylamino)methylene]-1H-benzotriazolium 3-oxide hexafluorophosphate HOBT: 1-hydroxybenzotriazole IMS: Industrial denatured alcohol MALDI-TOF-MS: Matrix-assisted laser desorption / ionization-time of flight mass spectrometry MeOH: Methanol MTBE: Methyl tertiary butyl ether NaOMe: Sodium methoxide TFA: Trifluoroacetic acid 1 H-NMR: Proton nuclear magnetic resonance spectrometry MS: Mass spectrometry HPLC: High-performance liquid chromatography

[0049] In the following Examples and Preparation Examples, "room temperature" generally refers to about 10° C. to about 35° C. Unless otherwise specified, % refers to weight or volume percent.

[0050] Proton nuclear magnetic resonance spectra chemical shifts are reported in δ units (ppm) relative to tetramethylsilane, coupling constants are reported in Hertz (Hz), patterns are s: singlet, d: doublet, t: triplet, q: quartet, quin: quintet, m: multiplet, br: broad, br.s: broad singlet.

[0051] Regarding silica gel column chromatography, silica gel was Merck's Silica Gel 60 (70-230 mesh or 230-400 mesh ASTM), Fuji Silysia Chemical's PSQ60B, or a prepacked column (column: Yamazen's Hi-Flash TM Column (Silicagel) or Biotage TM A SNAP Ultra Silica Cartridge was used.

[0052] Compound names were as displayed in "E-Notebook" version 12 or 13 (PerkinElmer) or "MarvinSketch" version 16 (ChemAxon), except for commonly used reagents.

[0053] The compounds can be produced by methods known to those skilled in the art with reference to the following Production Examples.

[0054] Synthesis scheme of compound 6 [ka]

[0055] Synthesis of compound 2 [ka]

[0056] DMF (200 mL) was added to hydrazine acetate (5.19 g, 56.4 mmol), and the mixture was stirred at 55° C. The reaction mixture was cooled to 15° C., and 1-O,2-O,3-O,4-O,6-O-pentaacetyl-β-D-mannopyranose (compound 1) (20.0 g, 51.2 mmol) was added, followed by stirring at 15° C. for 16 hours. The reaction mixture was added to water and extracted with EtOAc. The combined organic layer was washed with saturated saline, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give compound 2 (14.3 g). 1 H-NMR (400MHz, CDCl 3 )δ(ppm):1.97-2.00 (m, 3H), 2.03 (s, 3H), 2.08 (s, 3H), 2.14 (s, 3H), 4.11-4.15 (m, 1H), 4.16-4.32 (m, 3H), 5.14-5.34 (m, 3H), 5.37-5.45 (m, 1H).

[0057] Synthesis of compound 3 [ka]

[0058] Compound 2 (5.00 g, 14.4 mmol), 2,2,2-trichloroacetonitrile (20.7 g, 143 mmol) and Cs in DCM (50 mL) 2 CO 3(5.14 g, 15.8 mmol) was added and stirred at 25° C. for 2 hours. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (50:1-20:1 petroleum ether / EtOAc) to give compound 3 (2.70 g). 1 H-NMR (400MHz, CDCl 3 )δ(ppm):2.01 (s, 3H), 2.04 (s, 2H), 2.08 (d, J=6.8Hz, 6H), 2.20 (s, 3H), 4.15-4.22 (m, 2H), 4.24-4.32 (m, 1H), 5.37-5.42 (m, 2H), 5.46-5.49 (m, 1H), 6.28 (d, J=1.8Hz, 1H), 8.79 (s, 1H).

[0059] Synthesis of compound 4 [ka]

[0060] Compound 3 (14.0 g, 28.4 mmol), 6-(Cbz-amino)-1-hexanol (8.57 g, 34.1 mmol) and molecular sieves 4A (10.0 g) were added to DCM (140 mL) and stirred at 0° C. for 30 min. Then, trimethylsilyl trifluoromethanesulfonate (6.32 g, 28.4 mmol) was added dropwise to the reaction mixture at −65° C., and the reaction mixture was stirred at 25° C. for 16 h. The reaction mixture was diluted with DCM and filtered, after which the filtrate was washed with saturated aqueous sodium bicarbonate, water and brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (50:1-20:1 petroleum ether / EtOAc) to give compound 4 (7.50 g).

[0061] Synthesis of compound 5 [ka]

[0062] Compound 4 (7.50 g, 12.9 mmol) was dissolved in MeOH (300 mL), NaOMe (2.79 g, 51.6 mmol) was added, and the mixture was allowed to react at room temperature for 3 hours. The reaction mixture was partially concentrated, poured into cold water, and acetic acid was added until the pH became 5. The resulting crude product was purified by silica gel column chromatography (50:1-20:1 DCM / MeOH) to obtain compound 5 (3.90 g). 1 H-NMR (400MHz, CDCl 3 )δ(ppm):1.26-1.63 (m, 8H), 3.14 (d, J=6.3Hz, 2H), 3.34 (d, J=8.8Hz, 1H), 3.54-3.74(m, 2H), 3.80-4.01 (m, 4H), 4.78 (s, 1H), 5.01-5.12 (m, 2H), 5.16-5.47 (m, 6H), 7.28-7.41 (m, 5H).

[0063] Synthesis of compound 6 [ka]

[0064] Dry 10% palladium-carbon (0.40 g) was added with MeOH (40 mL) and compound 5 (3.90 g, 9.43 mmol), and the mixture was stirred at 25° C. under hydrogen pressure (50 psi) for 3 hours. The reaction mixture was filtered and concentrated under reduced pressure to give compound 6 (1.50 g). 1 H-NMR(400MHz,MeOD)δ(ppm): 1.38-1.49 (m, 4H), 1.61 (dquin, J=13.3, 6.9, 6.9, 6.9, 6.9Hz, 4H), 2.73-2.88 (m, 2H), 3.31 (dt, J=3.3, 1.6Hz, 1H), 3.43 (dt, J=9.7, 6.1Hz, 1H), 3.48-3.89 (m, 8H).

[0065] Synthesis of compound 8 [ka]

[0066] To a solution of 6-azidohexanoic acid (compound 7) (50.2 mg, 319 μmol) and compound 6 (268 mg, 959 μmol) in DMF (3 mL), HOBt (129 mg, 959 μmol), DIPEA (248 mg, 1.92 mmol) and EDCI (183 mg, 959 μmol) were added and stirred at 25° C. for 3 hours. The reaction mixture was purified by preparative HPLC (TFA condition) to obtain compound 8 (20.1 mg). 1 H-NMR(400MHz,DMSO-d6)δ(ppm): 1.24-1.54 (m, 14H) 2.04 (t, J=7.40Hz, 2H) 3.00 (q, J=6.53Hz, 2H) 3.23-3.40 (m, 12H) 3.55-3.66 (m, 2H) 4.57 (s, 1H) 7.75 (br s, 1H)

[0067] Synthesis scheme of compound 11 [ka]

[0068] Synthesis of compound 10 [ka] To a mixture containing CTC resin (0.50 g, 0.50 mmol, 1.00 mmol / g) and N-[(9H-fluoren-9-ylmethoxy)carbonyl]-5-tert-butyl glutamate (compound 9) (212 mg, 0.50 mmol), DCM (30 mL) and DIPEA (258 mg, 2.00 mmol) were added, and the reaction mixture was stirred for 2 hours. MeOH was added to the reaction mixture (0.2 mL) and stirred for 30 minutes. 6-azidohexanoic acid (compound 7) (118 mg, 0.75 mmol), DIPEA (258 mg, 2.00 mmol), HBTU (270 mg, 712 umol), and DMF (3 mL) were added to the reaction mixture and stirred at 25° C. for 1 hour. 90% TFA / 10% DCM was added to the reaction mixture and stirred for 2 hours. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to give compound 10 (110 mg).

[0069] Synthesis of compound 11 [ka]

[0070] To a solution of compound 10 (50.0 mg, 174 μmol) and compound 6 (292 mg, 1.05 mmol) in DMF (3 mL), HOBt (141 mg, 1.05 mmol), DIPEA (270 mg, 2.10 mmol) and EDCI (200 mg, 1.05 mmol) were added. The reaction mixture was stirred at 25° C. for 16 hours. Purification by preparative HPLC (TFA condition) gave compound 11 (18.1 mg). 1 H-NMR (400MHz, DMSO-d 6 ) δ(ppm): 1.16-1.56 (m, 24H), 1.62-1.73 (m, 1H), 1.76-1.87 (m, 1H), 2.00-2.07 (m, 2H), 2.09-2.15 (m, 2H), 2.95-3.07 (m, 4H), 3.22-3.41 (m, 18H), 3.65 (br s, 6H), 4.10-4.19 (m, 1H), 4.57 (d, J=1.00Hz, 2H), 7.74-7.93 (m, 3H).

[0071] Synthesis of compound 12 [ka]

[0072] To CTC resin (1.00 g, 1.00 mmol, 1.00 mmol / g) and 5-tert-butyl N-[(9H-fluoren-9-ylmethoxy)carbonyl]-glutamate (compound 9) (511 mg, 1.20 mmol) were added DCM (20 mL), DIPEA (516 mg, 4.00 mmol), and the reaction mixture was diluted with N 2 The mixture was stirred for 2 hours with bubbling. MeOH (1 mL) was added to the reaction mixture and stirred for 30 minutes. A solution of 20% piperidine in DMF (30 mL) was added to the reaction mixture and stirred for 30 minutes, the resin was washed with DMF, and a solution of N-[(9H-fluoren-9-ylmethoxy)carbonyl]-glutamic acid 1-tert-butyl ester (N-Fmoc-glutamic acid-1-tert-butyl ester) (851 mg, 2.00 mmol), HBTU (720 mg, 1.90 mmol) and DIPEA (516 mg, 4.00 mmol) in DMF (5 mL) was added and stirred at 25° C. for 1 hour. A solution of 20% piperidine in DMF (30 mL) was added and stirred for 30 minutes, and the resin was washed with DMF. Then, a solution of 6-azidohexanoic acid (compound 7) (0.30 g, 1.91 mmol), HBTU (720 mg, 1.90 mmol) and DIPEA (516 mg, 4.00 mmol) in DMF (5 mL) was added and stirred at 25° C. for 1 hour, and the resin was washed with DMF, washed with MeOH and dried under reduced pressure. 50% TFA / 50% DCM was added to the resulting residue (0.60 g) and stirred for 2 hours, and the reaction mixture was concentrated under reduced pressure to give compound 12 (240 mg).

[0073] Synthesis of compound 13 [ka] Compound 13 was synthesized according to the synthesis method of compound 12, except that Azido-PEG8-acid (CAS No. 1214319-92-2) was used instead of 6-azidohexanoic acid to obtain compound 13 (350 mg). 1 H-NMR (400MHz, CDCl 3 ) δ(ppm): 2.01 (s, 2H), 2.18 (s, 2H), 2.32-2.63 (m, 6H), 3.40 (t, J=5.0Hz, 2H), 3.54-3.71 (m, 32H), 4.49 (s, 2H), 7.67-8.03 (m, 2H).

[0074] Synthesis of compound 14 [ka]

[0075] To a solution of compound 12 (0.20 g, 481 μmol) and compound 7 (1.08 g, 3.85 mmol) in DMF (0.5 mL), DIPEA (996 mg, 7.70 mmol), HOBt (520 mg, 3.85 mmol) and EDCI (738 mg, 3.85 mmol) were added. The reaction mixture was stirred at 25° C. for 16 hours and purified by preparative HPLC (TFA condition) to give compound 14 (103 mg). 1 H-NMR(400MHz,MeOD)δ(ppm): 1.26-1.72 (m, 32H), 1.82-1.97 (m, 2H), 2.03-2.13 (m, 2H), 2.22-2.41 (m, 6H), 3.14-3.24 (m, 6H), 3.37-3.47 (m, 3H), 3.49-3.55 (m, 3H), 3.60 (t, J=9.5Hz, 3H), 3.65-3.86 (m, 15H), 4.22-4.42 (m, 2H), 4.73 (s, 3H).

[0076] Synthesis of compound 15 [ka]

[0077] To a solution of compound 13 (0.20 g, 275 μmol) and compound 7 (616 mg, 2.20 mmol) in DMF (0.5 mL), DIPEA (570 mg, 4.41 mmol), HOBT (298 mg, 2.20 mmol) and EDCI (423 mg, 2.20 mmol) were added, and the reaction mixture was stirred for 16 hours at 25° C. The reaction mixture was purified by preparative HPLC (TFA condition) to give compound 15 (127 mg). 1 H-NMR(400MHz,MeOD)δ(ppm): 1.23-1.68 (m, 24H), 1.78-1.95 (m, 2H), 2.04-2.19 (m, 2H), 2.23-2.31 (m, 2H), 2.33-2.42 (m, 2H), 2.48-2.60 (m, 2H), 3.10-3.26 (m, 6H), 3.35-3.46 (m, 5H), 3.48-3.56 (m, 3H), 3.57-3.84 (m, 50H), 4.23-4.41 (m, 2H), 4.73 (s, 3H).

[0078] Synthesis scheme of compound 17 [ka]

[0079] Synthesis of compound 16 [ka] To CTC resin (0.50 mmol, 0.50 g, 1.00 mmol / g) and N-[(9H-fluoren-9-ylmethoxy)carbonyl]-5-tert-butyl glutamate (compound 9) (212 mg, 0.50 mmol) was added DCM (30 mL) and DIPEA (258 mg, 2.00 mmol). The reaction mixture was stirred for 2 h, after which MeOH (0.2 mL) was added and stirred for 30 min. To the reaction mixture, a solution of HBTU (360 mg, 950 μmol) and DIPEA (258 mg, 2.00 mmol) in DMF (3 mL) and N-[(9H-fluoren-9-ylmethoxy)carbonyl]-glutamic acid 1-tert-butyl ester (N-Fmoc-glutamic acid-1-tert-butyl ester) (425 mg, 1.00 mmol) were added, and the mixture was stirred at 25 ° C. for 1 hour, followed by addition of a solution of N-[(9H-fluoren-9-ylmethoxy)carbonyl]-glutamic acid 1-tert-butyl ester (N-Fmoc-glutamic acid-1-tert-butyl ester) (425 mg, 1.00 mmol), HBTU (360 mg, 950 μmol) and DIPEA (258 mg, 2.00 mmol) in DMF (3 mL) at 25 ° C. for 1 hour. Next, 6-azidohexanoic acid (compound 7) (118 mg, 750 μmol), HBTU (360 mg, 950 μmol), and DIPEA (258 mg, 2.00 mmol) were added and stirred for 1 hour. 90% TFA / 10% DCM was added and stirred at room temperature for 2 hours. The reaction mixture was filtered and concentrated under reduced pressure to give compound 16 (200 mg).

[0080] Synthesis of compound 17 [ka]

[0081] To a solution of compound 16 (50.0 mg, 91.8 μmol) and compound 7 (256 mg, 918 μmol) in DMF (3 mL), HOBt (124 mg, 918 μmol), DIPEA (237 mg, 1.84 mmol) and EDCI (176 mg, 918 μmol) were added, and the reaction mixture was stirred for 16 hours at 25° C. The reaction mixture was purified by preparative HPLC (TFA condition) to obtain compound 17 (18.2 mg). 1 H-NMR (400MHz, DMSO-d 6 ) δ(ppm): 1.19-1.56 (m, 40H), 1.66 (br s, 3H), 1.83 (br s, 3H), 2.00-2.15 (m, 7H), 2.94-3.05 (m, 8H), 3.29 (br d, J=5.02Hz, 24H), 3.52-3.71 (m, 24H), 4.13 (br s, 3H), 4.57 (s, 4H), 7.74-7.99 (m, 7H).

[0082] Synthesis scheme of compounds 21 and 22 [ka]

[0083] Synthesis of compound 19 [ka]

[0084] Galactosamine pentaacetate Compound 18 (12.0 g, 30.8 mmol), 6-(Cbz-amino)-1-hexanol (10.3 g, 41.1 mmol) and ZnCl 2To a mixture of (5.60 g, 41.1 mmol) (dried under reduced pressure at 110° C. for 1 h before use), DCE (120 mL) was added, and the reaction mixture was stirred at 70° C. for 3 h. The reaction mixture was diluted with EtOAc and aqueous sodium bicarbonate, and the mixture was stirred for 10 min, filtered through Celite®, and washed with EtOAc. The filtrate was washed with water and brine. The aqueous layer was extracted with EtOAc, and the organic layer was dried over sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (2:1 petroleum ether / EtOAc) to give compound 19 (14.0 g). 1 H-NMR (400MHz, CDCl 3 )δ(ppm):1.35 (d, J=3.8Hz, 4H), 1.45-1.66 (m, 4H), 1.77 (s, 2H), 1.94 (s, 3H),2.00 (s, 3H), 2.05 (s, 3H), 2.14 (s, 3H), 3.20 (qq, J=13.4, 6.8Hz, 2H), 3.48 (dt, J=9.6, 6.5Hz, 1H), 3.79-4.03 (m, 3H), 4.08-4.19 (m, 2H), 4.65 (d, J=8.3Hz, 1H), 4.90 (s, 1H), 5.04-5.17 (m, 2H), 5.26 (dd, J=11.2, 3.1Hz, 1H), 5.30 (s, 1H), 5.34 (d, J=2.8Hz, 1H), 5.94 (d, J=8.8Hz, 1H), 7.28-7.42 (m, 5H).

[0085] Synthesis of compound 20 [ka]

[0086] Compound 19 (7.50 g, 12.9 mmol) was added with MeOH (300 mL) and NaOMe (2.79 g, 51.7 mmol) and stirred at room temperature for 3 hours. The reaction mixture was partially concentrated, poured into cold water, and acetic acid was added until pH=5. The crude product was then purified by silica gel column chromatography (50:0-50:1 DCM / MeOH) to give compound 20 (4.00 g). 1 H-NMR(400MHz,MeOD)δ(ppm): 1.19-1.35 (m, 4H), 1.40-1.56 (m, 4H), 1.93 (s, 2H), 3.06 (t, J=7.0Hz, 2H), 3.27 (dt, J=3.3, 1.6Hz, 1H), 3.31 (s, 1H), 3.37-3.49 (m, 2H), 3.55 (dd, J=10.8, 3.3Hz, 1H), 3.65-3.76 (m, 2H), 3.78-3.94 (m, 3H), 4.31 (d, J=8.5Hz, 1H), 5.02 (s, 2H), 7.17-7.44 (m, 5H).

[0087] Synthesis of compound 21 [ka]

[0088] Under an argon atmosphere, MeOH (25 mL) and compound 20 (2.40 g, 5.28 mmol) were added to dry 10% palladium-carbon (240 mg), and the mixture was stirred under hydrogen pressure (50 psi) at 25° C. for 3 hours. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to give compound 21 (0.66 g). 1 H-NMR(400MHz,MeOD)δ(ppm): 1.30-1.44 (m, 4H), 1.52-1.66 (m, 4H), 1.98 (s, 3H), 2.76-2.90 (m, 2H), 3.31 (dt, J=3.3, 1.6Hz, 2H), 3.44-3.51 (m, 2H), 3.58 (dd, J=10.8, 3.3Hz, 1H), 3.73-3.78 (m, 2H), 3.82-3.95 (m, 3H), 4.34 (d, J=8.5Hz, 1H).

[0089] Synthesis of compound 22 [ka] Ethanol (6 mL) and 10% palladium-carbon (50% water content) (42 mg) were added to compound 19 (202 mg, 0.35 mmol) and stirred at room temperature under a hydrogen atmosphere for 3 hours. The reaction mixture was filtered through Celite (registered trademark) and washed with ethanol. The filtrate was concentrated under reduced pressure to give compound 22 (159 mg). 1 H-NMR (600 MHz, DMSO-d6) δ(ppm): 0.64 (br s, 1H) 1.27 (br s, 5H) 1.33-1.52 (m, 4H) 1.67 (br s, 1H) 1.80 (s, 3H) 2.61 (br t, J=6.88Hz, MS (ESI+): m / z 321 [M+H]+.

[0090] Synthesis of compound 23 [ka]

[0091] To a solution of compound 12 (602 mg, 1.88 mmol) and compound 21 (601 mg, 1.88 mmol) in DMF (0.5 mL), DIPEA (485 mg, 3.76 mmol), HOBt (254 mg, 1.88 mmol) and EDCI (360 mg, 1.88 mmol) were added, and the reaction mixture was stirred for 16 hours at 25° C. The residue was purified by preparative HPLC (TFA condition) to give compound 23 (104 mg). 1 H-NMR(400MHz,MeOD)δ(ppm): 1.29-1.70 (m, 32H), 1.82-2.01 (m, 11H), 2.08 (s, 2H), 2.22-2.38 (m, 8H),3.18 (d, J=6.3Hz, 6H), 3.42-3.52 (m, 6H), 3.60 (d, J=10.5Hz, 3H), 3.71-3.95 (m, 14H), 4.28 (s, 1H), 4.36 (d, J=8.3Hz, 3H).

[0092] Synthesis of compound 24 [ka] Compound 24 was obtained in the same manner as in the synthesis of compound 23, except that compound 16 was used instead of compound 12. 1 H-NMR (600 MHz, DMSO-d6) δ(ppm): 1.15-1.59 (m, 44H) 1.68 (br d, J=9.90Hz, 3H) 1.79 (s, 12H) 1.82-1.92 (m, 5H) 1.97-2.22 (m, 9H) 3.02 (br d, J=5.50Hz, 10H) 3.32-3.58 (m, 19H) 3.61-3.72 (m, 13H) 4.06-4.27 (m, 8H) 4.43 (br s, 4H) 4.46-4.59 (m, 9H) 7.59 (br d, J=8.80Hz, 4H) 7.74 (br s, 1H) 7.83 (br s, 3H) 7.89 (br s, 3H); MS (ESI+): m / z 1754 [M+H]+.

[0093] Synthesis scheme of compound 28 [ka]

[0094] Synthesis of compound 25 [ka] To a mixture containing CTC resin (1.33 mmol / g, 0.375 g, 0.50 mmol) and compound 9 (212 mg, 0.50 mmol), DCM (30 mL) was added, DIPEA (0.35 mL, 2.00 mmol) was added dropwise, and the reaction was stirred for 2 h. MeOH (0.2 mL, 4.94 mmol) was added to the reaction and stirred for 35 min. The reaction was stirred with a solution of 20% piperidine in DMF (15 mL) for 30 min. The reaction mixture was filtered, and the resulting solid was washed with DMF. Then, N-Fmoc-D-glutamic acid-1-tert-butyl ester (213 mg, 0.499 mmol) and N-Fmoc-L-glutamic acid-1-tert-butyl ester (213 mg, 0.499 mmol), HBTU (360 mg, 0.949 mmol), DMF (3 mL) and DIPEA (0.35 mL, 2.00 mmol) were added, and the reaction mixture was stirred for 1 hour while bubbling with nitrogen. A solution of 20% piperidine in DMF (15 mL) was added and stirred for 30 minutes. The resin was then washed with DMF. Then, N-[(9H-fluoren-9-ylmethoxy)carbonyl]-D-glutamic acid 1-tert-butyl ester (N-Fmoc-D-glutamic acid-1-tert-butyl ester) (213 mg, 0.499 mmol), N-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-glutamic acid 1-tert-butyl ester (N-Fmoc-L-glutamic acid-1-tert-butyl ester) (213 mg, 0.499 mmol), HBTU (360 mg, 0.949 mmol) and DMF (3 mL) were added, and DIPEA (0.35 mL, 2.00 mmol) was slowly added dropwise. The reaction mixture was stirred for 80 minutes while bubbling with nitrogen. The resin was then washed with DMF, washed with MeOH and dried under reduced pressure. To the resulting residue was added 50% TFA / 50% DCM, stirred for 2 hours, filtered, and the filtrate was concentrated under reduced pressure to give compound 25 (307.3 mg). MS(ESI) M / Z:610〔M+H〕 + 1 H-NMR (600MHz, DMSO-d 6 ) δ(ppm): 1.77 (m, 5H), 1.94 (m, 3H), 2.12-2.23 (m, 6H), 2.26 (m, 2H), 2.38 (br t, J=7.34Hz, 2H), 4.13-4.21 (m, 3H), 5.09 (s, 2H), 7.30-7.40 (m, 5H), 8.05-8.15 (m, 3H).

[0095] Synthesis of compound 26 [ka] Compound 22 (154 mg, 0.344 mmol) and compound 25 (21.0 mg, 0.034 mmol) were dissolved in DMF (2 mL) and HOBt (52.8 mg, 0.344 mmol), DIPEA (0.12 mL, 0.689 mmol) and EDCI (66.0 mg, 0.344 mmol) were added. The reaction mixture was stirred at room temperature for 18 hours, extracted with water, EtOAc, and a small amount of methanol, and the organic layer was dried over sodium sulfate. After filtration, the mixture was concentrated under reduced pressure. The residue was purified by preparative HPLC to obtain compound 26 (16.7 mg). MS(ESI) M / Z:1162〔M+2H〕 + 1 H-NMR (600MHz, DMSO-d 6 ) δ(ppm): 1.23 (br s, 16H), 1.31-1.41 (m, 8H), 1.41-1.48 (m, 8H), 1.60-1.73 (m, 4H), 1.77 (s, 12H), 1.78-1.87 (m, 4H), 1.89 (s, 12H), 1.99 (s, 12H), 2.01-2.07 (m, 2H), 2.10 (s, 12H), 2.11-2.21 (m, 6H), 2.33-2.37 (m, 2H), 2.95-3.07 (m, 8H), 3.37-3.43 (m, 4H), 3.65-3.72 (m, 4H), 3.82-3.90 (m, 4H), 3.98-4.05 (m, 12H), 4.09-4.20 (m, 3H), 4.48 (d, J=8.44Hz, 4H), 4.97 (dd, J=11.19, 3.12Hz, 4H), 5.08 (s, 2H), 5.21 (d, J=3.30Hz, 4H), 7.30-7.39 (m, 5H), 7.80 (m, 8H), 7.86-7.95 (m, 3H).

[0096] Synthesis of compound 27 [ka] To a solution of compound 26 (19.0 mg, 8.18 μmol) in ethanol (3 mL), 10% palladium-carbon (50% water content) (5.3 mg) was added. The reaction mixture was stirred at room temperature under a hydrogen atmosphere for 3 hours. The reaction mixture was filtered through Celite (registered trademark) and washed with ethanol. The filtrate was concentrated under reduced pressure to give compound 27 (19.6 mg). 1 H-NMR (600MHz, DMSO-d 6 ) δ(ppm): 1.24 (br s, 22H), 1.30-1.40 (m, 8H), 1.45 (br s, 8H), 1.61-1.74 (m, 4H), 1.77 (s, 12H), 1.80-1.87 (m, 2H), 1.89 (s, 12H), 1.99 (s, 12H), 2.01-2.07 (m, 2H), 2.10 (s, 12H), 2.12-2.25 (m, 4H), 2.93-3.06 (m, 8H), 3.36-3.46 (m, 4H), 3.64-3.72 (m, 4H), 3.82-3.91 (m, 4H), 3.98-4.20 (m, 16H), 4.50 (br d, J=8.07Hz, 4H), 4.94-5.02 (m, 4H), 5.21 (d, J=2.93Hz, 4H) 7.73-8.03 (m, 11H).

[0097] Synthesis of compound 28 [ka] Under a nitrogen atmosphere, triethylamine (8.7 μL, 62 μmol) and pentafluorophenyl trifluoroacetate (5.4 μL, 31 μmol) were added to a solution of compound 27 (17.4 mg, 7.79 μmol) in DMF (2 mL), and the reaction mixture was stirred at room temperature for 1.5 hours. Water was added to the reaction solution, and the mixture was extracted with EtOAc. The organic layer was washed with saturated sodium bicarbonate, sodium hydrogen sulfate, and saturated saline. After concentration under reduced pressure, the residue was triturated with pentane, filtered, and dried under reduced pressure at 40 degrees to obtain compound 28 (11.72 mg). MS(ESI) M / Z:1200〔M+2H〕 + 1 H-NMR (600MHz, DMSO-d 6 ) δ(ppm): 1.24 (br s, 20H), 1.31-1.40 (m, 8H), 1.41-1.49 (m, 8H), 1.61-1.72 (m, 2H), 1.77 (s, 12H), 1.79-1.86 (m, 2H), 1.89 (s, 12H), 1.99 (s, 12H), 2.01-2.07 (m, 2H), 2.10 (s, 12H), 2.12-2.21 (m, 4H), 2.25-2.31 (m, 2H), 2.80 (br t, J=7.34Hz, 2H), 2.95-3.08 (m, 8H), 3.37-3.43 (m, 4H), 3.65-3.72 (m, 4H), 3.82-3.90 (m, 4H), 3.97-4.06 (m, 12H), 4.10-4.22 (m, 3H), 4.48 (d, J=8.44Hz, 4H), 4.97 (dd, J=11.00, 2.93Hz, 4H), 5.21 (d, J=2.93Hz, 4H), 7.70-7.86 (m, 8H), 7.86-8.01 (m, 3H).

[0098] Examples 1 to 7: Synthesis of nucleic acid complexes 1 to 7 SEQ-1 and SEQ-2 were synthesized by Gene Design. SEQ-2 (1.3 μmol) was added with sodium tetraborate buffer pH 8.5 (final concentration 40 mM), DBCO-NHS ester (CAS No. 1353016-71-3, 60 μmol) dissolved in DMSO was added, and the mixture was stirred at room temperature for 15 minutes. Water was added to the reaction solution, and the mixture was purified by gel filtration using a PD-10 column (GE Healthcare). The mixture was further purified and concentrated using an Amicon Ultra 3K (Millipore) to obtain a crude product (SEQ-3). Triethylammonium acetate pH 7.0 (final concentration 50 mM) was added to the crude product (40 nmol), and compounds 8, 11, 14, 15, 17, 23, and 24 (400 nmol) dissolved in water were added, and the mixture was stirred at room temperature for 15 minutes. The reaction solution was purified by gel filtration using a PD-10 column (GE Healthcare). Further purification and concentration were carried out using Amicon Ultra 3K (Millipore), thereby obtaining nucleic acid complexes, that is, nucleic acid complexes 1 to 7 corresponding to compounds 8, 11, 14, 15, 17, 23, and 24.

[0099] [ka]

[0100] Nucleic acid complex 1 [ka]

[0101] Nucleic acid complex 2 [ka]

[0102] Nucleic acid complex 3 [ka]

[0103] Nucleic acid complex 4 [ka]

[0104] Nucleic acid complex 5 [ka]

[0105] Nucleic acid complex 6 [ka]

[0106] Nucleic acid complex 7 [ka]

[0107] Example 8: Synthesis of Nucleic Acid Complex 8 SEQ-2 (26.6 nmol) was added with sodium tetraborate buffer pH 8.5 and compound 28 (120 nmol) dissolved in DMSO, and stirred at room temperature. Water was added to the reaction solution, and purification was performed using Amicon Ultra 3K (Millipore). Five times the amount of 28% ammonia water was added to the obtained crude product, and the mixture was allowed to stand at room temperature for 3 hours. Water was added to the reaction solution, and purification was performed using Amicon Ultra 3K (Millipore). Nucleic acid complex 8 was obtained by further purification using reverse phase HPLC.

[0108] Nucleic acid complex 8 [ka]

[0109] Description of Nucleic Acid Sequences The sequence of the nucleic acid used in this example (5'-3') is A(F)^G(M)^G(F)A(M)C(F)U(M)G(F)G(M)U(F)C(M)U(F)U(F)U(M)C(F)U(M)A(F)U(M)A(F)U(M)^C(F)^U(M), where the capital alphabet represents RNA, (M) represents 2'-O-methyl modified RNA, (F) represents 2'-fluoro RNA, and ^ represents phosphorothioate bond. The 5' end of each oligonucleotide is bound to a fluorescent cyanine dye Cy3 (excitation wavelength 555 nm, fluorescence wavelength 570 nm). The molecular weights of SEQ-1, 2, and the nucleic acid complexes synthesized in this example were measured by MALDI-TOF-MS, and the results are shown in Table 1.

[0110] [Table 1]

[0111] <Test Example 1> In vitro evaluation of uptake activity of nucleic acid complexes into human CD206-expressing Lenti-X 293T cells The nucleic acid complexes synthesized in the examples were introduced into human CD206-expressing Lenti-X 293T cells (Clontech) by the following method, and the uptake was evaluated. 4 The cells were seeded in a 96-well PDL-coated plate (Corning) at 100 μL / well and incubated at 37°C in 5% CO 2 The cells were cultured in an incubator for 2 days. SEQ-1 or a synthetic nucleic acid complex was added to the cells at a final concentration of 100 nmol / L, and the cells were incubated at 37°C in 5% CO 2The cells were incubated for 2 hours in an incubator. 4% paraformaldehyde / PBS (Wako) containing 10 μg / mL Hoechst (Life Technologies) was added, and the cells were fixed at room temperature for 30 minutes and washed four times with PBS. Fluorescence imaging analysis was performed using an IN Cell Analyzer 2200 (GE Healthcare), and the Cy3 fluorescence intensity in the well was corrected for the number of nuclei to calculate the average Cy3 fluorescence intensity per cell. In this case, the fluorescence intensity of the well to which SEQ-1, a nucleic acid without sugar ligand modification, was set as 1, and the fluorescence intensity of the well to which each nucleic acid complex was added was calculated as a relative value.

[0112] The results are shown in Table 2. The results demonstrated that, compared with SEQ-1 not containing a sugar ligand, nucleic acid complex 6 having GalNac was not efficiently taken up into cells expressing CD206, whereas nucleic acid complexes having mannose, nucleic acid complexes 1 to 5, were efficiently taken up into cells expressing CD206.

[0113] [Table 2]

[0114] <Test Example 2> In vitro uptake activity of nucleic acid complexes into human ASGR1-expressing Lenti-X293T cells The nucleic acid complexes synthesized in the examples were introduced into human ASGR1-expressing Lenti-X293T cells (Clontech) by the following method, and the uptake activity was evaluated. 4 The cells were seeded in a 96-well PDL-coated plate (Corning) at 100 μL / well and incubated at 37°C in 5% CO 2 The cells were cultured in an incubator for 1 or 2 days. SEQ-1 or a synthesized nucleic acid complex was added to the cells at a final concentration of 100 nmol / L, and the cells were incubated at 37°C in 5% CO 2The cells were incubated in an incubator for 2 hours, after which the fluorescence imaging analysis was carried out in the same manner as in Test Example 1.

[0115] The results are shown in Table 3. The results demonstrated that, compared with SEQ-1 containing no sugar ligand, nucleic acid complexes 1 to 5 having mannose were not efficiently taken up into cells expressing ASGR1, whereas nucleic acid complexes 6 to 8 having GalNac were efficiently taken up into cells expressing ASGR1.

[0116] [Table 3]

[0117] <Test Example 3> Evaluation of uptake of nucleic acid complexes in mouse hepatocytes and Kupffer cells (in vivo evaluation) SEQ-1, nucleic acid complexes 3 and 6 were subcutaneously administered at 1 mg / kg to BALB / c mice (n=3). Four hours after administration, the liver was perfused, and hepatic parenchymal cells and Kupffer cells were separated using a flow cytometer, and the fluorescence intensity was measured. At this time, the fluorescence intensity of the group administered with SEQ-1 was set to 1, and the fluorescence intensity of each nucleic acid complex administration group was calculated as a relative value. The results are shown in Table 4. These results demonstrate that, compared with SEQ-1 containing no sugar ligand, nucleic acid complex 3 having mannose was efficiently taken up into mouse Kupffer cells, which have been reported to be CD206 positive, and nucleic acid complex 6 having GalNac was efficiently taken up into mouse hepatic parenchymal cells, which have been reported to be ASGR1 positive.

[0118] [Table 4]

[0119] Synthesis scheme of compound 37 [ka]

[0120] Synthesis of compound 30 [ka]

[0121] A mixture of hydrazine acetate (1.29 g, 14.06 mmol) and DMF (50 mL) was heated at 50 °C. It was then cooled in a 15 °C water bath and α-D-mannose pentaacetate (4.99 g, 12.8 mmol) was added. The resulting solution was stirred in a 15-19 °C water bath for 16 h. The mixture was extracted with water and EtOAc, and the aqueous layer was further extracted with EtOAc. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography using Biotage Isolera (100 g KP-Sil, 20-100% EtOAc / cyclohexane) to give compound 30 (4.19 g) as a 9:1 mixture of α and β diastereomers. alpha isomer 1 H NMR (600 MHz, CDCl 3 ) δppm: 2.00 (s, 3H), 2.05 (s, 3H), 2.11 (s, 3H), 2.16 (s, 3H), 3.00 (d, J=4.03Hz, 1H), 4.15 (br d, J=10.64Hz, 1H), 4.22-4.28 (m, 2H), 5.23-5.33 (m, 3H), 5.43 (br dd, J=10.27, 3.30Hz, 1H)

[0122] Synthesis of compound 31 [ka] To a solution of compound 30 (3.64 g, 10.5 mmol) in DCM (218 mL) was added trichloroacetonitrile (10.48 mL, 104.5 mmol) and then DBU (0.158 mL, 1.05 mmol) dropwise under nitrogen atmosphere. The mixture was stirred at room temperature under nitrogen atmosphere for 2.25 h. Concentration under reduced pressure and the residue was purified by column chromatography using Biotage Isolera (100 g KP-Sil, 0-50% EtOAc / cyclohexane) to give compound 31 (4.567 g). 1 H NMR (600 MHz, CDCl 3 ) δppm: 2.01 (s, 3H), 2.07 (s, 3H), 2.08 (s, 3H), 2.20 (s, 3H), 4.14-4.23 (m, 2H), 4.28 (dd, J=12.47, 4.77Hz, 1H), 5.38-5.43 (m, 2H), 5.47 (br s, 1H), 6.28 (s, 1H), 8.79(s, 1H)

[0123] Synthesis of compound 33 [ka]

[0124] A solution of compound 31 (2.45 g, 4.96 mmol) and N-Cbz-6-amino-hexan-1-ol (1.87 g, 7.45 mmol) in DCM (90 mL) was cooled to 2° C. (internal temperature) and, under nitrogen atmosphere, boron trifluoride diethyl etherate (0.126 mL, 0.993 mmol) was added dropwise. The resulting mixture was stirred at 2° C. for 25 min, then at room temperature for 1 h 20 min. The mixture was extracted with saturated aqueous sodium bicarbonate, and the aqueous layer was extracted with DCM. The combined organic layers were washed with saturated brine, filtered through hydrophobic filter paper, and concentrated under reduced pressure. The residue was purified by column chromatography using Biotage Isolera (100 g KP-Sil, 0-100% EtOAc / cyclohexane) to give compound 33 (1.37 g). 1H NMR (600 MHz, CDCl 3 ) δppm: 1.32-1.43 (m, 4H), 1.49-1.53 ​​(m, 2H), 1.56-1.64 (m, 2H), 1.99 (s, 3H), 2.04 (s, 3H), 2.10 (s, 3H), 2.15 (s, 3H), 3.20 (q, J=6.72Hz, 2H), 3.40-3.48 (m, 1H), 3.64-3.71 (m, 1H), 3.97 (ddd, J=9.90, 5.32, 2.38Hz, 1H), 4.08-4.12 (m, 1H), 4.28 (dd, J=12.29, 5.32Hz, 1H), 4.79 (d, J=1.47Hz, 2H), 5.10 (s, 2H), 5.22 (dd, J=3.30, 1.83Hz, 1H), 5.28 (t, J=9.54Hz, 1H), 5.34 (dd, J=10.64, 3.67Hz, 1H), 7.29-7.33 (m, 1H), 7.36 (d, J=4.03Hz, 4H)

[0125] Synthesis of compound 34 [ka]

[0126] To a solution of compound 33 (1.35 g, 2.31 mmol) in IMS (38 mL), 10% palladium-carbon (0.293 g, 50% water content) was added under a nitrogen atmosphere, and the mixture was stirred at room temperature under a hydrogen atmosphere for 1.5 hours. The mixture was filtered through Celite (registered trademark) and washed with IMS. A 4N solution of hydrogen chloride in 1,4-dioxane (0.78 mL, 3.12 mmol) was added to the filtrate, and the mixture was concentrated under reduced pressure to obtain compound 34 (1.06 g). 1 H NMR (600 MHz, DMSO-d 6 ) δ ppm: 1.30-1.37 (m, 4H), 1.51-1.61 (m, 4H), 1.94 (s, 3H), 2.03 (s, 6H), 2.11 (s, 3H), 2.72-2.80 (m, 2H), 3.42-3.50 (m, 1H), 3.63 (dt, J=9.72, 6.88Hz, 1H), 3.88-3.96 (m, 1H), 4.06 (dd, J=12.10, 2.57Hz, 1H), 4.15 (dd, J=12.29, 5.32Hz, 1H), 4.87 (s, 1H), 5.04-5.15 (m, 3H), 7.80 (br s, 3H)

[0127] Synthesis of compound 35 [ka]

[0128] To a solution of compound 34 (510 mg, 1.05 mmol) and compound 25 (128.4 mg, 0.21 mmol) in DMF (3 mL), HOBt (161 mg, 1.05 mmol) and EDCI (202 mg, 1.05 mmol) were added in sequence. The resulting mixture was stirred at room temperature for 10 minutes, and then DIPEA (0.18 mL, 1.05 mmol) was added. The resulting solution was stirred at room temperature for 22 hours. A solution of compound 34 (505.9 mg, 1.04 mmol) in DMF (1.5 mL), HOBt (161 mg, 1.05 mmol) and EDCI (202 mg, 1.05 mmol) were added, and after 10 minutes, DIPEA (0.18 mL, 1.05 mmol) was added. The mixture was stirred for an additional 3 hours. The mixture was purified by preparative HPLC to give compound 34 (46.3 mg). 1 H NMR (600 MHz, DMSO-d 6 ) δ ppm: 1.21-1.45 (m, 24H), 1.49-1.59 (m, 8H), 1.61-1.89 (m, 8H), 1.93 (s, 12H), 2.01 (s, 24H), 2.10 (s, 12H), 2.07-2.23 (m, 8H), 2.33-2.37 (m, 2H), 2.96-3.08 (m, 7H), 3.41-3.48 (m, 4H), 3.57-3.65 (m, 4H), 3.88-3.94 (m, 4H), 4.05 (dd, J=12.10, 1.83Hz, 4H), 4.10-4.20 (m, 8H), 4.85 (s, 4H), 5.06-5.13 (m, 14H), 7.31-7.40 (m, 6H), 7.69-7.94 (m, 6H); LCMS (ESI+): m / z 1164.58 [M+2H] 2+ .

[0129] Synthesis of compound 36 [ka]

[0130] To a solution of compound 35 (40.7 mg, 0.02 mmol) in IMS (6 mL), 10% palladium-carbon (11.5 mg, 50% water content) was added under a nitrogen atmosphere, and the mixture was stirred at room temperature under a hydrogen atmosphere for 2 hours. The mixture was filtered through Celite (registered trademark) and washed with IMS. The combined filtrate was concentrated under reduced pressure to give compound 36 (43.8 mg). 1 H NMR (600 MHz, DMSO-d 6 ) δ ppm: 1.20-1.44 (m, 24H), 1.51-1.60 (m, 8H), 1.61-1.88 (m, 8H), 1.93 (s, 12H), 2.02 (s, 12H), 2.02 (s, 12H), 2.10 (s, 12H), 2.12-2.27 (m, 10H), 2.96-3.08 (m, 7H), 3.41-3.48 (m, 4H), 3.58-3.65 (m, 4H), 3.88-3.94 (m, 4H), 4.04 (br d, J=10.27Hz, 4H), 4.09-4.29 (m, 8H), 4.85 (s, 4H), 5.04-5.14 (m, 12H), 7.71-8.01 (br m, 7H)

[0131] Synthesis of compound 37 [ka]

[0132] To a solution of compound 36 (32.8 mg, 0.015 mmol) in DMF (3 mL), trimethylamine (16.3 μL, 0.12 mmol) was added, followed by dropwise addition of pentafluorophenyl trifluoroacetate (10.0 μL, 0.06 mmol), and the resulting mixture was stirred at room temperature for 1.25 hours. The reaction mixture was poured into water, saturated saline was added, and the mixture was extracted with EtOAc. The organic layer was washed successively with saturated aqueous sodium bicarbonate, 1M aqueous sodium hydrogen sulfate, and saturated saline. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was triturated with pentane. The solid was then dried under reduced pressure at 40° C. to give compound 37 (36.7 mg). 1 H NMR (600 MHz, DMSO-d 6 ) δ ppm: 1.20 - 1.47 (m, 24H), 1.51 - 1.61 (m, 8H), 1.62 - 1.91 (m, 8H), 1.93 (s, 12H), 2.01 (s, 24H), 2.10 (s, 12H), 2.12 - 2.33 (m, 8H), 2.80 (br t, J = 6.79Hz, 2H), 2.96 - 3.09 (m, 7H), 3.41 - 3.50 (m, 4H), 3.58 - 3.65 (m, 4H), 3.91 (br s, 4H), 4.04 (br d, J = 12.10Hz, 4H), 4.10 - 4.28 (m, 8H), 4.85 (br s, 4H), 5.06 - 5.15 (m, 12H), 7.68 - 8.03 (m, 7H); LCMS (ESI+): m / z 1202.46 [M + 2H] 2+ .

[0133] Synthesis scheme of Compound 44

Chem.

[0134] Synthesis of Compound 39

Chem.

[0135] Anhydrous zinc chloride (3.65 g, 26.8 mmol) was weighed into a 250 mL 3-neck flask under argon. The flask was dried under vacuum at 110° C. for 1 hour and then allowed to cool under vacuum overnight. The flask was then filled with argon and ((2S,3R,4R,5R,6R)-3-acetamido-6-(acetoxymethyl)tetrahydro-2H-pyran-2,4,5-triyl triacetate (7.85 g, 20.2 mmol), N-Cbz-6-amino-hexan-1-ol (6.74 g, 26.8 mmol) and DCE (85 mL) were added. After heating the mixture under argon at 70° C. (external temperature) for 3 h, the mixture was cooled and then diluted with ethyl acetate and saturated aqueous sodium bicarbonate. The mixture was stirred for 20 min, filtered through Celite® and washed with ethyl acetate. The combined filtrate was partitioned and the organic layer was washed with water followed by saturated brine. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by column chromatography using a CombiFlash (330 g PuriFlash Si, 20-100% EtOAc / cyclohexane) to give compound 39 (9.42 g). 1 H NMR (400 MHz, CDCl 3 ) δ ppm: 1.30-1.43 (m, 4H), 1.45-1.65 (m, 4H), 1.94 (s, 3H), 2.00 (s, 3H), 2.05 (s, 3H), 2.13 (s, 3H), 3.12-3.29 (m, 2H), 3.48 (dt, J=9.63, 6.50Hz, 1H), 3.79-4.02 (m, 3H), 4.06-4.21 (m, 2H), 4.65 (br d, J=8.19Hz, 1H), 4.78-4.93 (m, 1H), 5.06-5.18 (m, 2H), 5.27 (dd, J=11.25, 3.06Hz, 1H), 5.34 (d, J=2.81Hz, 1H), 5.77 (br d, J=8.44Hz, 1H), 7.29-7.42 (m, 5H) LCMS (m / z 581 [M+H] + )

[0136] Synthesis of compound 40 [ka]

[0137] To a solution of compound 39 (9.23 g, 15.9 mmol) in IMS (275 mL), 4 M hydrogen chloride in dioxane (5.17 mL, 20.7 mmol) and 10% palladium-carbon (1.895 g; 50% water content) were added under a nitrogen atmosphere, and the mixture was stirred at room temperature for 2.5 hours under a hydrogen atmosphere. The mixture was filtered through Celite (registered trademark) and washed with IMS. The filtrate was concentrated under reduced pressure to give compound 40 (8.07 g). 1 H NMR (400 MHz, DMSO-d 6 ) δ ppm: 1.22-1.35 (m, 4H), 1.39-1.62 (m, 4H), 1.78 (s, 3H), 1.89 (s, 3H), 2.00 (s, 3H), 2.10 (s, 3H), 2.69-2.82 (m, 2H), 3.41-3.47 (m, 2H), 3.64-3.77 (m, 1H), 3.81-3.93 (m, 1H), 3.95-4.12 (m, 2H), 4.49 (d, J=8.56Hz, 1H), 4.97 (dd, J=11.25, 3.42Hz, 1H), 5.22 (d, J=3.42Hz, 1H), 7.62-7.80 (m, 3H), 7.86 (d, J=9.29Hz, 1H) LCMS (m / z 447 [M+H] + )

[0138] Synthesis of compound 41 [ka]

[0139] To a mixture of CTC resin (1.33 mmol / g, 1.31 g, 1.74 mmol), Fmoc-Glu(OtBu)-OH (0.370 g, 0.87 mmol), Fmoc-D-Glu(OtBu)-OH (0.370 g, 0.87 mmol) and DCM (105 mL) in a 250 mL Quickfit® Erlenmeyer flask, DIPEA (1.215 mL, 6.96 mmol) was added dropwise. The flask was stoppered, attached to a plate shaker and shaken at 300 rpm for 2 h. MeOH (0.7 mL, 17.3 mmol) was added and the mixture was shaken for an additional 35 min. The mixture was filtered through a 70 mL plastic phase separation cartridge and treated with 20% piperidine in DMF (50 mL) for 30 min while bubbling with nitrogen. The solution was drained and the resin was washed with DMF. To the resin in the cartridge was added a solution of Fmoc-D-Glu-OtBu (0.740 g, 1.74 mmol), N-Fmoc-L-glutamic acid-1-t-butyl ester (0.740 g, 1.74 mmol) and HBTU (1.253 g, 3.30 mmol) in DMF (10 mL), followed by dropwise addition of DIPEA (1.22 mL, 6.96 mmol). The mixture was mixed for 1 hour while bubbling with nitrogen. The solution was then purged with a stream of nitrogen and the resin was treated with a 20% solution of piperidine in DMF (50 mL) for 35 minutes while mixing by bubbling with nitrogen. The solution was drained and the resin was washed with DMF. To the remaining resin in the cartridge, a solution of 1,5-pentanedioic acid monobenzyl ester (0.580 g, 2.61 mmol) and HBTU (1.253 g, 3.30 mmol) in DMF (10 mL) was added dropwise, followed by DIPEA (1.215 mL, 6.96 mmol). The mixture was mixed by bubbling nitrogen for 80 minutes. The solution was drained and the resin was washed with DMF and then MeOH. The resin was then sucked dry. The resin was transferred to a 25×150 mm test tube and dried under vacuum for 30 minutes, then treated with DCM (5 mL) and TFA (5 mL) and shaken on a plate shaker for 2 hours. The mixture was then filtered through a phase separation cartridge and washed with DCM. The filtrate was concentrated under reduced pressure to give compound 41 (0.5859 g). 1H NMR (400 MHz, DMSO-d 6 ) δ ppm: 1.68-1.84 (m, 4H), 1.86-2.01 (m, 2H), 2.13-2.23 (m, 4H), 2.23-2.29 (m, 2H), 2.38 (t, J=7.52Hz, 2H), 4.11-4.22 (m, 2H), 5.09 (s, 2H), 7.29-7.42 (m, 5H), 8.06-8.17 (m, 2H) LCMS (m / z 481 [M+H] + )

[0140] Synthesis of compound 42 [ka]

[0141] To a solution of compound 40 (4.06 g, 8.41 mmol) and compound 41 (0.5821 g, 1.21 mmol) in DMF (8 mL), HOBt (1.392 g, 9.09 mmol), EDCI (1.742 g, 9.09 mmol), and then DIPEA (1.587 mL, 9.09 mmol) were added sequentially. The mixture was stirred at room temperature for 20 hours. The mixture was purified by preparative HPLC to give compound 42 (0.48 g). 1 H NMR (600 MHz, DMSO-d 6 ) δ ppm: 1.23 (br s, 12H), 1.29-1.40 (m, 6H), 1.40-1.48 (m, 6H), 1.60-1.71 (m, 2H), 1.76 (s, 9H), 1.76-1.87 (m, 4H), 1.89 (s, 9H), 1.99 (s, 9H), 2.00-2.07 (m, 2H), 2.10 (s, 9H), 2.11-2.15 (m, 2H), 2.18 (br t, J=7.15Hz, 2H), 2.34-2.38 (m, 2H), 2.94-3.07 (m, 6H), 3.36-3.43 (m, 3H), 3.65-3.73 (m, 3H), 3.82-3.91 (m, 3H), 3.98-4.06 (m, 9H), 4.10-4.20 (m, 2H), 4.48 (d, J=8.44Hz, 3H), 4.97 (dd, J=11.19, 3.12Hz, 3H), 5.08 (s, 2H), 5.21 (d, J=3.30Hz, 3H), 7.30-7.39 (m, 5H), 7.71-7.76 (m, 1H), 7.77-7.85 (m, 5H), 7.87-7.93 (m, 2H) LCMS (m / z 1766 [M+H] + )

[0142] Synthesis of compound 43 [ka]

[0143] To a solution of compound 42 (0.43 g, 0.243 mmol) in IMS (85 mL), 10% palladium-carbon (0.158 g, 0.074 mmol; 50% water content) was added under a nitrogen atmosphere, and the mixture was stirred at room temperature under a hydrogen atmosphere for 2.75 hours. The mixture was filtered through Celite (registered trademark) and washed with IMS. The filtrate was concentrated under reduced pressure, and the residue was dissolved in DCM / EtOH (2:1), filtered through Celite (registered trademark), and washed with DCM-EtOH (2:1). The filtrate was concentrated under reduced pressure to give compound 43 (0.4252 g). 1 H NMR (600 MHz, DMSO-d 6) δppm: 1.18-1.29 (m, 12H), 1.31-1.41 (m, 6H), 1.41-1.49 (m, 6H), 1.62-1.75 (m, 4H), 1.77 (s, 9H), 1.81-1.87 (m, 2H), 1.89 (s, 9H), 1.99 (s, 9H), 2.01-2.08 (m, 2H), 2.10 (s, 9H), 2.12-2.24 (m, 6H), 2.95-3.07 (m, 6H), 3.37-3.45 (m, 3H), 3.66-3.73 (m, 3H), 3.82-3.92 (m, 3H), 3.98-4.06 (m, 9H), 4.09-4.20 (m, 2H), 4.49 (d, J=8.44Hz, 3H), 4.97 (dd, J=11.19, 2.38Hz, 3H), 5.21 (d, J=3.30Hz, 3H), 7.73-8.04 (m, 8H), 11.81-12.20 (m, 1H). LCMS (m / z 1676 [M+H] + ).

[0144] Synthesis of compound 44 [ka]

[0145] To a solution of compound 43 (0.4239 g, 0.253 mmol) in DMF (50 mL), triethylamine (0.282 mL, 2.024 mmol) was added dropwise, followed by pentafluorophenyl trifluoroacetate (0.174 mL, 1.012 mmol) under a nitrogen atmosphere. The mixture was stirred at room temperature for 1 hour and poured into water. Saturated saline was added to the aqueous layer, and the mixture was extracted with EtOAc. The combined organic layers were washed successively with saturated aqueous sodium hydrogen carbonate solution, 1M aqueous sodium hydrogen sulfate solution, and saturated aqueous sodium chloride solution. The organic layer was dried over anhydrous sodium sulfate, filtered, and then concentrated under reduced pressure to obtain a residue, to which EtOAc was added, and the residue was washed with 1M aqueous sodium hydrogen sulfate solution and then saturated aqueous sodium chloride solution. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain compound 44 (0.4513 g). 1 H NMR (600 MHz, DMSO-d 6 )δ ppm: 1.24 (br s, 12H), 1.31-1.40 (m, 6H), 1.41-1.48 (m, 6H), 1.61-1.73 (m, 2H), 1.76 (s, 9H), 1.79-1.93 (m, 13H), 1.99 (s, 9H), 2.01-2.06 (m, 2H), 2.10 (s, 9H), 2.11-2.18 (m, 2H), 2.27 (br t, J=7.34Hz, 2H), 2.80 (t, J=6.97Hz, 2H), 2.96-3.07 (m, 6H), 3.37-3.43 (m, 3H), 3.69 (dt, J=9.63, 6.19Hz, 3H), 3.82-3.91 (m, 3H), 3.98-4.06 (m, 9H), 4.09-4.22 (m, 2H), 4.48 (d, J=8.44Hz, 3H), 4.97 (dd, J=11.37, 3.30Hz, 3H), 5.21 (d, J=3.30Hz, 3H), 7.73 (br t, J = 5.14Hz, 1H), 7.77-7.85 (m, 5H), 7.91 (br dd, J = 11.19, 7.89Hz, 1H), 7.98 (br d, J = 8.07Hz, 1H) LCMS (m / z 1843.56 [M+H] + )

[0146] Synthesis of Compound 47

change

[0147] Synthesis of compound 45

change

[0148] To a solution of Compound 34 (1383 mg, 2.86 mmol) and Compound 41 (183.1 mg, 0.381 mmol) in DMF (3.0 mL) were sequentially added HOBt (438 mg, 2.86 mmol), EDCI (548 mg, 2.86 mmol), and DIPEA (0.50 mL, 2.86 mmol). The resulting mixture was stirred at room temperature for 24 hours. The mixture was purified by preparative HPLC to obtain Compound 45 (133.4 mg). 1 H NMR (600 MHz, DMSO-d 6 ) δ ppm: 1.23 - 1.34 (m, 12H), 1.34 - 1.43 (m, 6H), 1.50 - 1.59 (m, 6H), 1.62 - 1.72 (m, 2H), 1.74 - 1.80 (m, 2H), 1.81 - 1.89 (m, 2H), 1.93 (s, 9H), 2.01 (s, 18H), 2.03 - 2.07 (m, 2H), 2.10 (s, 9H), 2.11 - 2.15 (m, 2H), 2.16 - 2.21 (m, 2H), 2.33 - 2.38 (m, 2H), 2.97 - 3.07 (m, 6H), 3.41 - 3.48 (m, 3H), 3.57 - 3.64 (m, 3H), 3.87 - 3.94 (m, 3H), 4.05 (dd, J = 12.10, 2.57Hz, 3H), 4.10 - 4.19 (m, 5H), 4.85 (s, 3H), 5.0 - 5.13 (m, 11H), 7.29 - 7.39 (m, 5H), 7.71 (br t, J = 4.95Hz, 1H), 7.76 - 7.84 (m, 2H), 7.85 - 7.91 (m, 2H) LCMS (m / z 1769 [M+H] + )

[0149] Synthesis of Compound 46

Chemical Structure

[0150] To a solution of compound 45 (101.9 mg, 0.058 mmol) in IMS (20 mL), 10% palladium-carbon (37.4 mg; 50% water content) was added under a nitrogen atmosphere. The mixture was stirred at room temperature under a hydrogen atmosphere for 2.5 hours. The mixture was filtered through Celite® and then washed with IMS. The combined filtrate was concentrated under reduced pressure to give compound 46 (95.9 mg). 1 H NMR (600 MHz, DMSO-d 6 ) δ ppm: 1.28 (br s, 12H), 1.34-1.44 (m, 6H), 1.49-1.60 (m, 6H), 1.61-1.76 (m, 4H), 1.81-1.90 (m, 2H), 1.93 (s, 9H), 2.02 (s, 9H), 2.02 (s, 9H), 2.04-2.08 (m,2H), 2.10 (s, 9H), 2.12-2.23 (m, 6H), 2.96-3.07 (m, 6H), 3.40-3.48 (m, 3H), 3.57-3.65 (m, 3H), 3.86-3.94 (m, 3H), 4.05 (dd, J=12.10, 2.38Hz, 3H), 4.09-4.18 (m, 5H), 4.85 (s, 3H), 5.01-5.14 (m, 9H), 7.62-8.17 (m, 5H), 11.84-12.15 (m, 1H). LCMS (m / z 1679 [M+H] + ).

[0151] Synthesis of compound 47 [ka]

[0152] To a solution of compound 46 (94.6 mg, 0.056 mmol) in DMF (9 mL), triethylamine (62.8 μL, 0.451 mmol) was added under a nitrogen atmosphere, and pentafluorophenyl trifluoroacetate (38.7 μL, 0.225 mmol) was added dropwise. The resulting mixture was stirred at room temperature for 1 hour. The mixture was poured into water, saturated saline was added, and the mixture was extracted with EtOAc. The combined organic layer was washed successively with a saturated aqueous solution of sodium bicarbonate, a 1M aqueous solution of sodium hydrogen sulfate, and saturated saline. The organic layer was dried over anhydrous sodium sulfate, filtered, and then concentrated under reduced pressure to obtain compound 47 (109.0 mg). 1 H NMR (600 MHz, DMSO-d 6 ) δ ppm: 1.23-1.33 (m, 12H), 1.34-1.43 (m, 6H), 1.50-1.60 (m, 6H), 1.62-1.77 (m, 2H), 1.80-1.91 (m, 4H), 1.93 (s, 9H), 2.01 (s, 18H), 2.03-2.07 (m, 2H), 2.10 (s, 9H), 2.12-2.20 (m, 2H), 2.25-2.30 (m, 2H), 2.77-2.82 (m, 2H), 2.96-3.08 (m, 6H), 3.41-3.48 (m, 3H), 3.53-3.68 (m, 3H), 3.86-3.95 (m, 3H), 4.00-4.07 (m, 3H), 4.10-4.23 (m, 5H), 4.85 (s, 3H) 5.04-5.13 (m, 9H), 7.71 (br t, J=5.32Hz, 1H), 7.78-7.85 (m, 2H), 7.89 (dd, J=12.84, 8.07Hz, 1H), 7.96 (d, J=8.07Hz, 1H) LCMS (m / z 1845.47 [M+H] + )

[0153] Synthesis scheme of compounds 53a, 53b and 53c [ka]

[0154] Synthesis of compound 49a [ka]

[0155] Zinc chloride (285 mg, 2.09 mmol) was dried in a 50 mL three-neck flask at 110° C. under reduced pressure for 1 hour and allowed to cool under reduced pressure overnight. The flask was flushed with nitrogen and a solution of 38 (611 mg, 1.57 mmol) and 48a (500 mg, 2.09 mmol) in DCE (5 mL) was added. The mixture was heated at 70° C. (external temperature) for 3 hours under nitrogen. The mixture was allowed to cool and diluted with ethyl acetate (10 mL) / saturated aqueous sodium bicarbonate (5 mL). The mixture was stirred for 10 minutes, filtered through Celite® and washed with ethyl acetate. The combined filtrate was washed with water and saturated brine. The organic layer was filtered through hydrophobic filter paper and concentrated under reduced pressure. The residue was purified by column chromatography using Biotage Isolera (50 g KP-Sil, 0-50% (3:1 EtOAc-IMS) / MTBE) to give 49a (599.4 mg). 1 H NMR (600 MHz, CDCl 3 ) δ ppm: 1.90 (s, 3H), 2.00 (s, 3H), 2.04 (s, 3H), 2.13 (s, 3H), 3.26-3.86 (m, 8H), 3.87-4.00 (m, 2H), 4.05-4.19 (m, 2H), 4.76 (br d, J=7.70Hz, 1H), 5.10-5.20 (m, 2H), 5.26 (br dd, J=6.60, 3.30Hz, 1H), 5.32 (br s, 1H), 5.43 (br s, 1H), 5.75 (br d, J=7.34Hz, 1H), 7.28-7.42 (m, 5H). LCMS (m / z 569 [M+H] + ).

[0156] Synthesis of compound 50a [ka]

[0157] To a solution of compound 49a (594.2 mg, 1.05 mmol) in IMS (18 mL), 4 M hydrogen chloride in 1,4-dioxane (0.34 mL, 1.36 mmol) and 10% palladium-carbon (50% water content, 125 mg) were added under a nitrogen atmosphere. The mixture was stirred at room temperature under a hydrogen atmosphere for 2.5 hours. The mixture was filtered through Celite (registered trademark) and washed with IMS. The combined filtrate was concentrated under reduced pressure, and compound 50a (507.9 mg) was obtained by concentrating under reduced pressure. 1 H NMR (600 MHz, DMSO-d 6 ) δ ppm: 1.77-1.84 (m, 3H), 1.86-1.92 (m, 3H), 1.97-2.03 (m, 3H), 2.06-2.13 (m, 3H), 2.92-3.00 (m, 2H), 3.52-3.75 (m, 5H), 3.83 (ddd, J=11.28, 5.41, 3.85Hz, 1H), 3.86-3.93 (m, 1H), 3.95-4.17 (m, 3H), 4.57 (d, J=8.44Hz 1H), 4.99 (dd, J=11.19, 3.48Hz, 1H), 5.23 (d, J=3.67Hz, 1H), 7.81 (br s, 3H), 7.85-7.92 (m, 1H) LCMS (m / z 435 [M+H] + )

[0158] Synthesis of compound 51a [ka]

[0159] To a solution of compound 50a (506.5 mg, 1.08 mmol) and compound 41 (68.9 mg, 0.14 mmol) in DMF (2.0 mL), HOBt (165 mg, 1.08 mmol), EDCI (206 mg, 1.08 mmol) and DIPEA (188 μL, 1.08 mmol) were added sequentially. The resulting mixture was stirred at room temperature for 20 hours. The mixture was purified by preparative HPLC to give compound 51a (40.0 mg). 1 H NMR (600 MHz, DMSO-d 6 ) δ ppm 1.62-1.73 (m, 2H), 1.77 (s, 9H), 1.80-1.87 (m, 2H), 1.89 (s, 9H), 1.99 (s, 9H), 2.02-2.08 (m, 2H), 2.10 (s, 9H), 2.11-2.24 (m, 4H), 2.34-2.38 (m, 2H), 3.13-3.22 (m, 4H), 3.34-3.42 (m, 7H), 3.44-3.54 (m, 7H), 3.55-3.61 (m, 3H), 3.73-3.80 (m, 3H), 3.84-3.91 (m, 3H), 3.97-4.08 (m, 9H), 4.13-4.25 (m, 2H), 4.55 (dd, J=8.07, 3.67Hz, 3H), 4.99 (br d, J=11.00Hz, 3H), 5.07-5.10 (m, 2H), 5.22 (d, J=3.30Hz, 3H), 7.31-7.40 (m, 5H), 7.60-7.97 (m, 8H) LCMS (m / z 1730 / 1731 [M+H] + )

[0160] Synthesis of compound 52a [ka]

[0161] To a solution of compound 51a (42.7 mg, 0.03 mmol) in IMS (8 mL), 10% palladium-carbon (50% water content; 16.0 mg) was added under a nitrogen atmosphere. The mixture was stirred at room temperature under a hydrogen atmosphere for 2.5 hours. The mixture was filtered through Celite (registered trademark) and washed with IMS. The filtrate was concentrated under reduced pressure to give compound 52a (41.2 mg). 1 H NMR (600 MHz, DMSO-d 6 ) δ ppm 1.62-1.75 (m, 4H), 1.78 (s, 9H), 1.81-1.87 (m, 2H), 1.89 (s, 9H), 2.00 (s, 9H), 2.05-2.32 (m, 17H), 3.12-3.23 (m, 4H), 3.36-3.43 (m, 7H), 3.45-3.54 (m, 7H), 3.55-3.61 (m, 3H), 3.74-3.81 (m, 3H), 3.83-3.92 (m, 3H), 3.98-4.08 (m, 9H), 4.11-4.23 (m, 2H), 4.51-4.59 (m, 3H), 4.99 (br d, J=11.00Hz, 3H), 5.22 (d, J=3.30Hz, 3H), 7.62-8.04 (m, 8H) LCMS (m / z 1640 / 1641 [M+H] + )

[0162] Synthesis of compound 53a [ka]

[0163] To a solution of compound 52a (41.6 mg, 0.03 mmol) in DMF (4 mL), triethylamine (28 μL, 0.20 mmol) was added dropwise, followed by pentafluorophenyl trifluoroacetate (17 μL, 0.10 mmol) under a nitrogen atmosphere. The resulting mixture was stirred at room temperature for 1.5 hours, poured into water, saturated saline was added, and extracted with EtOAc. The combined organic layer was washed successively with saturated aqueous sodium bicarbonate, 1M aqueous sodium hydrogen sulfate, and saturated saline. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was transferred to a sample vial containing DCM / MeOH, and the solvent was evaporated, followed by drying under reduced pressure at 40° C. for 2 hours to obtain compound 52c (27.1 mg). 1 H NMR (600 MHz, DMSO-d 6 ) δ ppm: 1.57-1.75 (m, 4H), 1.77 (s, 9H), 1.86-1.95 (m, 11H), 2.00 (s, 9H), 2.05-2.33 (m, 15H), 2.77-2.88 (m, 2H), 3.11-3.24 (m, 4H), 3.41-3.54 (m, 14H), 3.55-3.62 (m, 3H), 3.74-3.81 (m, 3H), 3.84-3.91 (m, 3H), 3.98-4.07 (m, 9H), 4.11-4.23 (m, 2H), 4.55 (br dd, J=8.07, 3.67Hz, 3H), 4.99 (br d, J=11.00Hz, 3H), 5.22 (d, J=3.30Hz, 3H), 7.65-8.04 (m, 8H) LCMS (m / z 1806.83 [M+H] + )

[0164] Synthesis of compound 49b [ka]

[0165] Zinc chloride (0.466 g, 3.42 mmol) in a 50 mL three-neck flask was dried at 110° C. under reduced pressure for 95 minutes and allowed to cool under reduced pressure overnight. The flask was purged with nitrogen and a solution of compound 38 (1.00 g, 2.57 mmol) and compound 48b (0.97 g, 3.42 mmol) in DCE (10 mL) was added. The mixture was heated at 70° C. (external temperature) under nitrogen for 3 hours. The mixture was allowed to cool and diluted with ethyl acetate (20 mL) / saturated aqueous sodium bicarbonate solution (10 mL). The mixture was stirred for 15 minutes, filtered through Celite® and washed with ethyl acetate. The filtrate was washed with water and saturated brine. The organic layer was filtered through hydrophobic filter paper and concentrated under reduced pressure. The residue was purified by column chromatography using a Biotage Isolera (100 g Sfar Duo Si, 0-50% (3:1 EtOAc-IMS) / MTBE) to give compound 49b (1.14 g). 1 H NMR (600 MHz, CDCl 3 ) δppm: 1.92 (s, 3H), 1.98 (s, 3H), 2.03 (s, 3H), 2.14 (s, 3H), 3.32-3.50 (m, 2H), 3.53-3.73 (m, 8H), 3.77-3.94 (m, 3H), 4.02-4.22 (m, 3H), 4.74 (br d, J=8.44Hz, 1H), 5.03-5.08 (m, 1H), 5.11 (br s, 2H), 5.26 (br s, 1H), 5.40-5.53 (m, 1H), 6.11-6.21 (m, 1H), 7.30-7.40 (m, 5H). LCMS (m / z 613 [M+H] + ).

[0166] Synthesis of compound 50b [ka]

[0167] To a solution of compound 49b (1.14 g, 1.86 mmol) in IMS (32 mL), a 4 M solution of hydrogen chloride in 1,4-dioxane (0.60 mL, 2.42 mmol) and 10% palladium-carbon (50% water content; 0.221 g) were added under a nitrogen atmosphere. The mixture was stirred at room temperature under a hydrogen atmosphere for 2.5 hours. The mixture was filtered through Celite® and washed with IMS. The combined filtrate was concentrated under reduced pressure to give compound 50b (0.97 g). 1 H NMR (600 MHz, DMSO-d 6 ) δ ppm 1.79 (s, 3H), 1.89 (s, 3H), 2.00 (s, 3H,) 2.11 (s, 3H), 2.94-3.02 (m, 2H), 3.50-3.62 (m, 9H), 3.77-3.84 (m, 1H), 3.86-3.94 (m, 1H), 3.98-4.10 (m, 3H), 4.54 (d, J=8.44Hz, 1H), 4.97 (dd, J=11.19, 3.48Hz, 1H), 5.22 (d, J=3.67Hz, 1H), 7.70-7.93 (m, 4H) LCMS (m / z 479 [M+H] + )

[0168] Synthesis of compound 51b [ka]

[0169] To a solution of compound 50b (556 mg, 1.08 mmol) and compound 41 (74.1 mg, 0.15 mmol) in DMF (3 mL), HOBt (165 mg, 1.08 mmol), EDCI (207 mg, 1.08 mmol), and DIPEA (189 μL, 1.08 mmol) were added sequentially. The resulting mixture was stirred at room temperature for 19 hours. The mixture was purified by preparative HPLC to give compound 51b (67.5 mg). 1 H NMR (600 MHz, DMSO-d 6) δ ppm 1.61-1.74 (m, 2H), 1.77 (s, 9H), 1.80-1.85 (m, 2H), 1.89 (s, 9H), 2.00 (s, 9H), 2.03-2.08 (m, 2H), 2.10 (s, 9H), 2.12-2.24 (m, 4H), 2.35-2.38 (m, 2H), 3.10-3.23 (m, 4H), 3.36-3.42 (m, 7H), 3.44-3.63 (m, 22H), 3.74-3.81 (m, 3H), 3.83-3.91 (m, 3H), 3.99-4.08 (m, 9H), 4.13-4.25 (m, 2H), 4.55 (d, J=8.44Hz, 3H), 4.95-5.00 (m, 3H), 5.09 (s, 2H), 5.21 (d, J=3.30Hz, 3H), 7.31-7.41 (m, 5H), 7.72-7.86 (m, 4H), 7.86-7.99 (m, 4H) LCMS (m / z 1862 / 1863 [M+H] + )

[0170] Synthesis of compound 52b [ka]

[0171] To a solution of compound 51b (66.1 mg, 0.04 mmol) in IMS (13 mL), 10% palladium-carbon (50% water content; 23.1 mg) was added under a nitrogen atmosphere. The mixture was stirred at room temperature under a hydrogen atmosphere for 2.5 hours. The mixture was filtered through Celite® and washed with IMS. The combined filtrate was concentrated under reduced pressure to give compound 52b (61.3 mg). 1 H NMR (600 MHz, DMSO-d 6) δ ppm 1.63-1.75 (m, 4H), 1.77 (s, 9H), 1.81-1.87 (m, 2H), 1.89 (s, 9H),2.00 (s, 9H), 2.03-2.24 (m, 17H), 3.09-3.23 (m, 4H), 3.35-3.43 (m, 7H), 3.44-3.64 (m, 22H), 3.74-3.81 (m, 3H), 3.84-3.91 (m, 3H), 3.98-4.07 (m, 9H), 4.10-4.24 (m, 2H), 4.56 (br d, J=8.80Hz, 3H), 4.98 (br d, J=11.00Hz, 3H), 5.22 (d, J=2.93Hz, 3H), 7.70-8.06 (m, 8H). LCMS (m / z 1772 / 1773 [M+H] + )

[0172] Synthesis of compound 53b [ka]

[0173] To a solution of compound 52b (60.7 mg, 0.03 mmol) in DMF (6 mL), triethylamine (38 μL, 0.27 mmol) was added dropwise, followed by pentafluorophenyl trifluoroacetate (24 μL, 0.14 mmol) under a nitrogen atmosphere. The resulting mixture was stirred at room temperature for 77 minutes, poured into water, saturated saline was added, and extracted with EtOAc. The combined organic layer was washed successively with saturated aqueous sodium bicarbonate, 1M aqueous sodium hydrogen sulfate, 4% aqueous lithium chloride, and saturated aqueous sodium chloride. The organic layer was dried over anhydrous sodium sulfate, filtered, concentrated, and dried under reduced pressure at 40° C. for 4 hours to obtain compound 53b (29.3 mg). 1 H NMR (600 MHz, DMSO-d 6 ) δ ppm: 1.62-1.75 (m, 2H), 1.77 (s, 9H), 1.85-1.95 (m, 13H), 2.00 (s, 9H), 2.10 (s, 9H), 2.12-2.33 (m, 6H), 2.78-2.86 (m, 2H), 3.12-3.24 (m, 4H), 3.35-3.44 (m, 7H), 3.45-3.64 (m, 22H), 3.75-3.81 (m, 3H), 3.83-3.92 (m, 3H), 3.99-4.06 (m, 9H), 4.12-4.26 (m, 2H), 4.55 (d, J=8.44Hz, 3H), 4.98 (dd, J=11.19, 2.02Hz, 3H), 5.21 (d, J=3.67Hz, 3H), 7.64-8.03 (m, 8H) LCMS (m / z 1939.01 [M+H] + )

[0174] Synthesis of compound 49c [ka]

[0175] Zinc chloride (210 mg, 1.54 mmol) was dried in a 50 mL three-neck flask at 110° C. under reduced pressure for 75 min and allowed to cool under reduced pressure overnight. The flask was purged with nitrogen and a solution of compound 38 (450.2 mg, 1.16 mmol) and compound 48c (503.4 mg, 1.54 mmol) in DCE (5 mL) was added. The mixture was heated at 70° C. (external temperature) for 3 h under argon. The mixture was allowed to cool and diluted with ethyl acetate (10 mL) / saturated aqueous sodium bicarbonate (5 mL). The mixture was stirred for 15 min, filtered through Celite® and washed with ethyl acetate. The filtrate was washed with water and saturated brine. The organic layer was filtered through hydrophobic filter paper and concentrated under reduced pressure. The residue was purified by column chromatography using Biotage Isolera (50 g Sfar Si, 0-50% (3:1 EtOAc-IMS) / MTBE) to give compound 49c (469.0 mg). 1 H NMR (600 MHz, CDCl3 ) δ ppm: 1.95 (s, 3H), 1.96 (s, 3H), 2.04 (s, 3H), 2.15 (s, 3H), 3.39 (br d, J=4.77Hz, 2H), 3.52-3.72 (m, 12H), 3.79-3.94 (m, 3H), 4.09-4.14 (m, 1H), 4.14-4.18 (m, 1H), 4.18-4.25 (m, 1H), 4.77 (br d, J=8.44Hz, 1H), 4.98-5.06 (m, 1H), 5.10 (br s, 2H), 5.31 (br s, 1H), 5.34-5.45 (m, 1H), 6.29-6.39 (m, 1H), 7.29-7.33 (m, 1H), 7.36 (d, J=4.40Hz, 4H) LCMS (m / z 657 [M+H] + )

[0176] Synthesis of compound 50c [ka]

[0177] To a solution of compound 49c (0.45 g, 0.69 mmol) in IMS (12 mL), 4 M hydrogen chloride in 1,4-dioxane (225 μL, 0.90 mmol) and 10% palladium-carbon (50% water content; 0.083 g) were added under a nitrogen atmosphere. The mixture was stirred at room temperature under a hydrogen atmosphere for 2.5 hours. The mixture was filtered through Celite (registered trademark) and washed with IMS. The filtrate was concentrated under reduced pressure to give compound 50c (0.40 g). 1 H NMR (600 MHz, DMSO-d 6) δ ppm 1.78 (s, 3H), 1.89 (s, 3H), 2.00 (s, 3H), 2.11 (s, 3H), 2.94-3.01 (m, 2H), 3.48-3.62 (m, 13H), 3.76-3.82 (m, 1H), 3.85-3.93 (m, 1H), 3.99-4.08 (m, 3H), 4.55 (d, J=8.44Hz, 1H), 4.94-5.01 (m, 1H), 5.22 (d, J=3.67Hz, 1H), 7.75-7.88 (m, 4H) LCMS (m / z 423 [M+H] + )

[0178] Synthesis of compound 51c [ka]

[0179] To a solution of compound 50c (379.8 mg, 0.679 mmol) and compound 41 (47.2 mg, 0.10 mmol) in DMF (2 mL), HOBt (104 mg, 0.68 mmol), EDCI (130 mg, 0.68 mmol) and DIPEA (119 μL, 0.68 mmol) were added in sequence. The resulting mixture was stirred at room temperature for 19 hours. The mixture was purified by preparative HPLC to give compound 51c (53.8 mg). 1 H NMR (600 MHz, DMSO-d 6) δ ppm 1.61-1.73 (m, 2H), 1.77 (s, 9H), 1.79-1.85 (m, 2H), 1.89 (s, 9H), 2.00 (s, 9H), 2.03-2.08 (m, 2H), 2.10 (s, 9H), 2.12-2.22 (m, 4H), 2.33-2.37 (m, 2H), 3.07-3.23 (m, 4H), 3.34-3.42 (m, 7H), 3.43-3.55 (m, 30H), 3.55-3.62 (m, 4H), 3.75-3.81 (m, 3H), 3.85-3.92 (m, 3H), 3.99-4.07 (m, 9H), 4.13-4.24 (m, 2H), 4.56 (d, J=8.80Hz, 3H), 4.97 (dd, J=11.00, 3.30Hz, 3H), 5.06-5.11 (m, 2H), 5.21 (d, J=3.30Hz, 3H), 7.30-7.39 (m, 5H), 7.62-7.84 (m, 5H), 7.85-7.95 (m, 3H) LCMS (m / z 1994 / 1995 [M+H] + )

[0180] Synthesis of compound 52c [ka]

[0181] To a solution of compound 51c (84 mg, 0.04 mmol) in IMS (17 mL), 10% palladium-carbon (50% water content; 27.3 mg) was added under a nitrogen atmosphere. The mixture was stirred at room temperature under a hydrogen atmosphere for 2.5 hours. The mixture was filtered through Celite (registered trademark) and washed with IMS. The filtrate was concentrated under reduced pressure to give compound 52c (78.9 mg). 1 H NMR (600 MHz, DMSO-d 6) δ ppm 1.60-1.75 (m, 4H), 1.78 (s, 9H), 1.80-1.87 (m, 2H), 1.89 (s, 9H), 2.00 (s, 9H), 2.03-2.32 (m, 17H), 3.12-3.23 (m, 4H), 3.36-3.42 (m, 7H), 3.43-3.63 (m, 34H), 3.74-3.81 (m, 3H), 3.84-3.92 (m, 3H), 3.99-4.07 (m, 9H), 4.09-4.22 (m, 2H), 4.57 (br d, J=8.44Hz, 3H), 4.98 (dd, J=11.19, 3.12Hz, 3H), 5.21 (d, J=3.30Hz, 3H), 7.61-8.05 (m, 8H), 12.01 (br s, 1H) LCMS (m / z 1904 / 1905 [M+H] + )

[0182] Synthesis of compound 53c

change

[0183] To a solution of compound 52c (77.3 mg, 0.04 mmol) in DMF (3 mL) was added triethylamine (45 μL, 0.33 mmol) and then pentafluorophenyl trifluoroacetate (27 μL, 0.16 mmol) dropwise under nitrogen atmosphere. The resulting mixture was stirred at room temperature for 1.5 hours, poured into water, added with saturated saline, and then extracted with EtOAc. The organic layer was washed successively with saturated aqueous sodium bicarbonate, 1M aqueous sodium hydrogen sulfate, and then saturated saline. The organic layer was dried over anhydrous sodium sulfate, filtered, and then concentrated under reduced pressure. The residue was transferred to a sample vial containing DCM / MeOH, and the solvent was evaporated, followed by drying under reduced pressure at 40° C. for 2 hours to obtain a residue. This was dissolved in EtOAc (20 mL), washed with 4% aqueous lithium chloride (5 mL), then saturated saline (5 mL), dried over anhydrous sodium sulfate, filtered, and then evaporated. The residue was transferred to a sample vial containing EtOAc, evaporated, and dried under reduced pressure at 40° C. DCM was added to the residue, concentrated under reduced pressure, and the residue was dried under reduced pressure at 40° C. for 4 hours to give compound 53c (19.4 mg). 1 H NMR (600 MHz, DMSO-d 6 ) δ ppm: 1.59-1.73 (m, 2H), 1.77 (s, 9H), 1.85-1.95 (m, 13H), 2.00 (s, 9H), 2.10 (s, 9H), 2.12-2.34 (m, 6H), 2.77-2.89 (m, 2H), 3.11-3.23 (m, 4H), 3.35-3.44 (m, 7H), 3.45-3.63 (m, 34H), 3.75-3.80 (m, 3H), 3.84-3.91 (m, 3H), 4.00-4.07 (m, 9H), 4.16-4.26 (m, 2H), 4.56 (d, J=8.80Hz, 3H), 4.97 (dd, J=11.37, 3.30Hz, 3H), 5.21 (d, J=3.67Hz, 3H), 7.62-8.03 (m, 8H). QC LCMS (m / z 1035.97 [M+2H] 2+ )

[0184] Examples 9 to 12: Synthesis of Nucleic Acid Complexes 9 to 12 The sense strand (215 nmol) of B2M (β2-microglobulin)-targeted siRNA was added with sodium tetraborate buffer pH 8.5 and compound 28 or 44 (1000 nmol) dissolved in DMSO, and stirred at room temperature. Water was added to the reaction solution, and purification was performed using Vivaspin 3K (Sartorius). Five times the amount of 28% ammonia water was added to the obtained crude product, and the mixture was allowed to stand at room temperature for 2 hours. Water was added to the reaction solution, and purification was repeated using Vivaspin 3K (Sartorius), to obtain nucleic acid complexes 9 to 12. The molecular weight of the nucleic acid complex synthesized in this example was measured by ESI-MS or MALDI-TOF-MS. The sequence of the sense strand, the sequence of the antisense strand, and the molecular weight used are shown in Table 5.

[0185] [Table 5]

[0186] The sequences and molecular weights of the nucleic acid complexes are shown in Table 6.

[0187] [Table 6]

[0188] Nucleic acid complex 9 [ka]

[0189] Nucleic acid complex 10 [ka]

[0190] Nucleic acid complex 11 [ka]

[0191] Nucleic acid complex 12 [ka]

[0192] Examples 13 to 16: Preparation of nucleic acid complexes 13 to 16 (si-RNA) si-RNA targeting B2M was prepared by adding equimolar amounts of the antisense strand (siB2M-as) and nucleic acid complexes 9 to 12. Table 7 shows the sense and antisense strands of si-RNA.

[0193] [Table 7]

[0194] <Test Example 4> The nucleic acid complexes (si-RNA) prepared in Examples 13 to 16 were subcutaneously administered at 1.0 mg / kg or 5.0 mg / kg to BALB / c mice (3 mice per group), and the livers were harvested 7 days after administration. The mRNA expression levels of B2M in the liver and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) as an internal control were measured by qPCR using a TaqMan probe (Applied Biosystems). The B2M mRNA expression level in the liver of mice in the untreated group (Control) was set as 100%, and the B2M mRNA expression level (relative value) in the nucleic acid complex-administered group was calculated. The results are shown in Table 8.

[0195] [Table 8]

[0196] As is clear from the results shown in Table 8, siRNA-2, siRNA-3, siRNA-4 and siRNA-5 showed dose-dependent gene expression-suppressing effects. siRNA-1, which does not contain a sugar ligand, showed almost no gene expression-suppressing effect even at 5.0 mg / kg, whereas siRNAs containing a sugar ligand showed high gene expression-suppressing effects even at 1.0 mg / kg.

[0197] Examples 17 to 19: Synthesis of nucleic acid complexes 17 to 19 A nucleic acid complex was prepared in the same manner as in Examples 9 to 11, except that a gapmer-type LNA antisense having a 3-10-3 motif (ASO; ISIS-549148) was used as the nucleic acid, and compounds 44, 47, or 53a were used. The sequences of the sense strand and the antisense strand used, and their molecular weights are shown in Table 9.

[0198] [Table 9]

[0199] The sequences and molecular weights of the nucleic acid complexes are shown in Table 10.

[0200] [Table 10]

[0201] Nucleic acid complex 17 [ka]

[0202] Nucleic acid complex 18 [ka]

[0203] Nucleic acid complex 19 [ka]

[0204] <Test Example 5> In vitro evaluation of uptake activity of nucleic acid complexes into human CD206-expressing Lenti-X 293T cells Nucleic acid complexes 17 to 19 were evaluated in the same manner as in Test Example 1. In Test Example 5, the fluorescence intensity of the well to which ASO_3'Cy3, a nucleic acid not modified with a sugar ligand, was added was set to 1, and the fluorescence intensity of the well to which each nucleic acid complex was added was calculated as a relative value.

[0205] The results are shown in Table 11. These results demonstrated that, compared with ASO_3'Cy3, a nucleic acid containing no sugar ligand, nucleic acid complexes 17 and 19 having GalNac were not efficiently taken up into cells expressing CD206, whereas nucleic acid complex 18 having mannose was efficiently taken up into cells expressing CD206.

[0206] [Table 11]

[0207] <Test Example 6> In vitro uptake activity of nucleic acid complexes into human ASGR1-expressing Lenti-X293T cells Nucleic acid complexes 17 to 19 were evaluated in the same manner as in Test Example 2. In Test Example 6, the fluorescence intensity of the well to which ASO_3'Cy3, a nucleic acid not modified with a sugar ligand, was added was set to 1, and the fluorescence intensity of the well to which each nucleic acid complex was added was calculated as a relative value.

[0208] The results are shown in Table 12. These results demonstrated that, compared with ASO_3'Cy3, a nucleic acid without sugar ligand modification, nucleic acid complex 18 having mannose was not efficiently taken up into cells expressing ASGR1, whereas nucleic acid complexes 17 and 19 having GalNac were efficiently taken up into cells expressing ASGR1.

[0209] [Table 12]

Claims

1. The following formula (III): 【Chemistry 1】 [Z is a group that includes an oligonucleotide.] or a pharma- ceutically acceptable salt thereof.

2. The following formula (IV): 【Chemistry 2】 [Z is a group that includes an oligonucleotide.] or a pharma- ceutically acceptable salt thereof.

3. The following formula (V): 【Chemistry 3】 [Z is a group that includes an oligonucleotide.] or a pharma- ceutically acceptable salt thereof.

4. The following formula (VI): 【Chemistry 4】 [Z is a group that includes an oligonucleotide.] or a pharma- ceutically acceptable salt thereof.

5. The following formula (VII): 【Chemistry 5】 [Z is a group that includes an oligonucleotide.] or a pharma- ceutically acceptable salt thereof.

6. The following formula (VIII): 【Chemistry 6】 [Z is a group that includes an oligonucleotide.] or a pharma- ceutically acceptable salt thereof.

7. The following formula (IX): 【Chemistry 7】 [Z is a group that includes an oligonucleotide.] or a pharma- ceutically acceptable salt thereof.

8. The following formula (X): 【Chemistry 8】 [Z is a group that includes an oligonucleotide.] or a pharma- ceutically acceptable salt thereof.

9. The following formula (XI): 【Chemistry 9】 [Z is a group that includes an oligonucleotide.] or a pharma- ceutically acceptable salt thereof.

10. The nucleic acid complex or a pharma- ceutically acceptable salt thereof according to any one of claims 1 to 9, wherein the oligonucleotide is single-stranded.

11. The nucleic acid complex or a pharma- ceutically acceptable salt thereof according to claim 10, wherein the oligonucleotide is linked via its 3' end.

12. The nucleic acid complex or a pharma- ceutically acceptable salt thereof according to claim 10, wherein the oligonucleotide is linked via the 5' end.

13. The nucleic acid complex or a pharma- ceutically acceptable salt thereof according to any one of claims 1 to 9, wherein the oligonucleotide is double-stranded.

14. 14. The nucleic acid complex or a pharma- ceutically acceptable salt thereof according to claim 13, wherein the oligonucleotide is linked via the 3' end of one strand.

15. 14. The nucleic acid complex or a pharma- ceutically acceptable salt thereof according to claim 13, wherein the oligonucleotide is linked via the 5' end of one strand.

16. A pharmaceutical composition comprising the nucleic acid complex according to any one of claims 1 to 15 or a pharma- ceutically acceptable salt thereof.

17. The pharmaceutical composition of claim 16 , which regulates expression of a target gene in a cell.

18. 18. The pharmaceutical composition of claim 17, wherein the cell is a dendritic cell, a macrophage or a hepatic parenchymal cell.

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