Conjugates and methods for their preparation and use

Abstract

To provide a compound that forms a conjugate with an active agent, thereby delivering the active agent, and methods for preparation and use thereof.SOLUTION: Disclosed are a compound for forming a conjugate with an active agent such as an oligonucleotide, having a structure represented by Formula (321), and a corresponding conjugate. The conjugate can specifically target hepatocytes, thereby effectively solving the problems associated with delivery of oligonucleotide drugs in vivo, and has low toxicity and excellent delivery efficiency while maintaining high stability for the delivered oligonucleotide.SELECTED DRAWING: Figure 16

Description

[Technical field]

[0001] The present disclosure relates to the field of drugs, specifically to a method for forming a complex with an active drug to activate the drug. The present disclosure further relates to compounds for the delivery of sexually active drugs and methods for their preparation and use. It relates to a complex formed by a compound. [Background technology]

[0002] In the development of small molecule nucleic acid drugs, the delivery system is one of the key technologies. Summary of the Invention [Problem to be solved by the invention]

[0003] Among these is a small molecule nucleic acid delivery system, which is a targeted complex delivery technology for hepatocytes. [Means for solving the problem]

[0004] According to one aspect of the present invention, the present disclosure provides a compound having the structure shown in formula (321): to provide.

[0005] [ka] During the ceremony, n1 is an integer from 1 to 3, and n3 is an integer from 0 to 4. Each of m1, m2, and m3 is independently an integer of 2 to 10; R 10 , R 11 , R 12 , R 13 , R 14 and R 15 are each independently H, C1-C 10 Alkyl groups, C1-C 10 Halogenated alkyl groups and C1-C 10 alkoxy groups, R4 is a group capable of covalently binding to an active drug or agent; Each L1 is a linear alkylene group having a length of 1 to 70 carbon atoms, The carbon atoms are C(O), NH, O, S, CH=N, S(O)2, C2-C 10 Arken C2-C 10 Alkynylene group, C6-C 10 Arylene group, C3-C 18 Hetero Cyclylene group and C5-C 10 Any one selected from the group consisting of heteroarylene groups. L1 is optionally substituted with one or more of C1-C 10 Alkyl groups, C6-C 10 Aryl Group, C5-C 10 Heteroaryl groups, C1-C 10 Halogenated alkyl groups, -OC1-C 10 Alkyl group, -OC1-C 10 Alkylphenyl group, -C1-C 10 Alkyl-OH , -OC1-C 10 Halogenated alkyl groups, -SC1-C 10 Alkyl group, -SC1-C 10 Alkylphenyl group, -C1-C 10 Alkyl-SH, -SC1-C 10 Halogenation Alkyl groups, halogen substituents, -OH, -SH, -NH2, -C1-C 10 Alkyl-N H2, -N(C1-C 10 Alkyl group)(C1-C 10 alkyl group), -NH(C1-C 10 alkyl group), -N(C1-C 10 Alkyl group)(C1-C 10 Alkylphenyl group ), -NH(C1-C 10 alkylphenyl group), cyano group, nitro group, -CO2H, - C(O)O(C1-C 10 alkyl group), -CON(C1-C 10 Alkyl group)(C1- C 10 alkyl group), -CONH(C1-C 10 alkyl group), -CONH2, -NHC (O)(C1-C 10 alkyl group), -NHC(O)(phenyl group), -N(C1-C1 0 alkyl)C(O)(C1-C 10 alkyl group), -N(C1-C 10 Alkyl)C( O) (phenyl group), -C(O)C1-C 10 Alkyl group, -C(O)C1-C 10 Al alkylphenyl group, -C(O)C1-C 10 Halogenated alkyl groups, -OC(O)C1-C 10 Alkyl group, -SO2(C1-C 10 alkyl group), -SO2 (phenyl group), -S O2(C1-C 10 Halogenated alkyl group), -SO2NH2, -SO2NH(C1-C 10 alkyl group), -SO2NH (phenyl group), -NHSO2 (C1-C 10 Alkyl group), -NHSO2 (phenyl group) and -NHSO2 (C1-C 10 Alkyl Halides optionally having one or more substituents from the group consisting of Each S1 is independently M1, and any active hydroxy group, if any, is protected with an alkoxy protecting group, Each M1 is independently selected from a ligand capable of binding to a cell surface receptor.

[0006] In some embodiments, each L1 independently represents a group of A1 through A26, and any combination thereof. The compound is selected from the group consisting of:

[0007] [ka] In the formula, each j1 is independently an integer from 1 to 20; Each j2 is independently an integer from 1 to 20; Each R' is independently C1-C 10 is an alkyl group, Each Ra is independently selected from the group consisting of A27 to A45, and any combination thereof.

[0008] [ka] Each Rb is independently C1-C 10 is an alkyl group, TIFF0007662216000004.tif4153 represents the site at which the group is attached to the remainder of the molecule.

[0009] In one aspect of the present invention, the present disclosure provides a conjugate having a structure as shown in formula (1): Provide.

[0010] [ka] During the ceremony, n1 is an integer from 1 to 3, and n3 is an integer from 0 to 4. m1, m2, and m3 each independently represent an integer of 2 to 10; R 10 , R 11 , R 12 , R 13 , R 14 and R 15 are each independently H, C1-C 10 Alkyl groups, C1-C 10 Alkyl halide or C1-C 10 Selected from alkoxy groups Selected, R3 is an active drug, R2 is a linear alkylene group having a length of 1 to 20 carbon atoms, Carbon atoms are C(O), NH, O, S, CH=N, S(O)2, C2-C 10 Alkenira group, C2-C 10 Alkynylene group, C6-C 10 Arylene group, C3-C 18 Heterosi Crylene group and C5-C 10 Any one selected from the group consisting of heteroarylene groups or more optionally substituted, R2 being C1-C 10 Alkyl groups, C6-C 10 Aryl Group , C5-C 10 Heteroaryl groups, C1-C 10 Halogenated alkyl groups, -OC1-C1 0 alkyl group, -OC1-C 10 Alkylphenyl group, -C1-C 10 Alkyl-OH, -OC1-C 10 Halogenated alkyl groups, -SC1-C 10 Alkyl group, -SC1-C1 0 alkylphenyl group, -C1-C 10 Alkyl-SH, -SC1-C 10 Halogenated A alkyl groups, halogen substituents, -OH, -SH, -NH2, -C1-C 10 Alkyl-NH 2, -N(C1-C 10 Alkyl group)(C1-C 10 alkyl group), -NH(C1-C1 0 alkyl group), -N(C1-C 10 Alkyl group)(C1-C 10 alkylphenyl group) , -NH(C1-C 10 alkylphenyl group), cyano group, nitro group, -CO2H, -C (O)O(C1-C 10 alkyl group), -CON(C1-C 10 Alkyl group)(C1-C 10 alkyl group), -CONH(C1-C 10Alkyl group), -CONH2, -NHC( O)(C1-C 10 alkyl group), -NHC(O)(phenyl group), -N(C1-C 10 Alkyl)C(O)(C1-C 10 alkyl group), -N(C1-C 10 Alkyl)C(O ) (phenyl group), -C(O)C1-C 10 Alkyl group, -C(O)C1-C 10 Alki phenyl group, -C(O)C1-C 10 Halogenated alkyl groups, -OC(O)C1-C1 0 alkyl group, -SO2(C1-C 10 alkyl group), -SO2 (phenyl group), -SO 2(C1-C 10 Halogenated alkyl group), -SO2NH2, -SO2NH(C1-C1 0 alkyl group), -SO2NH (phenyl group), -NHSO2 (C1-C 10 Alkyl group ), -NHSO2(phenyl group) and -NHSO2(C1-C 10 Halogenated alkyl groups Optionally, one or more substituents from the group consisting of Each L1 is independently a straight chain alkylene group of 1 to 70 carbon atoms in length, or multiple carbon atoms are C(O), NH, O, S, CH=N, S(O)2, C2-C 10 Alkenylene group, C2-C 10 Alkynylene group, C6-C 10 Arylene group, C3-C1 8 Heterocyclylene groups and C5-C 10 Heteroarylene groups may be selected from the group consisting of L1 is optionally substituted with one or more of C1-C 10 Alkyl groups, C6-C 10 Aryl groups, C5-C 10 Heteroaryl groups, C1-C 10Halogenated alkyl groups, -O C1-C 10 Alkyl group, -OC1-C 10 Alkylphenyl group, -C1-C 10 Alki Ru-OH, -OC1-C 10 Halogenated alkyl groups, -SC1-C 10 Alkyl group, -S C1-C 10 Alkylphenyl group, -C1-C 10 Alkyl-SH, -SC1-C 10 Ha Halogenated alkyl groups, halogen substituents, -OH, -SH, -NH2, -C1-C 10 Al Kyl-NH2, -N(C1-C 10 Alkyl group)(C1-C 10 Alkyl group), -NH( C1-C 10 alkyl group), -N(C1-C 10 Alkyl group)(C1-C 10 Alkylph phenyl group), -NH(C1-C 10 alkylphenyl group), cyano group, nitro group, -CO 2H, -C(O)O(C1-C 10 alkyl group), -CON(C1-C 10 (alkyl group) (C1-C 10 alkyl group), -CONH(C1-C 10 alkyl group), -CONH2, -NHC(O)(C1-C 10 alkyl group), -NHC(O)(phenyl group), -N(C 1-C 10 Alkyl)C(O)(C1-C 10 alkyl group), -N(C1-C 10 Alki -C(O)C1-C 10 Alkyl group, -C(O)C1-C 10 Alkylphenyl group, -C(O)C1-C 10 Halogenated alkyl groups, -OC(O) C1-C 10 Alkyl group, -SO2(C1-C 10 alkyl group), -SO2(phenyl group ), -SO2(C1-C 10 Halogenated alkyl group), -SO2NH2, -SO2NH( C1-C 10 alkyl group), -SO2NH (phenyl group), -NHSO2 (C1-C 10 alkyl group), -NHSO2 (phenyl group) and -NHSO2 (C1-C 10 Halogenation alkyl group), Each M1 is a selected member of a ligand capable of binding to a cell surface receptor.

[0011] In some embodiments, each L1 is selected from the groups A1 through A26, and any combination thereof. are independently selected from the group consisting of:

[0012] [ka] In the formula, each j1 is independently an integer from 1 to 20; Each j2 is independently an integer from 1 to 20; Each R' is independently C1-C 10 is an alkyl group, Each Ra is independently selected from groups of formulas A27-A45, and any combination thereof.

[0013] [ka] Each Rb is independently C1-C 10 is an alkyl group, TIFF0007662216000008.tif3158 represents the site at which the group is attached to the remainder of the molecule.

[0014] In one aspect of the present invention, the present disclosure provides a method for the administration of the conjugates disclosed herein to hepatocytes. Drugs for the treatment and / or prevention of pathological conditions or diseases caused by the expression of specific genes in The present invention provides for use in the preparation of

[0015] In one aspect of the invention, the present disclosure provides a method for administering to a subject the use of a conjugate as disclosed herein. The method includes administering to a subject in need thereof an effective amount of Methods for treating pathological conditions or diseases resulting from expression are provided.

[0016] In one aspect of the present invention, the present invention provides a method for contacting a subject with a complex as disclosed herein. The present invention provides a method for suppressing the expression of a specific gene in a liver cell, comprising:

[0017] In one aspect of the invention, the present disclosure provides a kit comprising the conjugates disclosed herein. to provide.

[0018] Other features and advantages of the present disclosure are described in detail in the following text.

[0019] [Incorporation by reference] All publications, patents and patent applications mentioned herein are hereby incorporated by reference in their entirety. Each application is hereby incorporated by reference to the same extent as if each application was specifically and individually incorporated by reference. and is hereby incorporated by reference. [Brief description of the drawings]

[0020] The appended claims particularly set forth the novel features of the invention and point out certain characteristics and advantages thereof. The following detailed description and drawings set forth illustrative embodiments, among others, provide a more detailed understanding of the present invention. It will be readily understood that these illustrative embodiments utilize the principles of the present invention. do. [Figure 1A-1B]1 shows semi-quantitative results of a stability test of siRNA complexes in in vitro tritosomes. [Figure 2A-2B] 1 shows semi-quantitative results of an in vitro human plasma stability study of test siRNA complexes. [Figure 3A-3B] 1 shows semi-quantitative results of an in vitro monkey plasma stability study of test siRNA complexes. [Figure 4-11] The PK / TK plasma or tissue concentration time course curves of Complex 24 in rat plasma at a dose of 10 mg / kg (Figure 4), Complex 24 in rat liver and kidney at a dose of 10 mg / kg (Figure 5), Complex 24 in rat plasma at a dose of 50 mg / kg (Figure 6), Complex 24 in rat liver and kidney at a dose of 50 mg / kg (Figure 7), Complex 25 in rat plasma at a dose of 10 mg / kg (Figure 8), Complex 25 in rat liver and kidney at a dose of 10 mg / kg (Figure 9), Complex 25 in rat plasma at a dose of 50 mg / kg (Figure 10), and Complex 25 in rat liver and kidney at a dose of 50 mg / kg (Figure 11) are shown. [Figures 12A-12D] The IC50 value determination of complex 24 inhibiting the expression of GSCM is shown, in comparison with GSSM, PSCM and PSSM, respectively. [Figure 13-15] 1 shows the inhibitory effect of the complex of the present disclosure on HBV mRNA in vivo. [Figure 16] 1 shows the time-dependent inhibitory effect of the complex of the present disclosure on HBsAg expression in the serum of HBV transgenic mice. [Figure 17] 1 shows the time-dependent inhibitory effect of the complex of the present disclosure on HBV DNA expression in the serum of HBV transgenic mice. [Figure 18] 1 shows the time-dependent inhibitory effect of the complex 25 of the present disclosure on HBsAg expression in the serum of HBV transgenic mice. [Figure 19] 1 shows the time-dependent inhibitory effect of the complex of the present disclosure on HBsAg expression in an M-Tg model. [Figure 20]1 shows the time-dependent inhibitory effect of the complex of the present disclosure on HBsAg expression in an M-Tg model. [Figure 21] 1 shows the time-dependent inhibitory effect of the complex of the present disclosure on HBsAg expression in a 1.28 copy HBV-Tg model. [Figure 22-24] 1 shows the inhibitory effect of the complex of the present disclosure on target mRNA and off-target mRNA. [Figure 25-27] 1 shows the inhibitory effect of the complex of the present disclosure on HBV mRNA in vivo. [Figure 28] 1 shows the time-dependent inhibitory effect of the complex of the present disclosure on HBsAg expression in the serum of HBV transgenic mice. [Figure 29] 1 shows the time-dependent inhibitory effect of the complex of the present disclosure on HBsAg expression in the serum of HBV transgenic mice. [Diagram 30] 1 shows the in vivo suppressive effect of the complex of the present disclosure on HBV mRNA on day 85. [Diagram 31] 1 shows the time-dependent inhibitory effect of complex 43 on HBsAg expression in the serum of HBV transgenic mice. [Diagram 32] 1 shows the time-dependent inhibitory effect of complex 43 on HBV DNA expression in the serum of HBV transgenic mice. [Figure 33-34] 1 shows the stability of complex 167 in human and mouse lysosomal lysates. [Diagram 35] 1 shows the time-dependent inhibitory effect of complex 168 on HBsAg expression in a 1.28 copy HBV-Tg model. [Diagram 36] 1 shows the time-dependent inhibitory effect of complex 168 on HBeAg expression in a 1.28 copy HBV-Tg model. [Figure 37] 1 shows the time-dependent inhibitory effect of complex 168 on HBV DNA expression in a 1.28 copy HBV-Tg model. [Fig. 38A-38B] The inhibition rates of ANGPTL mRNA expression on days 14 and 28 are shown. [Figure 39A-39B] The inhibition rate of lipids by the complex of the present disclosure is shown, and is expressed as total cholesterol (CHO) and triglyceride (TG) in serum. [Fig. 40A-40B] The inhibition rate of lipids by complex 115 over time is shown, expressed as serum total cholesterol (CHO) and triglycerides (TG). [Fig. 41A-41B] The inhibition rate of lipids by conjugates 115 and 111 over time is shown, expressed as serum total cholesterol (CHO) and triglycerides (TG). [Fig. 42A-42D] The inhibition rate of lipids by complex 111 over time at different doses is shown as serum total cholesterol (CHO) and triglycerides (TG). [Fig. 43A-43B] The inhibition rate of lipids by conjugates 25 and 169 over time is shown, expressed as serum total cholesterol (CHO) and triglycerides (TG). [Figure 43C] The inhibition rate of ANGPTL mRNA expression is shown. [Figure 44A] The inhibition rate of APOC3 expression in the liver on day 14 is shown. [Fig. 44B-44C] The inhibition rates of lipids by different doses of complex 144 are shown, expressed as serum total cholesterol (CHO) and triglycerides (TG). [Fig. 45A-45B] The inhibition rate of lipids by different doses of complex 170 is shown, expressed as serum total cholesterol (CHO) and triglycerides (TG). [Fig. 46A-46D] The inhibition rate of lipids by the complex of the present disclosure is shown, and is expressed in terms of serum total cholesterol (CHO) and triglyceride (TG). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] Hereinafter, the embodiments of the present disclosure will be described in detail. The detailed description is merely for the purpose of explaining or interpreting the present disclosure and is not intended to be limiting of the present invention. It is not intended to limit the disclosure in any respect.

[0022] (definition) In the context of this disclosure, unless otherwise stated, the capital letters C, G, U and A are The lowercase letter m indicates that the adjacent nucleotide to the left of the letter m is 2. A lowercase letter f denotes a '-methoxy modified nucleotide, and the lowercase letter f is adjacent to the left of the letter f. A lowercase s indicates that one of the nucleotides is a 2'-fluoro modified nucleotide. The two nucleotides adjacent to the left and right of the letter s are bonded by a thiophosphate group. P1 is a nucleotide that is adjacent to the right of P1 and is 5' 5'-phosphate nucleotide or 5'-phosphate analog modified nucleotide, vinyl phosphate modified nucleotides (represented as VP in the examples below); 5'-phosphate nucleotides (represented by P in the examples below) or 5'-thiophosphate It refers to an ester-modified nucleotide (designated Ps in the examples below).

[0023] In the context of this disclosure, the terms "complement" and "reverse complement" as described may be used interchangeably. In general, the term "double stranded nucleic acid molecule" refers to the separation of bases in one strand from those in the other strand. It means that each base pairs with the base on the other strand in a complementary manner. The purine base adenine (A) is always paired with the pyrimidine base thymine (T) (or R In NA, uracil (U) is paired with the purine base guanine (G), which is usually Each base pair consists of one purine base, which pairs with the pyrimidine base cytosine (C). and one pyrimidine. An adenine on one strand is always replaced by a thymine (or uracil) on the other strand. If all nucleotides in the sequence are paired with cytosine, and guanine always pairs with cytosine, then both strands are complementary. It is believed that the base sequence of one strand can be inferred from the sequence of its complementary strand. A "match" is a sequence in which bases at corresponding positions in a double-stranded nucleic acid do not exist in a complementary pair. This means that...

[0024] In the context of this disclosure, unless otherwise specified, "essentially reverse complementary" refers to two This refers to the presence of three or fewer mismatches between the nucleotide sequences of the "Completely reverse complementary" means that there is not more than one base mismatch between the two nucleotide sequences. "Perfectly reverse complementary" refers to a pair of nucleotide sequences that have no base pairs. This means that there is no match.

[0025] In the context of this disclosure, a " A "nucleotide difference" is a change in the base of a nucleotide at the same position between nucleotide sequences. For example, if the nucleotide base in the second sequence is A and the nucleotide base in the first sequence is If the base at the same position is U, C, G, or T, then there is a nucleotidic error between the two sequences at that position. In some embodiments, a nucleotide difference at a position may exist. When an abasic nucleotide or nucleotide analog is used in place of a nucleotide, the nucleotide at that position is It is also believed that nucleotide differences exist.

[0026] In the context of the present disclosure, in particular the preparation of the composite molecules or siRNA complexes described in this disclosure. In describing the methods, unless otherwise stated, nucleoside monomers are used. The term "unmodified or modified RNA monomer" refers to the RNA sequence to be prepared. Phosphoramidites (unmodified or modified RNA phosphoramidites) These are used in solid-phase phosphoramidite synthesis, which is called Solid phase phosphoramidite synthesis is a well-known method in the art for synthesizing RNA. Elsewhere in this disclosure, RNA phosphoramidites are Also called nucleoside phosphoramidite All of the nucleoside monomers used in this disclosure are commercially available.

[0027] As used herein, a dash ("-") that is not between two letters or symbols is used to indicate the point of attachment of a substituent. For example, -C1-C 10 Alkyl-N H2 is C1-C 10 The bond is formed by an alkyl group.

[0028] As used herein, "any" or "optionally" refers to the event or events described thereafter. that circumstances may or may not occur and that such statements are based on the assumption that an event or circumstance will occur. For example, "optionally substituted alkyl" includes cases where the alkyl group is substituted or unsubstituted. , including "alkyl" and "substituted alkyl" as defined in the following sentence. With respect to any groups that contain groups, it is to be understood that these groups may have sterically impractical, synthetically impractical, or otherwise unfeasible effects. It is not intended to introduce any substitutions or substitution patterns that are inherently unstable. This will be understood by those skilled in the art.

[0029] As used herein, "alkyl" refers to both straight and branched chains having the specified number of carbon atoms. The specific number usually refers to 1 to 20 carbon atoms, for example, 1 to 10 carbon atoms. For example, C1-C6 alkyl is 1 to 6 Includes straight and branched chain alkyls of up to 10 carbon atoms. Refers to alkyl residues having a specific number of carbons. When used herein, it is intended to include all branched and straight chain forms having that number of carbons. Thus, for example, "butyl" includes n-butyl, sec-butyl, isobutyl and tert-butyl. -butyl, and "propyl" includes n-propyl and isopropyl. Alkylene is a subset of alkyl, just like alkyl, but with two points of attachment. It refers to a residue that

[0030] As used herein, "alkenyl" means an alkyl group having at least one carbon-carbon double bond. refers to an unsaturated branched or straight chain alkyl having a carbon-carbon double bond, The group is obtained by removing one hydrogen atom from adjacent carbon atoms of the The bonds may be in the cis or trans configuration. Exemplary alkenyls include vinyl and Prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl Propylene such as allyl, propanyl, propan-2-en-2-yl, and butan-1-en-1-yl. But-1-en-2-yl, 2-methylprop-1-en-1-yl, but-2-en-1 buta-1,3-dien-1-yl, buta-2-en-2-yl, buta-1,3-dien-1-yl, buta-1 , butenyl, such as 3-dien-2-yl, and the like. In certain embodiments, alkenyl has 2 to 20 carbon atoms, while in other embodiments, Alkenylene has 2 to 10, 2 to 8 or 2 to 6 carbon atoms. Alkenyl is a subset of aryl and refers to residues similar to alkenyl, but having two points of attachment.

[0031] As used herein, "alkynyl" means an alkynyl group having at least one carbon-carbon triple bond. The term refers to an unsaturated branched or straight chain alkyl group having a carbon-carbon triple bond, It is obtained by removing two hydrogen atoms from adjacent carbon atoms of an alkyl. The quinyls include ethynyl, prop-1-yn-1-yl, prop-2-yn-1-yl, and Propynyl, such as but-1-yn-1-yl, but-1-yn-3-yl, but-3-yn-2-yl, and butynyl, such as -yn-1-yl. In certain embodiments, alkynyl has 2 to 20 carbon atoms, but in other embodiments, 2 to 4 carbon atoms. Alkynylene is a subset of alkynyl having 10, 2 to 8 or 2 to 6 carbon atoms. It refers to a residue similar to alkynyl, but having two points of attachment.

[0032] As used herein, "alkoxy" refers to an alkyl group having a specified number of carbon atoms attached through oxygen bridges. alkyl of a carbon atom, for example, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, pentyloxy, 2-pentyl Oxy, isopentyloxy, neopentyloxy, hexyloxy, 2-hexyloxy Alkoxy is usually 1- It has 10, 1 to 8, 1 to 6 or 1 to 4 carbon atoms bonded by oxygen bridges.

[0033] As used herein, "aryl" refers to a group formed by removing a hydrogen atom from a ring carbon atom. and a group derived from an aromatic monocyclic or polycyclic hydrocarbon ring system. The formula or polycyclic hydrocarbon ring system contains only hydrogen and carbon from 6 to 18 carbon atoms, At least one of the rings in the ring system is fully unsaturated, i.e., it follows the Hückel theory Contains a cyclic, delocalized (4n+2) π-electron system. Aryl includes phenyl, fluorene, etc. Arylene is a substituted or unsubstituted aryl group. A aryl group is a subset of aryl and refers to a residue that is the same as aryl but has two points of attachment.

[0034] As used herein, "cycloalkyl" generally refers to a group having 3 to 7 ring carbon atoms. The ring may be saturated or may contain one or more carbon-carbon bicyclic rings. Examples of cycloalkyl include cyclopropyl, cyclobutyl, Cyclopentyl, cyclopentenyl, cyclohexyl and cyclohexenyl, and nor These include bridged and cage cyclic groups such as norbornane.

[0035] As used herein, a "halogen substituent" or "halogen" refers to any group including fluoro, chloro ... The term "halogen" refers to fluorine, chlorine, bromine, and iodine. include.

[0036] As used herein, "halogenated alkyl" refers to an alkyl group having a specified number of carbon atoms. is an alkyl group as defined above, substituted with up to the maximum permitted number of halogen atoms. Examples of halogenated alkyl groups include trifluoromethyl, difluoromethyl, and , 2-fluoroethyl, and pentafluoroethyl.

[0037] The term "heterocyclic group" refers to a group having 2 to 12 carbon atoms and 1 to 2 carbon atoms selected from nitrogen, oxygen, and sulfur. It refers to a stable 3- to 18-membered non-aromatic cyclic group containing 6 heteroatoms. Unless otherwise specified, heterocyclic groups may be monocyclic, bicyclic, tricyclic or tetracyclic ring systems, fused or bridged. The heteroatoms in the heterocyclic groups may be optionally oxidized. Nitrogen atoms, if present, are optionally quaternized. Heterocyclic groups may be partially saturated or completely Heterocyclic groups can be attached to the rest of the molecule through any atom of the ring. Examples of such heterocyclic groups include dioxanyl, thiophenyl[1,3]diphenyl. Sulfonyl (thienyl[1,3]dithianyl), decahydroisoquinolinyl Imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, Morpholinyl, Octahydroindole, Octahydroisoindole, 2-Oxapipe Razinyl, 2-oxapiperidyl, 2-oxapyrrolidinyl, oxazolidinyl, piperidinyl Dysyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidyl, quinuclidinyl , thiazolidinyl, tetrahydrofuryl, trisulfonyl, tetra tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxothiomorpholinyl Examples include, but are not limited to, 1,1-dioxothiomorpholinyl and 1,1-dioxothiomorpholinyl.

[0038] "Heteroaryl" means an alkyl group having 2 to 17 carbon atoms and a group selected from nitrogen, oxygen, and sulfur. and 1 to 6 heteroatoms, and a group derived from a 3 to 18-membered aromatic ring radical. As used herein, heteroaryl is a monocyclic, bicyclic, tricyclic, or tetracyclic ring system. At least one of the rings in the ring system may be fully unsaturated, i.e., it may be hysterically unsaturated. Heteroaryls contain cyclic delocalized (4n+2) π-electron systems according to the Kell theory. or bridged ring systems. The heteroatoms in heteroaryl are optionally oxidized. A number of nitrogen atoms, if present, are optionally quaternized. Heteroaryl is any one of the heteroaryls in the ring. An example of a heteroaryl is azepinyl. , acridinyl, benzimidazolyl, benzoindole, 1,3-benzodioxazolyl benzofuryl, benzoxazolyl, benzo[d]thiazolyl, benzothiadiazolyl Benzo[b][1,4]dioxepinyl inyl), benzo[b][1,4]oxazinyl, 1,4-benzodioxanyl, Zonaphthofuranyl, benzoxazolyl, benzodioxolyl yl), benzodioxinyl, benzopyranyl, ben Zopyronyl, benzofuryl, benzofuranonyl, benzothiophenyl, benzothieno[3 ,2-d]pyrimidinyl, benzotriazolyl, benzo[4,6]imidazo[1,2-a ]pyridyl, carbazolyl, cinnolyl, cyclopenta[d]pyrimidinyl, 6,7-di Hydro-5H-cyclopenta[4,5]thieno[2,3-d]pyrimidinyl, 5,6-di Hydrobenzo[h]quinazolinyl, 5,6-dihydrobenzo[h]cinnolyl, 6,7- Dihydro-5H-benzo[6,7]cyclohepta[1,2-c]pyridazinyl, dibenzo Furyl, dibenzothiophenyl, furyl, furanonyl, furo[3,2-c]pyridyl, 5 ,6,7,8,9,10-Hexahydrocycloocta[d]pyrimidinyl, 5,6,7, 8,9,10-Hexahydrocycloocta[d]pyridazinyl, 5,6,7,8,9,1 0-Hexahydrocycloocta[d]pyridyl, isothiazolyl, imidazolyl, indane Zolyl, indole, indazolyl, isoindole, indolinyl, isoindolinyl , isoquinolyl, indolizinyl, isoxazolyl, 5,8-methanol-5,6,7 ,8-Tetrahydroquinazolinyl, naphthyridinyl, 1,6-naphthyridinonyl, oxa Diazolyl, 2-oxazepinyl, oxazolyl, oxiranyl, 5,6,6a,7, 8,9,10,10a-Octahydrobenzo[H]quinazolinyl, 1-phenyl-1H- Pyrrolyl, phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, pteryl Dinyl, purinyl, pyrrolyl, pyrazolyl, pyrazolo[3,4-d]pyrimidinyl, pyri pyridyl, pyrido[3,2-d]pyrimidinyl, pyrido[3,4-d]pyrimidinyl, pyrazidinyl nyl, pyrimidinyl, pyridazinyl, pyrrolyl, quinazolinyl, quinoxalinyl, quinolinyl isoquinolyl, tetrahydroquinolyl, 5,6,7,8-tetrahydroquinazolinyl , 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidinyl, 6,7,8,9-Tetrahydro-5H-cyclohepta[4,5]thieno[2,3-d]pi Rimidinyl, 5,6,7,8-tetrahydropyrido[4,5-c]pyridazinyl, thiazo aryl, thiadiazolyl, triazolyl, tetrazolyl, triazinyl, thieno[2,3- d]pyrimidinyl, thieno[3,2-d]pyrimidinyl, thieno[2,3-c]pyridyl and thiophenyl (i.e., thienyl).

[0039] A variety of hydroxy protecting groups may be used in the present disclosure. Generally, the protecting groups are In this way, chemical functional groups can be rendered insensitive to certain reaction conditions while leaving the remainder of the molecule substantially can be added to and removed from the relevant functional groups in a molecule without damaging the A representative hydroxy protecting group is described in Beaucage et al., Tetrahedron 19 92, 48, 2223-2311, and Greene and Wuts, Protective Groups in Organic Synthesis, 2nd edition, Chapter 2, Joh Wiley & Sons, New York, 1991, Each of the above is incorporated by reference in its entirety. Thus, the protecting group is stable under basic conditions but can be removed under acidic conditions. In some embodiments, non-exclusive illustrative examples of hydroxy protecting groups that can be used herein include Dimethoxytrityl (DMT), monomethoxytrityl, 9-phenylxanthene- 9-yl(Pixyl) "9-phenylxanthen-9-yl(Pixyl)" and 9-(p-methoxyphenyl)xanthen-9-yl (Mox) Some examples include Non-exclusive illustrative examples of hydroxy protecting groups that can be used herein include Tr( trityl), MMTr (4-methoxytrityl), DMTr (4,4'-dimethoxytrityl) Trimethoxytrityl) and TMTr (4,4',4''-trimethoxytrityl).

[0040] The term "subject," as used herein, refers to any animal, e.g., a mammal. The subject of the present invention may be a human, a non-human primate (e.g., a rhesus macaque), monkeys or other species of macaques), mice, pigs, horses, donkeys, cattle, sheep, rats and poultry of any kind, but not limited to.

[0041] As used herein, the terms "treat," "alleviate," or "ameliorate" are used interchangeably herein. These terms refer to a method for obtaining a beneficial or desired result, including a therapeutic effect. "Therapeutic benefit" includes, but is not limited to, the eradication or amelioration of the underlying disorder being treated. This means that the effectiveness of treatment may not be affected by the possibility that the patient may still suffer from the underlying disorder. eradicate or ameliorate one or more physiological symptoms associated with the underlying disorder, despite the By doing so, improvements are observed and obtained in the patient.

[0042] As used herein, "prevention" and "prophylaxis" may be used interchangeably. The term refers to a method for obtaining a beneficial or desired result, including, but not limited to, a prophylactic effect. In order to obtain a "preventive effect," the disease may not have been diagnosed, The complex or composition is administered to a patient at risk of developing a particular disease, or to a patient suffering from one or more of the diseases. It may be administered to a subject who reports multiple physiological symptoms.

[0043] <Complex molecule> In one aspect, the present disclosure provides a conjugate molecule for delivering an active agent or drug. In some embodiments, the conjugate molecules disclosed herein are capable of inducing In some embodiments, the conjugate molecules disclosed herein are used to target specific The mAb binds to a cell surface receptor. For this purpose, any cell surface receptor or Biomarkers or portions thereof are also believed to be suitable. The conjugate molecules disclosed herein specifically bind to receptors specific to particular tissues, and thus exert tissue-specific functions. In some embodiments, the conjugate molecules disclosed herein are It specifically targets receptors on the surface of liver cells and specifically targets liver tissue. In the present specification, the conjugate molecule specifically binds to a cell surface receptor specific to hepatocytes. In some embodiments, the conjugate molecules disclosed herein target the hepatic surface Asialoglycoprotein receptor r, ASGPR).

[0044] As used herein, "active agent" and "active drug" may be used interchangeably. Both refer to molecules that can be delivered by the conjugate molecules disclosed herein. In some embodiments, the active agent is a reagent that is desired to be delivered to hepatocytes. These reagents are well known to those skilled in the art and include functional nucleotides, e.g. Examples of suitable oligonucleotides include, but are not limited to, oligonucleotides, particularly those disclosed herein.

[0045] In some embodiments, the present disclosure provides a conjugate having a structure shown in formula (321): Provide a child.

[0046] [ka] During the ceremony, n1 is an integer from 1 to 3, and n3 is an integer from 0 to 4. m1, m2, and m3 are each independently an integer of 2 to 10; R 10 , R 11 , R1 2. R 13 , R 14 and R 15 are each independently H, C1-C 10 Alkyl groups, C1- C 10 Halogenated alkyl groups and C1-C 10 alkoxy groups, R4 is a group capable of covalently binding to an active drug or agent; Each L1 is a linear alkylene group having a length of 1 to 70 carbon atoms, The carbon atoms are C(O), NH, O, S, CH=N, S(O)2, C2-C 10 Arken C2-C 10 Alkynylene group, C6-C 10 Arylene group, C3-C 18 Hetero Cyclylene group and C5-C 10 one or more selected from the group consisting of heteroarylene groups and L1 is C1-C 10 Alkyl groups, C6-C 10 Aryl group, C5- C 10 Heteroaryl groups, C1-C 10 Halogenated alkyl groups, -OC1-C 10 Alki group, -OC1-C 10 Alkylphenyl group, -C1-C 10 Alkyl-OH, -OC1 -C 10 Halogenated alkyl groups, -SC1-C 10 Alkyl group, -SC1-C 10 Alki phenyl group, -C1-C 10 Alkyl-SH, -SC1-C 10 Halogenated alkyl groups , halogen substituents, -OH, -SH, -NH2, -C1-C 10 Alkyl-NH2, -N (C1-C 10 Alkyl group)(C1-C 10alkyl group), -NH(C1-C 10 Alki ), -N(C1-C 10 Alkyl group)(C1-C 10 alkylphenyl group), -NH (C1-C 10 alkylphenyl group), cyano group, nitro group, -COH, -C(O)O (C1-C 10 alkyl group), -CON(C1-C 10 Alkyl group)(C1-C 10 Al kill group), -CONH(C1-C 10 alkyl group), -CONH2, -NHC(O)(C 1-C 10 alkyl group), -NHC(O)(phenyl group), -N(C1-C 10 Alkyl )C(O)(C1-C 10 alkyl group), -N(C1-C 10 Alkyl)C(O)(phenyl nyl group), -C(O)C1-C 10 Alkyl group, -C(O)C1-C 10 Alkylpheny group, -C(O)C1-C 10 Halogenated alkyl groups, -OC(O)C1-C 10 Alki group, -SO2(C1-C 10 alkyl group), -SO2(phenyl group), -SO2(C1 -C 10 Halogenated alkyl group), -SO2NH2, -SO2NH(C1-C 10 Alki -SO2NH (phenyl group), -NHSO2(C1-C 10 alkyl group), -N HSO2 (phenyl group) and -NHSO2 (C1-C 10 (halogenated alkyl group) and optionally having one or more substituents selected from the group consisting of Each S1 is independently M1, and any active hydroxy group, if any, is protected with an alkoxy protecting group, Each M1 is independently selected from a ligand capable of binding to a cell surface receptor.

[0047] In some embodiments, n1 may be an integer from 1 to 3, and n3 is an integer from 0 to 4. and wherein there are at least two S groups in the conjugated molecule. In some embodiments, n1+n3≧2, whereby the compound In the complex formed by the molecule, the number of M1 ligands is at least 3, and M 1) The ligand binds more easily to the asialoglycoprotein receptor on the surface of hepatocytes, allowing for intracellular uptake. The uptake of the complex into cells can be promoted by the uptake action of the complex. As shown in Fig. 1, when the number of M1 ligands is three or more, the M1 ligands and the auxin on the surface of hepatocytes are closely related. Since the improvement of the ease of binding to the alloglycoprotein receptor is not clear, the ease of synthesis, Considering various aspects such as structure / process cost and delivery efficiency, several embodiments are possible. In this embodiment, n1 is an integer of 1 to 2, n3 is an integer of 0 to 1, and n1+n3 =2~3.

[0048] In some embodiments, m1, m2, and m3 each independently represent an integer of 2 to 10. In the case where the number of M1 ligases is selected from the number of M1 ligases, the complex formed by the complex molecule is The spatial position of the M1 ligand on the hepatocyte surface is related to the binding of the M1 ligand to the asialoglycoprotein receptor. It is believed that the present disclosure provides a more simplified method for producing a complex molecule. In order to make some embodiments of the present disclosure easier to synthesize and / or less costly, According to one embodiment, m1, m2, and m3 are each independently an integer of 2 to 5, In terms of form, m1=m2=m3.

[0049] R 10 , R 11 , R 12 , R 13 , R 14 and R 15 are independently H, C1-C 10 Alkyl groups, C1-C 10 Halogenated alkyl groups and C1-C 10 Select from alkoxy In the case of the selection of the conjugate molecule, none of the above can be used without changing the properties of the conjugate molecule provided by the present disclosure. It will be understood by those skilled in the art that the objectives of the present disclosure can be achieved. In this state, R 10 , R 11 , R 12 , R 13 , R 14 and R 15 are each independently H, a methyl group, or an ethyl group. In some embodiments, R 10 , R1 1. R 12 , R 13 , R 14 and R 15 are both H.

[0050] R4 is a group capable of binding to an active agent to be delivered by the conjugate molecule of the present disclosure. In an embodiment, R4 is an oligonucleotide delivered by the conjugate molecule of the present disclosure. In some embodiments, R4 is a group capable of covalently linking an oligonucleotide to a In some embodiments, R4 is a group that can be attached to a phosphorus It is a group that can be attached to an oligonucleotide by an acid diester bond. In one embodiment, R4 provides a bond to an N atom on the nitrogen-containing backbone and is It is selected to provide suitable reactive sites for the synthesis of oxidase conjugates. In this context, the term "nitrogen-containing backbone" refers to a chain structure, and the R 10 , R 11 , R 12 , R 13 , R 14 and R 15 In some embodiments, the carbon atom and the N atom of wherein R4 is a group that can be attached to the N atom on the nitrogen-containing backbone in a suitable manner. In some embodiments, R4 may include a group bonded to an N atom on the nitrogen-containing backbone. The site that is reacted to be conjugated to an oligonucleotide via a phosphodiester bond. The term "functional group" includes any functional group that can

[0051] In some embodiments, R4 is a nucleotide on the oligonucleotide or nucleotide. a first functional group capable of reacting with a hydroxy group to form a phosphate ester bond; a second functional group capable of forming a covalent bond with a group or an amino group, In some embodiments, the first functional group comprises a covalently bonded solid support. is a phosphoramidite, a hydroxy group or a protected hydroxy group. In embodiments, the second functional group is a phosphoramidite, a carboxy group, or a carboxylic acid. In some embodiments, the second functional group is a hydroxy group or an amino group. A solid support that is attached to other parts of a molecule through covalent bonds formed by In one embodiment, the solid support is a phosphate ester bond, a carboxylate ester bond or In some embodiments, the solid support is a resin. It's fat.

[0052] In some embodiments, the first functional group is a hydroxy group, -OR k or the formula ( C3), and / or the second functional group comprises a group represented by formula (C1), (C2), (C3), C3), (C1') or (C3').

[0053] [ka] In the formula, q1 is an integer of 1 to 4, X is O or NH, and M + is a cation and R k is a hydroxy protecting group, SPS represents a solid phase support, TIFF0007662216000011.tif3143 represents the site to which the group is covalently attached.

[0054] In some embodiments, the first functional group is a phosphoramidite functional group, e.g., The phosphoramidite functional group comprises a group represented by formula (C3), and the phosphoramidite functional group is any one of the groups on the nucleotide. Hydroxy groups at any position, for example, hydroxy groups at the 2' position or 3' position and The oligonucleotides are then linked together to form phosphodiester bonds via oxidation, resulting in the formation of complex molecules called oligonucleotides. Thus, the absence of a second functional group does not prevent the The disclosed conjugate molecules can be conjugated to nucleotides. In this case, the conjugate molecules is suitable for reacting with the hydroxy group on the terminal nucleotide in a nucleotide sequence. In a subsequent oxidation step, the phosphodiester bond is formed, resulting in the formation of the The conjugated molecule is conjugated to an oligonucleotide.

[0055] In some embodiments, the first functional group comprises a protected hydroxy group. In some embodiments, the second functional group comprises a group reactive with a solid phase support; A conjugated molecule comprising a solid support is provided. In some embodiments, the second functional group is represented by the formula As shown in (C1), (C2) or (C3), a carboxy group, a carboxylate or a phosphine The carboxyl group or carboxylate can be attached to a solid support, e.g., a resin. The hydroxyl or amino group in the A complex comprising a solid support bonded by a tert-bond or a solid support bonded by an amide bond. Phosphoramidites can be attached to a variety of solid supports, such as resins. The hydroxy group in the amine reacts with the amine to form a phosphodiester bond through subsequent oxidation. This allows the formation of a solid phase carrier to which the antibody is bound. For example, methods for preparing the conjugates of the present disclosure using such conjugated molecules are provided. In an embodiment, the method first comprises subjecting the conjugate molecule and the solid support to a condensation or coupling reaction. The nucleoside monomers are then coupled by solid-phase phosphoramidite synthesis. In addition, a conjugate of the present disclosure comprising a conjugate molecule of the present disclosure conjugated to an oligonucleotide can be obtained. In some embodiments, in the phosphoramidite solid phase synthesis process, The functional group of 1 is deprotected and then reacted with the phosphoryl group on the nucleoside under coupling reaction conditions. Coupling with an amidite group.

[0056] In some embodiments, R4 is a hydroxy group or a protected hydroxy group. and a carboxylic acid ester bond, an amide bond or a phosphodiester bond. or by a carboxylate bond, an amide bond or a phosphodiester bond. In some embodiments, the second functional group comprises a bound solid support. The functional group is a group represented by formula (C1') or (C3'). When the second functional group includes a solid phase carrier, the conjugate molecule including the solid phase carrier is the conjugate molecule of the present disclosure. Thus, in one aspect of the present invention, the conjugated molecule is used to prepare the Methods for preparing the disclosed conjugates are provided. In some embodiments, the methods include The conjugated molecules containing the support and the nucleoside monomers were synthesized according to the phosphoramidite solid phase synthesis method. In some embodiments, the conjugation of the conjugate molecule of the present disclosure to the oligonucleotide is carried out by reacting the conjugate molecule with the oligonucleotide. In an embodiment, the solid support comprises a conjugated molecule having a carboxy group, a carboxylate, or a phosphonate. It can be obtained internally by reacting a conjugated molecule having a phoramidite with a solid support. In some embodiments, the conjugate molecule may be provided by a supplier.

[0057] In some embodiments, the carboxylate is -COO - M + This may be expressed as So, M + is a cation, for example a metal cation, the ammonium cation NH + or an organic ammonium cation. The ions are alkali metal ions, e.g., K + Or Na + It may be possible to improve the solubility. In order to facilitate the reaction, in some embodiments, an organic ammonium The cation is an ammonium cation formed by a tertiary amine or a quaternary ammonium cation. Ammonium cations, such as the ammonium ion formed by triethylamine or N,N -Diisopropylethylamine is the ammonium cation formed by In one embodiment, the carboxylate is triethylamine carboxylate or N,N-diethylamine carboxylate. isopropylethylamine carboxylate.

[0058] In some embodiments of the present disclosure, R4 is selected from the group consisting of formula (B9), (B10), (B9′) , (B10'), (B11), (B12), (B11') or (B12') It is.

[0059] [ka] In the formula, q1 is an integer of 1 to 4, q2 is an integer of 1 to 10, and X is O or NH. Ri, M + is a cation and R k is a hydroxy protecting group, SPS represents a solid phase support, represents the site at which the group is covalently attached. In some embodiments, q1 is 1 or In some embodiments, q2 is an integer from 1 to 5. In some embodiments, R4 comprises a group represented by formula (B9) or (B10). In one embodiment, R4 comprises a group shown in formula (B11) or (B12).

[0060] In some embodiments, R k Tr (trityl group), MMTr (4-methoxy trityl group), DMTr (4,4'-dimethoxytrityl group), TMTr (4,4',4 In some embodiments, R k is a DMTr.

[0061] L1 is a linear alkylene group having a length of 1 to 70 carbon atoms, Carbon atoms are C(O), NH, O, S, CH=N, S(O)2, C2-C 10 Alkenira group, C2-C 10 Alkynylene group, C6-C 10 Arylene group, C3-C 18 Heterosi Crylene group and C5-C 10 one or more selected from the group consisting of heteroarylene groups; L1 is optionally substituted, C1-C 10 Alkyl groups, C6-C 10 Aryl groups, C5-C 10 Heteroaryl groups, C1-C 10 Halogenated alkyl groups, -OC1-C 10 Alkyl group, -OC1-C 10 Alkylphenyl group, -C1-C 10 Alkyl-OH, -OC1- C 10 Halogenated alkyl groups, -SC1-C 10 Alkyl group, -SC1-C 10 Alkyl Phenyl group, -C1-C 10 Alkyl-SH, -SC1-C 10 Halogenated alkyl groups, Halogen Substituents: -OH, -SH, -NH2, -C1-C 10 Alkyl-NH2, -N( C1-C 10 Alkyl group)(C1-C 10 alkyl group), -NH(C1-C 10 Alkyl group), -N(C1-C 10 Alkyl group)(C1-C 10 Alkylphenyl group), -NH( C1-C 10 alkylphenyl group), cyano group, nitro group, -COH, -C(O)O( C1-C 10 alkyl group), -CON(C1-C 10Alkyl group)(C1-C 10 Alki ), -CONH(C1-C 10 alkyl group), -CONH2, -NHC(O)(C1 -C 10 alkyl group), -NHC(O)(phenyl group), -N(C1-C 10 Alkyl) C(O)(C1-C 10 alkyl group), -N(C1-C 10 Alkyl)C(O)(phenyl ), -C(O)C1-C 10 Alkyl group, -C(O)C1-C 10 Alkylphenyl Group, -C(O)C1-C 10 Halogenated alkyl groups, -OC(O)C1-C 10 Alkyl group, -SO2(C1-C 10 alkyl group), -SO2(phenyl group), -SO2(C1- C 10 Halogenated alkyl group), -SO2NH2, -SO2NH(C1-C 10 Alkyl group), -SO2NH (phenyl group), -NHSO2(C1-C 10 alkyl group), -NH SO2 (phenyl group) and -NHSO2 (C1-C 10 (halogenated alkyl group) Conveniently, L1 is a linear alkylene group. However, for example, amines and alkenes resulting from the above-mentioned replacement and / or substitution are also included. It will be understood by those skilled in the art that in some cases the groups may not be linear or may have different names. For purposes of this disclosure, the length of L1 is the number of atoms in the chain connecting the two attachment points. For this purpose, the ring obtained by substituting the carbon atoms of the linear alkylene (e.g., hexane) may be The heterocyclylene or heteroarylene) is one atom.

[0062] In some embodiments, L1 is a nitrogen-containing ligand in combination with an M1 ligand (or the corresponding S1 group). It serves to bind N atoms on the framework and provide liver targeting function to the complexes of the present disclosure. In some embodiments, L1 is any one of the groups of formulae A1 to A26 or any of them. In some embodiments, L1 is selected from any combination of A1, A4, A5, Any one or any combination of A6, A8, A10, A11 and A13. In some embodiments, L1 is at least two of A1, A4, A8, A10, and A11. In some embodiments, L1 is a combination of at least one of A1, A8, and A10. is also a combination of the two.

[0063] In some embodiments, the length of L1 is 3 to 25, 3 to 20, 4 to 15, or 5 to In some embodiments, the length of L1 may be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 , 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 2 0, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55 or 60 In some embodiments according to the present disclosure, j1 is an integer from 2 to 10; In some embodiments, j1 is an integer from 3 to 5. In some embodiments, j2 is an integer from 2 to 10, and in some embodiments, j2 is an integer from 3 to 5. R' is a C1-C4 alkyl group, and in some embodiments, R' is methyl. Ra is one of A27, A28, A29, A3 0 and A31, and in some embodiments, Ra is one of A27 or A28. Rb is a C1-C5 alkyl group, and in some embodiments, Rb is a methyl group. In some embodiments, the alkyl group is one of a methyl group, an ethyl group, an isopropyl group, and a butyl group. By selecting j1, j2, R', Ra, and Rb in each of the formulas A1 to A26, Therefore, in the oligonucleotide complex formed by the conjugated molecule, the M1 ligand and nitrogen The bond between the M1 ligand and the N atom on the nitrogen-containing backbone is realized, and the spatial position between the M1 ligand and the liver is determined. It is adapted by binding to the asialoglycoprotein receptor on the cell surface.

[0064] Each M1 is independently selected from ligands capable of binding to a cell surface receptor. In one embodiment, at least one M1 is a ligand capable of binding to a receptor on the surface of a hepatocyte. In some embodiments, at least one M1 is a mammalian cell surface In some embodiments, at least one M1 is a ligand that can bind to a receptor on the surface of human hepatocytes. At least one M1 binds to the asialoglycoprotein receptor (ASGPR) on the liver surface. It is a ligand that can bind.

[0065] In some embodiments, M1 is an asialoglycoprotein on the surface of mammalian hepatocytes. Any one of the ligands that may have affinity for the protein receptor (ASGPR) These types of ligands are known to those of skill in the art. In some embodiments, at least one M is a sugar. In some embodiments, each M is a sugar. In some embodiments, at least one M1 is a monosaccharide, a disaccharide, a trisaccharide, or a polysaccharide. In some embodiments, each M is a monosaccharide, a disaccharide, a trisaccharide, or a polysaccharide. In some embodiments, at least one M is a modified sugar. In some embodiments, each M is a modified sugar. In some embodiments, each M is independently a polysaccharide. , a modified polysaccharide, a monosaccharide or a monosaccharide derivative. Each or at least one of them is independently selected from glucose and its derivatives, mannose and its derivatives, derivatives thereof, galactose and its derivatives, xylose and its derivatives, ribose and its derivatives Derivatives, fucose and its derivatives, lactose and its derivatives, maltose and its derivatives The glycerol derivative is composed of arabinose and its derivatives, fructose and its derivatives, and sialic acid. It is possible to select from the group.

[0066] In some embodiments, each or at least one of M1 is independently a D-macro L-Mannopyranose, L-Mannopyranose, D-Arabinose, D-Xylofuranose, L -Xylofuranose, D-glucose, L-glucose, D-galactose, L-galactose D-mannose, α-D-mannofuranose, β-D-mannofuranose, α-D-mannopyranolate β-D-mannopyranose, α-D-glucopyranose, β-D-glucopyranose α-D-glucofuranose, β-D-glucofuranose, α-D-fructofuranose α-D-fructopyranose, α-D-galactopyranose, β-D-galactopyranose D-galactofuranose, α-D-galactofuranose, β-D-galactofuranose, glucosamine, Aryl acid, galactosamine, N-acetylgalactosamine, N-trifluoroacetylgalactosamine Galactosamine, N-propionylgalactosamine, N-butyrylgalactosamine, N- Isobutyrylgalactosamine, 2-amino-3-O-[(R)-1-carboxyethyl] -2-Deoxy-β-D-glucopyranose, 2-Deoxy-2-methylamino-L-glucose Leucopyranose, 4,6-dideoxy-4-formamido-2,3-di-O-methyl-D -Mannopyranose, 2-deoxy-2-sulfoamino-D-glucopyranose, N-Glucopyranose Lycoryl-α-neuraminic acid, 5-thio-β-D-glucopyranose, methyl 2,3,4 -Tris-O-acetyl-1-thio-6-O-trityl-α-D-glucopyranoside, 4 -Thio-β-D-galactopyranose, ethyl 3,4,6,7-tetra-O-acetyl- 2-Deoxy-1,5-dithio-α-D-glucoheptopyranoside, 2,5-anhydro -D-allononitrile (2,5-anhydro-D-allononitrile), Ribose, D-ribose, D-4-thioribose, L-ribose, L-4-thioribose In some embodiments, at least one M1 is N-acetylgalactosamine (GalNAc). In some embodiments, Each M1 is N-acetylgalactosamine (GalNAc). For the selection, reference may be made to the disclosure of, for example, CN105378082A. No. 6,399,623, the disclosure of which is hereby incorporated in its entirety.

[0067] CN105378082A describes compounds containing modified oligonucleotides and complex groups. The composite group is shown to have at least one phosphorus-linking group or a neutral-linking group and one or more Each ligand may be a polysaccharide, a modified polysaccharide, mannose, galactose, mannose, or mannose. Dose derivatives, galactose derivatives, D-mannopyranose, L-mannopyranose, D -Arabinose, D-xylofuranose, L-xylofuranose, D-glucose, L- Glucose, D-galactose, L-galactose, α-D-mannofuranose, β-D -Mannofuranose, α-D-mannopyranose, β-D-mannopyranose, α-D- Glucopyranose, β-D-glucopyranose, α-D-glucofuranose, β-D-glucose Leucofuranose, α-D-fructofuranose, α-D-fructopyranose, α-D- Galactopyranose, β-D-galactopyranose, α-D-galactofuranose, β- D-galactofuranose, glucosamine, sialic acid, α-D-galactosamine, N-acetate Tilgalactosamine, 2-amino-3-O-[(R)-1-carboxyethyl]-2-de Oxy-β-D-glucopyranose, 2-deoxy-2-methylamino-L-glucopyranose Nose, 4,6-dideoxy-4-formamido-2,3-di-O-methyl-D-manno Pyranose, 2-deoxy-2-sulfoamino-D-glucopyranose, N-glycolyl -α-Neuraminic acid, 5-thio-β-D-glucopyranose, methyl 2,3,4-tris( -O-Acetyl-1-thio-6-O-trityl-α-D-glucopyranoside, 4-thio- β-D-Galactopyranose, Ethyl 3,4,6,7-tetra-O-acetyl-2-deoxy 2,5-Anhydro-D-Aceto-1,5-dithio-α-D-glucoheptopyranoside ribose, D-ribose, D-4-thioribose, L-ribose or L- The compound is selected from the group consisting of 4-thioribose and 4-thioribose. It is said that it can reduce

[0068] WO2016077321A1 describes many siRNAs that specifically target HBV genes. A and a method for its delivery are disclosed, which modify the nucleotides of siRNA to This improves the serum stability of the siRNA. Furthermore, multiple types of siRNA complexes are specifically disclosed.

[0069] WO2016168286A1 describes a number of compounds that specifically target the ANGPTL3 gene. The present invention discloses siRNA and a method for its delivery, which includes modifying the nucleotides of the siRNA. This improves the serum stability of siRNA. This is disclosed in et al.

[0070] N-acetylgalactosamine (Ga 1NAc) is a ligand that binds to the asialoglycoprotein receptor on the liver surface. Asialoglycoprotein receptor (A The SGPR (SEQ ID NO: 1) is an endogenous receptor that is specifically expressed by hepatocytes. -Acetylgalactosamine (GalNAc) is used as a targeting molecule to bind small RNA molecules. For example, Alnylam Pharmaceuticals delivers cals, Inc. has demonstrated that siRNA based on GalNAc conjugation technology inhibits IL-1 expression in mice. It was reported for the first time that β-lactamase exhibits anti-inflammatory activity (Nair et al., J. Am. Chem. Soc. ,2014,136,16958-16961). The text contains three clusters of Gal siRNA complexed to the NAc showed good delivery activity in both in vivo and in vitro experiments. It was reported that a single dose of ED 50 1mg It was confirmed that the dose was 1 ml or less per single injection. By subcutaneously injecting the compound once a week, it was possible to obtain stable interference activity for up to nine months. can be done.

[0071] In some embodiments, S1 is independently M1. and S1 independently represents a hydroxyl group in M1 such that at least one active hydroxyl group is hydroxyl-protected. In some embodiments, S1 is independently a group protected by a protecting group in M1. All active hydroxy groups (if any) in the hydroxyl group are protected with hydroxy protecting groups. In some embodiments, any hydroxy protecting group familiar to one of skill in the art may be used as M It can be used to protect the active hydroxy group in 1. wherein the protected hydroxy group is represented by the formula YCOO-, and each Y is independently C- C 10 Alkyl and C6-C 10 aryl, halogen substituents and It may be optionally substituted with one or more substituents selected from C1-C6 alkyl. In some embodiments, each Y is a methyl group, a trifluoromethyl group, a difluoromethyl group, group, monofluoromethyl group, trichloromethyl group, dichloromethyl group, monochloromethyl group group, ethyl group, n-propyl group, isopropyl group, phenyl group, halophenyl group and C1 -C6 alkylphenyl groups.

[0072] In some embodiments, each S1 is independently selected from the groups of formulae A46-A54: is selected from the group consisting of:

[0073] [ka]

[0074] In some embodiments, S1 is of formula A49 or A50.

[0075] In some embodiments, each Y is independently a methyl group, a trifluoromethyl group, a difluoromethyl group, Fluoromethyl group, monofluoromethyl group, trichloromethyl group, dichloromethyl group, mono n-chloromethyl group, ethyl group, n-propyl group, isopropyl group, phenyl group, halophenyl group For the purposes of simplifying the conjugated molecules of this disclosure, In some embodiments, Y is a methyl group.

[0076] In some embodiments, the conjugated molecule of the present disclosure has the formula (403), (404), (4 05), (406), (407), (408), (409), (410), (411), (412), (413), (414), (415), (416), (417), (418) ), (419), (420), (421) or (422).

[0077] [ka] TIFF0007662216000015.tif236147TIFF0007662216000016.tif241152TIFF00076622160 00017.tif251144TIFF0007662216000018.tif195141TIFF0007662216000019.tif160143

[0078] In the above formulas (403) to (422), X is O or NH, and R k is a hydroxy protecting group And M +is formed by metal cations, ammonium cations, and tertiary amines. cation or a quaternary ammonium cation. In some embodiments, M + teeth, The file is TIFF0007662216000020.tif13136.

[0079] In some embodiments, the conjugated molecule of the present disclosure is represented by the formula (423), (424), (4 25), (426), (427), (428), (429), (430), (431), (432), (433), (434), (435), (436), (437), (438) ), (439), (440), (441) or (442). .

[0080] [ka] TIFF0007662216000022.tif241142TIFF0007662216000023.tif235143TIFF0007662216 000024.tif252154TIFF0007662216000025.tif201141TIFF0007662216000026.tif85156

[0081] In the above formulas (423) to (442), X is O or NH, and R k is a hydroxy protecting group where SPS stands for solid phase support.

[0082] In some embodiments, the conjugated molecule of the present disclosure has the formula (503), (504), (5 05), (506), (507), (508), (509), (510), (511), (512), (513), (514), (515), (516), (517), (518) ), (519), (520), (521) or (522).

[0083] [ka] TIFF0007662216000028.tif240149TIFF0007662216000029.tif235140TIFF0007662216 000030.tif250135TIFF0007662216000031.tif186142TIFF0007662216000032.tif89150

[0084] In the above formulas (503) to (522), DMTr represents 4,4'-dimethoxytrityl. ,structure TIFF0007662216000033.tif13143 represents the salt formed with the corresponding carboxylic acid and triethylamine.

[0085] In some embodiments, the conjugated molecule of the present disclosure has the formula (523), (524), (5 25), (526), ​​(527), (528), (529), (530), (531), (532), (533), (534), (535), (536), (537), (538) ), (539), (540), (541) or (542). .

[0086] [ka] TIFF0007662216000035.tif240141TIFF0007662216000036.tif235150TIFF0007662216 000037.tif255142TIFF0007662216000038.tif200151TIFF0007662216000039.tif89155

[0087] In the above formulas (523) to (542), SPS represents a solid phase support, and DMTr represents 4,4'- It stands for dimethoxytrityl.

[0088] Preparation of the Conjugate Molecules of the Present Disclosure One of skill in the art may prepare the conjugated molecules of the present disclosure by any reasonable synthetic route.

[0089] In some embodiments of the present disclosure, the method for preparing a conjugated molecule of formula (321) is In an organic solvent, under esterification reaction conditions, in the presence of an alkali and an esterification catalyst, the formula The compound represented by (313) is contacted with a cyclic acid anhydride, subjected to ion exchange, and isolated to give the compound represented by the formula The method includes obtaining a compound shown in (321).

[0090] [ka] During the ceremony, R6 is a group that provides R4 in formula (321). In some embodiments, For example, R6 has the structure shown in formula (A61).

[0091] [ka] (A61)

[0092] n1, n3, m1, m2, m3, R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , The definitions and selectable ranges of L1 and S1 are as described above. i is nitrogen-containing Bonds to N atoms on the framework, R k Any group that can be bonded to O to attach a free hydroxyl group Yes, R kis a hydroxy protecting group. In this case, R4 is the first functional group, hydroxy Formula (3) containing a protecting group and a group represented by formula (C1) or (C2) which is a second functional group 21) to obtain a compound of formula: In some embodiments, R6 is B7 or B8.

[0093] [ka] In the formula, q2 and R k The definitions of each are as described above.

[0094] The esterification reaction conditions are a reaction temperature of 0 to 100° C. and a reaction time of 8 to 4 hours. In one embodiment, the esterification reaction conditions include: The reaction temperature is 10 to 40°C, and the reaction time is 20 to 30 hours.

[0095] In some embodiments, the organic solvent is an epoxy solvent, an ether solvent, a halogen solvent, or a fluorine solvent. Alkyl chloride solvents, dimethyl sulfoxide, N,N-dimethylformamide and N,N- In some embodiments, the compound includes one or more of diisopropylethylamine. The epoxy solvent is dioxane and / or tetrahydrofuran. In one embodiment, the ether solvent is ethyl ether and / or methyl tert-butyl ether. and the halogenated alkyl solvent is dichloromethane, trichloromethane, and In some embodiments, the active ingredient is one or more of 1,2-dichloroethane. The organic solvent is dichloromethane. The dosage of the organic solvent is 3 to 50 L / mol, and in some embodiments, 5 to 20 L / mol. mol.

[0096] In some embodiments, the cyclic acid anhydride is succinic anhydride, glutaric anhydride. anhydride, adipic anhydride, or pimelic anhydride, and in some embodiments The cyclic acid anhydride is succinic anhydride. In some embodiments, the molar ratio of the compound to be treated is 1:1 to 10:1, and in some embodiments, 2: The ratio is 1~5:1.

[0097] The esterification catalyst may be any catalyst that catalyzes the esterification reaction, e.g. For example, the catalyst may be 4-dimethylaminopyridine. ) is 1:1 to 10:1 in some embodiments. , 2:1~5:1.

[0098] In some embodiments, the base is any inorganic base, organic base, or combination thereof. Considering the solubility and stability of the product, the base is preferably a tertiary amine. In some embodiments, the tertiary amine is triethylamine or N,N- The tertiary amine is diisopropylethylamine. The compound represented by the formula (313) In some embodiments, the molar ratio of It is.

[0099] The ion exchange reaction is carried out by reacting the compound of formula (321) with the desired carboxylic acid or carboxylate. The ion exchange method is known to those skilled in the art and is suitable for converting the ion exchange Using the ion exchange solution and exchange conditions, + It is possible to obtain a complex molecule having a cation. In some embodiments, the ion exchange reaction is carried out using a triethylamine phosphate solution. In some embodiments, the concentration of the triethylamine phosphate solution is In some embodiments, the triethylamine phosphate is 0.2 to 0.8 M. The concentration of the solution is 0.4 to 0.6 M. In some embodiments, the compound of formula (313) The amount of the triethylamine phosphate solution is 3 to 6 L / mol for the compound; In some embodiments, it is 4 to 5 L / mol.

[0100] The compound of formula (321) can be isolated from the reaction mixture by any suitable isolation method. In some embodiments, after the solvent is evaporated off, the chromatography method can be performed. The compound of formula (321) can be isolated by, for example, (1) normal phase purification: 200 to 3 00 mesh silica gel packing in dichloromethane containing 1 wt‰ triethylamine: Gradient elution with methanol = 100:18 to 100:20, or (2) Reverse phase purification: C1 8. C8 reverse phase packing material was dissolved in a gradient of methanol:acetonitrile = 0.1:1 to 1:0.1. In some embodiments, the compound can be isolated by chromatography under conditions that allow the compound to be isolated. The solvent can be directly removed to obtain the crude compound of formula (321), The product can be used as is in the subsequent reaction.

[0101] In some embodiments, the method for preparing a compound of Formula (321) comprises the steps of: In an organic solvent, in the presence of a condensation agent, a condensation catalyst and a tertiary amine, the above ion exchange reaction product is The method further comprises contacting the substance with a solid support that contains an amino or hydroxy group. R4 is a first functional group, a hydroxy-protecting group, and a second functional group, a compound represented by formula (C1'): A compound of formula (321) containing the group represented by the formula (321) is obtained.

[0102] The solid phase support is one of the supports used in solid phase synthesis of siRNA. Some are known to those of skill in the art. For example, the solid phase support may have an active hydroxy or amino group. In some embodiments, the solid support may be selected from a solid support that includes a functional group. In some embodiments, the amino resin is a hydroxy resin. The resin or hydroxy resin has a particle size of 100 to 400 mesh, and the surface The amino or hydroxyl group loading parameter is 0.2 to 0.5 mmol / g. The ratio of the compound represented by formula (321) to the solid support is 10 to 400 μmol compound / In some embodiments, the solid support of formula (32) is a 1 to 100 μmol / g. The ratio of the compound shown in 1) to the solid phase carrier is 50 to 200 μmol / g.

[0103] The organic solvent may be any suitable solvent or mixture of solvents known to those skilled in the art. In some embodiments, the organic solvent is acetonitrile, an epoxy solvent, an ether solvent, or the like. agents, halogenated alkyl solvents, dimethyl sulfoxide, N,N-dimethylformamide and and N,N-diisopropylethylamine. wherein the epoxy solvent is dioxane and / or tetrahydrofuran, The ether solvent is ethyl ether and / or methyl tert-butyl ether, The halogenated alkyl solvents are dichloromethane, trichloromethane and 1,2-dichloroethane. In some embodiments, the organic solvent is one or more of acetonitrile, acetone, ethyl alcohol ... The amount of the organic solvent is 20 to 200 L / L relative to the compound of formula (313). mol, and in one embodiment, 50 to 100 L / mol.

[0104] In some embodiments, the condensing agent is benzotriazol-1-yl-oxy Tripyrrolidinophosphonium hexafluorophosphate, 3-diethoxyphosphoryl- 1,2,3-benzotriazol-4(3H)-one and / or O-benzotriazol- It may also be tetramethyluronium hexafluorophosphate. In one embodiment, the condensing agent is O-benzotriazole-tetramethyluronium hexafluorophosphate. The molar ratio of the condensing agent to the compound represented by formula (313) is 1: :1 to 20:1, and in other embodiments, 1:1 to 5:1.

[0105] In some embodiments, the tertiary amine is triethylamine and / or N, N-diisopropylethylamine, and in some embodiments, N,N-diisopropyl propylethylamine, and the molar ratio of the tertiary amine to the compound represented by formula (313) The ratio of the fluorine atom to the fluorine atom is 1:1 to 20:1, and in some embodiments, 1:1 to 5:1.

[0106] In some embodiments, the method for preparing a compound of formula (321) comprises the steps of: The product is reacted with a capping reagent and an acylation catalyst in an organic solvent under capping reaction conditions. and isolating to obtain a compound of formula (321). The capping reaction is carried out to avoid the formation of unwanted by-products in subsequent reactions. The capping reaction is to remove all active functional groups that have not yet reacted. Regarding reaction conditions, the reaction temperature is 0 to 50° C., and in some embodiments, 15 to 20° C. The reaction temperature is 35° C., and the reaction time is 1 to 10 hours, and in some embodiments, 3 to 6 hours. The capping reagent may be any of the capping reagents known to those skilled in the art that are used in siRNA solid-phase synthesis. In some embodiments, the capping reagent may be The capping reagent consists of capping reagent A (capA) and capping reagent B (capB). Mapping reagent A is N-methylimidazole, and in some embodiments, N- Methylimidazole is provided as a solution in pyridine / acetonitrile. In some embodiments, the volume ratio of acetonitrile is 1:10 to 1:1. In some embodiments, the total volume of pyridine and acetonitrile is 3 to 1:1. The volume ratio of N-methylimidazole is 1:1 to 10:1, and in some embodiments In some embodiments, the capping reagent B is 3:1 to 7:1. acetic anhydride, and in some embodiments, capping reagent B is acetic anhydride. It is supplied as a solution in acetonitrile with a volume ratio of acetic anhydride to acetonitrile of 1:1. ~1:10, and in some other embodiments, 1:2 to 1:6.

[0107] In some embodiments, the N-methylimidazole is The ratio of the volume of the mixed solution to the mass of the compound of formula (313) is 5 ml / g to 50 ml / g. In some embodiments, the concentration of acetic anhydride is 15 ml / g to 30 ml / g. The ratio of the volume of the acetonitrile solution to the mass of the compound of formula (313) is 0.5 ml / g to 1 0 ml / g, and in some embodiments, 1 ml / g to 5 ml / g.

[0108] In some embodiments, the capping reagent is a mixture of equimolar amounts of acetic anhydride and N-methyl In some embodiments, the organic solvent is acetonitrile. , epoxy solvents, ether solvents, halogenated alkyl solvents, dimethyl sulfoxide, N, One or more of N-dimethylformamide and N,N-diisopropylethylamine In some embodiments, the organic solvent is acetonitrile. 3) For the compound, the amount of the organic solvent is 10 to 50 L / mol, and some In an embodiment, it is 5 to 30 L / mol.

[0109] In some embodiments, the acylation catalyst is used in an esterification or amidation reaction. The catalyst may be selected from any suitable catalyst, such as an alkali heterocyclic compound. In an embodiment, the acylation catalyst is 4-dimethylaminopyridine. The mass ratio of the compound represented by formula (313) is 0.001:1 to 1:1, and several In an embodiment, it is 0.01:1 to 0.1:1.

[0110] In some embodiments, the compound of Formula (321) is isolated from the reaction mixture by any suitable method. The compound can be isolated. In some embodiments, the compound can be isolated by extensive washing with an organic solvent. , and filtering to remove unreacted reactants, excess capping reagent, and other impurities. The compound of formula (321) can be obtained by the above-mentioned organic solvent, and the organic solvent is acetonitrile, dichloromethane, etc. In some embodiments, the organic solvent is selected from acetone or methanol. It is trinitrile.

[0111] In some embodiments, the method for preparing the conjugated molecule of formula (321) comprises the steps of: In the presence of a coupling reagent under coupling reaction conditions, The compound is contacted with a phosphorodiamidite and isolated to give the compound of formula (321). In this case, R4 is provided with a first functional group, a hydroxy protecting group, and a second functional group, A compound of formula (321) containing a functional group represented by formula (C3) is obtained.

[0112] In some embodiments, the coupling reaction conditions include a temperature of 0 to 50° C. For example, the reaction temperature is 15 to 35° C., and the mole ratio of the compound of formula (313) and phosphorodiamidite is The ratio may be 1:1 to 1:50, for example, 1:5 to 1:15. The molar ratio of the compound to the coupling reagent may be 1:1 to 1:100, for example 1: The reaction time may be 200 to 3000 seconds, for example, 500 to 1:80. The phosphorodiamidite is, for example, bis(diisopropylamino) (2-cyanoethoxy)phosphine, which may be purchased commercially; may be prepared by methods known in the art. The coupling reagent is 1H-tetrazo 1H-tetrazole, 5-ethylthio-1H-tetrazole, 5-benzylthio-1H-tetrazole The compound is selected from one or more of the above, for example, 5-ethylthio-1H-tetrazole. The coupling reaction may be carried out in an organic solvent, the organic solvent being anhydrous acetonitrile. anhydrous acetonitrile, anhydrous DMF, anhydrous dichloromethane, It is preferable that the amount of the organic solvent is 3 to 100% relative to the compound of formula (313). The amount may be from 1 to 50 L / mol, for example, from 5 to 20 L / mol. By carrying out a coupling reaction, the hydroxy group in the compound of formula (313) and the phosphorus group are In some embodiments, the aryl amide is reacted with a phosphate group to form a phosphoramidite group. The solvent can be directly removed to obtain the crude compound of formula (321), The product can be used as is in the subsequent reaction.

[0113] In some embodiments, the method for preparing a compound of Formula (321) comprises a coupling reaction The isolated product is reacted with a hydroxy group-containing amine in the presence of a coupling reagent in an organic solvent under the conditions The method further comprises contacting the solid phase carrier with the oxidizing agent. Then, the capping reaction and the oxidation reaction are carried out. and is isolated to obtain a compound of formula (321). In this case, R4 is provided with a first functional group, hydroxyl. A compound of formula (321) containing a aryl protecting group and a group represented by formula (C3') as a second functional group. A compound is obtained.

[0114] In some embodiments, the solid support is a solid support that can be used for solid phase synthesis of nucleic acids. For example, a commercially available general-purpose solid phase support (e.g., NittoPhase (R)HL UnyLinker TM 300 Oligonucleotide Synthesis Support, Kinovate Life Science s, the structure of which is shown in formula B80).

[0115] [ka]

[0116] Deprotection reactions are well known in the art. In some embodiments, deprotection conditions The reaction temperature is 0 to 50° C., for example, 15 to 35° C., and the reaction time is 30 to 30 0 seconds, for example, 50 to 150 seconds. The deprotection reagent is trifluoroacetic acid, trichloroacetic acid, In some embodiments, the acetic acid may be one or more selected from the group consisting of dichloroacetic acid, dichloroacetic acid, and chloroacetic acid. In an embodiment, the deprotection reagent is dichloroacetic acid. The molar ratio of DMTr (4,4'-dimethoxytrityl) protecting group is 2:1 to 100:1. The ratio may be, for example, 3:1 to 50:1. This provides reactive free hydroxyl groups on the surface of the substrate, allowing subsequent coupling reactions to be carried out. This can be done.

[0117] The coupling reaction conditions and coupling reagents may be selected as above. By carrying out a coupling reaction, the free hydroxyl group formed in the deprotection reaction and the phosphoryl group The amidite groups are reacted to form phosphite linkages.

[0118] In some embodiments, the capping reaction conditions include a temperature of 0 to 50° C. For example, the reaction temperature is 15 to 35° C., and the reaction time is 5 to 500 seconds, for example, 10 to 100 seconds. The capping agent and the dosage may be selected as above.

[0119] The oxidation reaction conditions may be a temperature of 0 to 50°C, for example, 15 to 35°C. The reaction time is 1 to 100 seconds, for example, 5 to 50 seconds, and the oxidation reagent is, for example, It may be iodine (provided as iodine water in some embodiments). In some embodiments, the molar ratio of the oxidation reagent to the nucleic acid sequence bound to the solid support is 1:1. In some embodiments, the ratio is 5:1 to 50:1. The oxidation reaction is carried out in a mixture of tetrahydrofuran, water, and pyridine in a ratio of 3:1:1 to 1:1:3. It is carried out in a mixed solvent.

[0120] In some embodiments, R6 is of formula B7 or B8, where The compound shown in (1) is a compound obtained by reacting amidation reaction condensation agent and amidation reaction condensation agent under amidation reaction conditions in an organic solvent. and a tertiary amine, a compound represented by formula (314) is reacted with a compound represented by formula (A-1) or a compound represented by formula (A-2), and isolating the compound represented by formula (313). The compound can be obtained by the preparation method of obtaining a compound.

[0121] [ka] In the formula, n1, n3, m1, m2, m3, R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , L1, S1, q2 and R k The definitions and available ranges of each are as described above. be.

[0122] The amidation reaction conditions are a reaction temperature of 0 to 100° C. and a reaction time of 1 to 48 hours. In some embodiments, the amidation reaction conditions may be reaction The reaction temperature is 10 to 40° C., and the reaction time is 2 to 16 hours.

[0123] In some embodiments, the organic solvent is an alcohol solvent, an epoxy solvent, an ether solvent, Terephthalic acid solvents, halogenated alkyl solvents, dimethyl sulfoxide, N,N-dimethylformamide In some embodiments, the amine is one or more of N,N-diisopropylethylamine and N,N-diisopropylethylamine. In the embodiment, the alcohol solvent is one of methanol, ethanol, and propanol. In some embodiments, the alcohol is ethanol. In an embodiment, the epoxy solvent is dioxane and / or tetrahydrofuran. In some embodiments, the ether solvent is ethyl ether and / or methyl ether. In some embodiments, the halogenated alkyl ether is tert-butyl ether. The solvent is one of dichloromethane, trichloromethane, and 1,2-dichloroethane. In some embodiments, the organic solvent is dichloromethane. For the compound of formula (314), the amount of organic solvent is 3 to 50 L / mol. In an embodiment, it is 3 to 20 L / mol.

[0124] In some embodiments, the amidation reaction condensing agent is benzotriazole-1- Il-oxytripyrrolidinophosphonium hexafluorophosphate, 3-diethoxy Phosphoryloxy-1,2,3-benzotriazin-4(3H)-one, 4-(4,6- Dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride, 2-ethoxy-1- Ethoxycarbonyl-1,2-dihydroquinoline (EEDQ) or O-benzotriazo In a further embodiment, 3-diethoxyphosphoryloxy-1,2,3-benzotriazine 4(3H)-o The molar ratio of the amidation reaction condensing agent to the compound represented by formula (314) is 1:1 It may be up to 10:1, and in some embodiments is from 2.5:1 to 5:1.

[0125] In some embodiments, the tertiary amine is triethylamine or N,N-diethylamine. isopropylethylamine, and in a further embodiment N,N-diisopropyl The molar ratio of the tertiary amine to the compound represented by formula (314) is 3 :1 to 20:1, and in one embodiment, 5:1 to 10:1.

[0126] In some embodiments, the compounds of formula (A-1) and formula (A-2) may be any suitable For example, R k When is a DMTr group, glyceric acid The compound of formula (A-1) can be prepared by reacting calcium with DMTrCl. Similarly, 3-amino-1,2-propanediol was contacted with a cyclic acid anhydride, and then DMTr was added. Cl to prepare a compound of formula (A-2), The acid anhydride may be a cyclic acid anhydride having 4 to 13 carbon atoms, and in one embodiment, 4 to 8 carbon atoms. As can be easily understood by those skilled in the art, the selection of the cyclic acid anhydride is based on the compound (A-2) For example, when the cyclic acid anhydride is succinic anhydride, When the cyclic acid anhydride is glutaric anhydride, q2=2. , the following can be inferred.

[0127] In some variations, the compound of formula (314) can be prepared by reacting the cyclic anhydride, 3- By reacting sequentially with amino-1,2-propanediol and DMTrCl, 313) can also be prepared. These modifications do not affect the structure and function of the compound of formula (313) and are within the scope of the present invention. This can be easily achieved by the vendor using the above method.

[0128] As above, the compound of formula (313) can be isolated from the reaction mixture by any suitable isolation method. In some embodiments, after the solvent is evaporated, the chromatograph may be The compound of formula (313) can be isolated by a chromatography method, for example, (1) normal phase purification. Preparation: 200-300 mesh silica gel packing is packed in petroleum ether: ethyl acetate: dichloromethane. Fluoromethane:N,N-dimethylformamide=1:1:1:0.5~1:1:1:0.6 Gradient elution. (2) Reverse-phase purification: C18, C8 reverse-phase packing, methanol:acetonitrile The compounds were isolated under two chromatographic conditions: gradient elution at pH 0.1:1 to 1:0.1. In some embodiments, the solvent can be removed directly to give the compound of formula (313). A crude product of the compound can be obtained, and the crude product can be used in the subsequent reaction as it is. do.

[0129] In some embodiments, the compound represented by Formula (314) is reacted in an organic solvent with The compound of formula (315) is contacted with haloacetic acid under deprotection reaction conditions and isolated to obtain the precursor compound. The compound can be obtained by a preparation method comprising obtaining a compound of formula (314).

[0130] [ka] In the formula, R7 is a group represented by the formula (330), (331), (332) or (333). In one embodiment, the structure of R7 is shown in formula (330).

[0131] [ka] n1, n3, m1, m2, m3, R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , The definitions and selectable ranges of L1 and S1 are as described above.

[0132] The haloacetic acid may be selected from the group consisting of dichloroacetic acid, trichloroacetic acid, chloroacetic acid and trifluoroacetic acid. In some embodiments, the dichloroacetic acid may be one or more selected from the group consisting of dichloroacetic acid, It is.

[0133] The deprotection reaction conditions are a reaction temperature of 0 to 100° C. and a reaction time of 0.1 to 2 In one embodiment, the reaction temperature is 10 to 40° C., and the reaction time is 4 hours. The time is 0.5 to 16 hours.

[0134] In some embodiments, the organic solvent is an epoxy solvent, an ether solvent, a halogen solvent, or a fluorine solvent. Alkyl chloride solvents, dimethyl sulfoxide, N,N-dimethylformamide and N,N- The epoxy solvent is one or more of diisopropyl ethylamine. In the form of dioxane and / or tetrahydrofuran, the ether solvent is In some embodiments, ethyl ether and / or methyl tert-butyl ether In some embodiments, the halogenated alkyl solvent is dichloromethane. one or more of chloroethane, trichloromethane, and 1,2-dichloroethane, In an embodiment, the organic solvent is dichloromethane. In a further embodiment, the amount of the organic solvent is 3 to 50 L / mol. 0 L / mol.

[0135] The molar ratio of the haloacetic acid to the compound represented by the formula (315) is 5:1 to 100:1. In some embodiments, the ratio is from 10:1 to 50:1.

[0136] Similarly as above, the compound of formula (314) can be isolated from the reaction mixture by any suitable isolation method. In some embodiments, after the solvent is evaporated, the chromatograph may be The compound of formula (314) can be isolated by a chromatography method, for example: (1) normal phase purification Preparation: 200-300 mesh silica gel packing material is dissolved in dichloromethane:methanol=10 Gradient elution at 0:30 to 100:40. (2) Reverse-phase purification: C18 and C8 reverse-phase packings. and gradient elution with methanol:acetonitrile = 0.1:1 to 1:0.1. In some embodiments, the solvent can be directly removed by chromatographic conditions. The crude product of the compound of formula (314) can be obtained by directly removing the It can be used in subsequent reactions.

[0137] The compound represented by formula (315) can be prepared by reacting amidation reaction condensation agent with tertiary amide in an organic solvent. In the presence of amine and under condensation reaction conditions, a compound represented by formula (317) is converted to a compound represented by formula (316). The compound can be obtained by a preparative process comprising contacting the compound with a compound to be treated and then isolating the compound.

[0138] [ka] In the formula, n1, n3, m1, m2, m3, R7, R 10 , R 11 , R 12 , R 13 , R1 4. R 15 The definitions and selectable ranges of L1, S1 are as described above.

[0139] As the compound of formula (316), for example, there is a compound described in J. Am. Chem. Soc. 20 14,136,16958-16961 may be used, or the compounds of the formula ( The compounds of the formula (316) can be prepared by a person skilled in the art by various methods, for example, US Pat. , 106,022 B2, several formulas (316) and the entire contents of the above references are incorporated herein by reference in their entirety. is incorporated into.

[0140] In some embodiments, the condensation reaction conditions include a reaction temperature of 0 to 100° C. In some embodiments, the reaction time is 0.1 to 24 hours, and in some embodiments, the reaction temperature is 1 The temperature is 0 to 40°C and the reaction time is 0.5 to 16 hours.

[0141] The molar ratio of the compound represented by the formula (316) to the compound represented by the formula (317) is It may be 2:1 to 10:1, and in some embodiments, it may be 2.5:1 to 5:1. be.

[0142] In some embodiments, the organic solvent is acetonitrile, an epoxy solvent, an acetonitrile, Terephthalic acid solvents, halogenated alkyl solvents, dimethyl sulfoxide, N,N-dimethylformamide The epoxy solvent is one or more of: amide and N,N-diisopropylethylamine. The agent, in some embodiments, is dioxane and / or tetrahydrofuran; In some embodiments, the ether solvent is ethyl ether and / or methyl t In one embodiment, the halogenated alkyl solvent is ert-butyl ether. One or more of dichloromethane, trichloromethane and 1,2-dichloroethane In some embodiments, the organic solvent is acetonitrile. 7) The amount of the organic solvent may be 3 to 50 L / mol relative to the compound, In one embodiment, the concentration is 5 to 20 L / mol.

[0143] In some embodiments, the amidation reaction condensing agent is benzotriazole-1- Il-oxytripyrrolidinophosphonium hexafluorophosphate, 3-diethoxy Phosphoryloxy-1,2,3-benzotriazin-4(3H)-one (DEPBT), O-Benzotriazole-tetramethyluronium hexafluorophosphate or 4- (4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride, In a further embodiment, 4-(4,6-dimethoxytriazin-2-yl)-4-methyl The amidation reaction condensation agent and the compound represented by formula (317) may be morpholine hydrochloride. The molar ratio of the compound is 2:1 to 10:1, and in some embodiments, 2.5:1 ~5:1.

[0144] The tertiary amine is N-methylmorpholine, triethylamine or N,N-diisopropylamine. In some embodiments, N-methylmorpholino The molar ratio of the tertiary amine to the compound represented by formula (317) is 3:1 to 20. :1, and in some embodiments, is between 5:1 and 10:1.

[0145] Similarly as above, the compound of formula (315) can be isolated from the reaction mixture by any suitable isolation method. In some embodiments, after the solvent is evaporated, the chromatograph may be The compound of formula (315) can be isolated by a chromatography method, for example: (1) normal phase purification Preparation: 200-300 mesh silica gel packing material is dissolved in dichloromethane:methanol=10 Gradient elution from 0:5 to 100:7. (2) Reverse-phase purification: C18 and C8 reverse-phase packings were used. Two chromatographic methods were used, with gradient elution of methanol:acetonitrile = 0.1:1 to 1:0.1. In some embodiments, the solvent can be directly isolated using chromatographic conditions. The crude product of the compound of formula (315) can be obtained by removing the crude product, which can be directly used for the subsequent It can be used in the reaction.

[0146] In some embodiments, a compound of formula (317) is combined with a sufficient amount of a compound of formula (316). The desired compound of formula (315) is produced by reacting the reactants in a single step, and the compound of formula (315) are the same for each S1-L1 group. In some embodiments, optionally, Compounds of formula (317) are reacted with compounds of formula (316) having different L1 and / or S1. The compound of formula (315) thus produced is reacted in batches with the compound of formula (316) An object can contain more than one type of S1 and / or L1. For example, the formula (3 17) is contacted with 2 eq of a first compound of formula (316), to give a compound of formula (317). The first S1-L1 group is then attached to the two terminal primary amine groups of the product, followed by (n3 +n1-1)eq (with the definitions and values ​​of n3 and n1 being taken together) The range is as described above), (n3+n1-1) units in the compound of formula (317) A second S1-L1 group can be attached to the secondary amine group of the

[0147] In some embodiments, the compound of Formula (317) is reacted with a compound represented by the formula (317) in the presence of an organic solvent. The compound of formula (318) is contacted with an aqueous methylamine solution under deprotection reaction conditions. and isolating the compound represented by formula (317). Cut.

[0148] [ka] In the formula, n1, n3, m1, m2, m3, R7, R 10 , R 11 , R 12 , R 13 , R1 4 and R 15 The definitions and selectable ranges of each are as described above.

[0149] The deprotection reaction conditions are a reaction temperature of 0 to 150° C. and a reaction time of 5 to 72 hours. In some embodiments, the reaction temperature is 20 to 80° C. The duration is 10 to 30 hours.

[0150] The organic solvent may be selected from alcohols, and in some embodiments, ethanol, ethanol, and isopropanol, and in some embodiments and methanol, and the amount of the organic solvent is 1 to 20 L relative to the compound of formula (318). / mol, and in one embodiment, it is 1.5 to 10 L / mol.

[0151] The concentration of the methylamine aqueous solution may be 30 to 40% by mass, and methylamine and a compound of the formula The molar ratio of the compound represented by (318) may be 10:1 to 500:1, In some embodiments, the ratio is from 50:1 to 200:1.

[0152] Similarly as above, the compound of formula (317) can be isolated from the reaction mixture by any suitable isolation method. In some embodiments, after the solvent is evaporated, the chromatograph may be The compound of formula (317) can be isolated by a chromatography method, for example: (1) normal phase purification Preparation: 200-300 mesh silica gel packing is mixed with dichloromethane:methanol:ammonium chloride. (2) Gradient elution with monia water (25 wt%) = 1:1:0.05 to 1:1:0.25. Reverse phase purification: C18 and C8 reverse phase packings were purified using methanol:acetonitrile = 0.1:1 to 1 The compound can be isolated using two chromatographic conditions: gradient elution at 0.05:0.1. In some embodiments, the solvent is directly removed to obtain a crude compound of formula (317). The crude product can be used as it is in the subsequent reaction.

[0153] In some embodiments, the compound of Formula (318) is reacted with a compound of Formula (318) in the presence of an organic solvent. , the compound represented by formula (319) is reacted with triphenylchloromethane (Tr Cl), diphenylethyl phenylchloromethane, phenyldiethyl phenylchloromethane or triethylphenylchloromethane, in some embodiments triphenylchloromethane It can be obtained by a preparation process which includes contacting it with trifluoromethane (TrCl) and isolating it. Cut.

[0154] [ka] In the formula, n1, n3, m1, m2, m3, R 10 , R 11 , R 12 , R 13 , R 14 and R 15 The definitions and selectable ranges of each are as described above.

[0155] The conditions for the substitution reaction are a reaction temperature of 0 to 100° C. and a reaction time of 5 to 72 hours. In one embodiment, the reaction temperature is 10 to 40° C. and the reaction time is 10 to 30 hours.

[0156] Triphenylchloromethane (TrCl), diphenylethylphenylchloromethane, Phenyldiethylphenylchloromethane or triethylphenylchloromethane is a commercially available product. It can be obtained by the above procedure, and triphenylchloromethane (TrCl), diphenylethyl fluoride (DHF), Phenylchloromethane, phenyldiethylphenylchloromethane or triethylphenylchloromethane The molar ratio of chloromethane to the compound represented by formula (319) may be 1:1 to 10:1. In some embodiments, the ratio is 1:1 to 3:1.

[0157] The organic solvent is an epoxy solvent, an ether solvent, an alkyl halide solvent, a dimethyl sulfoxide solvent, or the like. sulfoxide, N,N-dimethylformamide and N,N-diisopropylethylamine The epoxy solvent may be one or more. In some embodiments, the epoxy solvent may be a di In one embodiment, the ether solvent is oxane and / or tetrahydrofuran. ethyl ether and / or methyl tert-butyl ether, In one embodiment, the alkyl fluoride solvent is selected from the group consisting of dichloromethane, trichloromethane, and 1,2-dichloroethane, and in one embodiment, the organic solvent The solvent is dichloromethane. The amount of the organic solvent is 3 ~50 L / mol, and in one embodiment, 5-20 L / mol.

[0158] As above, the compound of formula (318) can be isolated from the reaction mixture by any suitable isolation method. In some embodiments, after the solvent is evaporated, the chromatograph may be The compound of formula (318) can be isolated by a chromatography method, for example: (1) normal phase purification Preparation: 200-300 mesh silica gel packing material, methanol:dichloromethane = 0. 01:1 to 0.5:1, or methanol:dichloromethane:ethyl acetate:petroleum ether = 0.1:1:1:1 to 1:1:1:1 gradient elution, (2) reverse phase purification: C18 and C 8. Elute the reversed-phase packing with a gradient of methanol:acetonitrile = 0.1:1 to 1:0.1. In some embodiments, the compound can be isolated using two chromatographic conditions: The solvent can be directly removed to obtain the crude compound of formula (318), The product can be used as is in the subsequent reaction.

[0159] In some embodiments, the compound represented by Formula (319) is reacted in an organic solvent by: contacting a compound of formula (320) with ethyl trifluoroacetate under displacement reaction conditions; and isolating the compound of formula (319) do.

[0160] [ka] In the formula, n1, n3, m1, m2, m3, R 10 , R 11 , R 12 , R 13 , R 14 and R 15 The definitions and selectable ranges of each are as described above.

[0161] In some embodiments, the organic solvent is acetonitrile, an epoxy solvent, an acetonitrile, Terephthalic acid solvents, halogenated alkyl solvents, dimethyl sulfoxide, N,N-dimethylformamide In some embodiments, the amine is one or more of N,N-diisopropylethylamine and N,N-diisopropylethylamine. In some embodiments, the epoxy solvent may be dioxane and / or is tetrahydrofuran, and in some embodiments, the ether solvent is one In an embodiment, ethyl ether and / or methyl tert-butyl ether. In some embodiments, the halogenated alkyl solvent is, in one embodiment, One or more of dichloromethane, trichloromethane and 1,2-dichloroethane In some embodiments, the organic solvent is acetonitrile. 0) For the compound, the amount of the organic solvent is 1 to 50 L / mol, and some In an embodiment, it is 1 to 20 L / mol.

[0162] The conditions for the substitution reaction are a reaction temperature of 0 to 100° C. and a reaction time of 5 to 72 hours. In some embodiments, the reaction temperature is 10 to 40° C., and the reaction time is The time span is 10 to 30 hours.

[0163] Compounds of formula (320) can be obtained commercially or prepared using methods known to those skilled in the art. For example, m1 = m2 = m3 = 3, n1 = 1, n3 = 2, and R1 0, R 11 , R 12 , R 13 , R 14 , R 15 When both are H, in equation (320), Compounds were purchased commercially from Alfa Aesar Inc. can be obtained.

[0164] The molar ratio of the ethyl trifluoroacetate to the compound represented by formula (320) is 2:1 to 1 It may be 0:1, and in one embodiment, it is 3:1 to 5:1.

[0165] Similarly as above, the compound of formula (319) can be isolated from the reaction mixture by any suitable isolation method. In some embodiments, after the solvent is evaporated, the chromatograph may be The compound of formula (319) can be isolated by a chromatography method, for example: (1) normal phase purification Preparation: 200-300 mesh silica gel packing material, methanol:dichloromethane = 0. 01:1 to 0.5:1, or methanol:dichloromethane:ethyl acetate:petroleum ether = 0.1:1:1:1 to 1:1:1:1 gradient elution, (2) reverse phase purification: C18 and C 8. Elute the reversed-phase packing with a gradient of methanol:acetonitrile = 0.1:1 to 1:0.1. In some embodiments, the compound can be isolated using two chromatographic conditions: The solvent can be directly removed to obtain the crude compound of formula (319), The product can be used as is in the subsequent reaction.

[0166] <Complex> In another aspect, the present disclosure provides a conjugate having a structure as shown in formula (1):

[0167] [ka] During the ceremony, n1 is an integer from 1 to 3, and n3 is an integer from 0 to 4. Each of m1, m2, and m3 is independently an integer of 2 to 10; Each R 10 , R 11 , R 12 , R 13 , R 14 and R 15 are each independently H, C1- C 10 Alkyl groups, C1-C 10 Alkyl halide or C1-C 10 From alkoxy groups In some embodiments, R 10 , R 11 , R 12 , R 13 , R 14 and R 15 are each independently selected from H, a methyl group, or an ethyl group; R3 is an active drug, and in some embodiments, R3 is a functional oligonucleotide. Including R2 is a linear alkylene group having a length of 1 to 20 carbon atoms, Carbon atoms are C(O), NH, O, S, CH=N, S(O)2, C2-C 10 Alkenira group, C2-C 10 Alkynylene group, C6-C 10 Arylene group, C3-C 18 Heterosi Crylene group and C5-C 10 Optionally substituted with one or more selected from heteroarylene groups And R2 is C1-C 10 Alkyl groups, C6-C 10 Aryl groups, C5-C 10 Hetero Aryl groups, C1-C 10 Halogenated alkyl groups, -OC1-C 10 Alkyl group, -OC 1-C 10 Alkylphenyl group, -C1-C 10 Alkyl-OH, -OC1-C 10 Halo Genated alkyl group, -SC1-C 10 Alkyl group, -SC1-C 10 Alkylphenyl group , -C1-C 10 Alkyl-SH, -SC1-C 10 Halogenated alkyl groups, halogenated Substituent, -OH, -SH, -NH2, -C1-C 10 Alkyl-NH2, -N(C1-C1 0 alkyl group) (C1-C 10 alkyl group), -NH(C1-C 10 alkyl group), -N (C1-C 10 Alkyl group)(C1-C 10 alkylphenyl group), -NH(C1-C1 0 alkylphenyl group), cyano group, nitro group, -CO2H, -C(O)O(C1-C1 0 alkyl group), -CON(C1-C 10 Alkyl group)(C1-C 10 alkyl group), - CONH(C1-C 10 alkyl group), -CONH2, -NHC(O)(C1-C 10 a -NHC(O)(phenyl group), -N(C1-C 10 Alkyl)C(O)( C1-C 10 alkyl group), -N(C1-C 10Alkyl)C(O)(phenyl group), - C(O)C1-C 10 Alkyl group, -C(O)C1-C 10 Alkylphenyl group, -C( O)C1-C 10 Halogenated alkyl groups, -OC(O)C1-C 10 Alkyl group, -SO 2(C1-C 10 alkyl group), -SO2(phenyl group), -SO2(C1-C 10 Halo -SO2NH2, -SO2NH(C1-C 10 alkyl group), -S O2NH (phenyl group), -NHSO2(C1-C 10 Alkyl group), -NHSO2( phenyl group) and -NHSO2(C1-C 10 halogenated alkyl group) optionally substituted with one or more groups; Each L1 is a linear alkylene group having a length of 1 to 70 carbon atoms, The carbon atoms are C(O), NH, O, S, CH=N, S(O)2, C2-C 10 Arken C2-C 10 Alkynylene group, C6-C 10 Arylene group, C3-C 18 Hetero Cyclylene group and C5-C 10 one or more selected from the group consisting of heteroarylene groups and L1 is C1-C 10 Alkyl groups, C6-C 10 Aryl group, C5- C 10 Heteroaryl groups, C1-C 10 Halogenated alkyl groups, -OC1-C 10 Alki group, -OC1-C 10 Alkylphenyl group, -C1-C 10 Alkyl-OH, -OC1 -C 10Halogenated alkyl groups, -SC1-C 10 Alkyl group, -SC1-C 10 Alki -C1-C 10 Alkyl-SH, -SC1-C 10 Halogenated alkyl groups , halogen substituents, -OH, -SH, -NH2, -C1-C 10 Alkyl-NH2, -N (C1-C 10 Alkyl group)(C1-C 10 alkyl group), -NH(C1-C 10 Alki ), -N(C1-C 10 Alkyl group)(C1-C 10 alkylphenyl group), -NH (C1-C 10 alkylphenyl group), cyano group, nitro group, -COH, -C(O)O (C1-C 10 alkyl group), -CON(C1-C 10 Alkyl group)(C1-C 10 Al kill group), -CONH(C1-C 10 alkyl group), -CONH2, -NHC(O)(C 1-C 10 alkyl group), -NHC(O)(phenyl group), -N(C1-C 10 Alkyl )C(O)(C1-C 10 alkyl group), -N(C1-C 10 Alkyl)C(O)(phenyl nyl group), -C(O)C1-C 10 Alkyl group, -C(O)C1-C 10 Alkylpheny group, -C(O)C1-C 10 Halogenated alkyl groups, -OC(O)C1-C 10 Alki group, -SO2(C1-C 10 alkyl group), -SO2(phenyl group), -SO2(C1 -C 10 Halogenated alkyl group), -SO2NH2, -SO2NH(C1-C10 Alki -SO2NH (phenyl group), -NHSO2(C1-C 10 alkyl group), -N HSO2 (phenyl group) and -NHSO2 (C1-C 10 (halogenated alkyl group) In some embodiments, L1 may optionally have any one or more substituents from the group consisting of may be selected from the groups of formulae A1 to A26 or any combination thereof, and the structures A1 to A26 The structure and definition are as described above.

[0168] n1, n3, m1, m2, m3, R 10 , R 11 , R 12 , R 13 , R 14 , R 15 Reach and M1 are defined as above.

[0169] In some embodiments, R2 is selected from the group R4 in the compound of formula (321) In some embodiments, R2 is a linking group formed by binding to an active drug via The R4 group in the compound of formula (321) is reacted to bind to a functional oligonucleotide. In some embodiments, the R2 group includes a linking group formed by The bond site bonded to the N atom of R1 and the bond site bonded to the P atom of R2 are both included. In some embodiments, R2 is bonded to an N atom on the nitrogen-containing backbone. The site bonded to the P atom in R3 forms an amide bond with the N atom, and the site bonded to the P atom in R3 is In some embodiments, R2 is selected from the group consisting of B5, It may be B6, B5' or B6'.

[0170] [ka] however, TIFF0007662216000053.tif4142 represents the site where the group is covalently bonded, and the selection and value range of q2 are as described above. It is.

[0171] In some embodiments, R3 is a group having the structure shown in A59.

[0172] [ka] wherein E1 is OH, SH, or BH2, and in some embodiments, E1 is OH or SH, and Nu is a functional oligonucleotide.

[0173] In the context of this disclosure, unless otherwise stated, a "composite" group or molecule refers to a group or molecule that is A complex group or molecule and its ligands can be Each has a specific function. Accordingly, a "conjugate" refers to the chemical group and its ligand. Furthermore, the term "oligonucleotide" refers to a compound formed by covalently bonding "Optide conjugates" are oligonucleotides in which one or more conjugated groups with specific functions are covalently attached to the oligonucleotide. In some embodiments, the compounds disclosed herein are The conjugate shown is an oligonucleotide conjugate. The "molecule" is reacted to be conjugated to an oligonucleotide, and finally the oligonucleotide of the present disclosure is obtained. In some embodiments, the nucleotide conjugate is a specific compound capable of forming a nucleotide conjugate. In this case, the oligonucleotide is an siRNA, in which case the complex of the present disclosure is It is an A complex.

[0174] In some embodiments, the conjugate is represented by formula (3), (4), (5), (6), (7) , (8), (9), (10), (11), (12), (13), (14), (15), ( The structure shown in (16), (17), (18), (19), (20), (21) or (22) It has a structure.

[0175] [ka] TIFF0007662216000056.tif220139TIFF0007662216000057.tif216142TIFF0007662216000058.tif240146TIFF0007662216000059.tif251142

[0176] In some embodiments, the oligonucleotides in the oligonucleotide complexes of the present disclosure A leutide is a functional oligonucleotide. A functional oligonucleotide is a nucleotide that binds to a target sequence. Stable and specific hybridization between the sequence results in RNA activation. (RNA activation, RNAa), RNA interference ence, RNAi), antisense nucleic acid technology, exon skipping (exon skipping By using the principle of gene-shifting (R-lipase) technology, the expression of the target gene is up-regulated or down-regulated, or an oligonucleotide capable of inducing alternative splicing of mRNA. In some aspects, the functional oligonucleotides are capable of binding to a target protein. The nucleic acid structure may be a nucleic acid structure capable of forming a stable and specific bond between the nucleic acid and the target nucleic acid. As can be easily understood, a polynucleotide (e.g., an mRNA itself or a fragment thereof) Similarly, the antibody can be conjugated to the conjugate molecule provided by the present disclosure to form a conjugate. This allows targeted delivery, for example to the liver, and allows the protein transcribed from mRNA to be delivered to the liver. Therefore, in the context, the term "functional oligonucleotide" is used to refer to a nucleic acid that modulates the expression of a nucleic acid. The term "tide" may also include mRNA or fragments thereof.

[0177] In some embodiments, the functional oligonucleotide interacts with a target sequence. They can be used to affect the normal function of target molecules, for example by cleaving mRNA or inhibiting translation. or selective splicing of mRNA due to exon skipping. In some embodiments, the functional oligonucleotide is In some embodiments, the functional oligonucleotide is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87% in the target sequence, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more base complementary, or completely complementary to the target sequence In some embodiments, the functional oligonucleotide is a target sequence. In some embodiments, the sequence may include 1, 2, or 3 bases that are not complementary to the sequence of the The functional oligonucleotide may be a deoxyribonucleotide or a ribonucleotide, and In some embodiments, the functional oligonucleotide comprises a nucleotide having a modification. The nucleic acids may be single-stranded DNA, RNA, or DNA-RNA chimeras, or is double-stranded DNA, RNA or a DNA-RNA hybrid.

[0178] This makes it possible, in some embodiments, to make oligonucleotide conjugates suitable for the present disclosure. The functional oligonucleotides used are small interfering RNA (siRNA), microRNA (mRNA), and icroRNA), anti-microRNA (antimiR), microRNA antagonist (an tagomirs, microRNA mimics, decoys Oligonucleotides (decoys), immune stimulators y), G-quadruplex, alternative splice variants (spl ice altering), single-stranded RNA (ssRNA), antisense nucleic acid (ant isense), Nucleic Acid Aptamer, low-grade Small activating RNA (saRNA), stem The nucleic acid may be one of stem-loop RNA or DNA. 15 / 006740A2 describes conjugates in which different ligands are conjugated to oligonucleotides. The present invention discloses a method for the preparation of a ligand-linked oligonucleotide comprising the steps of: The oligonucleotides are small interfering RNA (siRNA), microRNA (microRNA), and the like. NA (microRNA), anti-microRNA (antimiR), microRNA antagonism Antagomirs, microRNA mimics , decoy oligonucleotides, immune stimulants atory, G-quadruplex, alternative splice variants (splice altering), single-stranded RNA (ssRNA), antisense nucleic acid (antisense), aptamer, stem-loop RNA The complex is one selected from the group consisting of oligonucleotides, oligonucleotides, and oligonucleotides. In a further embodiment, the present invention shows good stability in the delivery of oligonucleotides to the body. Functional oligonucleotides suitable for the disclosed oligonucleotide complexes are described in WO20090 82607A2, WO2009073809A2 or WO2015006740A2 The oligonucleotides shown are those shown in Table 1, the contents of which are incorporated herein by reference in their entirety. Can be enjoyed.

[0179] The oligonucleotide conjugates of the present disclosure can be used to bind active agents, e.g., functional oligonucleotides. By improving the efficiency of liver targeting delivery, it is possible to target specific genes in specific cells, e.g., hepatocytes. Regulating abnormal expression of genes and enhancing the correlation between functional oligonucleotides and target sequences in cells In some embodiments, the specific gene can enhance the interaction of the liver. It may be an endogenous gene expressed in the liver, or a pathogen gene that reproduces in the liver. Genes abnormally expressed in hepatocytes include ApoB, ApoC, ANGPTL3, P CSK9, SCD1, TIMP-1, Col1A1, FVII, STAT3, p53, H In some embodiments, the hepatocytes may be infected with a gene that is a virulent virus (BV), a hepatitis C virus (HCV), or the like. The constantly expressed genes are the HBV gene, the ANGPTL3 gene, or the APOC3 gene. In the context of this disclosure, the HBV gene is Genbank Accession No. NC_0039 77.1, and the ANGPTL3 gene refers to a gene having a sequence shown in Genba nk accession number NM_014495.3, and A The POC3 gene is an mRNA sequence identified by the Genbank accession number NM_000040.1. It refers to a gene having the A sequence.

[0180] In some embodiments, a "target sequence" is a target mRNA. In the present specification, the term "target mRNA" refers to, for example, an mRNA corresponding to a gene abnormally expressed in liver cells. The term refers to a gene that is expressed in a specific region of the genome, and an overexpressed gene may be expressed in a specific region of the genome, or an underexpressed gene may be expressed in a specific region of the genome. In some embodiments, the disease may be caused by overexpression of an mRNA. In most cases, the target mRNA is the mRNA that corresponds to the overexpressed gene. In some embodiments of the present disclosure, the gene encoding the abnormally expressed gene is The target mRNAs are ApoB, ApoC, ANGPTL3, PCSK9, SCD1, and TI MP-1, Col1A1, FVII, STAT3, p53, HBV, HCV, etc. In some embodiments, the target mRNA may be mRNA transcribed from the corresponding HBV gene, ANGPTL3 gene, or APOC3 gene It may be NA.

[0181] The P atom in formula A59 is bonded to any available position in the oligonucleotide sequence. For example, it may be attached to any nucleotide of the oligonucleotide. In some embodiments, the functional oligonucleotide in the oligonucleotide complex of the present disclosure The nucleotide is a single-stranded oligonucleotide (e.g., a single-stranded RNA or an aptamer). In this case, the P atom in formula A59 is a terminal region of the single-stranded oligonucleotide. The terminal region of the single-stranded oligonucleotide may be linked to In some embodiments, the term refers to the four nucleotides preceding the nucleotide. In the formula A59, the P atom is bonded to any terminal of the single-stranded oligonucleotide. are combined.

[0182] In some embodiments, the functional oligonucleotides in the oligonucleotide conjugates of the present disclosure The oligonucleotide is a double-stranded oligonucleotide (e.g., siRNA, microRNA, or DNA), and the double-stranded oligonucleotide comprises a sense strand and an antisense strand. In some embodiments, the P atom in formula A59 is The nucleic acid sequence is linked to the terminal region of the sense strand or the antisense strand of the nucleotide, is the four nucleotides preceding the one end of the sense strand or the antisense strand. In some embodiments, the P atom in formula A59 is and attached to any terminus of the antisense strand, in some embodiments, The P atom in formula A59 is bound to the 3' end of the sense strand. When attached to the above positions of the sense strand of an oligonucleotide, After entering the cell, the oligonucleotide complex unwinds into a single double-stranded molecule. The antisense strand of the oligonucleotide is released and the target mRNA is translated into protein. It can block the process and suppress the expression of specific genes.

[0183] The P atom in formula A59 can be any atom on a nucleotide in the oligonucleotide sequence. Possible positions, e.g., 5' position of nucleotide, 2' position of nucleotide, 3' position of nucleotide In some embodiments, the nucleotide of formula A5 may be attached to the 'position or the base of the nucleotide. The P atom in 9 binds the oligonucleotide by forming a phosphodiester bond. It may be attached to the 2', 3' or 5' position of the nucleotide in the sequence. In a specific embodiment, the P atom in formula A59 is a double-stranded oligonucleotide sequence. The oxygen atom that is the dehydrogenated 3' hydroxyl group of the 3'-terminal nucleotide of the sense strand in the row or the P atom in formula A59 is bonded to By replacing the hydrogen in the 2'-hydroxy group of the nucleotide in the sense strand, or the P atom in formula A59 is linked to a double-stranded oligonucleotide sequence replaces the hydrogen atom in the 5' hydroxyl group of the 5'-terminal nucleotide of the sense strand in The nucleotide is then attached to the nucleotide.

[0184] Without wishing to be limited thereto, the following embodiments and examples illustrate the oligonucleotides of the present disclosure. Functional oligonucleotides in nucleotide complexes are small interfering RNA (siRNA) In this case, the oligonucleotide complex of the present disclosure is In the context of this specification, for convenience of explanation, The siRNA complex is also referred to as the siRNA complex of the present disclosure. It is shown that the oligonucleotide in the nucleotide complex can only be siRNA. On the contrary, the oligonucleotide, and thus the active drug, may be any of the alternatives disclosed herein. or other alternatives well known to those skilled in the art. By way of illustration, other active agents or functional oligonucleotides may be used in combination with the compounds provided by this disclosure. It can be expected to have a similar effect when conjugated to a synthetic molecule.

[0185] siRNA comprises a nucleotide group as a structural unit, the nucleotide group being a phosphate group, It is known to those skilled in the art that the ribose group and the base are included. The length of the iRNA is about 12-40 nucleotides, and in some embodiments, about 15 to 30 nucleotides, each nucleotide in the siRNA being independently modified. The siRNA may be modified or unmodified nucleotides to improve stability. At least one nucleotide in A is a modified nucleotide.

[0186] The inventors of the present disclosure have demonstrated that the siRNAs described in the following embodiments have high activity and / or stability. Therefore, it can be used as the siRNA of the present disclosure. We found that.

[0187] In some embodiments, the siRNA of the siRNA complex of the present disclosure (hereinafter, Each nucleotide in the siRNA (also referred to as siRNA) can be independently modified or unmodified. The siRNA comprises a sense strand and an antisense strand, The strand comprises nucleotide sequence 1 and the antisense strand comprises nucleotide sequence 2. The nucleotide sequence 1 and the nucleotide sequence 2 each have a length of 10, 11, or 12. , 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides, which form a double-stranded complementary region in at least a portion thereof in a reverse complementary manner; The nucleotide sequence 2 is at least partially complementary to the first nucleotide sequence fragment, The first nucleotide sequence fragment is a nucleotide sequence fragment in a target mRNA.

[0188] In some embodiments, the siRNA of the present disclosure is administered at a concentration of 3 mg / kg or less. At least 50% HBV gene expression, at least 50% ANGPTL3 gene expression or This refers to siRNA that can suppress APOC3 gene expression by at least 50%. In the present disclosure, the siRNA has a molecular weight of at least 5 mg / kg. 5%, 60%, 65%, 70%, 75% or 80% of HBV genes and ANGPTL3 genes It refers to those that can suppress the expression of the APOC3 gene.

[0189] In some embodiments, the nucleotide sequence 1 and the first nucleotide sequence Fragments are of equal length and differ by no more than three nucleotides from the nucleotide sequence 2. and nucleotide sequence B are equal in length and have no more than three nucleotide differences, The nucleotide sequence B is a nucleotide sequence completely reverse-complementary to the first nucleotide sequence fragment. Although not wishing to be limited, these specific nucleotide differences result in s These specific nucleotides were not significantly reduced in the inhibitory capacity of the iRNA complex. siRNA complexes containing variations are also within the scope of protection of this disclosure.

[0190] In some embodiments, the nucleotide sequence 1 and the nucleotide sequence 2 are Essentially reverse complementary, essentially completely reverse complementary or completely reverse complementary.

[0191] In some embodiments, the nucleotide sequence 1 and the first nucleotide sequence The fragments have no more than one nucleotide difference and / or are identical to said nucleotide sequence 2 and said Nucleotide sequence B has no more than one nucleotide difference. The nucleotide difference between the nucleotide sequence 2 and the nucleotide sequence B is 5 From the 3' end to the 1st nucleotide in said nucleotide sequence 2 In some embodiments, the difference is at the Z' position. Thus, the last nucleotide Z in the nucleotide sequence 1 is a nucleotide complementary to Z'. This is the punch line.

[0192] In some embodiments, the sense strand further comprises nucleotide sequence 3, The antisense strand further comprises a nucleotide sequence 4, and the nucleotide sequence 3 and the Nucleotide sequences 4 are equal in length, each consisting of 1 to 4 nucleotides. nucleotide sequence 3 is linked to the 5' end of nucleotide sequence 1; 4 is linked to the 3' end of the nucleotide sequence 2, and the nucleotide sequence 4 is linked to the 2 The second nucleotide sequence fragment is complementary to the nucleotide sequence fragment of the target m a fragment of said first nucleotide sequence adjacent to said RNA, said fragment having a length of said nucleotide sequence 4. In some embodiments, the nucleotide sequence 3 and the nucleotide sequence 4 are essentially completely reverse-complementary or completely reverse-complementary. Therefore, the length of the sense strand and the antisense strand is 19 to 23 nucleotides. Good too.

[0193] In some embodiments, the siRNA of the present disclosure further comprises nucleotide sequence 5. The nucleotide sequence 5 has a length of 1 to 3 nucleotides, and the antisense strand and the 3' overhang end of the antisense strand is In some embodiments, the nucleotide sequence 5 is 1 or 2 nucleotides in length. Thus, in some embodiments, the nucleotide sequence of the siRNA of the present disclosure is The ratio of the length of the sense strand to the antisense strand was 19 / 20, 19 / 21, 20 / 21, and 20 / 2. 2, 21 / 22, 21 / 23, 22 / 23, 22 / 24, 23 / 24 or 23 / 25 It is also possible.

[0194] In one embodiment, the nucleotide sequence 5 is 2 nucleotides in length and From the 3' end to the 3' end, the nucleotide sequence 5 is a sequence of two consecutive thymidine deoxyribonuclease (TDR) residues. or two consecutive uridine nucleotides, or a third nucleotide the third nucleotide sequence fragment is complementary to the target mRNA A is adjacent to the first nucleotide sequence fragment or the second nucleotide sequence It refers to a nucleotide sequence adjacent to the fragment and having a length equal to the nucleotide sequence 5. In some embodiments, the ratio of the length of the sense strand to the antisense strand of the siRNA of the present disclosure is 1. 9 / 21 or 21 / 23, in which the siRNAs of the present disclosure inhibited significant hepatocyte mRNA expression. A silencing activity.

[0195] In some embodiments, each of the nucleotides in the siRNA of the disclosure is In some embodiments, the nucleotides of the present disclosure are independently modified or unmodified. The siRNA does not include modified nucleotide groups and, in some embodiments, is a nucleotide sequence of the present disclosure. The iRNA contains modified nucleotide groups.

[0196] Currently, in the field, backbone modifications (also called internucleotide bond modifications, e.g., phosphorylation Several modifications that can be used to modify siRNA, including modifications of the ribose group and bases, etc. There are several methods (e.g., Watts, JK, GFDeleavey and MJDamha,Chemically Modified siRNA:tool s and applications. Drug Discov Today,20 08.13(19-20): p.842-55, and the contents are incorporated herein by quotation. (which is incorporated herein by reference).

[0197] In the context of this disclosure, the term "modified nucleotide" refers to a nucleotide whose ribose group is modified. For example, nucleotides in which the 2'-hydroxy group is replaced with another group, These include nucleotides with nucleotide analogs, or modified bases.

[0198] In some embodiments of the present disclosure, at least one of the sense strand or the antisense strand At least one nucleotide is a modified nucleotide and / or at least one nucleotide is a In other words, the acid ester group is a phosphate ester group having a modifying group. and in the antisense strand, at least one single strand of phosphate-ribose backbone At least a portion of the acid ester groups and / or ribose groups is a phosphate ester having a modifying group. Ribose groups having modified groups (or modified phosphate ester groups and / or modified ribosaccharide groups) In some embodiments of the present disclosure, the sense strand and / or the ace strand are All of the nucleotides in the antisense strand are modified nucleotides.

[0199] In some embodiments, each nucleotide in the sense strand and the antisense strand is , are independently a fluoro-modified nucleotide or a non-fluoro-modified nucleotide.

[0200] A "fluoro-modified nucleotide" refers to a nucleotide that has a 2' hydroxyl group at the 2' position of the ribose group. It refers to a nucleotide in which the group is substituted with fluorine, and has the structure shown in the following formula (207): do.

[0201] A "non-fluoro modified nucleotide" refers to a nucleotide that has a hydroxyl group at the 2' position of the ribose group. It refers to a nucleotide or nucleotide analog in which the fluorinated group is replaced by a non-fluorinated group. In some embodiments, each non-fluoro modified nucleotide is independently A nucleotide or nucleoside in which the hydroxy group at the 2' position of the bosyl group is replaced with a non-fluorinated group It is a chido analogue.

[0202] Nucleotides in which the hydroxy group at the 2' position of the ribose group is replaced with a non-fluorinated group are known in the art. These nucleotides are known to those skilled in the art, and include, for example, 2'-alkoxy modified nucleotides. , 2'-substituted alkoxy modified nucleotides, 2'-alkyl modified nucleotides, 2'-substituted 2'-substituted alkyl-modified nucleotides, 2'-amino-modified nucleotides, 2'-substituted amino-modified nucleotides It is a nucleotide or a 2'-deoxynucleotide.

[0203] In some embodiments, the 2'-alkoxy modified nucleotide is represented by formula (208): In some embodiments, the 2'-substituted methoxy-modified nucleotide is The alkoxy modified nucleotide is a 2'-O-methoxyethyl modified nucleotide, as shown in formula (209). In some embodiments, the 2'-amino modified nucleotide is , as shown in formula (210). In some embodiments, the 2'-deoxynucleotide (DNA) is shown in formula (211).

[0204] [ka]

[0205] Nucleotide analogs are compounds that can replace nucleotides in nucleic acids. , adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, It refers to a group different from uracil ribonucleotide or thymine deoxyribonucleotide. In some embodiments, the nucleotide analogs include, for example, isonucleotides, crosslinked nucleotides, Bridged nucleic acid (BNA) nucleotides or acyclic It may be a nucleotide.

[0206] BNA nucleotide refers to a constrained or inaccessible nucleotide. Have a "fixed" C3'-endo sugar puckering of five, six, or seven membered rings The bridges are usually incorporated at the 2'- and 4'-positions of the ribose ring. 2',4'-BNA nucleotides, for example, LNAs shown in formula (212), formula (213) ) and cET BNA shown in formula (214).

[0207] [ka]

[0208] Acyclic nucleotides are nucleotides in which the sugar ring of the nucleotide is opened, for example, nucleotides of the formula ( 215) and the unlocked nucleic acid (UNA) nucleotide represented by formula (216) Glycerol Nucleic Acid (GNA) is a nucleotide that is used to

[0209] [ka] where R is H, OH, or alkoxy (O-alkyl).

[0210] An isonucleotide is a nucleotide in which the position of the base in the ribose ring has been changed. For example, the base is represented by the formula (217) or (218), These are compounds in which the aryl group has migrated to the '- or 3'-position.

[0211] [ka] In the formula, Base represents a nucleic acid base such as A, U, G, C, or T, and R represents H, OH, F. or a non-fluorinated group as described above.

[0212] In some embodiments, the nucleotide analog is an isonucleotide, an LNA, In some embodiments, each of the non-fluoro The modified nucleotides are all methoxy-modified nucleotides, A ribose nucleotide is a nucleotide in which the 2'-hydroxyl group of the ribose group is replaced with a methoxy group. vinegar.

[0213] In the context, "fluoro-modified nucleotide", "2'-fluoro-modified nucleotide" "Nucleotides in which the 2'-hydroxy group of the ribose group is replaced with fluorine" and "2'- "Nucleotide having a fluororibose group" and "Nucleotide having a fluororibose group" have the same meaning. The 2'-hydroxy group of the aryl group is replaced by fluorine, and the structure is represented by the formula (207). "Methoxy-modified nucleotide", "2'-methoxy-modified nucleotide", "Lys "Nucleotides in which the 2'-hydroxy group of the bosyl group is replaced with methoxy" and "2'-Methoxy "Nucleotide having a xylibose group" and "Nucleotide having a xylibose group" have the same meaning. The 2'-hydroxy group of the bosyl group is replaced with methoxy, as shown in formula (208). Point.

[0214] In some embodiments, the siRNA of the present disclosure has the following modifications: That is, from the 5' end to the 3' end, The nucleotides at positions 7, 8, and 9 of said nucleotide sequence 1 are fluoro-modified nucleotides. and the nucleotides at other positions in the sense strand are methoxy-modified nucleotides. and / or in the antisense strand, the 2nd, 6th, 14th and 5th bases of the nucleotide sequence 2 the nucleotide at position 16 is a fluoro-modified nucleotide; and In some embodiments, the nucleotides at the other positions are methoxy-modified nucleotides. The siRNA of the present disclosure is an siRNA having the following modifications: nucleotide sequence 1 in the sense strand of the siRNA from the 3′ end toward the 3′ end the nucleotides at positions 5, 7, 8, and 9 of said sense strand are fluoro-modified nucleotides; and / or wherein the nucleotide at the other position is a methoxy-modified nucleotide. In the antisense strand, the nucleotides at positions 2, 6, 8, 9, 14, and 16 of the nucleotide sequence 2 are The nucleotide is a fluoro-modified nucleotide, and In some embodiments, the nucleotide is a 2'-methoxy modified nucleotide. The siRNA of the present disclosure is an siRNA having the following modifications: Towards the 3' end, the seventh nucleotide of nucleotide sequence 1 in the sense strand of the siRNA , the nucleotides at positions 8 and 9 are fluoro-modified nucleotides, The nucleotide at the other position is a methoxy-modified nucleotide and / or the nucleotide at the 3 'towards the end of said nucleotide sequence 2 in the antisense strand of said siRNA the nucleotides at positions 2, 6, 14 and 16 are fluoro-modified nucleotides; The nucleotides at other positions in the sense strand are methoxy modified nucleotides.

[0215] In some specific embodiments of the siRNA described in this disclosure, the nucleoside In the context of this disclosure, a modification on the phosphate group is one embodiment of In the form of the thiophosphoric acid shown in the following formula (201), te) modification, i.e., replacing a non-bridging oxygen atom in the phosphodiester bond with a sulfur atom. By replacing the phosphodiester bond with a phosphorothioate bond, In some embodiments, the modifications stabilize the structure of the siRNA and improve base pairing. Maintains high specificity and high affinity.

[0216] [ka]

[0217] According to some embodiments of the present disclosure, in the siRNA, a thiophosphate group The first and second nucleotides at either end of the sense or antisense strand are Between the nucleotides 2 and 3 of either end of the sense or antisense strand and any combination thereof. In some embodiments, the thiophosphate group is located at any position except the 5' end of the sense strand. In some embodiments, the thiophosphate is present bound to at least one of the above positions. Terminator groups are present attached at all of the above positions except the 3' end of the sense strand. In embodiments, the thiophosphate group is attached at at least one of the following positions: It exists as such. Between the first and second nucleotides of the 5' end of the sense strand, Between the second and third nucleotides of the 5' end of the sense strand, Between the first and second nucleotides of the 3' end of the sense strand, Between the second and third nucleotides of the 3' end of the sense strand, Between the first and second nucleotides of the 5' end of the antisense strand , Between the second and third nucleotides of the 5' end of the antisense strand , Between the first and second nucleotides of the 3' end of the antisense strand , and Between the second and third nucleotides at the 3' end of the antisense strand .

[0218] According to some embodiments of the present disclosure, the 5' of the antisense strand sequence of the siRNA molecule The terminal nucleotide is a 5'-phosphate nucleotide or a 5'-phosphate analog-modified nucleotide. This is the punch line.

[0219] In some embodiments, the 5'-phosphate nucleotide is represented by formula (202): The structure may be

[0220] [ka]

[0221] The types of commonly used 5'-phosphate analog modified nucleotides are known to those skilled in the art, e.g. For example, Anastasia Khvorova and Jonathan K. Wa tts,The chemical evolution of oligonucle otide therapies of clinical utility. Nat In Ure Biotechnology, 2017, 35(3):238-48, Four nucleotides shown in formulas (203) to (206) are disclosed.

[0222] [ka] where R represents a group selected from the group consisting of H, OH, F and a methoxy group; Base represents a base selected from A, U, C, G, or T.

[0223] In some embodiments, the 5'-phosphate nucleotide or 5'-phosphate analog modification The modified nucleotide is a vinyl phosphate ester, as shown in formula (203). sphonate (VP) modification, a nucleotide having a 5'-lysyl sphonate (VP) modification as shown in formula (202), or a 5'-thiophosphate modified nucleotide represented by formula (205): It is.

[0224] The inventors of the present disclosure have found that the siRNA complexes described in the present disclosure have significantly improved serum stability. The results showed that the target mRNA silencing activity was not significantly reduced and that the Surprisingly, we found that it also has the effect of suppressing gene expression in the body. The disclosed siRNA complexes have been shown to have high in vivo delivery efficiency. According to certain embodiments, the oligonucleotide conjugates of the present disclosure can be, for example, any of the oligonucleotide conjugates shown in Tables 1A-1F. The siRNA complex contains an siRNA.

[0225] [Table 1] siRNA sequences [Table 1A] JPEG0007662216000068.jpg182161 [Table 1B] JPEG0007662216000070.jpg182161 [Table 1C] [Table 1D] [Table 1E] JPEG0007662216000074.jpg222162JPEG0007662216000075.jpg147162 [Table 1F] JPEG0007662216000077.jpg182159 * S: sense strand, AS: antisense strand

[0226] In the above table, capital letters C, G, U, and A represent nucleotide base sequences, and lowercase letters m means that the nucleotide adjacent to the left of the letter m is a 2'-methoxy modified nucleotide. A lowercase letter f indicates that the nucleotide adjacent to the left of the letter f is 2. '-fluoro modified nucleotide, and the lowercase letter s is adjacent to the letter s. The two nucleotides that correspond to each other are linked by a phosphorothioate group, and P 1 is a nucleotide adjacent to the right side of the P1 that is a 5'-phosphate nucleotide or a 5 In some embodiments, vinyl phosphate modified nucleotide (represented as VP in the examples below), 5' -phosphate nucleotide (represented by P in the following examples) or thiophosphate modification It is a decorative nucleotide (represented as Ps in the examples below).

[0227] As will be apparent to those skilled in the art, nucleoside monomers having corresponding modifications can be used. The modified nucleotide groups can be introduced into the siRNAs described in this disclosure by using Methods for preparing nucleoside monomers having corresponding modifications and methods for preparing modified nucleoside monomers are provided. Methods for introducing nucleotide groups into siRNA are also well known to those skilled in the art. The leioside monomers may be commercially available or may be prepared by known methods.

[0228] <Preparation of Oligonucleotide Complex> The oligonucleotide conjugates of the present disclosure may be prepared by any rational synthetic route. For example, the oligonucleotide conjugates of the present disclosure can be prepared by the following method: The method comprises the steps of: synthesizing the oligonucleotides in a phosphoramidite solid-phase synthesis condition; Depending on the type and order of nucleotides, the nucleoside monomers are ordered from 3' to 5'. Each nucleoside monomer is then deprotected, coupled, capped, and oxidized. or sulfurization. In some embodiments, the method includes four reactions: Under coupling reaction conditions and in the presence of a coupling reagent, a compound of formula (321) can be obtained. with a nucleoside monomer or a nucleotide sequence bound to a solid support, 321) to bind to a nucleotide sequence by coupling reaction. Further includes:

[0229] In some embodiments, the method includes removing the protecting group to cleave the solid support and The method further comprises a step of isolating and purifying the product.

[0230] In some embodiments, the oligonucleotide is a double-stranded oligonucleotide. The preparation method comprises reacting a compound of formula (I) under coupling reaction conditions and in the presence of a coupling reagent. 321) as a nucleoside monomer at the 3' end of the sense strand or antisense strand. and attaching the compound of formula (321) to the first nucleotide in the sequence. The nucleoside monomers are sequentially linked from 3' to 5' to form a double-stranded oligonucleotide. A process for synthesizing a sense strand or an antisense strand of a nucleic acid, the compound of formula (321) being R4 is a first functional group, a protected hydroxy group, and a second functional group represented by the formula (C A compound represented by formula (321), which includes a group represented by (C1') or (C3'), Before coupling with the first nucleoside monomer, the compound of formula (321) is deprotected and each nucleoside is The binding of cleoside monomers can be done in four ways: deprotection, coupling, capping, oxidation or sulfurization. to obtain a sense strand or an antisense strand of a nucleic acid to which a conjugate molecule is bound; Double-stranded oligonucleotides are created by linking nucleoside monomers in order from 3' to 5'. The other strand is synthesized, and each nucleoside monomer bond is deprotected, coupled, capped, and The reaction involves four steps: deprotection, oxidation, or sulfurization, which removes the protecting group, cleaves the solid support, and isolates and purifies the product. The method includes the steps of preparing a sense strand and an antisense strand and annealing them.

[0231] In some embodiments, the oligonucleotide is a double-stranded oligonucleotide. The preparation method includes sequentially binding nucleoside monomers from 3' to 5' to form a bifunctional nucleotide. The sense and antisense strands of the double-stranded oligonucleotide were synthesized, and each nucleoside monomer was The bond between the solids involves four reactions: deprotection, coupling, capping, and oxidation or sulfurization. obtaining a solid support-bound sense strand and a solid support-bound antisense strand; Under coupling reaction conditions and in the presence of a coupling reagent, a compound of formula (321) can be obtained. The material is contacted with either the sense strand bound to a solid support or the antisense strand bound to a solid support. Then, a compound of formula (321) containing a first functional group that is a phosphoramidite in R4 is prepared. a step of binding the nucleic acid to the sense or antisense strand; a step of removing the protecting group to cleave the nucleic acid from the solid support; A process for isolating and purifying the sense strand or antisense strand of the oligonucleotide and annealing the sense strand or antisense strand. A conjugate molecule is bound to the sense strand or the antisense strand of the oligonucleotide. is.

[0232] In some embodiments, the P atom in formula A59 is a sense atom in the siRNA. and a method for preparing an siRNA complex of the present disclosure includes: (1) The compound of formula (321) to which the solid phase support is bound (hereinafter, referred to as solid phase support-bound compound) The protecting group R in the L complex molecule k The reaction mixture was removed and subjected to coupling reaction conditions. In the presence of a ring reagent, the solid support-bound L-complex molecule is reacted with a nucleoside monomer. contacting to obtain a nucleoside monomer bound to the solid support by an L conjugate molecule; (2) starting with a nucleoside monomer that is bound to a solid support by the L conjugate molecule, The sense strand of siRNA was synthesized by phosphoramidite solid-phase synthesis according to the 3'-5' direction. To synthesize, (3) Synthesize the antisense strand of siRNA by phosphoramidite solid-phase synthesis. To do so, (4) Isolating and annealing the sense and antisense strands of the siRNA to produce the siRNA of the present disclosure. obtaining an RNA complex.

[0233] In step (1), the protecting group R in the solid support-bound L conjugate molecule is k Remove The method includes contacting a compound of formula (321) with a deprotecting reagent under deprotecting conditions. The deprotection conditions are a temperature of 0 to 50° C., and in some embodiments, a temperature of 15 to 50° C. In some embodiments, the reaction time is 30 to 300 seconds, and in some embodiments, the reaction time is 50 to 35°C. The deprotection reagent was trifluoroacetic acid, trichloroacetic acid, dichloroacetic acid, and in some embodiments, one or more selected from dihydroacetic acid and The molar ratio of the deprotection reagent to the compound of formula (321) is 10:1 to 1000: 1, and in some embodiments, is between 50:1 and 500:1.

[0234] The coupling reaction conditions and coupling reagents are suitable for the above coupling reaction. In some embodiments, solid phase synthesis is employed. The same conditions and reagents are used as for the coupling reaction in the method.

[0235] In some embodiments, the coupling reaction conditions include a reaction temperature of 0 to 40° C. 50° C., and in some embodiments, 15 to 35° C. Compound of Formula (321) The molar ratio of the compound to the nucleoside monomer may be 1:1 to 1:50. In an embodiment, the ratio of the compound of formula (321) to the coupling reagent is 1:2 to 1:5. The molar ratio may be from 1:1 to 1:50, and in some embodiments, from 1:3 to 1: 10, and the reaction time may be 200 to 3000 seconds, and in some embodiments The coupling reagent is 1H-tetrazole, 5-ethyl thio 1H-tetrazole, 5-benzylthio 1H-tetrazole, or There may be more than one type, and in some embodiments, 5-ethylthio 1H-tetrazole. The coupling reaction may be carried out in an organic solvent, and the organic solvent may be anhydrous aldehyde. one or more selected from acetonitrile, anhydrous DMF, and anhydrous dichloromethane; In some embodiments, the compound is anhydrous acetonitrile. The amount of the organic solvent may be 3 to 50 L / mol. In some embodiments, the concentration is 5 to 20 L / mol.

[0236] In step (2), the nucleic acid prepared in the above step is synthesized by a phosphoramidite nucleic acid solid phase synthesis method. Starting with a nucleoside monomer bound to a solid support by a modified L-conjugate molecule, The sense strand S of the siRNA complex is synthesized according to the direction of the '. In this case, the L complex molecule is obtained The 3′ end of the sense strand is linked to the 3′ end of the

[0237] Other conditions for the solid phase synthesis in steps (2) and (3) include: Deprotection conditions, type and dosage of deprotection reagent, coupling reaction conditions, coupling reagent Type and dosage, capping reaction conditions, type and dosage of capping reagent, oxidation reaction conditions, The present specification includes the type and amount of the oxidation reagent, the sulfurization reaction conditions, and the type and amount of the sulfurization reagent. Various reagents, dosages and conditions commonly used in the field are employed.

[0238] For example, in some embodiments, in steps (2) and (3), the solid phase synthesis Then, the following conditions may be used:

[0239] The deprotection conditions for the nucleoside monomers are 0 to 50°C. In an embodiment, the temperature is 15 to 35° C., the reaction time is 30 to 300 seconds, and some experiments In an embodiment, the deprotection time is 50 to 150 seconds, and the deprotection reagent is trifluoroacetic acid, trichloroacetic acid, One or more selected from acetic acid, dichloroacetic acid, and chloroacetic acid may be used. In one embodiment, the deprotection reagent is dichloroacetic acid. The molar ratio of the methoxytrityl protecting group may be 2:1 to 100:1, and some In an embodiment, it is from 3:1 to 50:1.

[0240] The coupling reaction conditions are a temperature of 0 to 50° C., and in some embodiments The temperature is 15 to 35° C., and the nucleic acid sequence bound to the solid support and the nucleoside monomer are mixed. The ratio of the fluorine atom may be 1:1 to 1:50, and in some embodiments, 1:5 to 1:1 5, and the molar ratio of the nucleic acid sequence to be bound to the solid phase carrier to the coupling reagent is 1:1 to 1: 100, and in some embodiments, 1:50 to 1:80, The time and choice of coupling reagents are the same as above.

[0241] The capping reaction conditions are a temperature of 0 to 50° C., and in some embodiments, In some embodiments, the reaction temperature is 15 to 35° C., and the reaction time is 5 to 500 seconds. The capping time is 10 to 100 seconds, and the selection of the capping reagent is the same as above. The molar ratio of the total amount of the reagent to the nucleic acid sequence bound to the solid support was 1:100 to 100:1. In some embodiments, the ratio is 1:10 to 10:1. When equimolar amounts of acetic anhydride and N-methylimidazole are used as the reaction product, -The molar ratio of methylimidazole to the nucleic acid sequence bound to the solid support is 1:1:10 to 10 :10:1, and in some embodiments, 1:1:2 to 2:2:1. do.

[0242] The oxidation reaction conditions are a temperature of 0 to 50° C., and in some embodiments, 15 In some embodiments, the reaction temperature is 1 to 35° C., and the reaction time is 1 to 100 seconds, and in some embodiments, the reaction time is 5 to 5 0 seconds and the oxidation reagent is iodine in some embodiments (see further embodiments). In this embodiment, it is provided as iodine water. The molar ratio of the nucleic acid sequence to be bound to the nucleic acid sequence may be 1:1 to 100:1, and some In some embodiments, the ratio is 5:1 to 50:1. The reaction was carried out in a mixed solvent of tetrahydrofuran:water:pyridine = 3:1:1 to 1:1:3. The sulfurization reaction conditions are a temperature of 0 to 50° C., and in some embodiments, 5 to 35° C., and the reaction time is 50 to 2000 seconds. In some embodiments, In some embodiments, the sulfurization reagent is xanthan gum. The sulfurization reagent and the nucleic acid sequence that is bound to the solid support in the coupling step are coupled. The molar ratio may be from 10:1 to 1000:1, and in some embodiments, 10 In some embodiments, the sulfurization reaction is carried out using acetonitrile. The reaction was carried out in a mixed solvent of ethanol:pyridine = 1:3 to 3:1.

[0243] The method provided by the present disclosure allows for the conjugation of all nucleoside monomers, followed by aniline. Prior to injection, the method further comprises isolating the sense and antisense strands of the siRNA. Isolation methods are known to those of skill in the art and generally involve the isolation of synthetic nucleotide sequences. Cleavage from the solid support, removal of protecting groups on the bases, phosphates and ligands, purification and desalting This includes:

[0244] The synthesized nucleotide sequence is cleaved from the solid support and the bases, phosphate groups and ligands are removed. The above protecting groups can be removed by standard cleavage and deprotection methods used in siRNA synthesis. For example, the resulting solid support-bound nucleotide sequence may be dissolved in concentrated aqueous ammonia. In the deprotection process, the protecting group YCOO- of the groups A46 to A54 is converted to hydroxy and converting the S1 group to the corresponding M1 group to produce a conjugate as shown in formula (1). Here, the concentrated ammonia water may be ammonia water having a concentration of 25 to 30% by weight. The dose of concentrated ammonia water is usually 0.2 ml / μmol per siRNA sequence of interest. It may be 1 to 0.8 ml / μmol.

[0245] If the synthesized nucleotide sequence has at least one 2'-TBDMS protection, The method comprises the steps of: cleaving the nucleotide sequence from the solid support by cleaving the nucleotide sequence with triethylamine trihydrofluoride; This further comprises removing the 2'-TBDMS protection by contacting the In this case, the desired siRNA sequence obtained contains two free nucleosides in the corresponding nucleosides. The dose of pure triethylamine trihydrofluoride is The concentration may be 0.4 ml / μmol to 1.0 ml / μmol for the corresponding siRNA sequence. In this manner, the siRNA complex of the present disclosure can be obtained.

[0246] Methods of purification and desalting are well known to those skilled in the art. For example, preparative ion chromatography. Purification of nucleic acids using a chromatographic purification column and gradient elution with NaBr or NaCl The products are collected and combined, then desalted using a reverse phase chromatography purification column. It is possible.

[0247] During the synthesis process, the purity and molecular weight of the nucleic acid sequence can be constantly detected to better control the synthesis quality. Detection methods are known to those skilled in the art, for example, ion exchange chromatography. Nucleic acid purity was detected by PCR and analyzed by liquid chromatography tandem mass spectrometry (LC-MS). The molecular weight can be measured by

[0248] Annealing methods are also well known to those skilled in the art. For example, a simple synthesized sense strand (S The antibody strand (A strand) and the antisense strand (AS strand) were mixed in an equal molar ratio in water for injection and heated to 70-95°C. Then, the mixture is cooled to room temperature, whereby a double-stranded structure can be formed by hydrogen bonding. The siRNA complex of the present disclosure can be obtained in the following manner.

[0249] After obtaining the complex of the present disclosure, in some embodiments, the complex may be purified by, for example, liquid chromatography. The synthesized siRNP was analyzed by molecular weight detection using methods such as tandem mass spectrometry. The characteristics of the A complex were elucidated, and the synthesized siRNA complex was confirmed to be a target-designed siRNA complex. The sequence of the synthesized siRNA is identical to the sequence of the siRNA to be synthesized. For example, it may be confirmed that the sequence corresponds to one of the sequences shown in Table 1 above.

[0250] <Use of the Conjugate of the Present Disclosure> As shown in the present disclosure, the complex delivers an active agent to a cell, and such delivery The compounds can be used to treat or prevent diseases or conditions that may require treatment. Without wishing to be bound by any theory, it is believed that the spatial arrangement of the complex molecules is mediated by the receptors on the surface of the target cells. It is believed that the method is particularly effective in bringing the loaded active agent into contact with the cells. In embodiments, such conjugates are oligonucleotide conjugates that target hepatocytes. do.

[0251] In some embodiments, the oligonucleotide conjugates of the present disclosure have superior liver targeting characteristics. Since the oligonucleotide has isomerism, it can efficiently deliver the conjugated functional oligonucleotide to the liver and promote the delivery of the oligonucleotide to liver cells. Therefore, the present invention can effectively regulate the expression of specific genes in cells. The oligonucleotide conjugates have the potential for a wide range of applications.

[0252] According to some embodiments of the present disclosure, the present disclosure provides a method for the preparation of an oligonucleotide complex of the present disclosure. and / or prevention of pathological conditions or diseases by expression of specific genes in liver cells - Patents.com The specific gene is an endogenous gene expressed in the liver. The gene may be a gene of a pathogen that reproduces in the liver. In this embodiment, the specific gene is, for example, ApoB, ApoC, ANGPTL3, PC SK9, SCD1, TIMP-1, Col1A1, FVII, STAT3, p53, HB In some embodiments, the specific gene is Hepatitis B virus or HCV. rus gene, angiopoietin-like protein 3 gene, or apolipoprotein C3 gene Accordingly, the diseases include chronic liver disease, hepatitis, liver fibrosis, liver hyperplasia, and and dyslipidemia (blood lipid abnormalities). Usually it is hypercholesterolemia, hypertriglyceridemia or atherosclerosis.

[0253] In some embodiments, the present disclosure provides a method for administering the oligonucleotides of the present disclosure to a patient in need thereof. Pathological conditions caused by the expression of specific genes in liver cells, including administering a tide complex In some embodiments, the particular The genes include, for example, ApoB, ApoC, ANGPTL3, PCSK9, SCD1, TI MP-1, Col1A1, FVII, STAT3, p53, HBV or HCV. In some embodiments, the specific gene is a Hepatitis B virus gene, angiotensin II gene, or a nucleotide sequence. The gene is selected from the ethin-like protein 3 gene and the apolipoprotein C3 gene. Therefore, the diseases include chronic liver disease, hepatitis, liver fibrosis, liver hyperplasia and lipid abnormalities. In some embodiments, the lipid abnormality is selected from the group consisting of hypercholesterolemia, hyperlipidemia, and hypercalcemia. In some embodiments, the condition is atherosclerosis. The conjugates provided by the disclosure are useful in treating diseases characterized by unwanted cell proliferation, blood disorders, metabolic disorders, and the like. The present invention may also be used to treat other liver diseases, including those characterized by inflammation and disease. Proliferative disorders of the liver can be benign or malignant, e.g., cancer, hepatocellular carcinoma (HCC), liver metastasis, or hepatoblastoma. The hepatic hematological or inflammatory disease may be a blood clotting factor, a complement-mediated inflammation, The metabolic disease of the liver may be a disease related to lipid abnormalities and glucose metabolism. In some embodiments, the method includes determining whether a disorder is associated with liver disease. By using one or more oligonucleotides having a sequence highly homologous to the gene sequence of interest, Includes.

[0254] According to some embodiments of the present disclosure, the present disclosure provides a method for administering a siRNA complex of the present disclosure to said hepatocytes. The present invention provides a method for suppressing the expression of a specific gene in a liver cell, the method comprising contacting the liver cell with

[0255] The oligonucleotide complexes of the present disclosure can be administered to a patient in need thereof to inhibit the expression of the gene. The mechanism of expression regulation is to determine the pathological condition or disease caused by the expression of specific genes in liver cells. Therefore, the oligonucleotides of the present disclosure can achieve the purpose of preventing and / or treating diseases. The nucleotide complexes are useful in preventing and / or treating the pathological conditions or diseases disclosed herein. or for the prevention and / or treatment of a pathological condition or disease disclosed herein. It can be used to prepare the drug.

[0256] As used herein, the term "drug administration / administration" refers to administration of a complex, such as an oligonucleotide conjugate. Methods for localizing the combination, at least in part, to a desired site to produce a desired effect The term "delivery" refers to the delivery of a complex to a subject's body by a suitable route or route. Routes of administration include, but are not limited to, local and systemic administration. Upon administration of the desired oligonucleotide, the oligonucleotide conjugate is delivered to a specific site in greater amounts than is available in the subject's systemic circulation. However, systemic administration delivers the oligonucleotide conjugate into the patient's systemic circulation. The present disclosure relates to the prevention and treatment of pathological conditions or diseases by expression of specific genes in hepatocytes. Considering that the present invention is intended to provide a therapeutic means, in some embodiments Therefore, an administration method capable of delivering the drug to the liver is adopted.

[0257] Administration to a patient may be by any suitable route known in the art, including, but not limited to, oral administration. Oral or non-gastrointestinal (parenteral) routes, e.g., intravenous, intramuscular, subcutaneous, oral Dermal, intratracheal (aerosol), pulmonary, nasal, rectal and topical (oral) The frequency of administration may be, but is not limited to, once a day ... It may be once or more per week, fortnightly, monthly or yearly.

[0258] The dosages of the oligonucleotide conjugates described in this disclosure are those usual in the art. The dose may vary depending on various parameters, in particular the age, weight and sex of the patient. Toxicity and therapeutic efficacy may be measured in cell cultures or experimental animals using standard pharmaceutical procedures. For example, LD50 (the dose that kills 50% of the colony) and ED50 (the dose that kills 50% of the colony) are used. It refers to the dose that can induce 50% of the maximum reaction intensity in a qualitative response. (referring to the dose that can produce a positive response in 50% of experimental subjects) A range of human dosages may be derived based on data obtained from cell culture assays and animal studies. It is possible.

[0259] When administering the conjugates described in the present disclosure, for example, male or female, 6 to 12 weeks of age, The above-mentioned treatment was performed on C57BL / 6J or C3H / HeNCrlVr mice weighing 18 to 25 g. The amount of oligonucleotide in the oligonucleotide complex was calculated as the functional oligonucleotide. To deliver the oligonucleotide complex formed by the nucleotide and the conjugate molecule, The dose of oligonucleotide delivered by the complex is 0.001-100 mg / kg In some embodiments, the dose is 0.01 to 50 mg / kg of body weight. In some embodiments, the dose is 0.05 to 20 mg / kg body weight, and in some other embodiments, the dose is 0.05 to 20 mg / kg body weight. In some embodiments, the dose is 0.1 to 15 mg / kg body weight, and in some other embodiments, the dose is 0.1 to 15 mg / kg body weight. The oligonucleotide complexes described in the present disclosure are administered in an amount of 0.1 to 10 mg / kg body weight. When administering to the body, reference may be made to the above dosages.

[0260] In addition, the oligonucleotide complex of the present disclosure can be administered to liver cells in which a particular gene is abnormally expressed. By introducing the gene into the liver cells, the gene expression control mechanism is activated, and the specific gene in the liver cells is In some embodiments, the liver cell The cells are hepatitis cells, and in some embodiments, HepG2.2.15 cells. In some embodiments, the hepatocytes are selected from the group consisting of Hep3B, HepG2, Huh7, etc. Hepatoma cell lines or isolated primary hepatocytes, and in some embodiments , Huh7 hepatoma cells.

[0261] When the expression of a specific gene in liver cells is suppressed by the method provided by the present disclosure The dosage of the functional oligonucleotide in the provided oligonucleotide complex is Those skilled in the art can easily determine the effect to be obtained. In one embodiment, the oligonucleotide complex is a siRNA complex, The siRNA dose in the iRNA complex is sufficient to reduce the expression of the target gene. , 1 pM to 1 μM, 0.01 nM to 100 nM, or 0.05 nM to 50 nM or 0.0 The amount required to achieve an extracellular concentration of 5 nM to about 5 nM is , delivery method, delivery site, number of cell layers between the delivery site and the target cell or tissue, delivery route (local The concentration at the site of delivery varies depending on a variety of factors, including whether the drug is delivered locally or systemically. Or it may be significantly higher than the concentration at the surface of the tissue. (Effects of the Invention)

[0262] In some embodiments, the conjugates provided by the present disclosure have a higher oligonucleotide content in the body. have higher oligonucleotide delivery efficiency, lower toxicity, better stability and / or higher activity In some embodiments, the conjugates of the present disclosure have at least 20%, 30%, 40%, %, 50%, 60%, 70%, 80%, 90% or 95% of the target gene expression is suppressed. In some embodiments, the conjugates of the present disclosure have at least 20%, 30%, 40% , 50%, 60%, 70%, 80%, 90% or 95% suppression of HBV gene expression In some embodiments, the conjugates of the present disclosure have at least 20%, 30%, 40% , 50%, 60%, 70%, 80%, 90% or 95% suppression rate of intrahepatic HBV gene expression In some embodiments, the conjugates of the present disclosure have at least 20%, 30%, 40%, Intrahepatic H in 0%, 50%, 60%, 70%, 80%, 90% or 95% of animal models The figures show the BV gene expression suppression rate. In some embodiments, the complex of the present disclosure has at least In either case, the percentage of the HBV expression suppression rate in subjects. is at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or In some embodiments, the present disclosure provides a method for the suppression of HBV surface antigen expression by 95%. The provided siRNA, siRNA composition or siRNA complex comprises at least 20% 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% ANGPTL3 In some embodiments, the complex of the present disclosure has at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% intrahepatic A 4 shows the inhibition rate of NGPTL3 gene expression. In some embodiments, the complex of the present disclosure , at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 9 The 5% suppression rate of intrahepatic ANGPTL3 gene expression in an animal model was shown. In some embodiments, the complexes of the present disclosure are at least 20%, 30%, 40%, 50%, 60% , 70%, 80%, 90% or 95% suppression of ANGPTL3 gene expression in human subjects In some embodiments, the conjugates of the present disclosure have a .alpha.-tocopherol concentration of at least 20%, 30% or more. , 40%, 50%, 60%, 70%, 80%, 90% or 95% APOC3 gene expression In some embodiments, the complex of the present disclosure exhibits an inhibition rate of at least 20%, 3 0%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of intrahepatic APOC3 residues In some embodiments, the complex of the present disclosure has a gene expression suppression rate of at least 2 0%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of animal models 4 shows the intrahepatic APOC3 gene expression suppression rate in the present invention. The complexes shown are at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, The APOC3 gene expression suppression rate in human subjects is 90% or 95%. In embodiments, the conjugates of the present disclosure do not exhibit significant off-target effects. The target effect may be, for example, the suppression of expression of a gene that is not a target gene. Binding / suppression of target gene expression is 50% or 40% lower than on-target gene effects If the level is below 30%, 20%, or 10%, the off-target effect is not significant. It is believed not to be the case.

[0263] According to some embodiments of the present disclosure, the complex of the present disclosure effectively delivers siRNA to the liver. The antibody can be delivered to the HBV host and exhibit excellent HBV gene expression suppression properties. It also has a vasopressin-releasing effect on the liver of hepatitis B model mice at a dose of 1 mg / kg. In some embodiments, the HBV gene expression is suppressed by 87.4% to 92.2%. The siRNA complex of the present disclosure inhibits the expression of HBV surface antigen in a hepatitis B model mouse. At a dose of 3mg / kg, the HBV surface antigen expression was suppressed by 96.9% and A HBV DNA suppression rate of 5.4% can be achieved. The conjugates provided by the present invention are capable of inhibiting HBV at low doses for periods of up to 140 days. It exhibits excellent inhibitory effect on expression.

[0264] According to one embodiment of the present disclosure, the complex provided by the present disclosure delivers siRNA to the liver. It can be effectively delivered to the liver and exhibits excellent HBV gene expression suppression properties, for example, low It has off-target effects and inhibits the liver function of hepatitis B model mice at a dose of 1 mg / kg. Suppresses HBV gene expression in the liver by at least 69.3% or 78.1-89.1% In some embodiments, the complex of the present disclosure is capable of inhibiting hepatitis B in a mouse model of hepatitis B. It effectively reduced the expression of HBV surface antigen, suppressing 98.4% of HBV surface antigens at a dose of 3mg / kg. It achieves a 95.8% suppression rate of HBV DNA and a 99.9% suppression rate of HBV antigen expression. In some embodiments, the specific complexes provided by the present invention are more effective than the reference complexes for up to 84 days. It shows excellent inhibitory effects on HBV expression at low doses over a period of time.

[0265] According to one embodiment of the present disclosure, the complex provided by the present disclosure delivers siRNA to the liver. It can be effectively delivered to the liver and exhibits excellent HBV gene expression suppression properties, for example, low It has off-target effects and inhibits the liver function of hepatitis B model mice at a dose of 1 mg / kg. Suppresses HBV gene expression in the liver by at least 48.5% or by 76.8-80.0% In some embodiments, the conjugates of the present disclosure may further comprise an anti-Hepatitis B antibody. It effectively reduced the expression of HBV surface antigen in model mice, and thus At this dose, 84.6% of HBV surface antigen expression was suppressed and 85.6% of HBV DNA was suppressed. In some embodiments, the methods provided by the present invention achieve a The complex showed higher inhibition of HBV expression at lower doses for up to 21 days. It shows action.

[0266] According to one embodiment of the present disclosure, the complex provided by the present disclosure delivers siRNA to the liver. It can be effectively delivered to the liver and exhibits excellent HBV gene expression suppression properties, for example, low It has off-target effects and inhibits the liver function of hepatitis B model mice at a dose of 1 mg / kg. At least 60.1% of the total liver tissue, or in one embodiment, 80.7 to 84.5% of the total liver tissue. In some embodiments, the gene expression of the HBV X gene region can be suppressed. In the present disclosure, the complex further inhibits the expression of HBV surface antigen in a hepatitis B model mouse. The drug effectively reduced HBV surface antigen expression by 94.8% at a dose of 3 mg / kg. In some embodiments, the HBV DNA suppression rate is 95.8%. Compared to the reference conjugate, the conjugates provided by the present invention showed a significant improvement over a period of up to 56 days. It exhibits excellent inhibitory effects on HBV expression at low doses.

[0267] According to one embodiment of the present disclosure, the complex provided by the present disclosure delivers siRNA to the liver. It can be effectively delivered to the liver and exhibits excellent ANGPTL3 gene expression suppression properties. For example, at a dose of 1 mg / kg, it reduced at least 57.3% of the lipid profile in the liver of a hyperlipidemic mouse model. It inhibits ANGPTL3 gene expression, with gene inhibition rates up to 90% at a dose of 3mg / kg. In some embodiments, the conjugates provided by the present disclosure have a .4% or higher fold increase compared to a reference conjugate. The complex showed excellent ANGPT activity at low doses and with low frequency of use for periods up to 49 days. It suppresses L3 expression and has a lipid-lowering effect.

[0268] According to one embodiment of the present disclosure, the complex provided by the present disclosure delivers siRNA to the liver. It can be effectively delivered to the liver and exhibits excellent APOC3 gene expression suppression properties, for example: At a dose of 3 mg / kg, at least 75% of APOC in the liver of hyperlipidemic mice was suppressed. In some embodiments, the present disclosure provides a conjugate that inhibits gene expression in a gene sequence other than a reference conjugate. The complex provided by this product provides excellent results at low doses and with low frequency of use for periods of up to 65 days. It exhibits lipid-suppressing effects.

[0269] In certain embodiments, the conjugates described in the present disclosure exhibit low toxicity in animal models. For example, in some embodiments, the conjugates of the present disclosure In C57BL / 6J mice, the effective concentration was increased to 100 times (effective concentration was increased to 3 mg / kg). g), no obvious toxic reactions were observed.

[0270] From the above examples, it can be seen that the conjugates provided herein are effective against receptors on the surface of target cells. It has been shown that the conjugate delivers the loaded active agent to cells expressing the receptor. The molecule can accommodate other cell surface receptors and other activators, and these receptors It is believed that it is possible to target active agents to cells expressing the

[0271] <Kit> In another aspect, the present disclosure provides a kit comprising such a complex.

[0272] In some embodiments, the kit provided herein comprises, in one container, In some embodiments, the kits provided herein include a conjugate comprising a drug In some embodiments, the method of the present invention further comprises administering to the patient a pharmaceutical composition comprising the pharmaceutical composition of the present invention. The kit provided herein may further comprise a pharma- ceutically acceptable additive, such as a stabilizer or preservative. In some embodiments, the kit provided herein comprises at least In some embodiments, the kit comprises one additional therapeutic agent. The container contains at least one additional therapeutic agent different from the therapeutic agent comprising the complex. In some embodiments, the kit comprises a conjugate and a pharma- ceutically acceptable excipient or other component. Includes instructions for mixing (if present).

[0273] In the kit of the present disclosure, the conjugate and / or pharma- ceutically acceptable additive may be any of It may be provided in any suitable form, for example in liquid, dried or lyophilized form. In one embodiment, the conjugate and / or the pharma- ceutically acceptable excipient are essentially clean. and / or sterile. In some embodiments, the kits of the present disclosure provide sterile water. do. EXAMPLES

[0274] The present disclosure will be described in detail below with reference to examples. The reagents and media used are all commercially available. All operations such as PCR were performed using Molecular Cloning (Cold Spring Harbor Boratory Press (1989) This is done with reference to the method described above.

[0275] HEK239A cells were provided by the Nucleic Acid Technology Laboratory, Institute of Molecular Medicine, Peking University, China. % fetal bovine serum (FBS, Hyclone), 0.2v% cyanococcid double resistant (c Penicillin-streptomycin (P enicillin-streptomycin), Gibco, Invitrogen The cells were incubated in DMEM complete medium (Hyclone) at 37°C in 5% CO2 / 95% air. The cells were cultured in an incubator.

[0276] Huh7 cells were purchased from the Stem Cell Bank of the Chinese Academy of Sciences and supplemented with 10% fetal bovine serum (FB S, Hyclone), DME containing 1% non-essential amino acids (NEAA, Corning) In M complete medium (Hyclone), culture in an incubator containing 5% CO2 / 95% air at 37°C. The cells were cultured in .

[0277] Unless otherwise specified, the siRNA complexes synthesized in Preparation Examples 12 to 15 below were used. If cells were transfected, Lipofec was used as the transfection agent. tamine TM 2000 (Invitrogen). For specific operations, Refer to the instructions provided by the manufacturer.

[0278] Unless otherwise stated, all reagent proportions provided below are by volume (v / v). It is calculated.

[0279] Unless otherwise stated, the animal models used are as follows: C57BL / 6J mice: purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd. SD rats: provided by Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd. HBV transgenic mice C57B / 6N-Tg(1.28HBV) / Vst( Genotype A: Purchased from Beijing Weitongda Biotechnology Co., Ltd. COI> 10 4 We selected mice with this gene (hereinafter, simply referred to as 1.28copy mice). HBV transgenic mouse C57BL / 6J-Tg(Alb1HBV)44Br i / J: Purchased from the Department of Experimental Animal Science, School of Medicine, Peking University. We selected the service. HBV transgenic mice: named M-TgHBV, and were developed at the Shanghai Public Health Center. - The method for producing transgenic mice was described in Ren J. et al., J. Medical Virology. 2006, 78:551-560 . AAV-HBV transgenic mice: Methods from literature (Dong Xiaoyan et al., Chin JB Prepared by iotech 2010, May 25, 26(5):679~686 Ta. rAAV8-1.3HBV, type D (ayw) virus (owned by Beijing Wukawa Molecular Medical Research Purchased from the company, 1×10 12 Viral genome (vg) / mL, Lot No. 20 16123011), 5 × 10 rAAV8-1.3HBV in sterile PBS. 11 v .g. / mL and 200 mL of diluted rAAV8-1.3HBV was administered per mouse. μL per mouse (i.e., 1 × 10 11 VG was injected. On the 28th day after injection, all mice were bled via the orbit (approximately 100 μL) and serum was collected. The samples were collected and used to detect HBsAg and HBV DNA. Low-concentration AAV-HBV transgenic mice: Before the experiment, incubate the virus in sterile PBS. 1×10 11 The virus was diluted to 100 µg / mL and 100 µL was injected per mouse. , i.e., 1 × 10 per mouse 10 Except for the VG injection, this is almost the same model as above. The ring method was used. BALB / c mice: 6-8 weeks old, purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd. ob / ob mice: 6-8 weeks old, purchased from Changzhou Cavens Laboratory Animal Co., Ltd. Human APOC3 transgenic mice: B6, CBA-Tg(APOC3)370 Purchase from 7Bres / J, Jackson Lab. Metabolic syndrome monkeys: all males, non-human primate study at the Institute of Molecular Medicine, Peking University Provided by the Center.

[0280] (Preparation Example 1) Preparation of L-9 Complex Molecule (Complex Molecule 1) In this preparation example, conjugate molecule 1 (hereinafter also referred to as L-9 conjugate molecule) was synthesized according to the following method. Successful.

[0281] (1-1) Synthesis of GAL-5 (terminal segment of the L-9 complex molecule)

[0282] [ka]

[0283] (1-1a) Synthesis of GAL-2 100.0 g of GAL-1 (N-acetyl-D-galactosamine hydrochloride, CAS number: 1772-03-8, purchased from Ningbo Hongxiang Biochemical Co., Ltd., 463.8 mmol) 1000 ml 100 ml of anhydrous pyridine, and 540 ml of acetic anhydride (purchased from Enox, The reaction mixture was stirred at room temperature for 1.5 hours. The reaction mixture was poured into 10 L of ice water. The cake was washed with 2 L of ice water and then poured into a flask until it was completely dissolved. Add acetonitrile / toluene mixed solvent (acetonitrile:toluene volume ratio = 1:1) The solvent was evaporated to dryness to obtain 130.0 g of a white solid product, GAL-2.

[0284] (1-1b) Synthesis of GAL-3 213 ml of GAL-2 (35.1 g, 90.0 mmol) obtained in step (1-1a) 1,2-dichloroethane, and 24.0 g of TMSOTf (CAS number: 27607-77-8, purchased from Macklin, 108.0 (mmol) was added and the reaction was carried out at room temperature overnight.

[0285] The reaction mixture was diluted with 400 ml of dichloromethane, filtered through diatomaceous earth, and then added to 1 L of saturated charcoal. Add an aqueous solution of sodium hydrogen carbonate, stir evenly, separate the organic phase, and dichloroethane into the aqueous phase. Extract twice with 300 ml each time, combine the organic phases, and add 300 ml of saturated Wash with 300 ml of aqueous sodium bicarbonate solution and 300 ml of saturated saline solution, then wash The organic phase was separated, dried over anhydrous sodium sulfate, and the solvent was evaporated to dryness under reduced pressure. As a result, a pale yellow, viscous starch syrup-like product GAL-3 was obtained.

[0286] (1-1c) Synthesis of GAL-4 The GAL-3 (26.9 g, 81.7 mmol) obtained in step (1-1b) was dissolved in 136 ml Dissolve in 1 l of anhydrous 1,2-dichloroethane, add 30 g of dried 4 Å molecular sieve powder, and 9. 0g of 5-hexen-1-ol (CAS number: 821-41-0, Adamas-be The mixture was stirred at room temperature for 30 minutes and then protected with nitrogen under ice bath. 9.08 g of TMSOTf (40.9 mmol) was added thereto under the above conditions, and the mixture was reacted at room temperature overnight with stirring. The 4Å molecular sieve powder was removed by filtration, and the filtrate was diluted with 300 ml of dichloroethane. Filter through soil, add 500 ml of saturated aqueous sodium bicarbonate solution, stir for 10 minutes, and wash. The organic phase was separated and the remaining aqueous phase was extracted once with 300 ml of dichloroethane. The phases were combined and each was diluted with 300 ml of saturated aqueous sodium bicarbonate solution and 300 ml of saturated aqueous sodium bicarbonate solution. After washing with brine, the organic phase obtained from the washing was separated and dried over anhydrous sodium sulfate. The solvent was evaporated to dryness under reduced pressure to obtain 41.3 g of a yellow syrup-like product, GAL-4, which was then purified. The next oxidation reaction was carried out without further treatment.

[0287] (1-1d) Synthesis of GAL-5 GAL-4 (14.9 g, 34.7 g) obtained by the method described in step (1-1c) (mmol) was dissolved in a mixture of 77 ml of dichloromethane and 77 ml of acetonitrile. Add 103 ml of deionized water and 29.7 g of sodium periodate (CAS no. No. 7790-28-5, purchased from Aladdin, 138.8 mmol) was added and cooled on ice. Stir in a water bath for 10 minutes and add ruthenium(III) chloride (CAS number: 14898-67- 0, purchased from Energy Co., 238 mg, 1.145 mmol) was added and reacted at room temperature overnight. The reaction mixture was diluted with 300 ml of water and stirred, and saturated sodium bicarbonate was added. The pH was adjusted to about 7.5 using 100% sodium hydroxide, the organic phase was separated, and the remaining aqueous phase was diluted with dichloromethane in one portion. The mixture was extracted three times with 200 ml of water, and the organic phase was discarded. The pH was adjusted to about 3 with citric acid, and the solution was extracted three times with 200 ml of dichloromethane each time. The organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was evaporated to dryness under reduced pressure to obtain 6.85 g Thus, a white foamy solid product GAL-5 was obtained.

[0288] The GAL-5 compound obtained by the above method is used to prepare L-9 by the following process: The complex molecule was synthesized.

[0289] [ka]

[0290] (1-2) Synthesis of M-11-T3:

[0291] [ka] J-0 (1.883 g, 10 mmol, purchased from Alfa Acer Inc.) Dissolve in acetonitrile and add triethylamine (4.048 g, 40 mmol). Cool to 0°C in an ice-water bath and add ethyl trifluoroacetate (5.683 g, 40 mmol). The mixture was then reacted at room temperature for 22 hours, the solvent was evaporated to dryness under reduced pressure, and the residue was foamed for 18 hours using a vacuum oil pump. After drying, 5.342 g of solid crude product M-11-T3 was obtained, which was used without further purification. The product was used as is in the subsequent reaction. MS m / z:C 15 H 22 F9N4O3, [M+H] + , reason Theoretical value: 477.35, actual value: 477.65.

[0292] (1-3) Synthesis of M-11-T3-Tr:

[0293] [ka] M-11-T3 crude product (5.342 g, 10 mmol) was dissolved in 50 ml of dichloromethane. The reaction mixture was dissolved in 100 ml of TrCl (3.345 g, 12 mmol) and triethylamine. Add 1.518 g (15 mmol) of amine and stir at room temperature for 20 h. Wash twice with 20 ml of saturated sodium bicarbonate and then with 20 ml of saturated saline. The organic phase was dried over anhydrous sodium sulfate, filtered, and the organic solvent was evaporated under reduced pressure. The residue was dried overnight using a vacuum oil pump to obtain 7.763 g of solid crude product M-11. -T3-Tr was obtained. MS m / z:C 34 H 36 F9N4O3, [M+Na] + ,theory Value: 741.25, Measured value: 741.53. The solid crude product M-11-T3-Tr was purified. This was used in the subsequent synthesis of M-18-Tr without any further purification.

[0294] (1-4) Synthesis of M-18-Tr:

[0295] [ka] The crude M-11-T3-Tr product obtained in step (1-3) (7.763 g, 10 mmol) ) was dissolved in 100 ml of methanol, and 100 ml of a methylamine aqueous solution (40% by mass) was added. ) was added, and the mixture was stirred at 50°C for 23 hours. Insoluble particles were removed by filtration, and the solvent was evaporated to dryness under reduced pressure. Add 200 ml of a 1:1 volume ratio DCM:methanol mixed solvent to the residue, and then add 50 ml of saturated sodium bicarbonate, and the aqueous phase was washed three times with 50 ml of dichloromethane each time. After extraction, all the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was removed under reduced pressure. The residue was evaporated to dryness, and then foam-dried overnight using a vacuum oil pump. The mixture was purified using a silica gel column, petroleum ether was added to the column, and 1 wt% triethylamine was added. The acidity of the silica gel was neutralized with dichloromethane:methanol:ammonia water (25wt%). Gradient elution was performed at a ratio of 1:1:0.05 to 1:1:0.25, the eluate was collected, and the solvent was evaporated under reduced pressure. The residue was dried by a vacuum oil pump to obtain 2.887 g of pure M-18-Tr. Got it. 1 H NMR (400 MHz,DMSO) δ7.47-7.39 (m,6 H),7.32-7.24 (m,6H),7.19-7.12 (m,3H),2.6 0-2.47 (m,4H),2.46-2.19 (m,13H),1.70-1.5 5 (m,4H),1.40 (p,J =6.8 Hz,2H). MS m / z: C 28 H 39 N4, [M+H] + , theoretical value: 431.65, measured value: 432.61.

[0296] (1-5) Synthesis of L-5-Tr:

[0297] [ka] M-18-Tr (2.02 g, 4.69 mmol) obtained in step (1-4) and step ( Mix GAL-5 (6.93 g, 15.48 mmol) obtained in 1-1) and add to 47 ml N-methylmorpholine (3.13 g, 30.96 mmol) was dissolved in acetonitrile. ) and 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride (DMTMM, 4.28 g, 15.48 mmol) was added, and the reaction was stirred at room temperature for 2 hours. Dilute the reaction mixture with 200 ml of dichloromethane and add 100 ml of saturated sodium bicarbonate solution. The organic phase was washed with 100 ml of saturated saline solution, and the organic phase was then washed with anhydrous sodium sulfate. The mixture was dried over sodium, filtered, and the solvent was evaporated to dryness under reduced pressure to obtain a crude product. The mixture was purified using a 100-mesh normal phase silica gel column, and petroleum ether was added to the column, followed by 1 wt% trichloroethylene. Neutralize the acidity of the silica gel with ethylamine, and use dichloromethane:methanol = 100:5~ Gradient elution at 100:7 was performed, and the product eluate was collected and evaporated to dryness under reduced pressure to obtain 7.49 g of pure product. L-5-Tr was obtained. 1 H NMR (400 MHz,DMSO) δ7.83-7. 10 (m,4H),7.67-7.60 (m,1H),7.44-7.34 (m, 6H),7.33-7.24 (m,6H),7.20-7.15 (m,3H),5. 22 (s,3H),4.97 (d,J =11.3 Hz,3H),4.49 (d ,J =8.4 Hz,3H),4.06-3.07 (m,9H),3.95-3.8 3 (m,3H),3.77-3.64 (m,3H),3.45-3.35 (m,3 H),3.12-2.87 (m,8H),2.30-2.15 (m,3H),2.1 1-1.98 (m,22H),1.95-1.84 (m,11H),1.81-1. 61 (m,14H),1.54-1.36 (m,14H).MS m / z:C 85 H 119 N7O 30 , [M+H] + , Theoretical value: 1718.81, Measured value: 1718.03 .

[0298] Synthesis of (1-6)L-8:

[0299] [ka] The L-5-Tr (5.94 g, 3.456 mmol) obtained in step (1-5) was dissolved in 69 ml 1 of dichloromethane, and dichloroacetic acid (13.367 g, 103.67 mmol) was dissolved in ) was added and reacted at room temperature for 2 hours. 100 ml of dichloromethane was added to the resulting reaction solution. Dilute, wash with saturated sodium bicarbonate solution and adjust to pH 7-8. The isolated aqueous phase was extracted six times with dichloromethane, each time with 30 ml of dichloromethane, and all the organic phases were evaporated. The mixture was dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated to dryness under reduced pressure to obtain a crude product. For purification, 200-300 mesh normal phase silica gel was used, and 10 wt% triethylamine was used. The acidity of the silica gel was neutralized with amine, the column was equilibrated with 1 wt‰ triethylamine, and then the column was cooled to room temperature. Gradient elution was performed with chloromethane:methanol = 100:30 to 100:40, and the product eluate was collected. The mixture was collected, and the solvent was evaporated to dryness under reduced pressure to obtain 4.26 g of pure product L-8. 1 H NMR (40 0 MHz,DMSO) δ 7.84 (d,J =9.0 Hz,3H),7.27 -7.23 (m,1H),7.13-7.18 (m,1H),5.22 (d,J =3.1 Hz,3H),4.97 (dd,J =11.3,3.1 Hz,3H), 4.48 (d,J =8.4 Hz,3H),4.09-3.98 (m,9H),3 .88 (dd,J =19.3,9.3 Hz,3H),3.75-3.66 (m, 3H),3.44-3.38 (m,3H),3.17-3.30 (m,4H),3. 10-2.97 (m,4H),2.35-2.20 (m,6H),2.15-2.0 8 (m,9H),2.07-1.98 (m,13H),1.94-1.87 (m, 9H),1.81-1.74 (m,9H),1.65-1.42 (m,18H). MS m / z:C 85 H 119 N7O 30 , [M+H] + , Theoretical value: 1477.59, Actual value: Measurement: 1477.23.

[0300] (1-7a) Synthesis of A-1

[0301] [ka] DMTrCl (4,4'-bismethoxytrityl chloride, 38.12 g, 112.5 (mmol) was dissolved in 450 ml of anhydrous pyridine and DL-calcium glycerate hydrate was added. The reaction mixture (12.88 g, 45.0 mmol) was added and reacted at 45° C. for 22 hours. The liquid was filtered, the cake was rinsed with 200 ml of DCM, and the filtrate was concentrated to dryness under reduced pressure. The residue was dissolved again in 500 ml of dichloromethane and diluted with 0.5 M triethylamine. The isolated aqueous phase was washed twice with 200 ml of dichloromethane phosphate (pH 7-8). Extract twice with methane (200 ml each time), combine the organic phases, and dry over anhydrous sodium sulfate. The mixture was dried, filtered, the solvent was evaporated to dryness under reduced pressure, and the residue was passed through a 200-300 mesh normal phase silica gel column. Purified with gel column, petroleum ether:ethyl acetate:dichloromethane:methanol=1:1 Gradient elution was performed with a gradient of 1:0.35 to 1:1:1:0.55, and the product eluate was collected and reduced in volume. The residue was redissolved in 500 ml of dichloromethane and diluted with 200 ml of 0. Wash once with 0.5 M triethylamine phosphate and dilute the aqueous phase with 20 mL of dichloromethane. Extract twice with 0.0 ml of water, combine all the organic phases, dry over anhydrous sodium sulfate, filter, The solvent was evaporated to dryness under reduced pressure, and the residue was dried under reduced pressure overnight using a vacuum oil pump to obtain 20.7 g of a white solid. The product A-1 was obtained. 1 H NMR (400 MHz,DMSO-d6) δ 7.4 6 (ddd,J =6.5,2.3,1.1 Hz,1H),7.40-7.28 ( m,7H),6.89-6.81 (m,4H),4.84 (d,J =5.0 Hz ,1H),4.36-4.24 (m,1H),4.29 (s,6H),3.92 ( dd,J =12.4,7.0 Hz,1H),3.67 (dd,J =12.3,7 .0 Hz,1H),2.52 (q,J =6.3 Hz,6H),1.03 (t, J = 6.3 Hz, 9H). MS m / z: C 24 H 23 O6, [MH] - ,theory Value: 407.15, Actual value: 406.92.

[0302] (1-7b) Synthesis of L-7:

[0303] [ka] L-8 (2.262 g, 1.532 mmol) obtained in step (1-6) and A-1 (2.342 g, 4.596 mmol) obtained in -7a) was mixed and added to 16 mL of Dissolve in dichloromethane and 3-diethoxyphosphoryloxy-1,2,3-benzotriazole Azin-4(3H)-one (DEPBT, 1.375 g, 4.596 mmol) was added, and Diisopropylethylamine (1.188 g, 9.191 mmol) was then added and the mixture was heated at 25°C. The reaction was stirred at RT for 2 h, the organic phase was washed with 10 ml of saturated sodium bicarbonate, and the aqueous phase was Extract with chloromethane three times, 10 ml each time, combine all the organic phases, and add 10 ml of saturated The organic phase was washed with brine and the isolated aqueous phase was extracted twice with 10 ml of dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was reduced. The mixture was evaporated to dryness under reduced pressure, and the residue was foam-dried overnight using a vacuum oil pump to obtain 4,900 g of a crude product. The crude product was purified by column chromatography using 120 g of 200-300 mesh normal phase silica gel. , neutralize the acidity of the silica gel with 20 ml of triethylamine, and The column was equilibrated with petroleum ether containing: petroleum ether:ethyl acetate:dichloromethane: Gradient elution with N,N-dimethylformamide = 1:1:1:0.5 to 1:1:1:0.6 The product eluate was collected, and the solvent was evaporated to dryness under reduced pressure to obtain 2.336 g of pure product L-7. 1 H NMR (400 MHz,DMSO) δ7.90-7.78 (m,4H), 7.75-7.64 (m,1H),7.38-7.18 (m,9H),6.91-6 .83 (m,4H),5.25-5.10 (m,4H),4.97 (dd,J = 11.2,3.2 Hz,3H),4.48-4.30 (m,4H),4.02 (s ,9H),3.93-3.84 (m,3H),3.76-3.66 (m,9H),3 .45-3.35 (m,3H),3.24-2.98 (m,10H),2.30-2 .20 (m,2H),2.11-1.88 (m,31H),1.80-1.40 ( m, 28H). MS m / z: C 90 H 128 N7O 35 , [M-DMTr]+ ,theory Value: 1564.65, Actual value: 1564.88.

[0304] Synthesis of (1-8)L-9:

[0305] [ka] L-7 (2.300 g, 1.26 mmol) obtained in step (1-7b), Aqueous solution (0.378 g, 3.78 mmol) and 4-dimethylaminopyridine (DMAP, 0.462g, 3.78mmol) were mixed and dissolved in 13ml of dichloromethane, DIPEA (0.814 g, 6.30 mmol) was then added and the mixture was stirred at 25° C. for 24 hours. The reaction mixture was washed with 5 ml of 0.5 M triethylamine phosphate, and the isolated aqueous phase was Extract with dichloromethane three times, each time using 5 ml of dichloromethane, combine all the organic phases, and evaporate to dryness under reduced pressure. The crude product was purified by column chromatography and 60 g of 200-300 MW elution was obtained. The acidity of the silica gel was neutralized with 1 wt% triethylamine. The column was equilibrated with dichloromethane and then diluted with 1 wt‰ triethylamine in dichloromethane. Elute with a gradient of 100:18 to 100:20 methanol, collect the product eluate, and use the solvent The mixture was evaporated to dryness under reduced pressure to obtain 1.874 g of pure L-9 complex molecule (complex molecule 1). 1 H NMR (400 MHz,DMSO) δ 8.58 (d,J =4.2 Hz,1 H),7.94-7.82 (m,3H),7.41-7.29 (m,5H),7.2 2 (d,J =8.1 Hz,5H),6.89 (d,J =8.3 Hz,4H) ,5.49-5.37 (m,1H),5.21 (d,J =3.0 Hz,3H), 4.97 (d,J =11.1 Hz,3H),4.49 (d,J =8.2 Hz ,3H),4.02 (s,9H),3.88 (dd,J =19.4, 9.4 Hz ,3H),3.77-3.65 (m,9H),3.50-3.39 (m,6H),3 .11-2.90 (m,5H),2.61-2.54 (m,4H),2.47-2. 41 (m,2H),2.26-2.17 (m,2H),2.15-1.95 (m, 22H),1.92-1.84 (m,9H),1.80-1.70 (m,10H), 1.65-1.35 (m,17H), 1.31-1.19 (m,4H),0.96 (t, J = 7.1 Hz, 9H). MS m / z: C 94 H 132 N7O 38 , [M -DMTr] + , Theoretical value: 1664.72, Actual value: 1665.03. The obtained L-9 complex The structure of the combined molecule is shown in formula (503).

[0306] (Preparation Example 2) Preparation of P-9 Conjugate Molecule (Conjugate Molecule 2) In this preparation example, conjugate molecule 2 (hereinafter also referred to as P-9 conjugate molecule) was synthesized according to the following method. Successful.

[0307] [ka]

[0308] (2-1) Synthesis of GAL5-C4-1 In 40 ml of N,N-dimethylformamide, GAL-5 (13.43 g, 30.0 mmol), 4-amino acid tert-butyl Ester hydrochloride (5.87 g, 30.0 mmol), O-benzotriazole-tetramethyl Thiuronium hexafluorophosphate (13.65 g, 36.0 mmol) and di Add isopropylethylamine (11.63 g, 90.0 mmol) and dissolve uniformly. After that, the reaction was stirred at room temperature for 5 hours. 300 ml of saturated sodium bicarbonate was added to the reaction solution. Add aqueous ethyl acetate solution, extract the isolated aqueous phase with ethyl acetate three times with 200 ml each time, and wash. The organic phases obtained by washing were combined and washed once with 200 ml of saturated saline solution. The organic phase was separated and further dried over anhydrous sodium sulfate and the solvent was reduced until dry. After distillation under reduced pressure, 30.3 g of oily crude product GAL5-C4-1 was obtained, which was directly used in the next reaction. Ta.

[0309] (2-2) Synthesis of GAL5-C4-2 The crude GAL5-C4-1 product (30.3 g, 30 mmol) obtained in step (2-1) was The mixture was dissolved in 180 ml of formic acid and reacted at room temperature for 16 hours with stirring. The solvent was evaporated to dryness. The crude product was purified by column chromatography (200-300 mesh normal phase column). Using silica gel, gradient elution was performed with dichloromethane:methanol = 100:18 to 100:20. The eluate was collected, and the solvent was removed by concentration to obtain 14.84 g of the desired product GAL5- Obtained C4-2.

[0310] (2-3) Synthesis of P-6: M-18-Tr (2.02 g, 4.6 g) obtained by the method described in step (1-4) 9 mmol) and GAL5-C4-2 (8.24 g, 15.48 mmol) obtained in step (2-2). The mixture was dissolved in 47 ml of acetonitrile and then N-methylmorpholin Add 4-(4,6-dimethoxytrimethylsilyl)propane (3.13 g, 30.96 mmol) and finally D-methylmorpholine hydrochloride (DMTMM, 4.28 g, 15.48 The resulting mixture was stirred at room temperature for 2 hours. The reaction mixture was diluted with 100 ml of saturated sodium bicarbonate solution and 100 ml of saturated sodium bicarbonate solution. The organic phase was washed with saturated saline, and all the organic phases were combined, dried over anhydrous sodium sulfate, and filtered. After filtration, the solvent was evaporated to dryness under reduced pressure to obtain a crude product, which was then passed through a 200-300 mesh normal phase silica gel column. The column was purified with petroleum ether, and the silica gel was treated with 1 wt% triethylamine. The acidity of the column was neutralized, and the column was eluted with a gradient of dichloromethane:methanol = 100:5 to 100:7. The product eluate was collected and evaporated to dryness under reduced pressure to obtain a total of 8.27 g of pure product P-6.

[0311] (2-4) Synthesis of P-7: The P-6 (6.82 g, 3.456 mmol) obtained in (2-3) above was added to a 69 mL flask. Dissolve in chloromethane and add dichloroacetic acid (13.367 g, 103.67 mmol). The reaction mixture was diluted with 100 ml of dichloromethane. Then, add saturated sodium bicarbonate solution to wash and adjust the pH to 7-8. The isolated aqueous phase was extracted six times with dichloromethane, each time with 30 ml of dichloromethane, and all the organic phases were collected. The mixture was combined, dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated to dryness under reduced pressure to obtain a crude product. The product was purified using 200-300 mesh normal phase silica gel and diluted with 10 wt% triethylamine. The acidity of the silica gel was neutralized, the column was equilibrated with 1 wt‰ triethylamine, and dichloromethane was added. Gradient elution was performed with ethanol:methanol = 100:30 to 100:40, and the product eluate was collected. The solvent was evaporated to dryness under reduced pressure to obtain a total of 4.82 g of P-7. MS m / z:C 78 H127 N 10 O 33 , [M+H] + , theoretical value: 1732.91, actual value: 1735.73.

[0312] (2-5) Synthesis of P-8:

[0313] [ka] P-7 (2.653 g, 1.532 mmol) and A-1 (2.342 g, 4.596 mmol) mol) were mixed and dissolved in 16 ml of dichloromethane, and 3-diethoxyphosphoryl- 1,2,3-Benzotriazol-4(3H)-one (DEPBT) (1.375 g, 4. 596mmol) was added, followed by diisopropylethylamine (1.188g, 9.19 1mmol) was added and the mixture was stirred at 25°C for 2 hours. The organic phase was washed with 10 ml of dichloromethane, and the aqueous phase was extracted three times with 10 ml of dichloromethane. The organic phases were combined and washed with 10 mL of saturated saline, and the isolated aqueous phase was washed once with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, and After filtration, the solvent was evaporated to dryness under reduced pressure, and the residue was foam-dried overnight using a vacuum oil pump to obtain a crude product. The crude product was purified by column chromatography using 120 g of 200-300 mesh normal phase silica gel. The acidity of the silica gel was neutralized with 20 ml of triethylamine, and 1 wt% triethylamine was added. The column was equilibrated with petroleum ether containing amines, and the mixture was diluted with petroleum ether:ethyl acetate:dichloromethane. Tantalum:N,N-dimethylformamide=1:1:1:0.5 to 1:1:1:0.6 with gradient The eluate was collected, and the solvent was evaporated to dryness under reduced pressure to obtain 2.793 g of pure product P-8. .

[0314] (2-6) Synthesis of P-9: P-8 (490 mg, 0.231 mmol), succinic anhydride (69 mg, 0.693 mmol) and 4-dimethylaminopyridine (DMAP, 68 mg, 0.554 mmol) ) were mixed and dissolved in 2.3 ml of dichloromethane, and then diisopropyl ethyl amine was added. The mixture was stirred at 25°C for 21 h. The reaction mixture was diluted with 50 ml of dichloromethane and then added with 100 ml of 0.5 The reaction mixture was washed with M triethylamine phosphate, and the isolated aqueous phase was washed once with dichloromethane. All the organic phases were combined and evaporated to dryness under reduced pressure to obtain the crude product. The product was purified by column chromatography using 80 g of 200-300 mesh normal phase silica gel, 1 w The acidity of the silica gel was neutralized with t% triethylamine, and the column was equilibrated with dichloromethane. Dichloromethane containing 1wt‰ triethylamine:methanol=100:18-100 Gradient elution was performed at 0.05 to 0.20, the eluate was collected, and the solvent was evaporated to dryness under reduced pressure to obtain 200 mg of pure P. -9 complex molecule (complex molecule 2) was obtained. MS m / z: C 106 H 153 N 10 O 41 , [ M-DMTr] + ,Theoretical value: 1921.05,Actual value: 1920.97. The obtained P-9 The structure of the complex molecule is shown in formula (504).

[0315] (Preparation Example 3) Preparation of R-4 Complex Molecule (Complex Molecule 3) In this preparation example, conjugate molecule 3 (hereinafter also referred to as R-4 conjugate molecule) was synthesized according to the following method. Successful.

[0316] [ka]

[0317] (3-1) Synthesis of GAL-C7-1 GAL-3 (26.4 g, 80.2 g) obtained by the method described in step (1-1b) (mmol) was dissolved in 134 ml of anhydrous 1,2-dichloroethane and 60 g of 4 Å molecular sieves were added. Add the flour, then add 7-octen-1-ol (11.3 g, 88.2 mmol). The reaction was stirred at room temperature for 10 minutes, and then trifluoromethanesulfonic acid trihydrate was added under nitrogen protection in an ice bath. Methylsilyl (Trimethylsilyl trifluoromethanesu lphonate, TMSOTf, 8.9 g, 40.1 mmol) was added and the mixture was left at room temperature for 24 hours. The mixture was stirred for 1 hour and the reaction was continued. The 4 Å molecular sieve powder was removed by filtration, and the filtrate was added with 500 ml of saturated sodium bicarbonate. The organic phase was separated and the remaining aqueous phase was diluted with 100 ml of dichloromethane. All the organic phases were combined and washed once with 250 ml of saturated saline, then The organic phase obtained by washing was separated, dried over anhydrous sodium sulfate, and then soaked in solvent until dry. The yellow syrup-like product GAL-C7-1 (33.3 g) was obtained by distillation under reduced pressure. The next oxidation reaction was carried out as it was.

[0318] (3-2) Synthesis of GAL-C7-2 The GAL-C7-1 (33.3 g, 72.8 mmol) obtained in step (3-1) was mixed with Dissolve in a mixture of 100 ml of dichloromethane and 160 ml of acetonitrile. Add 216 ml of water and solid sodium periodate (62.3 g, 291.2 mmol). The mixture was stirred in an ice-water bath for 10 minutes, and the catalyst, ruthenium(III) chloride (498 mg, 2 The mixture was allowed to warm to room temperature and react for 23 hours with stirring. Dilute with 200 ml of water, stir, and add saturated sodium bicarbonate to adjust the pH to 7. The temperature was adjusted to 5, the organic phase was separated, and the remaining aqueous phase was extracted three times with dichloromethane. The organic phase was discarded, and the aqueous phase obtained by extraction was adjusted to pH 3 with solid citric acid and dichloromethane was added. Extract with 200 ml of hexane three times, combine the organic phases, and add sodium sulfate anhydride. The solvent was removed under reduced pressure, and the mixture was then subjected to column chromatography (200-300 Using normal phase silica gel with mesh, dichloromethane:methanol=100:18-10 0:20) to obtain 22.4 g of white foamy solid product GAL-C7- 2 was obtained. MS m / z: C 21 H 32 NO 11 , [M+H] + , theoretical value: 476.50 , Actual value: 475.94.

[0319] (3-3) Synthesis of R-1: M-18-Tr (2.02 g, 4.6 g) obtained by the method described in step (1-4) 9 mmol) and GAL-C7-2 (7.36 g, 15.48 mmol) were mixed to obtain a 47 ml 1 of acetonitrile, and N-methylmorpholine (3.13 g, 30.96 mmol) and finally 4-(4,6-dimethoxytriazin-2-yl)-4-methyl Add dimorpholine hydrochloride (DMTMM, 4.28 g, 15.48 mmol) and let the mixture stand at room temperature for 2 The reaction mixture was stirred for 1 h. The reaction mixture was diluted with 200 ml of dichloromethane. The organic phase was washed with 100 ml of saturated sodium bicarbonate solution and 100 ml of saturated saline solution. All the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated to dryness under reduced pressure. The crude product was solidified and purified using a 200-300 mesh normal phase silica gel column. The ter was placed in a column, the acidity of the silica gel was neutralized with 1 wt% triethylamine, and dichloromethane was added. Gradient elution was performed with methane:methanol = 100:5 to 100:7, and the product eluate was collected and reduced. After evaporation to dryness under reduced pressure, 7.82 g of pure product R-1 was obtained.

[0320] (3-4) Synthesis of R-2: R-1 (6.23 g, 3.456 mmol) was dissolved in 69 ml of dichloromethane. Dichloroacetic acid (13.367 g, 103.67 mmol) was added and reacted at room temperature for 2 h. The reaction mixture was diluted with 100 ml of dichloromethane and saturated sodium bicarbonate was added. The pH was adjusted to 7-8 by adding ethanol solution, and the isolated aqueous phase was diluted with dichloromethane. All organic phases were combined and dried over anhydrous sodium sulfate. After drying and filtering, the solvent was evaporated to dryness under reduced pressure to obtain a crude product. The acidity of the silica gel was neutralized with 10 wt% triethylamine. The column was equilibrated with 1 wt‰ triethylamine and diluted with dichloromethane:methanol = 10 Gradient elution was performed at 0:30 to 100:40, and the solvent was evaporated to dryness under reduced pressure to obtain 4.49 g of pure product R- I got 2.

[0321] (3-5) Synthesis of R-3: R-2 (2.391 g, 1.532 mmol) and A-1 (2.342 g, 4.596 mmol) mol) were mixed and dissolved in 16 ml of dichloromethane, and 3-diethoxyphosphoryl Dioxy-1,2,3-benzotriazin-4(3H)-one (DEPBT, 1.375 g, 4 0.596mmol) was added, followed by diisopropylethylamine (1.188g, 9.1 91 mmol) was added and the mixture was stirred at 25°C for 2 hours. The organic phase was washed with ethyl acetate and the isolated aqueous phase was extracted three times with dichloromethane, each time with 10 ml of dichloromethane. All the organic phases were combined and washed with 10 ml of saturated saline, and the isolated aqueous phase was diluted with dichloromethane. Extract twice with 10 ml of toluene each time, combine the resulting organic phases, and add anhydrous sodium sulfate. After drying at 40°C and filtering, the solvent was evaporated to dryness under reduced pressure, and the residue was dried overnight under reduced pressure using a vacuum oil pump. The crude product was purified using a column of 120 g of 200-300 mesh normal phase chromatograph. Silica gel was used, and the acidity of the silica gel was neutralized with 20 ml of triethylamine, and 1 wt% The column was equilibrated with petroleum ether containing triethylamine, and the mixture was diluted with petroleum ether:ethyl acetate: Dichloromethane:N,N-dimethylformamide=1:1:1:0.5~1:1:1:0 Gradient elution with 0.6 and the solvent was evaporated to dryness under reduced pressure to give 2.642 g of pure product R-3.

[0322] (3-6) Synthesis of R-4: R-3 (795 mg, 0.4074 mmol), succinic anhydride (82 mg, 0.81 48mmol) and 4-dimethylaminopyridine (DMAP, 100mg, 0.8148 (mmol) were mixed and dissolved in 4 ml of dichloromethane, and then diisopropyl ethyl Add amine (DIPEA, 100 mg, 0.8148 mmol) and stir at 25°C for 18 h. The reaction solution was washed with 5 ml of 0.5 M triethylamine phosphate and then diluted with water. The phases were extracted three times with dichloromethane, each time with 5 ml, and all the organic phases were combined and the solvent was reduced. The crude product was purified by column chromatography to obtain 30 g of 200-300 mesh. The acidity of the silica gel was neutralized with 1 wt% triethylamine. The column was equilibrated with dichloromethane, and then diluted with dichloromethane:methylene chloride containing 1 wt. Elute with a gradient of ethanol = 100:18 to 100:20, collect the eluate, and evaporate the solvent under reduced pressure. After drying, 505 mg of pure R-4 complex (complex 3) was obtained. The molecular structure is shown in formula (507).

[0323] (Preparation Example 4) Preparation of LA-4 conjugate molecule (conjugate molecule 4) The conjugate molecule 4 (hereinafter also referred to as the LA-4 conjugate molecule) can be synthesized by the following process route: The structure of the resulting LA-4 conjugate molecule is shown in formula (512).

[0324] [ka]

[0325] (Preparation Example 5) Preparation of LB-4 Complex Molecule (Complex Molecule 5) In this preparation example, conjugate molecule 5 (hereinafter also referred to as LB-4 conjugate molecule) was prepared according to the following method. It was synthesized.

[0326] [ka]

[0327] (5-1) Synthesis of LB-1: L-8 (5.0 g, 3.386 mmol) obtained by the method described in step (1-6) l), adipic anhydride (870 mg, 6.772 mmol) and 4-dimethylaminopyridine Lysine (DMAP, 827 mg, 6.772 mmol) was mixed in 130 ml of dichloromethane. Dissolve in methane and diisopropylethylamine (DIPEA, 2.2 g, 16. The reaction mixture was stirred at 25°C for 4 hours. The reaction mixture was diluted with chloromethane and washed with 0.5M triethylamine phosphate. The separated aqueous phase was extracted four times with dichloromethane, each time with 10 ml of dichloromethane, and all the organic phases were combined. The solvent was evaporated to dryness under reduced pressure to obtain a crude product. The crude product was purified using a column to obtain 120 g of 200- Using 300 mesh normal phase silica gel, acidify the silica gel with 1 wt% triethylamine. The column was neutralized with dichloromethane and then equilibrated with petroleum ether:ethyl acetate:dichloromethane. Gradient elution was performed with ethanol:methanol = 1:1:1:0.2 to 1:1:1:1. The solvent was evaporated under reduced pressure. After drying, 4.267 g of pure product LB-1 was obtained.

[0328] (5-2) Synthesis of LB-2: LB-1 (4.697 g, 2.753 g) obtained by the method described in step (5-1) mmol, two batches of product combined), 3-amino-1,2-propanediol (313mg, 3.442mmol), 4-(4,6-dimethoxytriazin-2-yl )-4-Methylmorpholine hydrochloride (DMTMM, 953 mg, 3.442 mmol) and N-Methylmorpholine (700 mg, 6.884 mmol) in 30 mL of acetonitrile and 3 ml of methanol, and the mixture was stirred at room temperature overnight. The solvent was evaporated and the residue was subjected to column chromatography (normal phase silica gel, 200-300 mesh). Gradient elution was performed using gel with dichloromethane:methanol = 1:0.07 to 1:0.5. The eluate was collected and the solvent was removed by concentration to obtain 3.27 g of the desired product LB- I got 2.

[0329] (5-3) Synthesis of LB-3: LB-2 (2.27 g, 1.353 mmol) was dissolved in 14 ml of anhydrous pyridine. In addition, 4,4'-bismethoxytrityl chloride (688 mg, 2.03 mmol) The mixture was stirred at room temperature overnight. 150 ml of methanol was added to quench the reaction. The solvent was evaporated to dryness. The residue was purified by column chromatography (200-300 Using normal phase silica gel with mesh, dichloromethane:methanol = 1:0.05 to 1: The eluate was collected and the solvent was removed by concentration to give 1.647 g of the desired product LB-3 was obtained.

[0330] (5-4) Synthesis of LB-4: LB-3 (822 mg, 0.415 mmol), succinic anhydride (83 g, 0.83 m mol) and 4-dimethylaminopyridine (DMAP, 102 mg, 0.83 mmol) The mixture was mixed and dissolved in 4 ml of dichloromethane, and DIPEA (270 mg, 2.0 The reaction mixture was stirred at 25° C. overnight. The isolated aqueous phase was washed three times with diethylamine phosphate, and then diluted with dichloromethane (2 ml each time). All organic phases were combined and the solvent was evaporated to dryness under reduced pressure to obtain the crude product. Purify with a column, use 200-300 mesh normal phase silica gel, and add 5 wt% triethyl ether. The acidity of the silica gel was neutralized with amine, the column was equilibrated with petroleum ether, and 1 wt‰ triethylamine was added. Gradient elution with dichloromethane:methanol = 100:5 to 100:20 containing ethylamine The solvent was evaporated to dryness under reduced pressure to obtain 787 mg of the pure LB-4 compound represented by the formula (513). The composite molecule (composite molecule 5) was obtained.

[0331] (Preparation Example 6) Synthesis of V-7 Complex Molecule (Complex Molecule 6) The following process pathway leads to the complex molecule 6 (hereinafter referred to as V-7) with the structure shown in formula (514): They anticipated that it would be possible to synthesize a complex molecule (also called a complex molecule).

[0332] [ka]

[0333] (Preparation Example 7) Preparation of W-7 Complex Molecule (Complex Molecule 7) In this preparation example, complex molecule 7 (hereinafter also referred to as W-7 complex molecule) was synthesized according to the following method. Successful.

[0334] [ka]

[0335] (7-1) Synthesis of W-1: W-0 (2.024 g, 10 mmol) was dissolved in 25 ml of acetonitrile, and Add triethylamine (4.048 g, 40 mmol) and cool to about 0°C in an ice bath. Ethyl trifluoroacetate (5.683 g, 40 mmol) was added and the mixture was reacted at room temperature for 22 hours. The solvent was evaporated to dryness under reduced pressure, and the residue was foam-dried using a vacuum oil pump for 18 hours. g of solid crude product W-1 was obtained.

[0336] (7-2) Synthesis of W-2: W-1 crude product (5.835 g, 10 mmol) was dissolved in 50 ml of dichloromethane. The reaction mixture was added with TrCl (3.345 g, 12 mmol) and triethylamine (1.518 g, 15 mmol) was added and the mixture was stirred at room temperature for 20 hours. The mixture was washed twice with saturated sodium bicarbonate and once with 20 ml of saturated saline. All the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the organic solvent was evaporated under reduced pressure. The mixture was dried and the residue was foam-dried overnight using a vacuum oil pump to obtain 8.012 g of solid crude product W-2. The solid crude product W-2 was used in the next deprotection reaction without further treatment.

[0337] (7-3) Synthesis of W-3: W-2 crude product (8.012 g, 10 mmol) was dissolved in 100 ml of methanol. Add 100 ml of methylamine aqueous solution (40 wt%) and react at 50℃ for 23 hours with stirring. The insoluble particles were removed by filtration, the solvent was evaporated to dryness under reduced pressure, and the residue was diluted with 200 ml of water. A 1:1 DCM-methanol mixed solvent was added, and the resulting organic phase was diluted with 50 ml of saturated bicarbonate. The aqueous phase was washed with sodium chloride and extracted three times with 50 ml each with dichloromethane. All the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure. The residue was dried overnight using a vacuum oil pump and then passed through a 200-300 mesh normal phase filter. The mixture was purified using a silica gel column, petroleum ether was added to the column, and silyl chloride was added to the column. Neutralize the acidity of the silica gel by dichloromethane:methanol:ammonia water (25wt%) = Gradient elution was performed at 1:1:0.05 to 1:1:0.25, the product eluate was collected, and the solvent was removed under reduced pressure. The mixture was evaporated to dryness, and the residue was foam-dried using a vacuum oil pump to obtain 3.062 g of pure product W-3. .

[0338] (7-4) Synthesis of W-4: W-3 (675 mg, 1.517 mmol) and GAL-C7-2 (2.60 g, 5.4 6 mmol) were mixed and dissolved in 47 ml of acetonitrile, and then diisopropyl ether was added. Add ethylamine (1.57 g, 12.14 mmol) and finally 3-diethoxyphosphoryl Peroxy-1,2,3-benzotriazin-4(3H)-one (DEPBT, 1.816 g, 6.04 mmol) was added and the mixture was stirred at room temperature for 2.5 hours. 0 ml of dichloromethane. 80 ml of saturated sodium bicarbonate solution and The organic phase was washed with 80 ml of saturated saline and 100 ml of sodium sulfate. The crude product was dried over ice, filtered, and the solvent was evaporated to dryness under reduced pressure to obtain a crude product. The mixture was purified using a normal phase silica gel column of 0.00 mesh, and petroleum ether was added to the column to obtain 1 wt% Neutralize the acidity of the silica gel with triethylamine and dissolve it in dichloromethane:methanol = 100: Gradient elution was performed at 5 to 100:7, and the eluate was collected and evaporated to dryness under reduced pressure to obtain 1.610 g of pure product. I got a W-4.

[0339] (7-5) Synthesis of W-5: W-4 (1.61 g, 0.886 mmol) was dissolved in 125 ml of dichloromethane. Dichloroacetic acid (3.5 ml, 42.43 mmol) was then added and the mixture was allowed to react at room temperature for 1 hour. The reaction mixture was neutralized by adding 150 ml of pyridine, and the solvent was evaporated to dryness under reduced pressure to obtain a crude product. The crude product was purified by column chromatography using 200-300 mesh normal phase silica gel. Neutralize the acidity of the silica gel with 10 wt% triethylamine, and then use 1 wt‰ triethylamine. The column was equilibrated and gradient elution was performed with dichloromethane:methanol = 100:30 to 100:40. The product eluate was collected, and the solvent was evaporated to dryness under reduced pressure to obtain 1.26 g of pure product W-5. .

[0340] (7-6) Synthesis of W-6: W-5 (1.25 g, 0.793 mmol) and the method described in step (1-7a) The resulting A-1 (1.21 g, 2.38 mmol) was mixed with 12 mL of dichloromethane. Dissolve 3-diethoxyphosphoryloxy-1,2,3-benzotriazine-4( 3H)-one (DEPBT, 0.712 g, 2.38 mmol) was added and diisopropyl Add propylethylamine (0.615g, 4.76mmol) and react at 25℃ for 3h with stirring. The organic phase was washed with 80 ml of saturated sodium bicarbonate, and the isolated aqueous phase was diluted with dichloromethane. The mixture was extracted three times with 10 ml of methane. All organic phases were combined and diluted with 10 ml of saturated aqueous broth. The organic phases were combined, dried over anhydrous sodium sulfate, and filtered. The solvent was evaporated to dryness under reduced pressure, and the residue was foam-dried overnight using a vacuum oil pump to obtain a crude product. The product was purified in a column, and 200-300 mesh normal phase silica gel was used. Neutralize the acidity of the silica gel with ml of triethylamine, and use a solution containing 1 wt% triethylamine. Equilibrate the column with petroleum ether and use petroleum ether:ethyl acetate:dichloromethane:N,N -Dimethylformamide = 1:1:1:0.1 to 1:1:0.7 gradient elution, and the eluate The mixture was recovered, and the solvent was evaporated to dryness under reduced pressure to obtain 1.57 g of pure product W-6.

[0341] (7-7) Synthesis of W-7: W-6 (1.238 g, 0.63 mmol), succinic anhydride (0.189 g, 1.8 9 mmol) and 4-dimethylaminopyridine (DMAP, 0.231 g, 1.89 mm ol) were mixed and dissolved in 7 ml of dichloromethane, and DIEA (0.407 g, The reaction mixture was stirred at 25°C for 24 hours. Wash with 0.5M triethylamine phosphate and extract the isolated aqueous phase once with dichloromethane. All the organic phases were combined and evaporated to dryness under reduced pressure to obtain the crude product. The product was purified in a column, and 30 g of 200-300 mesh normal phase silica gel was used to separate 1 wt The acidity of the silica gel was neutralized with % triethylamine, and the column was equilibrated with dichloromethane. Dichloromethane containing 1wt‰ triethylamine:methanol=100:18~100: The product eluate was collected and the solvent was evaporated to dryness under reduced pressure to give 1.033 g of pure 1,2-dichloro-2,2-tetrahydrofuran. The product W-7 complex (complex 7) was obtained. MS m / z:C 101 H 146 N7O 38 , [M-DMTr] + ,Theoretical value: 1763.92,Actual value: 1763.21. The obtained W- The structure of the seven-complex molecule is shown in formula (515).

[0342] (Preparation Example 8) Preparation of X-7 Complex Molecule (Complex Molecule 8) The following process pathway leads to the complex molecule 8 (hereafter referred to as X-7) with the structure shown in formula (521): They anticipated that it would be possible to synthesize a complex molecule (also called a complex molecule).

[0343] [ka]

[0344] (Preparation Example 9) Preparation of K-3 Complex Molecule (Comparative Complex Molecule 1) In this preparation example, K-3 composite molecule (hereinafter also referred to as comparative composite molecule 1) was prepared according to the following method. was synthesized.

[0345] [ka]

[0346] Synthesis of (9-1)K-1: In 60 ml of dichloromethane, A-1 obtained by the method described in step (1-7a) was added. (3.0g, 6.0mmol), PyBOP (6.2g, 12.0mmol), HOBt (1.6 g, 2.0 mmol) and diisopropylethylamine (DIPEA, 3.9 g , 30.0 mmol) was added and the mixture was stirred at room temperature for 10 minutes. The reaction mixture was added to 30 ml of 1.6 g of 100 ml of 100% ethanol and reacted at room temperature for 1 hour and 50 minutes. 1 of saturated sodium bicarbonate solution, and the isolated aqueous phase was diluted with dichloromethane in one portion. The organic phases were combined, washed with saturated sodium chloride solution, and then extracted with 30 ml of water. After drying over sodium sulfate, the crude product was filtered and concentrated. The mixture was purified on a 0.00 mesh normal phase silica gel column and diluted with dichloromethane:methanol:ammonia. Gradient elution was performed with 10:2:0.1 to 4:4:1 aqueous solution (25 wt%), and the product eluate was collected. The solvent was removed by concentration, and the residue was foam-dried using a vacuum oil pump to obtain 2.2 g of a white solid product. Got the K-1. 1 H NMR (400 MHz,DMSO-d6) δ 8.02 ( s,1H),7.43 (d,J =7.8 Hz,2H),7.34-7.17 (m ,7H),6.87 (d,J =8.6 Hz,4H),4.05 (d,J =5. 2 Hz, 1H), 3.74 (s, 6H), 3.20-3.01 (m, 5H), 2.6 0-2.38 (m,12H),1.60-1.39 (m,8H),1.24 (s, 1H). MS m / z: C 33 H 47 N4O5, [M+H] + , theoretical value: 579.35 , Actual value: 579.26.

[0347] (9-2) Synthesis of K-2: In 3 ml of dichloromethane, GAL-5 (4 83mg, 1.08mmol), 3-diethoxyphosphoryloxy-1,2,3-benzoxanthate Triazin-4(3H)-one (359 mg, 1.2 mmol), diisopropyl ethyl An amine (DIPEA, 310 mg, 2.4 mmol) was added and stirred at room temperature for 30 min. K-1 (174 mg, 0.3 mmol) was added and the reaction was carried out at room temperature for 16 hours. Pour into 10 ml of saturated sodium bicarbonate solution and wash the isolated aqueous phase once with dichloromethane. Extract three times with 10 ml of each solution, combine all the organic phases, and add 10 ml of saturated sodium chloride solution. The mixture was washed with ethyl acetate, dried over anhydrous sodium sulfate, filtered, and concentrated. The mixture was purified on a normal phase silica gel column and eluted with dichloromethane:methanol=20:1. The eluate was collected, the solvent was removed by concentration, and the mixture was dried and foamed using a vacuum oil pump to obtain 205 mg of a yellow solid. The shaped product K-2 was obtained.

[0348] (9-3) Synthesis of K-3: 1.1 ml of dichloromethane, K-2 (205 mg, 0.11 mmol), succinic acid free Water (22 mg, 0.22 mmol), 4-dimethylaminopyridine (DMAP, 27 mmol) g, 0.22 mmol) and diisopropylethylamine (DIPEA, 71 mg, 0. The reaction mixture was stirred at room temperature overnight. Wash three times with phosphate solution, 0.5 ml each time, and remove the aqueous phase from each wash. Back-extraction was performed once with 0.5 mL of dichloromethane. All organic phases were combined and washed with anhydrous sodium sulfate. The mixture was dried over an oven, the solvent was removed by concentration, and the mixture was dried and foamed using a vacuum oil pump to obtain 218 mg of pale yellow solid. The solid product K-3 conjugate molecule (comparative conjugate molecule 1) was obtained.

[0349] (Preparation Example 10) This preparation example illustrates the synthesis of a (GalNAc)3 conjugate molecule (comparative conjugate molecule 2). It is meant to explain In this preparation example, the preparations described in Examples 1 to 4 in WO2014025805A1 are The method produced compound 47 (referred to herein as (GalNAc)3 conjugate) having the structure shown below: A molecule (referred to as comparative composite molecule 2) was synthesized.

[0350] [ka]

[0351] (Preparation Example 11) This preparation example describes the preparation of FIN-2 conjugate molecule (comparative conjugate molecule 3). It is for In this preparation example, Rajeev et al., ChemBioChem 2015,16,903- 908, the FIN-2 conjugate molecule can be prepared by the following process: (Comparative complex molecule 3) was synthesized.

[0352] (11-1) Synthesis of PRO-10 Compound

[0353] [ka]

[0354] (11-1-1) Synthesis of PRO-7 2.93 g of PRO-6 (L-hydroxyproline, CAS number: 51-35-4, E (purchased from Nervous System, 22.4 mmol) in 22.5 ml of 1,4-dioxane (1, 4-dioxane) and 34 ml of 10% (w / w) Na2CO3 aqueous solution was added. In addition, 6.954 g of Fmoc-Cl (9-fluorenylmethyl chloroformate, CAS no. No.: 28920-43-6, purchased from Energy Co., 26.8 mmol) in 56.5 ml Dissolve in 1,4-dioxane and add to the reaction mixture in an ice bath. Allow to warm to room temperature. The reaction mixture was poured into 150 ml of ice water and methyl tert-butyl ether was added. The organic phase was discarded and the remaining aqueous phase was extracted with concentrated HCl three times with 100 ml each time. The pH was adjusted to ≦5, and the mixture was extracted twice with 100 ml of ethyl acetate. The mixture was combined, dried over anhydrous sodium sulfate, and the solvent was evaporated to dryness under reduced pressure to give 7.83 g of a white foam. A solid product PRO-7 was obtained. 1 H NMR (400 MHz, DMSO-d6) δ 7.91 (t,J =7.2 Hz,2H),7.67 (d,J =7.5 Hz, 2H),7.48-7.39 (m,2H),7.38-7.27 (m,2H),5. 17 (s,1H),4.27 (s,2H),4.23-4.11 (m,2H),3 .55-3.41 (m,3H),2.31-2.10 (m,1H),2.08-1. 88 (m,1H). HRMS (ESI) m / z theoretical value (calcd for) C 20 H 19 NO5[MH] - 352.1190, measured value 352.1033.

[0355] (11-1-2) Synthesis of PRO-8 7.83 g of PRO-7 (22.2 mmol) was dissolved in 80 ml of THF and heated in an oil bath. Heat to 65℃ and reflux 36.6ml of 2mol / L BH3-Me2S in THF solution. liquid (CAS number 13292-87-0, from J&K Scientific) (purchased, 73.2 mmol) was added and the reaction was continued under reflux for 3 hours. The reaction solution was drained and methanol was added. Dissolve the remaining solids in methanol, and add methanol under stirring until no gas remains in the reaction mixture. The mixture was stirred for 30 minutes, the solvent was removed under reduced pressure, and the residue was purified with petroleum ether three times. 7.1 g of a white solid product, PRO-8, was obtained. 1 H NMR (400 MHz, DMS O-d6) δ 7.91 (t,J =6.7 Hz,2H),7.67 (d,J =7.2 Hz,2H),7.49-7.39 (m,2H),7.38-7.26 ( m,2H),5.18 (dd,J =6.1,3.8 Hz,1H),4.28 (s ,2H),4.23-4.13 (m,2H),3.55-3.38 (m,2H),2 .32-2.11 (m,1H),2.08-1.89 (m,1H). HRMS ( ESI) m / z theoretical value(calcd for) C 20 H 21 NO4[M+Na] + 362.1368, actual value 362.1012.

[0356] (11-1-3) Synthesis of PRO-9 7.1 g of PRO-8 (21 mmol) was dissolved in 100 ml of pyridine, and 14.2 g DMTr-Cl (4,4'-bismethoxytrityl chloride, 42 mmol) was added, The reaction was stirred at room temperature for 5 hours. The solvent was removed under reduced pressure, and the crude product was dissolved in ethyl acetate and then dehydrated. The impurities were removed by filtration, the solvent was distilled off under reduced pressure, and the residue was purified using a silica gel column. For purification, the crude product dissolved in DCM was pretreated with pyridine for alkalinization. The DMTr-Cl was extracted with DCM containing 1% (v / v) pyridine. After eluting with ethyl acetate, the product was eluted, the product eluate was collected, and the solvent was distilled off under reduced pressure. After drying, 8.2 g of white solid product PRO-9 was obtained. HRMS (ESI) m / z Theoretical value (calcd for) C 41 H 39 NO6[M+Na] + 664.267 5. Actual value 664.2348, C18 RP-HPLC (Lot No. JJS160324 -1) Purity 94.20%.

[0357] (11-1-4) Synthesis of PRO-10 Dissolve 8.2 g of PRO-9 (12.8 mmol) in 64 ml of DMF and add 40 ml Piperidine (384 mmol) was added, and the reaction was allowed to proceed with stirring at room temperature for 30 minutes. The mixture was poured into 0.0 ml of ice water and extracted three times with ethyl acetate, each time at 150 ml. The phases were combined and washed with 200 ml of saturated saline, and the organic phase obtained by washing was then dissolved in sulfuric anhydride. After drying over sodium, the solvent was removed under reduced pressure, and the residue was purified using a silica gel column. For preparation, the crude product dissolved in DCM was pretreated with pyridine for alkalinization. The mixture was loaded onto a silica gel column. Fmoc was eluted with DCM containing 1% (v / v) pyridine. After that, the product was eluted with ethyl acetate, the product eluate was collected, and the solvent was evaporated to dryness under reduced pressure. 4.65 g of a white solid product, PRO-10, was obtained. 1 H NMR (400 MHz, DMSO-d6) δ 7.40 (d,J =7.2 Hz,2H),7.35-7. 18 (m,7H),6.93-6.84 (m,4H),4.56 (d,J =3. 9 Hz,1H),4.12 (s,1H),3.74 (s,6H),3.46-3. 37 (m,1H),2.88 (ddd,J =18.5,10.0,5.5 Hz, 2H),2.75 (dd,J =8.7,5.8 Hz,1H),2.62 (dd, J =11.0,2.7 Hz,1H),1.74-1.65 (m,1H),1.40 (ddd,J =12.9,8.5,5.9 Hz,1H),HRMS (ESI) m / z theoretical value (calcd for) C 26 H 29 NO4[M+Na] + 442. 1994, Actual value 442.1999, C18 RP-HPLC (Lot No. JJS160 329-1) Purity 97.07%.

[0358] (11-2) Synthesis of FIN-1

[0359] [ka] GAL-5 (4.5 g, 10 mmol) obtained by the method described in (1-1) was Dissolve 3.9 g of DIPEA (N,N-diisopropyl ethyl acetate) in 40 ml of DMF. Ming, CAS number: 7087-68-5, purchased from Aladdin, 30 mmol) and and 3.8 g of HBTU (benzotriazole-N,N,N',N'-tetramethyluronidase). Umium hexafluorophosphate, CAS number: 94790-37-2, Aladdin (11 mmol) were added in order, and the mixture was stirred at room temperature for 10 minutes to obtain a reaction solution. Dissolve PRO-10 (4.2 g, 10 mmol) obtained in 12-4) in 40 ml of DMF. After this, the reaction mixture was added to the above reaction mixture, dried with anhydrous sodium sulfate, and then incubated at room temperature for 2 The reaction mixture was poured into 120 ml of ice water and diluted with ethyl acetate in portions of 60 ml each. Extract three times, combine the organic phases, and wash with 20 ml of water and 20 ml of saturated saline. The organic phase obtained by washing was separated, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The residue was purified on a silica gel column. The column was loaded with 1% (v / v) triethylene glycol (TGE) and 1% (v / v) triethylene glycol (TGE) on a silica gel column pretreated with pyridine for elution. The mixture was eluted with a dichloromethane (DCM) solution containing dimethylamine and 1% (v / v) methanol. The eluate was collected, and the solvent was evaporated to dryness under reduced pressure to obtain 6.5 g of a pale yellow foamy solid product, FIN-1. Ta. 1 H NMR (400 MHz,DMSO-d6) δ 7.83 (d,J = 9.2 Hz,1H),7.32 (t,J =6.6 Hz,4H),7.20 (t d,J =8.9,3.5 Hz,5H),6.93-6.84 (m,4H),5.2 1 (d,J =3.2 Hz,1H),5.04-4.90 (m,2H),4.49 (s,1H),4.40 (d,J =4.4 Hz,0.8H),4.31 (d, J =5.0 Hz,0.2H),4.15 (s,1H),4.03 (s,3H), 3.93 (s,1H),3.74 (s,7H), 3.59 (dt,J =12.0 ,6.0 Hz,1H),3.50-3.40 (m,1H),3.39-3.25 ( m,3H),3.13 (dd,J =8.9,5.2 Hz,1H),3.00 (d q,J =9.3,5.3,4.3 Hz,1H),2.22 (s,2H),2.07 (s,3H),1.99 (s,3H),1.90 (s,4H),1.74 (s, 3H),1.50 (s,3H),1.36 (s,1H). C18 RP-HPLC( (Lot number LJ160422) Purity 95.45%.

[0360] (11-3) Synthesis of FIN-2

[0361] [ka] FIN-1 (3.0 g, 3.53 mmol) obtained in step (11-2) was added to 10 mL of Dissolve 2.13 g of PA (bis(diisopropylamino) (2 -Cyanoethoxy)phosphine, Adamas, product number 11356B, 7.06 mm ol), 346 mg of tetrazole (CAS number: 288-94-8, Aladdin (purchased from Sigma-Aldrich Co., Ltd., 4.94 mmol) was added, the mixture was stirred at room temperature, and 10 ml of DMF was added. The reaction was continued with stirring for 1 hour. The solvent was removed under reduced pressure, and the residue was subjected to silica gel column chromatography. For purification, the crude product dissolved in DCM was purified by alkalizing. The mixture was loaded onto a silica gel column pretreated with pyridine for elution with ethyl acetate. The resulting solution was collected, and the solvent was removed by distillation under reduced pressure to obtain 4.5 g of a colorless syrup-like crude product. The crude product was dissolved in 50% (v / v) acetonitrile aqueous solution until the product was dissolved. The sample was purified on a 30 g, 300 Å (medium pressure purification column) and the column was preloaded with 1% (v / v) pi. The product was collected by gradient elution using a solution of lysine in acetonitrile and the solvent was reduced. After distillation under reduced pressure, 2.2 g of a white powder product, FIN-2 conjugate molecule (comparative conjugate molecule 3), was obtained. 31 P NMR (162 MHz,CDCl3) δ 148.04,147.94,14 7.62,147.19, phosphorus spectrum purity 92%, C18 RP-HPLC purity 90 .54%.

[0362] Preparation Example 12 This preparation is used to illustrate the preparation of Z-4 conjugate molecule (conjugate molecule 153). It is of In this preparation example, conjugate molecule 153 (hereinafter also referred to as Z-4 conjugate molecule) was prepared according to the following method. was synthesized.

[0363] [ka]

[0364] (12-1) Synthesis of Z-1: W-3 (1.50 g, 3.37 mmo) obtained by the method described in step (7-3) 1) and GAL5-C4-2 (7.18 g) obtained by the method described in step (2-2). , 13.48 mmol) were mixed and dissolved in 34 ml of dichloromethane, and then diisopropyl Propylethylamine (3.48 g, 26.96 mmol) was added, followed by 3-diethoxyethylamine. Dioxyphosphoryloxy-1,2,3-benzotriazin-4(3H)-one (DEPBT The mixture was stirred at room temperature for 4.5 hours to react. The reaction mixture was diluted with 100 ml of dichloromethane and added 80 ml of saturated sodium bicarbonate solution. The organic phase was washed with 80 ml of saturated aqueous sodium chloride solution and 100 ml of saturated aqueous sodium chloride solution. All the organic phases were combined and washed with anhydrous sulfuric acid. The crude product was obtained by drying over sodium hydroxide, filtering, and then evaporating the solvent under reduced pressure to dryness. The mixture was purified on a 00-300 mesh normal phase silica gel column, and petroleum ether was added to the column. Neutralize the acidity of the silica gel with 1 wt% triethylamine, and dissolve in dichloromethane:methanol = Gradient elution was performed from 30:1 to 15:1, and the eluate was collected and evaporated to dryness under reduced pressure to obtain 3.97 g of pure toluene. Product Z-1 was obtained. MS m / z:C 98 H 143 N 10 O 33 , [M+H]+ , Theoretical value: 1987.98, Actual value: 1987.90.

[0365] (12-2) Synthesis of Z-2: Z-1 (3.97 g, 2.00 mmol) was dissolved in 250 ml of dichloromethane. Dichloroacetic acid (10.941 g, 84.85 mmol) was further added and reacted at room temperature for 1 hour. The reaction solution was neutralized by adding pyridine until it became neutral, and the solvent was evaporated to dryness under reduced pressure. The crude product was obtained by adding 200 g of 200-300 mesh normal phase silica gel to the column. The acidity of the silica gel was neutralized with 10% pyridine, and the column was equilibrated with 1‰ pyridine. Gradient elution was performed with chloromethane:methanol = 10:1 to 2:1, and the eluate was collected and the solvent was reduced in pressure. After evaporation to dryness, 3.49 g of pure Z-2 was obtained. MS m / z:C 79 H 129 N 10 O 33 , [M+H] + , theoretical value: 1746.94, actual value: 1746.90.

[0366] (12-3) Synthesis of Z-3: Z-2 (3.49 g, 2.0 mmol) obtained by the method described in step (1-7a) and A-1 (3.06 g, 6.0 mmol) were mixed and dissolved in 30 ml of dichloromethane. 3-(diethoxyphosphoryloxy)-1,2,3-benzotriazine-4(3H After adding diisopropyl ethyl ether (DEPBT, 1.80 g, 6.0 mmol), Aminopropylamine (1.55 g, 12.0 mmol) was added and the reaction was carried out at 25° C. for 3 hours with stirring. The reaction mixture was diluted with 100 ml of dichloromethane and washed with saturated sodium bicarbonate. The organic phase was washed twice with 30 ml portions and the aqueous phase was extracted with 10 ml portions of dichloromethane. All the organic phases were combined, washed with 50 ml of saturated saline, and the resulting organic phases were combined and washed with water. After drying with sodium sulfate and filtering, the solvent was evaporated to dryness under reduced pressure, and the residue was extracted with a vacuum oil pump. The crude product was purified in a column and 200 g of 200-300 ml of ethyl acetate was added. 00 mesh normal phase silica gel was used, and the silica gel was acidified with 20 ml of triethylamine. The column was neutralized with petroleum ether containing 1 wt% triethylamine, and dichloromethane was added. Gradient elution was performed with methane:methanol = 25:1 to 15:1, the eluate was collected, and the solvent was distilled off under reduced pressure. After drying, 2.2 g of pure product Z-3 was obtained. MS m / z:C 103 H 151 N 10 O3 8, [M+H] + , theoretical value: 2136.02, actual value: 2136.20.

[0367] (12-4) Synthesis of Z-4: Z-3 (2.10 g, 0.983 mmol) was dissolved in DIEA (635 mg, 4.915 mmol). mol) in 14.8 ml of dichloromethane and 4-dimethylaminopyridine (DMAP, 240 mg, 1.966 mmol) was added, stirred and clarified, and then succinic acid Anhydride (197 mg, 1.966 mmol) was added and the reaction was stirred at 25° C. for 18 hours. The reaction mixture was diluted with 50 ml of dichloromethane and diluted with 80 ml of 0.5 M triethylamine. Wash the organic phase with ethylamine phosphate and dilute the aqueous phase with dichloromethane in 2 portions of 50 ml each. All the organic phases were combined and evaporated to dryness under reduced pressure to obtain the crude product. Purify and separate using 188 g of 200-300 mesh normal phase silica gel. The acidity of the silica gel was neutralized with diethylamine, and the column was equilibrated with dichloromethane. Gradient elution with dichloromethane:methanol = 10:1 to 3:1 containing triethylamine was used. The solution was collected, and the solvent was evaporated to dryness under reduced pressure to obtain 1.95 g of the pure 422-formula 422. A product Z-4 composite molecule (complex molecule 12) was obtained. MS m / z:C 107 H 155 N 10 O4 1, [M+H] + , theoretical value: 1935.07, actual value: 1935.29.

[0368] (Preparation Example 13) Preparation of L10-siHBa1 complex (complex 9) In this preparation example, starting from the L-9 conjugate molecule (conjugate molecule 1), L10 was synthesized according to the following method. -siHBa1 complex (hereinafter also referred to as complex 9) was prepared.

[0369] (13-1) Synthesis of compound L-10:

[0370] [ka]

[0371] In this step, the L-9 conjugate molecule is bound to a solid support to form the L-10 compound. was prepared.

[0372] The L-9 complex molecule (0.233 g, 0.1126 mmol) obtained in step (1-8), O-Benzotriazole-tetramethyluronium hexafluorophosphate (HBT U, 0.064 g, 0.1689 mmol) and diisopropylethylamine (DIPE A, 0.029 g, 0.2252 mmol) were mixed and dissolved in 19 ml of acetonitrile. The mixture was stirred at room temperature for 5 minutes. Aminomethyl resin (0.901 g, 100-200 mesh) was added. (amino group loading: 400 μmol / g, purchased from Nankai Wasei Co., Ltd.) was added to the reaction solution and the mixture was stirred at 25°C. The reaction was carried out in a shaker at 220 rpm for 15 hours and then filtered. Rinse the extract twice with 30 ml of DCM and once with 30 ml of acetonitrile. Rinse three times with 10 ml of ethyl ether, rinse once with 30 ml of ethyl ether, and dry for 2 h using a vacuum oil pump. The capping reaction was carried out according to the blending ratios shown in Table 2.

[0373] [Table 2] Here, Cap1 and Cap2 are capping reagent solutions, Cap1 is 20% (v / v) Solution of N-methylimidazole in pyridine / acetonitrile mixture. The volume ratio of pyridine to acetonitrile was 3:5, and Cap2 was 20% (v / v ) A solution of acetic anhydride in acetonitrile.

[0374] Cap1, Cap2, 4-dimethylaminopyridine (DMAP) and acetonitrile Add the above reaction mixture and react in a shaker at 25°C, rotating at 200 rpm for 5 The reaction mixture was filtered, and the residue was diluted with acetonitrile three times, each time at 30 ml. Rinse, evaporate the solvent to dryness, and dry the mixture overnight under reduced pressure with a vacuum oil pump. , 1.100 g, loading amount 90.8 μmol / g, L-1 having the structure shown in formula (523) 0 compound (i.e., the L-9 conjugate molecule bound to a solid support).

[0375] (13-2) Synthesis of the sense strand of L10-siHBa1 complex In this step, the siRNA in the siRNA complex had the sequence number siHBa1. . siHBa1 Sense strand: 5'-CCUUGAGGCAUACUUCAAA-3' (SEQ ID NO: 1), Antisense strand: 5'-UUUGAAGUAUGCCUCAAGGUU-3' (sequence no. No. 2)

[0376] Starting from the L-10 compound prepared in the above step, the phosphoramidite solid phase synthesis method was used. Then, nucleoside monomers were linked one by one in the 3'-5' direction in the above sequence. Each time a nucleoside monomer is attached, it is subjected to deprotection, coupling, capping and oxidation. The synthesis conditions were as follows:

[0377] Nucleoside monomers were provided in acetonitrile at 0.1 M concentration, and deprotection was performed at each step. The reaction conditions were the same, that is, the temperature was 25° C. and the reaction time was 70 seconds. The reagent is a solution of dichloroacetic acid in dichloromethane (3% v / v), which is The molar ratio of the 4,4'-dimethoxytrityl protecting group in the support was 5:1.

[0378] The coupling reaction conditions were the same for all of them, with the temperature being 25°C and the binding to the solid support. The molar ratio of the nucleic acid sequence to the nucleoside monomers is 1:10, and the nucleic acid is bound to a solid support. The molar ratio of the nucleic acid sequence to the coupling reagent was 1:65, and the reaction time was 600 seconds. The coupling reagent was 5-ethylthio-1H-tetrazole in 0.5M acetonite. The solution was distillate.

[0379] The capping conditions were the same for all of them, with the temperature at 25°C and the reaction time at 15 seconds. The capping reagent solution was a mixture of Cap1 and Cap2 in a molar ratio of 1:1. The molar ratio of the capping reagent to the nucleic acid sequence to be bound to the solid support is acetic anhydride. The ratio of N-methylimidazole to the nucleic acid sequence bound to the solid support was 1:1:1.

[0380] The oxidation reaction conditions were the same for each reaction, with the temperature at 25°C and the reaction time at 15 seconds. The reagent was 0.05M iodine water. The iodine and the iodine bound to the solid support in the coupling step were The molar ratio of the nucleic acid sequence to be combined was 30:1. The reaction was carried out in tetrahydrofuran:water:pyrifos. The experiment was carried out using a mixed solvent of 3:1:1 ethylenediaminetetraacetate (EDTA) and 3:1:1 ethylenediaminetetraacetate (EDTA).

[0381] The cleavage and deprotection conditions are as follows: The column was added to 25 wt% ammonia water, and the dosage of ammonia water was 0.5 ml / μmol. The mixture was reacted at 55°C for 16 hours, the liquid was removed, and the residue was concentrated under vacuum until dry. After treatment with ammonia water, the amount of single-stranded nucleic acid was 0.4 ml / μmol of N-methylcellulose. After dissolving the product in diethylpyrrolidone, 0.3 ml / μmol of triethylamine and 0 Add 0.6 ml / μmol of triethylamine trihydrofluoride to the 2' end of ribose. -TBDMS protection was removed. Purification and desalting: Preparative ion chromatography column (S Using our 15Q, we were able to purify nucleic acids using gradient elution with NaCl. Specifically, eluent A was 20 mM sodium phosphate (pH 8.1), with the solvent being water / acetonite. eluent B: 1.5 M sodium chloride, 20 mM sodium phosphate Thorium (pH 8.1), solvent is water / acetonitrile = 9:1 (volume ratio), elution Gradient: Eluent A:Eluent B = 100:0 to 50:50. Product eluate was collected. Then, the mixture was combined and desalted using a reverse phase chromatography purification column. was desalted using a Sephadex column, the packing material being Sephadex-G25; Elution was carried out with deionized water.

[0382] Detection: Ion exchange chromatography (IEX-HPLC) was used to detect The purity was 92.4%, and the molecular weight was determined by liquid chromatography-mass spectrometry (LC-MS). When analyzed, the theoretical value was 7253.96 and the measured value was 7253.12.

[0383] This allows the L-9 conjugate molecule to bind to the 3' end of the sense strand obtained in this step. Thus, the sense strand S of siRNA was obtained, in which the L-9 conjugated molecule was conjugated to the 3' end of the siRNA.

[0384] (13-3) Synthesis of antisense strand In this process, a general-purpose solid phase support (UnyLinker TM Loaded Nitt oPhase(R) HL Solid Supports, Kinovate L The antisense oligonucleotide L10-siHB1 complex was synthesized by Fabry-Perot Sciences Inc. The antisense strand AS of siRNA was synthesized under the same conditions as the synthesis of the sense strand. , deprotection, coupling, capping, oxidation reaction conditions, deprotection and Includes cleavage and isolation conditions.

[0385] Detection: Ion exchange chromatography (IEX-HPLC) was used to detect the The purity was 93.2%, and the molecular weight was determined by liquid chromatography-mass spectrometry (LC-MS). The theoretical value was 6675.04, and the measured value was 6674.50.

[0386] (13-4) Synthesis of L10-siHBa1 complex The S chain and the AS chain were each dissolved in an aqueous solution for injection to obtain a 40 mg / ml solution. These were mixed in an equimolar ratio, heated at 50°C for 15 minutes, cooled to room temperature, and then hydrogen-bonded. The double-stranded structure was formed by merging.

[0387] After the above synthesis was completed, ultrapure water (homemade with Milli-Q ultrapure water system, resistivity 18.2M The complex was diluted to a concentration of 0.2 mg / mL using a liquid chromatograph. chromatography mass spectrometer (LC-MS, Liquid Chromatography- Mass Spectrometry, purchased from Waters, model number: LCT Pre The molecular weight was detected by a spectrophotometer. As a result, the theoretical molecular weight S: 7253.96, A S: 6675.04, measured molecular weight S: 7253.24, AS: 6674.61 The measured values ​​were consistent with the theoretical values, and the synthesized complex was the intended design containing the L-9 complex molecule. The double-stranded nucleic acid sequence of the L10-siHBa1 complex (complex 9) was determined to be: The structure is shown in formula (3).

[0388] (Preparation Example 14) Complexes 16-18, 24-26, 42-43, 62, 78, 105, 1 Preparation of 09, 111, 115, 144 and 154-171 1) The complex siRNA is a complex 16 to 18, 24 to 26, or 42 shown in Tables 3A to 3G. ~43, 62, 78, 105, 109, 111, 115, 144, 157~163 and 1 67 to 171, and 2) a thiol between two nucleotides in the target sequence. When linked by a phosphate ester, the latter of the two nucleotides The following sulfurization reaction steps are used instead of the oxidation reaction step in the bond, and the sulfurization reaction conditions for each step are The conditions were the same, the temperature was 25°C, the reaction time was 300 seconds, and the sulfurizing agent was xanthan gum. The sulfurizing reagent is a thiol hydride, and the nucleic acid sequence that is bound to the solid support in the coupling step is The molar ratio of the column is 120:1, and the reaction is carried out in a mixed solvent of acetonitrile:pyridine = 1:1. 3) modifying the 2'-position of all nucleotides in the target sequence; If the cleavage and deprotection conditions are such that the 2'-TBDMS protection on the ribose is removed, The title complex was prepared by the same method as in Preparation Example 13, except that the step of Complexes of 16-18, 24-26, 42-43, 62, 78, 105, 109, 111 , 115, 144, 157-163 and 167-171 were prepared.

[0389] In addition, the following conjugate molecules were used instead of the L-9 conjugate molecule, and the The P-9 conjugate molecule (conjugate molecule 2) obtained in Preparation Example 7 was used to prepare conjugate 164. The W-7 conjugate molecule (conjugate molecule 7) was used to prepare conjugates 155, 156 and 165, Using the Z-4 ​​conjugate molecule (conjugate molecule 12) obtained in Preparation Example 12 above, conjugates 154 and 1 66 was prepared, and the complexed siRNA was prepared as complexes 154 to 15 shown in Tables 3A to 3G, respectively. 6 and 164 to 166, and the above complexes are identified according to Tables 3A to 3G, These structures are numbered as formula (3), formula (4), formula (15), and formula (22), respectively. Except as otherwise indicated, conjugates 154 to 156 and 164 were prepared in the same manner as in Preparation Example 13. The molecular weight of the complex was detected by liquid chromatography mass spectrometry. The theoretical value of complex 142 was S: 7649.55, AS: 6991.46, and the measured value S : 7649.1, AS: 6991, Theoretical value of complex 170 S: 7649.55, AS: 6 995.47, measured value S: 7648.8, AS: 6994.8, theoretical value S of complex 171 : 7649.55, AS: 7011.53, Observed S: 7648.8, AS: 7010. 9. Complex 115: theoretical value S: 7584.5, AS: 7007.46, actual value S: 758 4, AS: 7006.2, Theoretical value of complex 109 S: 7584.5, AS: 6931.4 7, Actual value S: 7584, AS: 6930.9, Theoretical value S for complex 106: 7572.4 7, AS: 6907.41, Measured S: 7571.8, AS: 6906.9, Complex 11 3 Theoretical value S: 7584.5, AS: 7011.47, Actual value S: 7584, AS: 70 11.3, Complex 17 theoretical value S: 7504.34, AS: 6961.52, measured value S: 7503.4, AS: 6960.9, Theoretical value of complex 25 S: 7504.34, AS: 7 037.51, measured S: 7503.6, AS: 7036.9, theoretical S for complex 18: 8218.83, AS:7703.05, Actual value S:8218, AS:7702.5, Complex Theoretical value of combined 16: S: 7516.37, AS: 6985.58, measured value: S: 7516.5 , AS: 6984.9, Theoretical value of complex 159 S: 7504.34, AS: 7057.5 8, Actual value S: 7503.6, AS: 7057, Theoretical value S of complex 160: 7504.3 4, AS: 7041.52, Measured S: 7503.6, AS: 7040.8, Complex 16 Theoretical value of 1: S: 7516.37, AS: 7065.58, Measured value: S: 7516.6, AS : 7064.5, theoretical value of complex 162 S: 7504.34, AS: 7139.68, actual Measured value S: 7515.6, AS: 7138.9, theoretical value S for complex 163: 7516.37 , AS:7081.64, Actual value S:7515.6, AS:7080.9, Complex 62 Theoretical value S: 7485.3, AS: 7161.7, Measured value S: 7484.4, AS: 716 0.9, Complex 78 theoretical value S: 7423.22, AS: 7207.78, Measured value S: 7 422.6, AS: 7207.2, Theoretical value of complex 42 S: 7407.22, AS: 72 08.77, measured value S: 7406.4, AS: 7208.1, theoretical value S for complex 43: 7 407.22, AS:7170.72, measured value S:7406.5, AS:7170.1 The results showed that the measured values ​​were consistent with the theoretical values.

[0390] [Table 3] siRNA complex [Table 3A] JPEG0007662216000105.jpg222161JPEG0007662216000106.jpg223156JPEG0007662216000107.jpg89161

[0391] [Table 3B] JPEG0007662216000109.jpg223165JPEG0007662216000110.jpg107155

[0392] [Table 3C] JPEG0007662216000112.jpg176156

[0393] [Table 3D] JPEG0007662216000114.jpg176157

[0394] [Table 3E] JPEG0007662216000116.jpg223156JPEG0007662216000117.jpg223158JPEG0007662216000118.jpg235161JPEG0007662216000119.jpg36154

[0395] [Table 3F] JPEG0007662216000121.jpg223164JPEG0007662216000122.jpg148156

[0396] [Table 3G] JPEG0007662216000124.jpg229159 * S: sense strand, AS: antisense strand Note: The capital letters C, G, U, and A represent the base sequence of nucleotides. dT stands for deoxyribonucleic acid. A lowercase letter m represents a nucleotide adjacent to the left of the letter m. represents a 2'-methoxy modified nucleotide, and a lowercase letter f is The adjacent nucleotide is a 2'-fluoro modified nucleotide. The letter s is a nucleotide sequence in which the two adjacent nucleotides on either side of the letter s are separated by a thiophosphate group. VP means that one nucleotide to the right of the letter VP is a vinyl P represents one nucleotide to the right of the letter P. The nucleotide is a phosphate nucleotide, and Ps is the letter Ps. The nucleotide on the right is a phosphorothioate-modified nucleotide, T moe represents a 2'-methoxyethoxy modified thymine nucleotide.

[0397] Vinyl phosphate and 2'-methoxy modified uridine monomers (VP-Um) are It was synthesized according to the method below.

[0398] [ka]

[0399] (14-1) Synthesis of VP-U-2 The VP-U-2 molecule was synthesized according to the following method.

[0400] [ka] 2'-Methoxy modified uracil nucleoside (2'-OMe-U, 51.30 g, 91. 6mmol), tert-butyldiphenylchlorosilane (TBDPSCl, 50.35 g, 183.2 mmol), imidazole (12.47 g, 183.2 mmol) mixed The mixture was dissolved in 450 ml of N,N-dimethylformamide (DMF) and incubated at room temperature for 20 hours. The mixture was stirred and reacted. DMF was removed by distillation, and the residue was dissolved in 600 ml of dichloromethane. The mixture was washed with 300 ml of saturated sodium bicarbonate and the isolated aqueous phase was washed with dichloromethane ( The organic phases were combined and diluted with 5% oxalic acid (DCM) for three times at 300 ml each. The aqueous phase was washed with 100 ml of 10 ... The product was obtained and used as is in the subsequent synthesis of VP-U-2.

[0401] The crude VP-U-1 product was dissolved in 100 ml of dichloromethane, and then cooled in an ice bath for 10 minutes. The mixture was stirred for 1 h and then added with 450 ml of 2% p-toluenesulfonyl chloride, which had been cooled in a refrigerator at 4°C. Add the phosphoric acid solution (the solvent is a methanol-dichloromethane mixed solvent with a volume ratio of 3:7). The reaction was continued for 10 minutes. An additional 200 ml of saturated sodium bicarbonate was added to quench the reaction. The organic phase was diluted with saturated sodium bicarbonate solution until the pH reached 8. The aqueous phases were combined and extracted twice with dichloromethane, each time with 200 ml of dichloromethane. The organic phases were combined, washed once with 200 ml of saturated saline, and then dried by removing the solvent. The residue was purified on a 200-300 mesh normal phase silica gel column and purified with petroleum ether. The ether was placed in a column and diluted with petroleum ether:ethyl acetate:dichloromethane:methanol in a ratio of 1:1. Gradient elution was performed with a gradient of 1:0.05 to 1:1:1:0.25, and the product eluate was collected and the solvent was reduced. The residue was dried by vacuum oil pump to obtain 40.00g of pure VP-U. -2 was obtained. 1H NMR (400 MHz, DMSO-d6) δ 7.96 (d ,J =7.8 Hz,1H),7.64 (dtd,J =5.1,4.0,2.2 Hz,4H),7.41-7.30 (m,6H),6.79 (d,J =4.7H z,1H),5.73 (d,J =7.6 Hz,1H),4.94 (t,J =7 .0 Hz,1H),4.12 (td,J =4.6,3.9 Hz,1H),4.0 5 (dd,J =4.8, 4.0 Hz,1H),3.96 (t,J =4.7H z,1H),3.68 (ddd,J =11.8,7.0,4.6 Hz,1H),3 .57-3.46 (m,1H),3.39 (s,3H),1.05 (s,8H). MS m / z:C 26 H 33 N2O6Si, [M+H]+ ,Theoretical value: 497.21,Actual value: Measurement: 497.45.

[0402] (14-2) Synthesis of VP-U-4:

[0403] [ka] VP-U-2 (19.84 g, 40.0 mmol), dicyclohexylcarbodiimide (DCC, 16.48 g, 80.0 mmol), pyridine (4.20 g, 53.2 mmol), Mix 100 ml of dichloromethane (6.61 g, 53.2 mmol) and trifluoroacetic acid (6.61 g, 53.2 mmol) in 200 ml of dichloromethane. The mixture was dissolved in dimethyl sulfoxide (DMSO) and reacted with stirring at room temperature for 20 hours to obtain a reaction solution. Separately, tetraethyl methylenediphosphonate (21.44 g, 74.4 mmol) was added to The mixture was dissolved in 100 ml of THF, cooled in an ice bath, and t-BuOK (11.36 g, 1 01.2 mmol) was added, and the mixture was reacted at ice bath temperature for 10 minutes, then the temperature was raised to room temperature and 0.5 After reacting for 1 hour, add it to the reaction solution mentioned above over about 1 hour and react at ice bath temperature for 1 hour. The mixture was then heated to room temperature and reacted for 18 hours. Water was added to quench the reaction. The organic phases were extracted three times with 200 ml each with dichloromethane. All organic phases were combined and After washing once with 000 ml of saturated saline, the solvent was evaporated until dry. The mixture was purified using a 0.0 mesh normal phase silica gel column, petroleum ether was added to the column, and the petroleum ether was Gradient elution was performed with ethyl acetate: 1:1 to 1:4, and the product eluate was collected and the solvent was evaporated under reduced pressure. The mixture was dried and the residue was foam-dried using a vacuum oil pump to obtain a total of 14.00 g of pure VP-U-4. Obtained. 1H NMR (400 MHz, DMSO-d6) δ 7.96 (d,J =7.8 Hz,1H),7.64 (dtd,J =5.1,4.0,2.2 Hz, 4H),7.41-7.30 (m,6H),6.82-6.71 (m,2H),5. 90 (ddd,J =25.9, 15.0,1.0 Hz,1H),5.73 (d, J =7.6 Hz,1H),4.36-4.21 (m,3H),4.18 (t,J =4.9 Hz,1H),4.05 (ddq,J =9.7,8.5,6.9 Hz ,2H),3.87 (t,J =4.8 Hz,1H),3.39 (s,3H),1 .32 (td,J =6.9,0.7 Hz,6H),1.05 (s,8H). M S m / z:C 31 H 42 N2O8PSi, [M+H] + ,Theoretical value: 629.24,Actual value Value: 629.51.

[0404] (14-3) Synthesis of VP-U-5:

[0405] [ka] VP-U-4 (14.00 g, 22.29 mmol) was dissolved in 100 mL of tetrahydrofuran. The solution was dissolved in ethanol and triethylamine trihydrofluoride (17.96 g, 111.45 mmol) was added. l) was added and stirred at room temperature for 20 hours until the reaction was complete. The solvent was then dried. The solution was evaporated to dryness, and then dissolved in 50 ml of dichloromethane twice. The crude product was purified by elution with a 200-300 mesh normal phase silica gel column. Purify, put petroleum ether into the column, and mix petroleum ether:ethyl acetate:dichloromethane:methane. The product eluate was diluted with methanol (1:1:1:0.05 to 1:1:1:0.25) and the resulting solution was The solvent was evaporated to dryness under reduced pressure, and the residue was foam-dried using a vacuum oil pump to obtain a total of 6.70 g. Pure VP-U-5 was obtained. 1H NMR (400 MHz, DMSO-d6) δ 7.96 (d,J =7.8 Hz,1H),6.77 (dd,J =15.0,6 .2 Hz,1H),5.99-5.82 (m,2H),5.73 (d,J =7. 6 Hz,1H),5.27 (d,J =5.1 Hz,1H),5.10 (dd, J =5.3,4.7 Hz,1H),4.29 (ddq,J =9.8,8.6,7 .0 Hz,2H),4.17 (ddd,J =6.2,5.2,1.0 Hz,1H ),4.12-3.98 (m,3H),3.39 (s,2H),1.32 (td, J = 6.9, 0.6 Hz, 6H). MS m / z: C 15 H 24 N2O8P, [M +H] + , theoretical value: 391.13, measured value: 391.38.

[0406] (14-4) Synthesis of VP-U-6:

[0407] [ka] VP-U-5 (391 mg, 1. 0 mmol), pyridine trifluoroacetate (0.232 g, 1.2 mmol), N -Methylimidazole (0.099 g, 1.2 mmol), bis(diisopropylamino )(2-cyanoethoxy)phosphine (0.452 g, 1.5 mmol) was added and the mixture was stirred at room temperature. The reaction was stirred for 5 hours. The solvent was distilled off until dry, and the residue was subjected to column chromatography. The mixture was purified by dichloromethane using normal phase silica gel of 200-300 mesh. Gradient elution with acetonitrile (containing 0.5 wt% triethylamine) = 3:1 to 1:3 The product eluate was collected, the solvent was removed by concentration, and a total of 508 mg of the target product VP-U- 6 was obtained. 31P NMR (161 MHz, DMSO-d6) δ 150.34, 150.29, 17.07, 15.50. MS m / z:C 24 H 41 N4O9P2, [M+H] + , Theoretical value: 591.23, Actual value: 591.55. VP-U-6 is the target generation The compound VP-Um has been shown to be involved in the synthesis of RNA strands as a nucleoside monomer. Ta.

[0408] A 5'-phosphate modification was attached to the 5' end of the antisense strand using the following method.

[0409] The raw material is a phosphorylated monomer having the structure of the following formula CPR-I, which is manufactured by Suzhou Jiama Co., Ltd. The product was provided by and had the number Cat#13~2601-XX.

[0410] [ka]

[0411] After all nucleoside monomers of the antisense strand are coupled, phosphoramidite nucleic acid Using solid-phase synthesis, four reactions were carried out: deprotection, coupling, capping, and oxidation. A monomer of formula CPR-I was bound to the 5' end of the antisense strand. After cleavage and deprotection, the antisense strand was obtained.

[0412] The synthesized nucleotide sequence bound to the carrier was added to 25 wt% ammonia water. For nucleotide sequences, the dose of aqueous ammonia was 0.5 ml / μmol, and 55 The reaction was allowed to proceed at 50° C. for 16 hours, the liquid was removed, and the residue was concentrated to dryness in vacuum. After treatment with NEA, the amount of single-stranded nucleic acid was 0.4 ml / μmol of N-methyl After dissolving the product in pyrrolidone, 0.3 ml / μmol of triethylamine and 0.6 Add 1 ml / μmol of triethylamine trihydrofluoride to the 2'-Tribosomal ribose. The BDMS protection was removed. Purification and desalting: A preparative ion chromatography column (Soup) was used. Nucleic acids were purified by gradient elution with NaCl using a column chromatography column (rce 15Q). Extractant A: 20 mM sodium phosphate (pH 8.1), solvent: water / acetonitrile = 9: 1 (volume ratio), and eluent B: 1.5 M sodium chloride, 20 mM sodium phosphate ( pH 8.1), the solvent was water / acetonitrile = 9:1 (volume ratio), and the elution gradient was: Gradient elution was performed with eluent A and eluent B = 100:0 to 50:50. The mixture was then desalted using a reverse phase chromatography purification column. Desalted using a hadex column, the packing material is Sephadex-G25, and deionized water is used. It was eluted at .

[0413] When the target product has a 5'-thiophosphate modification, the above oxidation occurs during binding. The sulfurization reaction was carried out by the same procedure as above, except that the sulfurization reaction conditions were used instead of the reaction conditions. went.

[0414] The synthesized sense strand and antisense strand were subjected to ion exchange chromatography. Purity was detected using IEX-HPLC and liquid chromatography tandem mass spectrometry The molecular weight was analyzed by LC-MS, and the synthesized nucleic acid sequences were confirmed to be identical to those of the Examples and It was confirmed that the siRNA corresponded to the comparative example.

[0415] (Preparation Example 15) Preparation of other conjugates in Tables 3A to 3G Also, 1) Complexes 31-37, 52-58, 68-74, 84-90, 121-127 , 146-152 and comparative complexes 4-5, 7-8, 10-13, L-9 complex molecule Instead of the conjugated molecules 2 to 8 or 12 obtained in the above Preparation Examples 2 to 10 or 12, and the comparative conjugated molecules Using molecules 1 and 2 (e.g., using P-9 complex molecule (complex molecule 2) instead of L-9 complex molecule) When used, P10 is added to the numbers 31, 52, 68, 84, 121, and 146. We expected to obtain the complex shown in Fig. 1, and instead of the L-9 complex, we obtained the R-4 complex (complex 3). When using the compound 32, 53, 69, 85, 122, and 147, R5 was added to the compound numbers. 2) the complex siRNA is obtained according to the method of Table 3A to Complexes 10–15, 19–23, 27–41, 44–61, and 63–77 shown in 3G; 79-104, 106-108, 110, 112-114, 116-141, 143-1 52 and comparative complexes 4-5, 7-8, and 10-17; and 3) a target sequence. When two nucleotides in a nucleic acid are linked by a phosphorothioate, the two nucleotides In place of the oxidation reaction step in the subsequent nucleotide bond of the nucleotide in Preparation Example 13, 4) the 2'-position of every nucleotide in the sequence of interest is In the case of modified hydroxy groups, the cleavage and deprotection conditions are such that the 2'- The same method as in Preparation Example 13 was used to prepare the above-listed compounds, except that the step of removing the TBDMS protection was not included. It is expected that other siRNA complexes of 3A-3G can be prepared. 10~15, 19~23, 27~41, 44~61, 63~77, 79~104, 106 ~108, 110, 112~114, 116~141, 143~152 and comparative complex 4 It is expected that compounds 10 to 17 can be prepared according to Tables 3A to 3G. The structures of these complexes of the present disclosure are represented by formulas (3), (4), (7), Each comparative composite shown in (12), (13), (14), (15), (21) or (22) The structures of the bodies are shown in equations (901) and (902), respectively.

[0416] [ka] where Nu is the siRNA in comparative complexes 4-5, 7-8, and 10-13. .

[0417] (Preparation Example 16) Synthesis of Comparative Complexes 6 and 9 Comparative complexes 6 and 9 were synthesized using FIN-2 obtained in step (11-3) above. General-purpose solid support (UnyLinker TM Loaded NittoPhase (Registered The HL Solid Supports were used for three cycles of reaction in FIN-2. After conjugating FIN_FIN_FIN to the 3' end of the RNA sense strand, solid-phase synthesis was performed. Except for the above, the conditions for RNA solid-phase synthesis and deprotection were the same as those described in the above step (11-2). This is consistent with the solid-phase synthesis of nucleic acids described previously.

[0418] Rajeev et al., ChemBioChem 2015, 16, 903-908 The FIN-FIN-FIN complex group is bonded by referring to the method described above. Specifically, first, The hydroxy protecting group in the above-mentioned general-purpose solid support is removed, and the coupling reaction conditions and coupling reaction are carried out. The capping reagent is then contacted with the FIN-2 complex to couple the FIN-2 complex. After carrying out the binding and oxidation reactions, the FIN conjugate molecule bound to the solid support was obtained. The hydroxy protecting group DMTr in the FIN conjugate molecule bound to the solid phase support is removed, and another The FIN-2 complex molecule is contacted with the FIN-2 complex molecule, and the coupling is performed to carry out the capping reaction and the oxidation reaction. The above deprotection-coupling-capping-oxidation process was repeated once again. The FIN-2 conjugate molecule was then attached to the solid support, resulting in a conjugate group (FIN_F The nucleotide monomers were then separated from the conjugated groups bound to the solid support. It has a composite group (FIN_FIN_FIN) that is finally conjugated to the 3' end. The RNA sense strand was obtained.

[0419] In the above reaction, the conditions of the deprotection, coupling, capping and oxidation reactions, solvents, etc. The reagents and agents were the same as those used in the solid phase nucleic acid synthesis method described in step (11-2).

[0420] Then, cleavage, deprotection, purification, desalting, detection, and annealing were performed in the same manner as in Preparation Example 12. Finally, comparative complexes 6 and 9 (hereinafter sometimes simply referred to as FIN complexes) were obtained. Liquid Chromatography Mass Spectrometer (LC-MS) aphy-Mass Spectrometry, purchased from Waters Corp., type The molecular weight was detected by LCT Premier. As a result, the measured value was consistent with the theoretical value. The synthesized FIN-siHBa1M1SP complex corresponds to the following formula (903): It was determined that the compound was of the intended design shown.

[0421] [ka] where Nu is the siRNA in comparative complex 6 or 9.

[0422] Once the complex is prepared, it is lyophilized by standard means and stored as a solid powder. If necessary, dissolve in water for injection or saline to the desired concentration. It may also be used as a solution.

[0423] (Experimental Example 1) This experiment demonstrates the toxicity of the complex of the present disclosure. In C57BL / 6J mice, 100 mg / kg or 200 mg / mouse / kg (calculated as siRNA, in the form of 0.9% sodium chloride solution, 10 ml each Conjugate 24 (10 mL / kg at 20 mg / mL or 20 mg / mL) was administered once. During the subcutaneous administration and continuous observation period, no animals died, and no clinical symptoms related to drug side effects were observed. No symptoms were observed, and 24 hours after administration, no clinical pathological findings or gross dissections As is clear from the above results, the complex of the present disclosure has The toxicity of the drug is low.

[0424] (Experimental Example 2) This experiment demonstrates the stability of the complex of the present disclosure. (Experimental Example 2-1) Stability in in vitro lysosomal lysate Complexes 24, 25, 42, 43, 62, 78 and comparative sequence 1 (siRNA concentration The solution was provided as a 0.9% sodium chloride solution containing 20 μM of 12 μl per group. Each was treated with 27.2 μL of sodium citrate solution (pH 5.0) and 4.08 μL of deionized water. Water and 2.72 μL of mouse tritosome (purchased from Xenotech Inc., no. R0610LT, No. 1610069, final concentration 0.2mU / μL) and mix evenly. The incubation was performed at 37℃ for 0 h, 5 min, 15 min, 30 min, 1 h, and 2 h. 5 μl of sample was taken at each time point, 4 h, and 8 h, and each was diluted with 15 μL of 9M After denaturation by addition to urea water, 4 μl of sample loading buffer (Solarbio, no. 20160830) was added and immediately frozen in a -80℃ refrigerator to stop the reaction. The time between the measurements indicates the time when the test sample and the lysosomal lysate were taken out immediately after being mixed uniformly. Preparation of reference samples not treated with lysosomal lysate: 1.5 μl equimolar amounts of each The above complexes (20 μM) were each dissolved in 7.5 μL of sodium citrate solution (pH 5 0), mixed uniformly with 1 μL of deionized water, and added to 15 μL of 9M urea aqueous solution. After denaturation with 500 mM NaCl, 4 μL of sample loading buffer (Solarbio, no. 20160830) was added. ) was added and mixed uniformly, and the mixture was immediately frozen in a refrigerator at -80°C to stop the reaction. The reference sample from the body is marked Con on the electropherogram.

[0425] A 16% by weight non-denaturing polyacrylamide gel was added to each group of frozen samples. All samples were loaded with 4 μl of sample loading buffer (purchased from Solarbio, 2 0 mM EDTA, 36 wt% glycerol, 0.06 wt% bromphenol blue in water After mixing with the 100 μl solution, 20 μl of the mixture was loaded onto the gel and electrophoresed at 20 mA for 10 min. After electrophoresis, the gel was electrophoresed at 40 mA for 30 minutes. Imaging after staining with dye (BioTium, Cat. 13G1203) for 10 min. The results are shown in FIG. 1A.

[0426] The comparison sequence 1 is as follows: Sense strand: 5'-CCUUGAGGCAUACUUCAAA-3' (SEQ ID NO: 250) , Antisense strand: 5'-UUUGAAGUAUGCCUCAAGGUU-3' (sequence no. No. 251).

[0427] By the same method, the stability of complexes 105, 109, 111, and 115 within 6 h was The activity was measured and the results are shown in FIG. 1B.

[0428] 1A-1B show semi-quantitative detection of the stability of siRNA complexes in vitro in tritosomes. The results are shown in Table 1. The results show that the complex of the present disclosure is maintained in tritosome for a long period of time and is not decomposed. It was shown that the composition exhibited excellent stability without any deterioration.

[0429] (Experimental Example 2-2) In vitro stability in human plasma Complexes 24, 25, 42, 43, 62, 78 and comparative sequence 1 (siRNA concentration (provided as a 0.9 wt% sodium chloride solution with 20 μM of 12 μl per group) Each of these was diluted with 108 μL of 90% human plasma (diluted with PBS) and The mixture was mixed uniformly and incubated at 37°C. At each time point, 10 μL samples were removed and immediately flash frozen in liquid nitrogen and stored at −80°C. The mixture was frozen and stored in a refrigerator at 37 °C for use. l) was mixed evenly with 8 μl of 1×PBS, and 10 μL of untreated human plasma samples were A pool was obtained (denoted as Con). A 20% by weight non-denaturing polyacrylamide gel was added. All samples from each group of frozen samples were loaded with 4 μL of sample loading buffer (2 0 mM EDTA, 36 wt% glycerol, 0.06 wt% bromphenol blue in water After mixing with the above solution, the mixture was loaded onto the gel and electrophoresed at a constant current of 80 mA for 60 minutes. After electrophoresis, the gel was washed with 1x Sybr Gold dye (Invitrogen, The results are shown in Figure 2A. The comparative sequence 1 is as follows: Sense strand: 5'-CCUUGAGGCAUACUUCAAA-3' (SEQ ID NO: 250) , Antisense strand: 5'-UUUGAAGUAUGCCUCAAGGUU-3' (sequence no. No. 251).

[0430] The same method was used to determine the stability of complexes 105, 109, 111, and 115 within 72 h. Qualitative measurements were performed and the results are shown in FIG. 2B.

[0431] Figures 2A and 2B show semi-quantitative results of the stability of the test siRNA complexes in human plasma in vitro. As can be seen from the results, the complex of the present disclosure was able to be detected in human plasma for up to 72 hours. The compound was not decomposed, demonstrating excellent stability in human plasma.

[0432] (Experimental Example 2-3) In vitro stability in monkey plasma Complexes 24, 25, 42, 43, 62, 78 and comparative sequence 2 (siRNA concentration was provided as a 0.9 wt% sodium chloride solution containing 20 μM of Comparative sequence 2 has a sense strand and an antisense strand corresponding to SEQ ID NO: 80 and SEQ ID NO: 81, respectively. (the siRNA having the sequence shown but not complexed with any of the complex molecules) 108 μL of 90% cynomolgus monkey plasma (Kosen Biosciences, Tokyo, Japan) was used for each group. The mixture was homogenously mixed with 100% ethanol (purchased from Eppendorf, HQ70082, diluted with PBS) and incubated at 37°C. 10 μL samples were taken at each time point: 2, 4, 6, 8, 24, 48, and 72 hours. The tissue was then immediately flash frozen in liquid nitrogen and stored in a refrigerator at -80°C. After sampling, each sample was diluted 5-fold with 1× PBS (pH 7.4) and then 10 μL was added. At the same time, equimolar amounts of each of the above complexes (2 μM, 2 μl) were added to 8 μl of the The resulting solution was mixed evenly with 1× PBS to obtain 10 μL of untreated monkey plasma sample ( Con) 20% by weight of non-denaturing polyacrylamide gel was mixed and the diluted All samples in each group were loaded with 4 μL of sample loading buffer (20 mM EDT A, 36% by weight glycerin, 0.06% by weight bromphenol blue aqueous solution). After that, the mixture was loaded onto the above-mentioned gel and electrophoresed at a constant current of 80 mA for 60 minutes. After completion, the gel was washed with 1× Sybr Gold dye (Invitrogen, Cat. 1149 4) for 15 minutes, followed by imaging, and the results are shown in Figure 3A.

[0433] By the same method, complexes 105, 109, 111 and 115 and comparison sequence 2 were The stability was measured within 2 h, and the results are shown in Figure 3B.

[0434] Figures 3A and 3B show semi-quantitative results of the stability of the test siRNA in monkey plasma in vitro. The siRNA complexes of the present disclosure were detectable in cynomolgus monkey plasma for up to 72 hours. It was found that the compound was not degraded, indicating excellent stability in monkey plasma.

[0435] (Experimental Example 3) In this experiment, the pharmacokinetics of complexes 24 and 25 in rats was explained. It is clear In this experiment, rats were used in each experimental group (10 rats per group, 10 males and 10 females). Conjugate 24 and Conjugate 25 were administered subcutaneously to each of the 10 and 15 mice (half each) at a single dose of 10 mg / kg and The drug concentrations in the plasma and liver of rats were then measured at each time point. The drug concentration in kidney tissue was detected.

[0436] First, SD rats were analyzed using the PRISTIMA 7.2.0 data system. The subjects were randomly divided by gender according to body weight, and then administered the conjugates at the designated doses. All animals were administered a single subcutaneous dose, calculated according to body weight, at doses of 10 and 15 mg / kg. and 50 mg / kg, and 1 mg / ml and 5 mg / ml of 0.9% sodium chloride aqueous solution. The liquid was administered in a volume of 10 ml / kg. 0 seconds), 30 minutes (±1 minute), 1 hour (±2 minutes), 2 hours (±2 minutes), 6 hours (± 5 minutes), 24 hours (±10 minutes), 48 hours (±20 minutes), 72 hours (±20 minutes) , 120 hours (± 30 minutes), and 168 hours (± 30 minutes) were injected into the jugular vein of rats. Whole blood samples were then centrifuged at 1800 × g for 10 minutes at 2–8°C. Plasma was isolated by centrifugation, and approximately 70 μL of the plasma sample was left in the tube, and the remaining identical sample was then poured into a separate tube. The tubes were then frozen at -70 to -86°C until detection. Liver and kidney tissues were collected from the rats at approximately 24, 48, 72, 120, and 168 hours after injection. The rats were anesthetized with sodium pentobarbital (60 mg / kg) depending on their body weight. / kg intraperitoneal injection), blood was collected from the abdominal aorta, and the rats were euthanized and dissected macroscopically. The liver and kidney of each rat were sampled and stored in 1 mL cryopreservation tubes. The samples were stored at -68°C or below until extraction and analysis.

[0437] Rat plasma and liver were analyzed by HPLC-FLD (high performance liquid chromatography with fluorescence detection). The concentrations of complexes 24 and 25 in kidney tissue were quantitatively detected by the following procedure. Depends on the process. (1) The tissue was ground until the tissue mass was 80 mg or less, and then the tissue and cell lysis solution (T issue and Cell Lysis Solution, Supplier: Epicen tre, No. MTC096H) was added to obtain a tissue homogenate of 66.7 mg / mL. Prepared. (2) The tissue homogenate was ultrasonically treated (150 W, 30 s) to disrupt the cells. (3) For each group of tissue samples, 75 μL of the tissue sample was added to a 96-well PCR plate. In addition to the plate, 5 μL of proteinase K (supplier: Invitrogen, no.: 255 30-015), 10 μL of 10 wt% acetonitrile and 0.01 wt% Tween 20 A mixed aqueous solution of was added. For plasma samples, 20 μL of plasma was added to a 96-well PCR plate and 45 μL of L of tissue and cell lysate, 5 μL of proteinase K and 20 μL of 10 wt% acetonitrile A mixture of 0.01 wt% Tween 20 and 100% ethanol was added. (4) The plate is sealed and placed in a PCR machine (supplier: Applied Biosystems) s, model number: GeneAmp (registered trademark) PCR system 9700), Incubation was carried out at 65°C for 45 minutes. (5) After the incubation, 10 μL of 3 M KCl solution (supplier: Sigma-Aldrich) was added to the culture. Add the mixture, shake to homogenize, and incubate at 4°C for 320 Centrifuge at 0 rcf for 15 minutes. (6) For each group of tissue samples, 80 μL of the supernatant was added to 120 μL of the hybrid mixture. compound (formulation method: 0.5mL of 6μM PNA probe (supplier: Hangzhou Taihe Biological Technology Co., Ltd. Company), 1mL of 200mM Trizma / pH=8, 5mL of 8M urea solution, 3. 5 mL of H2O, 2 mL of acetonitrile). For each group of plasma samples, 40 μL of the supernatant was added to 160 μL of the hybridization mixture ( Mixture: 0.5 mL of 6 μM PNA probe, 1 mL of 200 mM Trizma / pH=8, 5mL of 8M urea solution, 7.5mL of HO, 2mL of acetonitrile) Added. (7) Seal the plate, place it in the PCR machine, incubate it at 95°C for 15 minutes, and then immediately place it on ice. Left on top for 5 minutes. (8) Transfer the samples to another conical-bottom 96-well plate, shake to homogenize, and incubate for 320 Centrifuge at 0 rcf for 1 minute. (9) Samples were loaded, detected, and quantitatively analyzed using HPLC-FLD (liquid phase System supplier: Thermo Fisher, chromatograph model number: ultimate 30 00).

[0438] The analytical results are shown in Figures 4 to 11. Figures 4 to 7 show the results of the analysis of the complex 24 in rat plasma, respectively. Time-course metabolism curve of PK / TK plasma concentration in rats and PK / TK in the liver and kidney of rats The time course metabolism curves of tissue concentration are shown in Figures 8 to 11. Time-course metabolism curve of K / TK plasma concentration and PK / TK tissue concentration in rat liver and kidney The time course metabolism curve of FIG. 4 shows the PK / gap ratio of conjugate 24 in rat plasma at a dose of 10 mg / kg. 1 shows a metabolic curve of TK plasma concentration over time. FIG. 5 shows the effect of Complex 24 on the liver and kidney of rats at a dose of 10 mg / kg. Figure 1 shows the time course of metabolic curves of PK / TK tissue concentrations. FIG. 6 shows the PK / gap ratio of conjugate 24 in rat plasma at a dose of 50 mg / kg. 1 shows a metabolic curve of TK plasma concentration over time. FIG. 7 shows the effect of Complex 24 on the liver and kidney of rats at a dose of 50 mg / kg. Figure 1 shows the time course of metabolic curves of PK / TK tissue concentrations. FIG. 8 shows the PK / gap ratio of conjugate 25 in rat plasma at a dose of 10 mg / kg. 1 shows a metabolic curve of TK plasma concentration over time. FIG. 9 shows the effect of Complex 25 on the liver and kidney of rats at a dose of 10 mg / kg. Figure 1 shows the time course of metabolic curves of PK / TK tissue concentrations. FIG. 10 shows the PK of conjugate 25 in rat plasma at a dose of 50 mg / kg. 1 shows a time-dependent metabolic curve of / TK plasma concentration. FIG. 11 shows the effect of Complex 25 on the liver and kidney of rats at a dose of 50 mg / kg. Figure 1 shows the time course of PK / TK tissue concentration in

[0439] As can be seen from the results in Figures 4 to 11, even at a low dose (10 mg / kg), Even at 50 mg / kg, the plasma concentrations of conjugates 24 and 25 in rats were In liver tissue, all of the α-amyloides decreased rapidly to below the detection limit in the first 1 h. The concentration of L-9 was maintained at a high and stable level for 68 h. As a result, the resulting siRNA complex was significantly enriched and stabilized specifically in the liver. This demonstrated a high degree of targetability.

[0440] (Experimental Example 4) In this experiment, the effect of a complex formed by different complex molecules on the proliferation and proliferation of C57BL We have demonstrated an inhibitory effect on target mRNA in / 6J mice. First, C57BL / 6J mice (all female) were randomly divided into experimental groups. The control complexes were numbered 158 (5 mice) and 14 (5 mice), and the control complexes were numbered 158 (5 mice). The BS control group (8 mice) was numbered. All animals were dosed according to body weight. were calculated and administered subcutaneously at different single doses of 1 mg / kg or 0.1 mg / kg, respectively. The administration volume was 5 ml / kg. To obtain different doses, the complex was diluted at 0.2 mg / ml The drugs were dissolved in 0.9% sodium chloride solution at 0.01 mg / ml and 0.02 mg / ml. The animals were sacrificed at 72 h, the livers were collected, and the liver tissue was homogenized using a tissue homogenizer. Then, total RNA was extracted using RNAVzol (VIGOROUS, lot:161G). Total RNA was obtained by extraction according to standard operating procedures.

[0441] The expression level of TTR mRNA in liver tissue was measured by fluorescent quantitative real-time PCR. Specifically, the Reverse Transcription System ( Promega, Lot#0000223677, REF:A3500) The total RNA extracted according to the instructions was reverse transcribed into cDNA. BI Step One) was used to measure the expression of mTTR mRNA in liver tissue by siRNA. The detection method detects the inhibition efficiency of the GAPDH gene as an endogenous reference gene. The primers for TTR and GAPDH were used as vectors. mTTR and GAPDH were detected.

[0442] See Table 4 for the sequences of detector primers.

[0443] [Table 4]

[0444] In this real-time PCR method, the expression level of mTTR mRNA was measured by the TTR gene expression level. The percentage was calculated according to the following equation: Residual TTR gene expression = (TTR gene copy number in test group / GAPDH copy number in test group) (number of copies of TTR gene in control group / number of copies of GAPDH in control group) × 100%

[0445] Then, the inhibition rate of the complex against mRNA was calculated by the following formula. mRNA suppression rate = (1-TTR gene expression level) x 100%

[0446] The control group was a control group of mice that received PBS in this experiment. The mice in each treatment group were administered with a different siRNA complex. The results are shown in Table 5.

[0447] [Table 5]

[0448] As can be seen from the results, although the siRNA sequences and modifications used were the same, The inhibition rate of the target mRNA by complex 158 was significantly higher than that of the control complex 14. Ta.

[0449] In the following Experimental Examples 5 to 11, siRNA target positions and sequence relatedness experiments were performed. The properties and effects of the siRNA complexes in Tables 3A to 3F were examined.

[0450] (Experimental Example 5) Experiment to verify the effect of siRNA complexes in Table 3A (Experimental Example 5-1) Experiment to verify the off-target effect of Complex 26 Kumico Ui-Tei et.al.,Functional dissect ion of siRNA sequence by systematic DNA substitution:modified siRNA with a DNA s eed arm is a powerful tool for mammalian gene silencing with significantly reduced ed off-target effect. Nucleic Acids Rese The detection process was carried out using the method described in arch, 2008.36(7), 2136-2151. The plasmid was constructed and cotransfected with the test siRNA complex into HEK293A cells. The dual luciferase reporter gene detection kit was used to detect the dual luciferase. The expression level of the luciferase reporter gene was detected. The specific steps were as follows: be.

[0451] 1. Construction of detection plasmid psiCHECK TM -2(Promega TM ) plasmid to generate four recombinant plasmids. The plasmids were constructed. GSCM stands for on-target plasmid, PSCM, GSSM , PSSM represents the off-target plasmid. (1) GSCM matches all 21 nucleotide sequences of the antisense strand of the test complex. Contains a perfectly complementary target sequence. (2) PSCM perfectly matches all 21 nucleotide sequences of the sense strand of the test complex. Contains a complementary sequence of interest. (3) GSSM is nucleotides 1 to 8 from the 5' end of the antisense strand of the test complex. The sequence is completely complementary to the target sequence. The nucleotide sequence from positions 9 to 21 was mismatched complementary to the corresponding target sequence. The mismatch rule is that the 5′ end of the antisense strand of the test complex is any position between 9 and 21. A G, C, A or U nucleotide at the corresponding position of the target sequence is The mismatch is with T, A, C or G. (4) PSSM is a nucleotide sequence consisting of nucleotides 1 to 8 from the 5' end of the sense strand of the test complex. Contains a completely complementary target sequence. The nucleotide sequence and its corresponding target sequence are mismatched in a complementary manner. The target complex is a complex consisting of a G, C, A or C residue at any position between 9 and 21 from the 5' end of the sense strand of the target complex. each of which is a U nucleotide, and each of which is a T, A, C or G nucleotide at the corresponding position of the target sequence. The problem was that it was a mismatch.

[0452] psiCHECK the target sequence TM The fusion protein was inserted into the XhoI / NotI site of the -2 plasmid. It got crowded.

[0453] 2. Transfection Lipofectamine in 96-well plates TM 2000(Invitro gen) instructions, 11 groups of constant siRNA and plasmid were prepared. The above plasmids were co-transfected with the corresponding siRNAs at the concentrations listed above. Specifically, 10 ng of plasmid was transfected per well, and 0.01 mg of plasmid was added to each well. 0.2 μL Lipofectamine TM 2000, and cotransfect each well. The final concentrations of the injected siRNA ranged from 100 nM to 0.0001 nM (4-fold series). The wells were diluted 100% and 100% ethanol (dilution of each well) with three replicate wells per group.

[0454] 3. Detection After 24 hours of co-transfection, dual luciferase reporter gene assay Dual luciferase reporter gene assay kit Kit (Promega, cat. E2940) was used according to the manufacturer's instructions. 93A cells were lysed and the expression level of the dual luciferase reporter gene was detected. In each specific concentration test group, no complex treatment was used as the control (con). Renilla luciferase protein level (Fr) versus Renilla luciferase protein level (Fr) Normalization was performed using Ren. Different concentrations of siRNA The activity results were plotted using the dose-effect curves and analyzed using Graphad 5.0 software. The function log(Inhibitor) and the response-change slope of the software are used, and the following equation is used: Curve modeling was performed.

[0455]

number

[0456] Based on the dose-effect curve, the IC5 of the test complex corresponding to the GSCM was calculated. A zero value was established and compared with PSCM, GSSM or PSSM.

[0457] For complex 24, the results are shown in Figures 12A-12D, which correspond to GSCM and for complex 2 4 IC 50 The calculated value was 0.0019 nM, which was not significant for PSCM, GSSM, or P Compared with SSM, complex 24 had no obvious inhibitory effect at any siRNA concentration. The siRNA complexes disclosed herein have high in vitro activity and low off-target effects. was shown.

[0458] (Experimental Example 5-2) In this experiment, the HBV m Demonstrating suppression efficiency on RNA expression levels In this experiment, complexes 16 and 24 were used to infect HBV transgenic mice C57BL HBV mRNA expression in / 6J-Tg(Alb1HBV)44Bri / J The inhibitory efficiency was investigated.

[0459] First, C57BL / 6J-Tg(Alb1HBV)44Bri / J mice were treated with serum HBs They were randomly divided according to the Ag content (all females, 4 mice per group), and each group was A saline (NS) control group was added, numbered as complex 16 and complex 24. The dosage was calculated according to the body weight, and a single dose of 1 mg / kg was administered subcutaneously. The concentrations of the complexes were 0.2mg / ml and 0. The dose was 0.02 mg / ml and the administration volume was 5 ml / kg. On the 14th day after drug administration, the animals The animals were killed, and the livers were harvested and stored in RNA Later (Sigma Aldrich). The liver tissue was homogenized using a tissue homogenizer. Total RNA was extracted using Trizol. Total RNA was obtained by extraction according to standard operating procedures.

[0460] HBV mRNA expression levels in liver tissue were measured using fluorescent quantitative real-time PCR. Specifically, ImProm-II TM Using a reverse transcription kit (Promega), Total RNA extracted from the ELISA kit was reverse tran...

Claims

1. A conjugate having a structure as shown in formula (1): 【Chemistry 1】 During the ceremony, n1 is an integer from 1 to 3, and n3 is an integer from 0 to 4, Each of m1, m2, and m3 is independently an integer from 2 to 10; Each R １０ , R １１ , R １２ , R １３ , R １４ and R １５ are each independently H, C １ -C １０ Alkyl group, C １ -C １０ Halogenated alkyl group and C １ -C １０ alkoxy groups, R ３ is a group having a structure represented by formula A59; 【Chemistry 2】 where E 1 is OH, SH or BH 2 , and Nu is a functional oligonucleotide; R ２ is a straight chain alkylene group of 1 to 20 carbon atoms in length, one or more of which is selected from the group consisting of C(O), NH, O, S, CH=N, S(O) ２ , C ２ -C １０ Alkenylene group, C ２ -C １０ Alkynylene group, C ６ -C １０ Arylene group, C ３ -C １８ Heterocyclylene group and C ５ -C １０ heteroarylene groups, R ２ is C １ -C １０ Alkyl group, C ６ -C １０ Aryl group, C ５ -C １０ Heteroaryl group, C １ -C １０ Halogenated alkyl group, -OC １ -C １０ Alkyl group, -OC １ -C １０ Alkylphenyl group, -C １ -C １０ Alkyl-OH, -OC １ -C １０ Halogenated alkyl group, -SC １ -C １０ Alkyl group, -SC １ -C １０ Alkylphenyl group, -C １ -C １０ Alkyl-SH, -SC １ -C １０ Halogenated alkyl groups, halogen substituents, -OH, -SH, -NH ２ , -C １ -C １０ Alkyl-NH ２ , -N(C １ -C １０ Alkyl group) (C １ -C １０ alkyl group), -NH(C １ -C １０ alkyl group), -N(C １ -C １０ Alkyl group) (C １ -C １０ alkylphenyl group), -NH(C １ -C １０ alkylphenyl group), cyano group, nitro group, -CO ２ H, -C(O)O(C １ -C １０ alkyl group), -CON(C １ -C １０ Alkyl group) (C １ -C １０ alkyl group), -CONH(C １ -C １０ alkyl group), -CONH ２ , -NHC(O)(C １ -C １０ -NHC(O) (phenyl group), -N(C １ -C １０ alkyl)C(O)(C １ -C １０ alkyl group), -N(C １ -C １０ alkyl)C(O)(phenyl group), -C(O)C １ -C １０ Alkyl group, -C(O)C １ -C １０ Alkylphenyl group, —C(O)C １ -C １０ Halogenated alkyl groups, -OC(O)C １ -C １０ Alkyl group, -SO ２ (C １ -C １０ alkyl group), -SO ２ (phenyl group), -SO ２ (C １ -C １０ halogenated alkyl group), -SO ２ N.H. ２ , -SO ２ NH (C １ -C １０ alkyl group), -SO ２ NH (phenyl group), -NHSO ２ (C １ -C １０ alkyl group), -NHSO ２ (phenyl group) and -NHSO ２ (C １ -C １０ and optionally having one or more substituents selected from the group consisting of halogenated alkyl groups, Each L １ are independently a straight chain alkylene group of 1 to 70 carbon atoms in length, one or more of which is selected from the group consisting of C(O), NH, O, S, CH=N, S(O) ２ , C ２ -C １０ Alkenylene group, C ２ -C １０ Alkynylene group, C ６ -C １０ Arylene group, C ３ -C １８ Heterocyclylene group and C ５ -C １０ heteroarylene groups; L １ is C １ -C １０ Alkyl group, C ６ -C １０ Aryl group, C ５ -C １０ Heteroaryl group, C １ -C １０ Halogenated alkyl group, -OC １ -C １０ Alkyl group, -OC １ -C １０ Alkylphenyl group, -C １ -C １０ Alkyl-OH, -OC １ -C １０ Halogenated alkyl group, -SC １ -C １０ Alkyl group, -SC １ -C １０ Alkylphenyl group, -C １ -C １０ Alkyl-SH, -SC １ -C １０ Halogenated alkyl groups, halogen substituents, -OH, -SH, -NH ２ , -C １ -C １０ Alkyl-NH ２ , -N(C １ -C １０ Alkyl group) (C １ -C １０ alkyl group), -NH(C １ -C １０ alkyl group), -N(C １ -C １０ Alkyl group) (C １ -C １０ alkylphenyl group), -NH(C １ -C １０ alkylphenyl group), cyano group, nitro group, -CO ２ H, -C(O)O(C １ -C １０ alkyl group), -CON(C １ -C １０ Alkyl group) (C １ -C １０ alkyl group), -CONH(C １ -C １０ alkyl group), -CONH ２ , -NHC(O)(C １ -C １０ -NHC(O) (phenyl group), -N(C １ -C １０ alkyl)C(O)(C １ -C １０ alkyl group), -N(C １ -C １０ alkyl)C(O)(phenyl group), -C(O)C １ -C １０ Alkyl group, -C(O)C １ -C １０ Alkylphenyl group, —C(O)C １ -C １０ Halogenated alkyl groups, -OC(O)C １ -C １０ Alkyl group, -SO ２ (C １ -C １０ alkyl group), -SO ２ (phenyl group), -SO ２ (C １ -C １０ halogenated alkyl group), -SO ２ N.H. ２ , -SO ２ NH (C １ -C １０ alkyl group), -SO ２ NH (phenyl group), -NHSO ２ (C １ -C １０ alkyl group), -NHSO ２ (phenyl group) and -NHSO ２ (C １ -C １０ and optionally having one or more substituents selected from the group consisting of halogenated alkyl groups, Each M １ is one selected from ligands capable of binding to the asialoglycoprotein receptor (ASGPR) on the liver surface.

2. Each L １ is independently selected from the group consisting of groups A1-A26 and any combination thereof: 【Chemistry 3】 wherein each j1 is independently an integer from 1 to 20; each j2 is independently an integer from 1 to 20; Each R' is independently C １ -C １０ is an alkyl group, each Ra is independently selected from the group consisting of A27-A45, and any combination thereof; 【Chemistry 4】 【change】 Each Rb is independently C １ -C １０ is an alkyl group, 【Chemistry 5】 represents the site at which the group is attached to the remainder of the molecule.

3. L １ is selected from the group consisting of A1, A4, A5, A6, A8, A10, A11, A13 groups and combinations thereof; or L １ The complex of claim 2, wherein is a combination of at least two bonds among groups A1, A4, A8, A10 and A11.

4. L １ The complex according to any one of claims 1 to 3, wherein the length of said complex is 3 to 25 atoms, or 4 to 15 atoms.

5. The complex according to claim 1, wherein m1, m2 and m3 are each independently an integer of 2 to 5, or m1 = m2 = m3.

6. Each M １ is independently selected from the group consisting of D-glucose, L-glucose, galactosamine, and N-acetylgalactosamine.

7. R ２ But, R ３ The complex according to claim 6, which forms a phosphate bond with a P atom in

8. 2. The complex of claim 1 having a structure as shown in formula (3), (4), (5), (6), (7), (8), (9), (10), (11), (12), (13), (14), (15), (16), (17), (18), (19), (20), (21) or (22): 【Chemistry 6】 【change】 【change】 【change】 【change】 【change】

9. 2. The complex of claim 1, wherein the functional oligonucleotide is selected from the group consisting of small interfering RNA, microRNA, anti-microRNA, microRNA antagonist, microRNA mimic, decoy oligonucleotide, immune stimulant, G-quadruplex, alternative splice variant, single stranded RNA, antisense nucleic acid, nucleic acid aptamer, stem loop RNA, mRNA fragment, activator RNA or DNA.

10. The functional oligonucleotide is a single-stranded oligonucleotide, and the P atom in formula A59 is attached to the 3' end of the single-stranded oligonucleotide; or The complex of claim 9, wherein the functional oligonucleotide is a double-stranded oligonucleotide, the double-stranded oligonucleotide comprises a sense strand and an antisense strand, and the P atom in formula A59 is bound to the 3' end of the sense strand.

11. The complex of claim 10, wherein each nucleotide in the siRNA is independently modified or unmodified, the siRNA comprises a sense strand and an antisense strand, the sense strand comprises nucleotide sequence 1, the antisense strand comprises nucleotide sequence 2, the nucleotide sequence 1 and the nucleotide sequence 2 are both 19 nucleotides in length and form a double-stranded complementary region in a reverse complementary manner at least in part, the nucleotide sequence 2 is at least in part complementary to a first nucleotide sequence fragment which is a nucleotide fragment in a target mRNA, and the target mRNA refers to the mRNA of a gene abnormally expressed in liver cells.

12. 12. The complex of claim 11, wherein the target mRNA is an mRNA corresponding to ApoB, ApoC, ANGPTL3, PCSK9, SCD1, TIMP-1, Col1A1, FVII, STAT3, p53, HBV or HCV.

13. The complex of claim 11, wherein the nucleotide sequence 1 and the first nucleotide sequence fragment are equal in length and have no more than three nucleotide differences, the nucleotide sequence 2 and the nucleotide sequence B are equal in length and have no more than three nucleotide differences, and the nucleotide sequence B is a nucleotide sequence that is completely reverse complementary to the first nucleotide sequence fragment.

14. 14. The complex of claim 13, wherein the nucleotide difference between nucleotide sequence 2 and nucleotide sequence B comprises a difference at the first nucleotide position Z' in nucleotide sequence 2 from the 5' end to the 3' end.

15. The complex according to claim 11, wherein the sense strand further comprises nucleotide sequence 3, the antisense strand further comprises nucleotide sequence 4, the nucleotide sequence 3 and the nucleotide sequence 4 are equal in length and each is 1 to 4 nucleotides, the nucleotide sequence 3 is bound to the 5' end of the nucleotide sequence 1, the nucleotide sequence 4 is bound to the 3' end of the nucleotide sequence 2, the nucleotide sequence 4 is complementary to a second nucleotide sequence fragment, the second nucleotide sequence fragment refers to a nucleotide sequence adjacent to the first nucleotide sequence fragment in the target mRNA and having the same length as the nucleotide sequence 4, and the nucleotide sequence 3 and the nucleotide sequence 4 are essentially completely reverse complementary or completely reverse complementary.

16. The complex of claim 11 or 15, wherein the siRNA further comprises nucleotide sequence 5, the nucleotide sequence 5 being 1 to 3 nucleotides in length and attached to the 3' end of the antisense strand and constituting a 3' overhanging end of the antisense strand.

17. The complex of claim 11, wherein each nucleotide in the sense strand and the antisense strand is independently a fluoro-modified nucleotide or a non-fluoro-modified nucleotide, the fluoro-modified nucleotide refers to a nucleotide in which the hydroxy group at the 2' position of the ribose group of the nucleotide is replaced with fluorine, and the non-fluoro-modified nucleotide refers to a nucleotide or nucleotide analog in which the hydroxy group at the 2' position of the ribose group of the nucleotide is replaced with a non-fluorinated group, and the nucleotide analog is a group that can replace a nucleotide in a nucleic acid and has a structure different from any one of adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, or thymine deoxyribonucleotide.

18. The complex of claim 17, wherein both the sense strand and the antisense strand contain fluoro-modified and non-fluoro-modified nucleotides, the fluoro-modified nucleotides are present in nucleotide sequence 1 and nucleotide sequence 2, the number of fluoro-modified nucleotides in nucleotide sequence 1 is 5 or less, the nucleotides at positions 7, 8, and 9 of nucleotide sequence 1 from the 5' end to the 3' end are fluoro-modified nucleotides, the number of fluoro-modified nucleotides in nucleotide sequence 2 is 7 or less, and the nucleotides at positions 2, 6, 14, and 16 of nucleotide sequence 2 from the 5' end to the 3' end are fluoro-modified nucleotides.

19. 18. The complex of claim 17, wherein each non-fluoro modified nucleotide is a methoxy modified nucleotide, said methoxy modified nucleotide referring to a nucleotide in which the 2'-hydroxy group of the ribose group is replaced with methoxy.

20. In the siRNA, at least one phosphate group is a thiophosphate, the thiophosphate being Between the first and second nucleotides of the 5' end of the sense strand, Between the second and third nucleotides of the 5' end of the sense strand, Between the first and second nucleotides of the 3' end of the sense strand, Between the second and third nucleotides of the 3' end of the sense strand, Between the first and second nucleotides at the 5' end of the antisense strand, between the second and third nucleotides at the 5' end of the antisense strand, Between the first and second nucleotides at the 3' end of the antisense strand, and The complex according to claim 11, wherein the complex is bound to at least one nucleotide selected from the group consisting of between the second and third nucleotides at the 3' end of the antisense strand.

21. The complex according to claim 11, wherein the 5'-terminal nucleotide of the antisense strand is a 5'-phosphate nucleotide or a 5'-phosphate analog-modified nucleotide.

22. A therapeutic agent for treating a pathological condition or disease caused by expression of a specific gene in liver cells in a subject in need thereof, comprising administering to the subject an effective amount of a complex according to any one of claims 1 to 21.

23. The therapeutic agent according to claim 22, wherein the specific gene is selected from the group consisting of a hepatitis B virus gene, angiopoietin-like protein 3 gene, and an apolipoprotein C3 gene.

24. The therapeutic agent according to claim 22, wherein the disease is selected from the group consisting of chronic liver disease, hepatitis, liver fibrosis, hepatic hyperplastic disease and dyslipidemia.

25. The therapeutic agent according to claim 22, wherein the lipid abnormality is hypercholesterolemia, hypertriglyceridemia or atherosclerosis.

26. An agent for inhibiting the expression of a specific gene in a hepatocyte, comprising the complex according to any one of claims 1 to 21, for contacting the hepatocyte.

27. The expression inhibitor according to claim 26, wherein the specific gene is ApoB, ApoC, ANGPTL3, PCSK9, SCD1, TIMP-1, Col1A1, FVII, STAT3, p53, HBV or HCV.

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