Nucleic acids, compositions and complexes containing the same, and methods of preparation and use

Abstract

To provide an siRNA composite that suppresses angiopoietin-like protein 3 gene expression, and a pharmaceutical composition including the siRNA composite.SOLUTION: An siRNA composite includes an siRNA and a complex group complexed to the siRNA, where the siRNA includes a sense strand and an antisense strand, each nucleotide of the siRNA is each independently a modified or unmodified nucleotide, the sense strand includes 5'-CCAAGAGCACCAAGAACUZA-3', the antisense strand includes 5'-Z'BAGUUCUUGGUGCUCUUGG-3', wherein, ZA is selected from A, U, G and C, Z'B is a first nucleotide at the 5' terminal of the antisense strand and complementary with ZA, and the length of the sense strand is 19 to 23 nucleotides, and the length of the antisense strand is 20 to 26 nucleotides.SELECTED DRAWING: Figure 12C

Description

[Technical Field]

[0001] The present disclosure relates to nucleic acids, compositions and complexes comprising such nucleic acids, and methods of preparation and use. [Background technology]

[0002] Abnormal lipids (blood lipids), also known as hyperlipidemia, are caused by abnormalities in fat metabolism or function, resulting in plasma It is a systemic disease in which lipid levels are elevated above normal levels, posing a serious threat to the health of patients worldwide. Conventional drugs for treating dyslipidemia are mainly statins, cholesterol absorption inhibitors, and These include fatty acids, probucol, fibrates, and nicotinic acid and its derivatives.

[0003] Angiopoietin-like protein 3 (ANGPTL3) is a secreted protein expressed primarily in the liver. It is a protein and is named after its similar gene structure to angiopoietin. Studies have shown that dyslipidemia is associated with high expression of ANGPTL3, and that ANGPTL3 regulates lipid metabolism. It regulates lipid metabolism by binding to adipose tissue and inhibiting the activity of lipoprotein lipase. It was demonstrated that low expression of ANGPTL3 inhibits atherosclerosis due to dyslipidemia. Therefore, silencing gene expression at the gene level can reduce Therefore, blocking the production of ANGPTL3 would be the most ideal therapeutic approach. There is no doubt that small interfering RNA (SMIR) , siRNA) is called RNA interference (RNAi). Mechanistically based, sequence-specific inhibition or blocking of expression of any target gene of interest Therefore, the purpose of treating the disease can be achieved. Summary of the Invention [Problem to be solved by the invention]

[0004] In the development of small RNA drugs, stabilizing modifications of siRNA and their delivery systems are two key factors. It's technology. [Means for solving the problem]

[0005] In some embodiments, the present disclosure provides a method for inhibiting ANGPTL3 gene expression. providing an RNA, the siRNA comprising a sense strand and an antisense strand; each nucleotide in is independently a modified or unmodified nucleotide, The sense strand comprises nucleotide sequence I, and the antisense strand comprises nucleotide sequence I nucleotide sequence I and nucleotide sequence II, forming a double-stranded region in a reverse complementary manner, wherein the nucleotide sequence I comprises the nucleotide sequence A; The nucleotide sequence A is equal in length to the nucleotide sequence shown in SEQ ID NO: 1, The nucleotide sequence II has three or fewer nucleotide differences, and the nucleotide sequence II does not contain the nucleotide sequence B. The nucleotide sequence B is equal in length to the nucleotide sequence shown in SEQ ID NO: 2. , with three or fewer nucleotide differences; 5'-CCAAGAGCACCAAGAACUZ-3' (SEQ ID NO: 1), 5'-Z'AGUUCUUGGUGCUCUUGG-3' (SEQ ID NO: 2) where Z is A and Z' is U. The nucleotide sequence A contains a nucleotide Z at position Z. A The said Nu Nucleotide sequence B contains nucleotide Z' at position Z' B and Z' B is the first nucleotide at the 5' end of the antisense strand.

[0006] In some embodiments, the present disclosure provides a method for the preparation of a medicament comprising administering to a subject a medicament the siRNA of the present disclosure and a pharmaceutically acceptable carrier. A pharmaceutical composition is provided that includes a carrier.

[0007] In some embodiments, the present disclosure provides siRNAs and the The present invention provides an siRNA complex (conjugate) containing a conjugated group that is conjugated and bound to the siRNA. To provide.

[0008] In some embodiments, the present disclosure provides siRNA and / or drug compositions of the present disclosure. and / or the siRNA complex, The present invention provides a use in the preparation of a medicament for treatment and / or prevention.

[0009] In some embodiments, the present disclosure provides siRNA and / or drug compositions of the present disclosure. and / or administering an effective amount of the siRNA complex to a subject suffering from dyslipidemia. The present invention provides a method for treating and / or preventing dyslipidemia, comprising:

[0010] In some embodiments, the present disclosure provides siRNA and / or drug compositions of the present disclosure. and / or contacting an effective amount of the siRNA complex with the hepatocyte. The present invention provides a method for suppressing the expression of the ANGPTL3 gene in a mammal.

[0011] In some embodiments, the present disclosure provides siRNA and / or drug compositions of the present disclosure. and / or a kit comprising the siRNA complex.

[0012] [Incorporated by reference] All publications, patents, and patent applications mentioned herein are the property of their respective owners. The patent applications are incorporated herein by reference to the same extent as if they were specifically and individually incorporated by reference. and is incorporated herein by reference. [Effects of the Invention]

[0013] The siRNA and siRNA complexes provided by the present disclosure have good stability, high gene expression, and It has inhibitory activity, very low off-target effects, and can significantly reduce lipid levels. can.

[0014] In some embodiments, the siRNA provided by the present disclosure The composition or siRNA complex containing the siRNA has higher stability and / or higher activity in the body. In some embodiments, the siRNA provided by the present disclosure may The siRNA composition or siRNA complex is capable of absorbing at least 20%, 30%, 40%, or The target gene expression suppression rate is 50%, 60%, 70%, 80%, 90%, or 95%. In some embodiments, the siRNA, siRNA composition or The siRNA complexes are expressed in vivo at least 20%, 30%, 40%, 50%, 60%, 70%, and The results show that the ANGPTL3 gene expression suppression rates are 80%, 90%, or 95%. In embodiments, the present disclosure provides an siRNA, an siRNA composition, or an siRNA. The complex is absorbed by the body at least 20%, 30%, 40%, 50%, 60%, 70%, 80% , 90% or 95% of the intrahepatic ANGPTL3 gene expression is suppressed. The siRNA, siRNA composition, or siRNA complex provided by the present disclosure is at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% in the body. % or 95% of the intrahepatic ANGPTL3 gene expression suppression rate in the animal model. In some embodiments, the siRNA, siRNA composition or siGe gene provided by the present disclosure RNA complexes are at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% suppression rate of intrahepatic ANGPTL3 gene expression in human subjects In some embodiments, the siRNA provided by the present disclosure Compositions or siRNA complexes containing A show no obvious off-target effects. An off-target effect may be, for example, the suppression of normal expression of a gene that is not the target gene. Binding / suppression of off-target gene expression is 50% less than on-target gene effects. If the off-target effect is less than 40%, 30%, 20%, or 10%, the off-target effect is not significant. It is believed that there is no

[0015] In some embodiments, the siRNAs provided by the present disclosure are effective ANGP TL3 gene expression suppression properties and siRNA detection in the psiCHECK system. C 50 The concentration is 3 to 30 pM, and even at 5 nM, the modified siRNA of the present disclosure has an off-target activity. No mitotic effect was observed, and the modified siRNA of the present disclosure exhibited a potent cytotoxic effect in an in vitro lysosomal lysate. It has good stability and does not decompose for at least 24 hours.

[0016] In some embodiments, the siRNA complexes provided by the present disclosure have good stability. The results were consistent in in vitro lysosomal lysates, human plasma, and monkey plasma. Maintain stability.

[0017] In some embodiments, the siRNA complexes provided by the present disclosure have excellent A It showed NGPTL3 mRNA suppression efficiency and significantly reduced lipid levels. In some embodiments, ANGPTL3 mRNA levels in mice are elevated 14 days after a single subcutaneous administration. In some embodiments, the A suppression rate is as high as 95% or more. The maximum suppression rate of triglycerides (TG) was 93% and total cholesterol (CHO) was 100%. The maximum suppression rate for TG was 83%, and on the 154th day after drug administration, the suppression rate for TG was over 55%. The inhibition rate against CHO can be maintained at 40% or more. Compared to the complexes consisting of multiple molecules provided by the prior art, the siRNs provided by the present disclosure Complex A showed superior gene suppression rate and lipid-lowering ability, and was effective at low doses. It maintained its excellent lipid-lowering effect over a long experimental period of 189 days with low-frequency administration. can be done.

[0018] Thus, the siRNAs, drug compositions and siRNA complexes provided by the present disclosure suppresses ANGPTL3 gene expression and prevents lipid abnormalities caused by ANGPTL3 gene overexpression can effectively treat and / or prevent diseases, and has bright prospects for application .

[0019] Other features and advantages of the present disclosure are described in the detailed description section below. Let me explain in detail. [Brief explanation of the drawings]

[0020] In order to more clearly describe the embodiments of the present invention and the technical solutions of the prior art, the following is a summary: The drawings used in the examples and the prior art will be briefly explained. The drawings described below are merely for illustrative purposes only. These are merely some examples of the invention, and a person skilled in the art can easily implement these without any creative effort. Furthermore, it is clear that other figures can be derived from these figures. [Figures 1A-1D] 1 shows the on-target activity and off-target effects of siRNA 8 in the in vitro psiCHECK system. [Figures 2A-2D] 1 shows the on-target activity and off-target effects of siRNA 1 in an in vitro psiCHECK system. [Figure 3A] 1 shows the inhibitory effect of siRNA of the present disclosure on the expression level of ANGPTL3 mRNA in a Huh7 cell line. [Figure 3B] 1 shows the inhibitory effect of the siRNA complex of the present disclosure on the expression level of ANGPTL3 mRNA in a Huh7 cell line. [Figure 4A] 1 shows the results of semi-quantitative detection of the stability of siRNAs of the present disclosure in vitro in mouse-derived lysosomal lysates. [Figure 4B] 1 shows the results of semi-quantitative detection of the stability of siRNA complexes of the present disclosure in vitro in mouse-derived lysosomal lysates. [Figure 4C] 1 shows the results of semi-quantitative detection of the stability of siRNA complexes of the present disclosure in human plasma in vitro. [Figure 4D] 1 shows the results of semi-quantitative detection of the stability of siRNA complexes of the present disclosure in monkey plasma in vitro. [Figures 5A-5D] 1 shows the inhibitory effect of the siRNA complex of the present disclosure on lipid levels in normal BALB / c mice. [Figures 6A-6D] 1 shows the inhibitory effect of the siRNA complex of the present disclosure on the amount of ANGPTL3 mRNA expression in the liver of normal BALB / c mice. [Figures 7A-7D] 1 shows the inhibitory effect of the siRNA complex of the present disclosure on lipid levels in normal BALB / c mice. [Figure 8A-8B]1 shows the inhibitory effect of the siRNA complex of the present disclosure on lipid levels in ob / ob mice. [Figure 8C] 1 shows the inhibitory effect of the siRNA complex of the present disclosure on the expression level of ANGPTL3 mRNA in the liver of ob / ob mice. [Figure 9A-9B] 1 shows the inhibitory effect of the siRNA complex of the present disclosure on lipid levels in ob / ob mice. [Figure 9C] 1 shows the inhibitory effect of the siRNA complex of the present disclosure on the expression level of ANGPTL3 mRNA in the liver of ob / ob mice. [Figures 10A-10B] 1 shows the time-dependent inhibitory effect of complex 1 on serum triglycerides (TG) and total cholesterol (CHO) in hyperlipidemic model mice. [Figures 11A-11D] 1 shows the time-dependent suppressive effects of different doses of complex 2 on serum total cholesterol (CHO) and triglycerides (TG) in hyperlipidemic model mice. [Figures 12A-12B] 1 shows the time-dependent inhibitory effect of Complex 2 on serum triglycerides (TG) and total cholesterol (CHO) in monkeys with metabolic syndrome. [Figure 12C] 1 shows the inhibitory effect of complex 2 on the expression level of ANGPTL3 mRNA in monkeys with metabolic syndrome. DETAILED DESCRIPTION OF THE INVENTION

[0021] The following describes in detail the embodiments of the present disclosure. The detailed description is merely for the purpose of explaining or interpreting the present disclosure, and It should be understood that this is not intended to be limiting.

[0022] In the present disclosure, the ANGPTL3 gene refers to the mRNA sequence of which is Genbank accession no. Refers to the gene represented by NM_014495.3.

[0023] [Definition] Unless otherwise stated in the context, the capital letters C, G, U, and A represent the base sequences of nucleotides. A lowercase m indicates that one nucleotide adjacent to the left of the m is methoxy-modified. A lowercase letter f indicates a nucleotide adjacent to the left of the letter f. The nucleotide is a fluoro-modified nucleotide, and the lowercase letter s indicates that the nucleotide is a fluoro-modified nucleotide. It indicates that two adjacent nucleotides are linked by a thiophosphate group. P1 is a nucleotide in which one nucleotide adjacent to the right of P1 is a 5'-phosphate nucleotide or represents a 5'-phosphate analog modified nucleotide, and the combination letter VP represents the combination The nucleotide immediately to the right of the VP is a vinyl phosphate-modified nucleotide. The combination letter Ps indicates that the combination letter Ps is a digit adjacent to the right of the combination letter Ps. The capital letter P indicates that the nucleotide is a phosphorothioate-modified nucleotide. The nucleotide immediately to the right of the letter P is a 5'-phosphate nucleotide. represent.

[0024] In this context, the term "fluoro-modified nucleotide" refers to a nucleotide having a ribose group of This refers to a nucleotide in which the hydroxy group at the 2' position is replaced with fluorine, and is called a "non-fluoro-modified nucleotide." "Retide" refers to a nucleotide in which the hydroxy group at the 2' position of the ribose group is replaced with a non-fluorine group. "Nucleotide analog" refers to a nucleotide or nucleotide analog that is Adenine ribonucleotides, which can substitute for nucleotides in nucleic acids, Guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide or refers to a group whose structure is different from thymine deoxyribonucleotide, e.g., isonucleotide , bridged nucleic acid (abbreviated as BNA) ) or acyclic nucleotides. The "methoxy-modified nucleotide" refers to a nucleotide having a ribose group It refers to a nucleotide in which the 2'-hydroxy group is replaced with methoxy.

[0025] In the context of this specification, the terms "complementary" or "reverse complementary" are used interchangeably. This may be used in the sense well known to those skilled in the art, i.e., in a double-stranded nucleic acid molecule, the bases of one strand are separated from the bases of the other strand. It means that the bases on the chain are complementary to each other. In DNA, the purine bases Adenine (A) in the nucleobase is always replaced by thymine (T) (or thymine in RNA), a pyrimidine base. The purine base guanine (C) always pairs with the pyrimidine salt Each base pair consists of one purine and one pyrimidine. Adenine on one strand always pairs with thymine (or uracil) on the other strand. If guanine always pairs with cytosine, then both strands are complementary, or It is believed that the sequence of the strand can be inferred from the sequence of its complementary strand. In the art, the term "match" refers to a complementary pairing of bases at corresponding positions in a double-stranded nucleic acid. This means that it does not exist.

[0026] Unless otherwise explained in the context, "essentially reverse complementary" refers to the complementary nature of two related sequences. "Substantially reverse complementary" refers to the presence of three or fewer base mismatches between the peptide sequences. " refers to the presence of no more than one base mismatch between two nucleotide sequences; "Perfectly complementary" means that there are no base mismatches between two nucleotide sequences. Point.

[0027] In this context, one nucleotide sequence has a "nucleotide difference" with another nucleotide sequence. The presence of "difference" means that the type of base at the same position in the former is different from that in the latter. For example, when one nucleotide base in the latter is A, the same If the corresponding nucleotide base at the same position is U, C, G, or T, Nucleotide differences between the two nucleotide sequences are recognized. In embodiments, an abasic nucleotide or its equivalent is substituted for the nucleotide at the original position. When a nucleotide difference is used, it is considered that a nucleotide difference has occurred at that position.

[0028] In this context, the siRNA, siRNA-containing composition or siRNA complex of the present disclosure In describing the preparation method of the compound, unless otherwise specified, the nucleoside monomer (nuc) The leoside monomer is used in the siRNA or siRNA complex to be prepared. The modifications or modifications used in solid-phase phosphoramidite synthesis depend on the type of nucleotide and the procedure used. represents unmodified nucleoside phosphoramidite monomers (unmodified or m modified RNA phosphoramidites. RNA phospho Phosphoramidites are also called Nucleoside phosphoramidites. Phosphoramidite solid-phase synthesis is a method used for RNA synthesis known to those skilled in the art. The nucleoside monomers used in this disclosure are all commercially available products. It is available for purchase.

[0029] In the context of this disclosure, unless otherwise specified, "composite" means that each component has a specific function. refers to the covalent bond between two or more chemical moieties, and accordingly, "Conjugate" refers to a compound formed by covalent bonding between the chemical moieties. Furthermore, an "siRNA complex" refers to a complex that contains one or more chemical moieties with specific functions. In the following text, the term "covalently linked siRNA" refers to a compound formed by covalently linking siRNA to the siRNA of the present disclosure. The siRNA complex may also be simply referred to as a "complex." The siRNA complexes represented by formula (305) and formula (307) are generic names for the siRNA complexes. A general term for the complex, or the siRNA complex represented by formula (305), formula (307), or formula (308). In the context of this disclosure, a "composite molecule" is understood to be a molecule that is formed by reacting Specific siRNAs that can be conjugated to iRNA to ultimately form siRNA complexes of the present disclosure. It should be understood that the compound is a compound.

[0030] As used herein, a dash that is not between two alphabetic characters or symbols The slash ("-") is used to indicate the location of the attachment point of a substituent. For example, -C1 -C 10 Alkyl-NH2 is C1-C 10 Attached via an alkyl group.

[0031] As used herein, "any" or "optionally" means any event or The circumstances may or may not occur, and the foregoing description may or may not be true unless the event or circumstances occur. For example, "optionally substituted alkyl" means , including "alkyl" and "substituted alkyl" as defined in the following sentence. For any group containing groups, it is important to understand that these groups may result in sterically impractical, synthetically impractical, and / or unfeasible reactions. It is not intended to introduce any substitutions or substitution patterns that are inherently unstable and / or unstable. It will be understood by those skilled in the art that

[0032] As used herein, "alkyl" refers to both straight and branched chain groups 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 carbon atoms. Name alkyl residues with a specific number of carbon atoms, including straight-chain and branched-chain alkyls. When used, 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. "Propyl" includes n-propyl and isopropyl. Alkylene is a subset of alkyl, similar to alkyl but with two points of attachment. It refers to the residue that

[0033] 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 molecule from the adjacent carbon atom of the The double bonds may be in the cis or trans configuration. Typical alkenyls include vinyl and , prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1- propenyl such as prop-2-en-2-yl and but-1-en-1-yl yl, but-1-en-2-yl, 2-methylprop-1-en-1-yl, but-2- buta-1,3-dien-1-yl, buta-2-en-2-yl, buta- butenyl, such as 1,3-dien-2-yl. In some embodiments, alkenyl has 2 to 20 carbon atoms, but in other embodiments Alkenylene has 2 to 10, 2 to 8, or 2 to 6 carbon atoms. Alkenyl is a subset of alkynyl and refers to residues similar to alkenyl but having two points of attachment.

[0034] 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 molecules from adjacent carbon atoms of a cyclohexyl. The alkyl groups include ethynyl, prop-1-yn-1-yl, prop-2-yn-1-yl, and prop-1-yn-1-yl. propynyl, but-1-yn-1-yl, but-1-yn-3-yl, but- butynyl, such as 3-yn-1-yl. In embodiments, alkynyl has 2 to 20 carbon atoms, but in other embodiments, 2 to Alkynylene is a subset of alkynyl having 10, 2 to 8, or 2 to 6 carbon atoms. It refers to residues similar to alkynyl but with two points of attachment.

[0035] As used herein, "alkoxy" refers to a group having a specified number of carbon atoms joined by oxygen bridges. alkyl of a carbon atom, such as 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.

[0036] As used herein, "aryl" refers to an aromatic monocyclic or polycyclic hydrocarbon ring system. The aromatic group refers to a group formed by removing a hydrogen atom from a ring carbon atom, which is derived from the aromatic group The cyclic or polycyclic hydrocarbon ring system contains only hydrogen and carbon atoms of 6 to 18 carbon atoms, At least one of the rings in the ring system is fully unsaturated, i.e., it is The aryl group includes phenyl, fluoro, and cyclic alkyl groups. Arylene includes, but is not limited to, groups such as phenyl and naphthyl. A subset of aryl, it refers to residues similar to aryl but with two points of attachment.

[0037] As used herein, "cycloalkyl" refers to a group typically having 3 to 7 ring carbon atoms. The ring may be saturated or may contain one or more carbon-carbon dimers. Examples of cycloalkyl include cyclopropyl and cyclobutyl. , cyclopentyl, cyclopentenyl, cyclohexyl and cyclohexenyl, and These include bridged and cage-like cyclic groups such as norbornane.

[0038] As used herein, a "halogen substituent" or "halo" refers to any group including fluoro, chloro, , bromo, and iodo, and the term "halogen" includes fluorine, chlorine, bromine, and iodine. .

[0039] As used herein, "halogenated alkyl" refers to a group having one or more of the specified number of carbon atoms. is alkyl as defined above substituted with multiple, up to the maximum permitted number of halogen atoms. Examples of halogenated alkyl groups include trifluoromethyl and difluoromethyl. , 2-fluoroethyl, and pentafluoroethyl.

[0040] The term "heterocyclic group" refers to a heterocyclic group having 2 to 12 carbon atoms and 1 to 10 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 heterocyclic radicals may optionally be oxidized. One or more nitrogen atoms (if present) are optionally quaternized. The heterocyclic group may be partially saturated. Heterocyclic groups may be bonded to the rest of the molecule through any atom of the ring. Examples of such heterocyclic groups include dioxanyl, thiophenyl[1 ,3]disulfonyl, decahydroisoquinolinyl, imidazolinyl, imidazolidinyl, Isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindole, Octahydroisoindole, 2-oxapiperazinyl, 2-oxapiperidyl, 2-o oxapyrimidinyl, oxazolidinyl, piperidyl, piperazinyl, 4-piperidonyl, Pyrrolidinyl, pyrazolidyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, Trisulfonyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-octyl Examples include, but are not limited to, 1,1-dioxothiomorpholinyl and 1,1-dioxothiomorpholinyl. I can't.

[0041] "Heteroaryl" means an alkyl group having 2 to 17 carbon atoms and a group selected from nitrogen, oxygen, and sulfur. refers to a group derived from a 3- to 18-membered aromatic ring radical containing 1 to 6 heteroatoms As used herein, heteroaryl refers to a monocyclic, bicyclic, tricyclic, or tetracyclic ring system. and at least one of the rings in the ring system is fully unsaturated, i.e., it is Heteroaryls include cyclic delocalized (4n+2) π-electron systems that conform to the Hellenistic theory. includes bridged ring systems. The heteroatoms in heteroaryl are optionally oxidized. One or more The nitrogen atom (if present) of is optionally quaternized. Heteroaryl is a heteroaryl group having any of the following ring structures: The bond to the rest of the molecule through an atom. An example of a heteroaryl is azepinyl. , acridinyl, benzimidazolyl, benzindole, 1,3-benzodioxazolyl benzofuryl, benzoxazolyl, benzo[d]thiazolyl, benzothiadiazolyl Benzo[b][1,4]dioxazolyl, Benzo[b][1,4]oxazolyl, 1 ,4-benzodioxazolyl, benzonaphthofuranyl, benzodiazolyl, benzodiox Benzophenyl, benzopyranyl, benzopyranonyl, benzofuryl, benzofuranonyl, Benzothiophenyl, benzothieno[3,2-d]pyrimidinyl, benzotriazolyl, Benzo[4,6]imidazo[1,2-a]pyridyl, carbazolyl, cinnolyl, cyclopentyl pentane[d]pyrimidinyl, 6,7-dihydro-5H-cyclopenta[4,5]thieno[2 ,3-d]pyrimidinyl, 5,6-dihydrobenzo[h]quinazolinyl, 5,6-dihydro benzo[h]cinnolinyl, 6,7-dihydro-5H-benzo[6,7]cyclohepta [1,2-c]pyridazinyl, dibenzofuryl, dibenzothiophenyl, furyl, furano furo[3,2-c]pyridyl, 5,6,7,8,9,10-hexahydrocyclohexyl buta[d]pyrimidinyl, 5,6,7,8,9,10-hexahydrocycloocta[d] pyridazinyl, 5,6,7,8,9,10-hexahydrocycloocta[d]pyridyl, Isothiazolyl, indazolyl, imidazolyl, indole, isoindole, indole nyl, isoindolinyl, isoquinolyl, indolizinyl, isoxazolyl, 5,8- Methano-5,6,7,8-tetrahydroquinazolinyl, naphthyridinonyl, 1,6-naphtho Thyridinonyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl, oxiranyl , 5,6,6a,7,8,9,10,10a-octahydrobenzo[H]quinazolinyl, 1-phenyl-1H-pyrrolyl, phenazinyl, phenothiazinyl, phenoxazinyl, Phthaloyl, pteridinyl, purinyl, pyrrolyl, pyrazolyl, pyrazolo[3,4-d] Pyrimidinyl, pyridyl, pyrido[3,2-d]pyrimidinyl, pyrido[3,4-d]pyridinyl Rimidinyl, pyrazinyl, pyrimidinyl, pyridazinyl, pyrrolyl, quinazolinyl, quino Xalinyl, quinolyl, isoquinolyl, tetrahydroquinolyl, 5,6,7,8-tetra Hydroquinazolinyl, 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3- d]pyrimidinyl, 6,7,8,9-tetrahydro-5H-cyclohepta[4,5]thieno pyrimidinyl, 5,6,7,8-tetrahydropyrido[4,5-c]pyrimidinyl Ridazinyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl, triazinyl , thieno[2,3-d]pyrimidinyl, thieno[3,2-d]pyrimidinyl, thieno[2 ,3-c]pridinyl and thiophenyl.

[0042] A variety of hydroxy protecting groups may be used in the present disclosure. Generally, the protecting groups are , rendering chemical functionalities insensitive to certain reaction conditions and can be attached to that functionality in the molecule without substantially damaging other parts of the molecule. and can be removed therefrom. Representative hydroxy protecting groups include Beaucage et al., Tetrahedron 1992, 48, 2223-2311, and Greene and Wuts,Protective Groups in Organic Sy nthesis,Chapter 2,2d ed,John Wiley & Son s, New York, 1991, the entire contents of which are incorporated by reference. In some embodiments, the protecting group is stable under basic conditions. In some embodiments, the present invention provides a method for the preparation of hydroxybenzoates, which can be removed under acidic conditions. Non-exclusive examples of hydroxy protecting groups that can be used herein include dimethoxytrityl (DMTP). ), monomethoxytrityl, 9-phenylxanthen-9-yl (Pixyl) "9-p henylxanthen-9-yl(Pixyl) and 9-(p-methoxyphenyl) ) Xanthen-9-yl (Mox) 9-(p-methoxyphenyl)xanthen In some embodiments, the compounds that can be used herein include phenyl-9-yl(Mox). Non-exclusive examples of hydroxy protecting groups that can be used include Tr (trityl), MMTr (4-methionine), dimethyltrityl), DMTr (4,4'-dimethoxytrityl) and TMTr (4,4', 4''-trimethoxytrityl).

[0043] The term "subject," as used herein, refers to any animal, e.g., a mammal. The subject matter of this disclosure includes humans, non-human primates (e.g., rhesus monkeys), and marsupials. or other species of macaques), mice, pigs, horses, donkeys, cattle, sheep, rats and and any type of poultry.

[0044] 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 effect" includes, but is not limited to, eradicating or ameliorating the underlying disorder being treated. In addition, the therapeutic effect does not necessarily mean that the subject may still suffer from the underlying disorder. eradicating or ameliorating one or more physiological symptoms associated with the underlying disorder, despite the possibility of By doing so, improvements are observed and obtained in the subject.

[0045] As used herein, "prevention" and "prevention" may be used interchangeably. The term refers to a method of 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 subject at risk of developing a particular disease, or to a subject who is at risk of developing one or more of the diseases. can be administered to a subject who has reported multiple pathological symptoms.

[0046] <sirna> The present disclosure provides siRNAs that can suppress ANGPTL3 gene expression.

[0047] The siRNA of the present disclosure contains a nucleotide group as a basic structural unit, and the nucleotide It is known to those skilled in the art that a phosphate group includes a phosphate group, a ribose group, and a base. The explanation will be omitted.

[0048] The siRNA of the present disclosure comprises a sense strand and an antisense strand, and each of the siRNAs wherein each nucleotide is independently a modified or unmodified nucleotide, comprises nucleotide sequence I, and the antisense strand comprises nucleotide sequence II; The nucleotide sequence I and the nucleotide sequence II are at least partially reverse-complementary to each other. forming a double-stranded region, said nucleotide sequence I comprising nucleotide sequence A, The nucleotide sequence A is equal in length to the nucleotide sequence shown in SEQ ID NO: 1 and is The difference is three or less, and the nucleotide sequence II contains the nucleotide sequence B, and the nucleotide sequence II Nucleotide sequence B is equal in length to the nucleotide sequence shown in SEQ ID NO: 2, There are three or fewer chido differences. 5'-CCAAGAGCACCAAGAACUZ-3' (SEQ ID NO: 1), 5'-Z'AGUUCUUGGUGCUCUUGG-3' (SEQ ID NO: 2) where Z is A and Z' is U. In the nucleotide sequence A, a nucleotide Z at position Z corresponds to A The said nucleus In the nucleotide sequence B, the nucleotide Z' at position Z' corresponds to B and Z' B teeth, It is the first nucleotide at the 5' end of the antisense strand.

[0049] In this context, "corresponding in position" means that the nucleotides are aligned from the same end of the nucleotide sequence. For example, the first nucleotide at the 3' end of nucleotide sequence A is The second nucleotide corresponds to the first nucleotide at the 3' end of SEQ ID NO: 1. It is leotide.

[0050] In some embodiments, the sense strand comprises only nucleotide sequence I, The antisense strand contains only nucleotide sequence II.

[0051] In some embodiments, the nucleotide sequence A and the nucleotide sequence set forth in SEQ ID NO:1 and / or the nucleotide sequence has no more than one nucleotide difference between the nucleotide sequence There is no more than one nucleotide difference between column B and the nucleotide sequence shown in SEQ ID NO:2. do.

[0052] In some embodiments, the nucleotide sequence B and the nucleotide sequence set forth in SEQ ID NO:2 The nucleotide difference between the nucleotide sequence Z' is selected from A, C, or G. B The difference in position In some embodiments, the nucleotide difference is selected from A, C, or G. Selected Z' B In some embodiments, the difference is in the position of Z A is Z' B and These nucleotide differences allow for the siRNA complex to The nucleotide differences were not significantly reduced in the target gene silencing capacity. iRNA complexes are also within the scope of protection of the present disclosure.

[0053] In some embodiments, the nucleotide sequence A and the nucleotide sequence B are Basically reverse complementary, substantially reverse complementary or completely reverse complementary, refers to the presence of three or fewer base mismatches between two nucleotide sequences, Substantially reverse complementary means that there is no more than one base mismatch between the two nucleotide sequences. Perfectly reverse complementary means that there are no mismatches between the two nucleotide sequences. This refers to the following.

[0054] In some embodiments, nucleotide sequence A is the nucleotide sequence set forth in SEQ ID NO:3. nucleotide sequence B is the nucleotide sequence shown in SEQ ID NO: 4 . 5'-CCAAGAGCACCAAGAACUZ A -3' (SEQ ID NO: 3), 5'-Z' B AGUUCUUGGUGCUCUUGG-3' (SEQ ID NO: 4) Here, Z' B is the first nucleotide at the 5' end of the antisense strand, and Z A is selected from A, U, G or C, and Z' B is Z A is a nucleotide complementary to In some embodiments, Z A is A and Z' B is U, The sense strand and the antisense strand may have the same or different lengths, and the length of the sense strand is The length of the antisense strand is 19 to 23 nucleotides, and the length of the antisense strand is 20 to 26 nucleotides. Thus, the length ratio of the sense strand to the antisense strand of the siRNA provided by the present disclosure However, 19 / 20, 19 / 21, 19 / 22, 19 / 23, 19 / 24, 19 / 25, 19 / 26, 20 / 20, 20 / 21, 20 / 22, 20 / 23, 20 / 24, 20 / 25, 20 / 26, 21 / 20, 21 / 21, 21 / 22, 21 / 23, 21 / 24, 21 / 2 5, 21 / 26, 22 / 20, 22 / 21, 22 / 22, 22 / 23, 22 / 24, 22 / 25, 22 / 26, 23 / 20, 23 / 21, 23 / 22, 23 / 23, 23 / 24, In some embodiments, the siRNP may be 23 / 25 or 23 / 26. The length ratio of the sense strand to the antisense strand of A is 19 / 21, 21 / 23, or 23 / 25 .

[0055] In some embodiments, the present disclosure provides a method for inhibiting ANGPTL3 gene expression. The siRNA comprises a sense strand and an antisense strand, and the siRNA each nucleotide in A is independently a modified or unmodified nucleotide; The sense strand comprises the nucleotide sequence I and the antisense strand comprises the nucleotide sequence II, comprising the nucleotide sequence shown in SEQ ID NO: 3; and Nucleotide sequence II, which contains the nucleotide sequence shown in number 4, is double-stranded in reverse complementarity. Form a region. 5'-CCAAGAGCACCAAGAACUZ A -3' (SEQ ID NO: 3), 5'-Z' B AGUUCUUGGUGCUCUUGG-3' (SEQ ID NO: 4) Here, Z' B is the first nucleotide at the 5' end of the antisense strand, and Z A is selected from A, U, G or C, and Z' B is Z A is a nucleotide complementary to In some embodiments, Z A is A and Z' B is U, The sense strand and the antisense strand may have the same or different lengths, and the length of the sense strand is The length of the antisense strand is 19 to 23 nucleotides, and the length of the antisense strand is 20 to 26 nucleotides. Thus, the length ratio of the sense strand to the antisense strand of the siRNA provided by the present disclosure However, 19 / 20, 19 / 21, 19 / 22, 19 / 23, 19 / 24, 19 / 25, 19 / 26, 20 / 20, 20 / 21, 20 / 22, 20 / 23, 20 / 24, 20 / 25, 20 / 26, 21 / 20, 21 / 21, 21 / 22, 21 / 23, 21 / 24, 21 / 2 5, 21 / 26, 22 / 20, 22 / 21, 22 / 22, 22 / 23, 22 / 24, 22 / 25, 22 / 26, 23 / 20, 23 / 21, 23 / 22, 23 / 23, 23 / 24, In some embodiments, the siRNP may be 23 / 25 or 23 / 26. The length ratio of the sense strand to the antisense strand of A is 19 / 21, 21 / 23, or 23 / 25 .

[0056] In some embodiments, the sense strand and the antisense strand are the same length. wherein the nucleotide sequence I further comprises a nucleotide sequence III, Sequence II further comprises nucleotide sequence IV and is identical to nucleotide sequence III. nucleotide sequences IV and V, each independently having a length of 1 to 4 nucleotides, String III is attached to the 5' end of nucleotide sequence A, and nucleotide sequence IV is the nucleotide sequence III is bound to the 3' end of the nucleotide sequence B; The sequence IV is of equal length.

[0057] The nucleotide sequence III and the nucleotide sequence IV may be complementary to each other. In some embodiments, to improve the stability of the siRNA, Nucleotide sequence III and nucleotide sequence IV are at least partially complementary to each other, In some embodiments, nucleotide sequence III and nucleotide sequence IV are 80% identical. or more than 90% or more of the bases are complementary, and in some embodiments, Sequence III and nucleotide sequence IV are substantially reverse complementary or completely reverse complementary. Substantially reverse complementary means that there is no more than one base mismatch between the two nucleotide sequences. Perfectly reverse complementary means that there are no mismatches between the two nucleotide sequences. and in some embodiments, nucleotide sequence III and nucleotide sequence I V is completely reverse complementary, so that the sense and antisense strands of the siRNA , are equal in length and the length ratio is 20 / 20, 21 / 21, 22 / 22 or 23 / 23. In some embodiments, the length ratio of the sense strand to the antisense strand of the siRNA is is 21 / 21 or 23 / 23.

[0058] In some embodiments, the nucleotide sequence III and the nucleotide sequence IV are , each of which is 1 nucleotide in length, the base of nucleotide sequence III is G, The base of nucleotide sequence IV is C, and the length ratio of the sense strand to the antisense strand is 20 / 20, or nucleotide sequences III and IV are both 2 nucleotides in length. From the 5' end to the 3' end, the base sequence of nucleotide sequence III is AG, and the base sequence of nucleotide sequence IV is CU, and in this case, the sense strand and the anti-sense strand are The length ratio of the sense strand is 21 / 21, or the nucleotide sequences III and IV are The length of each is 3 nucleotides, and the nucleotide sequence is The base sequence of nucleotide sequence III is AAG and the base sequence of nucleotide sequence IV is CUU; In this case, the length ratio of the sense strand to the antisense strand is 22 / 22, or Sequences III and IV are both 4 nucleotides in length and extend from the 5' to the 3' end. The base sequence of nucleotide sequence III is CAAG, and the base sequence of nucleotide sequence IV is The base sequence is CUUG, and the length ratio of the sense strand to the antisense strand is 23 / 2 In some embodiments, nucleotide sequence III and nucleotide sequence IV are Column IV is 2 nucleotides in length, from the 5' end to the 3' end, the base sequence of nucleotide sequence III is AG, and the base sequence of nucleotide sequence IV is CU; In this case, the length ratio of the sense strand to the antisense strand is 21 / 21.

[0059] In some embodiments, nucleotide sequence III and nucleotide sequence IV are The bases of nucleotide sequence III are the same length and are completely reverse complementary. Then, the base of nucleotide sequence IV is also determined.

[0060] In some embodiments, the sense and antisense strands are different lengths, Nucleotide sequence II further comprises a nucleotide sequence V, and nucleotide sequence V has a length a length of 1 to 3 nucleotides, and is attached to the 3' end of the antisense strand, This forms the 3' overhanging end of the siR strand provided by the present disclosure. The length ratio of the sense strand to the antisense strand of NA was 19 / 20, 19 / 21, 19 / 22, and 2 0 / 21, 20 / 22, 20 / 23, 21 / 22, 21 / 23, 21 / 24, 22 / 23 , 22 / 24, 22 / 25, 23 / 24, 23 / 25 or 23 / 26. In some embodiments, the nucleotide sequence V is 2 nucleotides in length; This allows the length ratio of the sense strand to the antisense strand of the siRNA provided by the present disclosure to be , 19 / 21, 21 / 23 or 23 / 25.

[0061] Each nucleotide in the nucleotide sequence V may be any nucleotide. In order to reduce the cost of synthesis and to facilitate synthesis, the nucleotide sequence V is preferably composed of two consecutive thymine deoxyribonucleotides (TT) or two consecutive uracil ribonucleoside It is a nucleotide sequence (UU) that increases the affinity of the antisense strand of siRNA to the target mRNA. To achieve this, nucleotide sequence V is complementary to the nucleotide at the corresponding position of the target mRNA. Thus, in some embodiments, the sense strand and the amino acid sequence of the siRNA of the present disclosure are The ratio of the lengths of the antisense strands is 19 / 21 or 21 / 23, and in this case, the siR NA has better mRNA silencing activity.

[0062] In some embodiments, the sense strand of the siRNA is the nucleic acid sequence set forth in SEQ ID NO:3. The antisense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO:5. Contains the nucleotide sequence. 5'-CCAAGAGCACCAAGAACUZ A -3' (SEQ ID NO: 3), 5'-Z' B AGUUCUUGGUGCUCUUGGCU-3' (SEQ ID NO: 5) Alternatively, the sense strand of the siRNA comprises the nucleotide sequence set forth in SEQ ID NO:6. The antisense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO:7. 5'-AGCCAAGAGCACCAAGAACUZ A -3' (SEQ ID NO: 6), 5'-Z' B AGUUCUUGGUGCUCUUGGCUUG-3' (SEQ ID NO: 7) However, the above Z' B is the first nucleotide at the 5' end of the antisense strand, and Z A is selected from A, U, G or C, and Z' B is Z A is a nucleotide complementary to

[0063] In some embodiments, the siRNA described in this disclosure is siAN1 or siA It's N2. siAN1 Sense strand: 5'-CCAAGAGCACCAAGAACUA-3' (SEQ ID NO: 8) Antisense strand: 5'-UAGUUCUUGGUGCUCUUGGCU-3' (SEQ ID NO: No. 9) siAN2 Sense strand: 5'-AGCCAAGAGCACCAAGAACUA-3' (SEQ ID NO: 10 ) Antisense strand: 5'-UAGUUCUUGGUGCUCUUGGCUUG-3' (sequence Column number 11)

[0064] As described above, the nucleotides in the siRNA of the present disclosure can each be independently modified. or unmodified nucleotides. In some embodiments, the siRNA of the present disclosure In some embodiments, the nucleotides in the Some or all of the nucleotides in the siRNA are modified nucleotides, These modifications on the tide group inhibit the ANGPTL3 gene expression of the siRNA complex of the present disclosure. does not appreciably weaken or eliminate the inhibitory function of the

[0065] In some embodiments, the siRNA of the present disclosure comprises at least one modified nucleoside. The term "modified nucleotide" as used in the context of this disclosure refers to a nucleotide A nucleotide or nucleoside in which the 2' hydroxyl group of the ribose group of the nucleotide is replaced with another group nucleotide analogs, or nucleotides in which the base on the nucleotide is a modified base The modified nucleotides clearly weaken the gene expression suppression function of siRNA. For example, JK Watts, GF Deleav ey,and MJ Damha,Chemically modified si RNA:tools and applications. Drug Discov Modified nucleotides disclosed in Today, 2008, 13(19-20):842-55 You may select the

[0066] In some embodiments, the sense strand or The antisense strand has at least one modified nucleotide, and / or At least one of the phosphate ester groups is a phosphate ester group having a modifying group. In other words, at least one single-stranded phosphate in the sense strand and the antisense strand At least some of the phosphate groups and / or ribose groups in the sugar backbone have a modified group. The ribose group has a phosphate group and / or a modifying group.

[0067] In some embodiments, nucleic acids in the sense strand and / or the antisense strand In some embodiments, the nucleotides are all modified nucleotides. Each nucleotide in the sense strand and the antisense strand of the siRNA provided is unique. They are either fluoro-modified or non-fluoro-modified nucleotides.

[0068] The inventors of the present disclosure have surprisingly found that the siRNAs described in the present disclosure can be used in animal experiments. Achieve a high balance between plasma stability and gene silencing efficiency found.

[0069] In some embodiments, the fluoro-modified nucleotide is and nucleotide sequence B, and from the 5' end to the 3' end, Nucleotides at positions 7, 8, and 9 in column A are fluoro-modified nucleotides, and nucleotides at positions 2, 6, 14, and 16 of said nucleotide sequence B towards the 3' end; is a fluoro-modified nucleotide.

[0070] In some embodiments, the fluoro-modified nucleotide is and a fluoro-modified nucleotide in nucleotide sequence A located at nucleotide sequence B; nucleotide sequence A, the nucleotides at positions 7, 8, and 9 are fluoro-modified nucleotides, B has 7 or fewer fluoro-modified nucleotides, and the second The nucleotides at positions 6, 14, and 16 are fluoro-modified nucleotides.

[0071] In some embodiments, from the 5' end to the 3' end, in the sense strand and the nucleotides at positions 7, 8, and 9 or 5, 7, 8, and 9 of the nucleotide sequence A are and the nucleotides at the remaining positions in the sense strand are non-fluoro-modified nucleotides. the antisense strand is a fluoro-modified nucleotide from the 5' end to the 3' end. In the nucleotide sequence B, The nucleotides at positions 4 and 16 are fluoro-modified nucleotides, and in the antisense strand and the nucleotides at the remaining positions are non-fluoro-modified nucleotides.

[0072] Fluoro-modified nucleotides are nucleotides in which the hydroxy group at the 2' position of the ribose group is It refers to a nucleotide having the structure shown in the following formula (7) which is substituted with fluorine. A fluoro-modified nucleotide is a nucleotide in which the hydroxyl group at the 2' position of the ribose group is a non-fluoro group. In some embodiments, the nucleotide or nucleotide analog is substituted with a fluorine group. In the formula, each non-fluoro-modified nucleotide has a hydroxyl group at the 2' position of the ribose group of the nucleotide. Independently from nucleotides or nucleotide analogs in which the hydroxy group is replaced with a non-fluorine group It is the one to be selected.

[0073] Nucleotides in which the hydroxyl group at the 2' position of the ribose group is replaced with a non-fluorine group are , known to those skilled in the art, and these nucleotides include 2'-alkoxy modified nucleotides, 2'-substituted alkoxy-modified nucleotides, 2'-alkyl-modified nucleotides, 2'-substituted Alkyl-modified nucleotides, 2'-amino-modified nucleotides, 2'-substituted amino-modified nucleotides The nucleotide may be one selected from the group consisting of nucleotides, nucleotides, and 2'-deoxynucleotides.

[0074] In some embodiments, the 2'-alkoxy modified nucleotide is shown in formula (8): In some embodiments, the nucleotide is a methoxy-modified nucleotide (2'-OMe). The 2'-substituted alkoxy modified nucleotide is, for example, a 2'-O-methyl nucleotide represented by formula (9): In some embodiments, the nucleotides may be 2'-methyl-2'-hydroxyethyl modified nucleotides (2'-MOE). In the formula (10), the 2'-amino modified nucleotide (2'-NH2) is shown. In some embodiments, the 2'-deoxynucleotide (DNA) is shown in formula (11): do.

[0075] [ka]

[0076] Nucleotide analogs are those that can replace nucleotides in nucleic acids. , adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, Refers to a group whose structure is different from that of uracil ribonucleotide or thymine deoxyribonucleotide In some embodiments, the nucleotide analog is an isonucleotide, a bridged nucleotide, or bridged nucleic acid (BNA) or acyclic nucleotides It may also be a

[0077] BNA refers to constrained or inaccessible nucleotides. BNAs are five-membered, six-membered, The structure contains a bridged structure with a "fixed" C3'-endo sugar puckering, either a ring or a seven-membered ring. Typically, the bridge is introduced at the 2'- and 4'-positions of the ribose to form 2',4'-BN. In some embodiments, the BNA is provided as a B nucleotide, as shown in formula (12): LNA shown in equation (13), ENA shown in equation (14), cET BNA shown in equation (15), etc. It is also possible.

[0078] [ka]

[0079] Acyclic nucleotides are nucleotides in which the sugar ring of the nucleotide is opened. In some embodiments, the acyclic nucleotide is an unlocked nucleic acid represented by formula (15): (UNA), or glycerol nucleic acid (GNA) shown in formula (16).

[0080] [ka]

[0081] In the above formulas (15) and (16), R represents H, OH, or an alkoxy group (O-alkyl). is selected from.

[0082] Isonucleotides are nucleotides in which the position of the base in the ribose ring changes. In some embodiments, the isonucleotide is represented by formula (17): 18), in which the base is transferred from the 1'-position to the 2'- or 3'-position of the ribose ring. It may also be a mixture.

[0083] [ka]

[0084] In the compounds of the above formulas (17) and (18), Base is A, U, G, C, T, etc. wherein R is selected from H, OH, F or the non-fluorine groups described above.

[0085] In some embodiments, the nucleotide analog is an isonucleotide, an LNA, In some embodiments, the antibody is one selected from ENA, cET, UNA, and GNA. In this case, each non-fluoro-modified nucleotide is a methoxy-modified nucleotide. In the context of methoxy-modified nucleotides, the 2'-hydroxy group of the ribose group is methyl. It refers to a nucleotide substituted with hydroxy.

[0086] In the context, "fluoro-modified nucleotide", "2'-fluoro-modified nucleotide" "A nucleotide in which the 2'-hydroxy group of the ribose group is substituted with fluorine" and "2'- The fluororibose group and the 2'-hydroxy group of a nucleotide have the same meaning. It refers to a compound having the structure shown in formula (7) in which the methoxy group is substituted with fluorine. "2'-Methoxy-modified nucleotide," "2'-hydroxyl group of ribose group" "Nucleotide in which the 2'-methoxy group is substituted with methoxy" and "2'-methoxyribose group" are The taste is the same, and in both cases the 2'-hydroxy group of the ribose group of the nucleotide is methoxy. It refers to a compound having a structure shown in formula (8) below.

[0087] In some embodiments, the siRNA of the present disclosure has the following modifications: That is, from the 5' end to the 3' end, in the sense strand, Nucleotides at positions 7, 8, and 9 or positions 5, 7, 8, and 9 of nucleotide sequence A are fluoro-modified nucleotides at the remaining positions in the sense strand, and the nucleotides at the remaining positions in the sense strand are methoxy-modified nucleotides. nucleotides of the nucleotide sequence B, The nucleotides at positions 14 and 16 or positions 2, 6, 8, 9, 14, and 16 are fluoro-modified nucleotides. and the nucleotides at the remaining positions in the antisense strand are methoxy-modified nucleotides. It is a nucleotide.

[0088] In some embodiments, the siRNA of the present disclosure has the following modifications: That is, from the 5' end to the 3' end, the sequence of the sense strand of the siRNA is The nucleotides at positions 5, 7, 8 and 9 of the nucleotide sequence A are fluoro-modified nucleotides. and the remaining nucleotides in the sense strand of the siRNA are methoxy-modified nucleotides. and the antisense strand of the siRNA from the 5' end to the 3' end. The nucleotides at positions 2, 6, 8, 9, 14 and 16 of nucleotide sequence B in modified nucleotides, and the nucleotides at the remaining positions of the antisense strand of the siRNA are is a methoxy-modified nucleotide, Alternatively, from the 5' end to the 3' end, the nucleotides in the sense strand of the siRNA The nucleotides at positions 5, 7, 8, and 9 of nucleotide sequence A are fluoro-modified nucleotides. and the nucleotides at the remaining positions in the sense strand of the siRNA are methoxy-modified nucleotides. , and from the 5' end to the 3' end, the nucleic acid in the antisense strand of the siRNA Nucleotides at positions 2, 6, 14, and 16 of nucleotide sequence B are fluoro-modified nucleotides. and the remaining nucleotides in the antisense strand of the siRNA are methoxy-modified nucleotides. It is leotide, Alternatively, from the 5' end to the 3' end, the nucleotides in the sense strand of the siRNA the nucleotides at positions 7, 8 and 9 of the nucleotide sequence A are fluoro-modified nucleotides; The nucleotides at the remaining positions of the sense strand of the iRNA are methoxy-modified nucleotides, or In addition, the nucleotides in the antisense strand of the siRNA from the 5' end to the 3' end Nucleotides at positions 2, 6, 14, and 16 of nucleotide sequence B are fluoro-modified nucleotides. and the nucleotides at the remaining positions of the antisense strand of the siRNA are methoxy-modified nucleotides. It's Chido.

[0089] In other words, the ribose group in the phosphate-sugar backbone of the siRNA is modified as follows: From the 5' end to the 3' end, the nucleic acid in the sense strand of the siRNA The glycosyl groups at positions 5, 7, 8, and 9 of nucleotide sequence A are 2'-fluororibose groups. The glycosyl groups of the remaining nucleotides in the sense strand of the siRNA are 2'-methoxy. The 5'-end to 3'-end of the siRNA is a siliboside group. Glycosyl groups at positions 2, 6, 8, 9, 14, and 16 of nucleotide sequence B in the sense strand The nucleotide groups are 2'-fluororibose groups, and the remaining positions of the antisense strand of the siRNA The glycosyl group of the ribonucleotide is a 2'-methoxyribose group, Alternatively, from the 5' end to the 3' end, the nucleotides in the sense strand of the siRNA The glycosyl groups at positions 5, 7, 8, and 9 of the nucleotide sequence A are 2'-fluororibose groups. The glycosyl groups of the remaining nucleotides in the sense strand of the siRNA are 2'-methoxyl groups. The antisense group of the siRNA is a bosen group, and the antisense group of the siRNA is a bosen group. The glycosyl groups at positions 2, 6, 14, and 16 of nucleotide sequence B in the base strand are 2'- The fluororolibose group of the nucleotides at the remaining positions of the antisense strand of the siRNA. the lycosyl group is a 2'-methoxyribose group, Alternatively, from the 5' end to the 3' end, the nucleotides in the sense strand of the siRNA The glycosyl groups at positions 7, 8, and 9 of the nucleotide sequence A are 2'-fluororibose groups, The glycosyl groups of the remaining nucleotides in the sense strand of the iRNA are 2'-methoxyribonucleotides. and from the 5' end to the 3' end of the antisense strand of the siRNA. The glycosyl groups at positions 2, 6, 14 and 16 of nucleotide sequence B in rolibose group, which is the glycosylation site of the nucleotides at the remaining positions of the antisense strand of the siRNA. The silyl group is a 2'-methoxyribose group.

[0090] In some embodiments, the siRNA provided by the present disclosure is siAN1-M 1, siAN2-M1, siAN1-M2, siAN2-M2, siAN1-M3, si It is one of AN2-M3. siAN1-M1 Sense strand: 5'-CmCmAmAmGfAmGfCfAfCmCmAmAmGmAmA mCmUmAm-3' (SEQ ID NO: 12) Antisense strand: 5'-UmAfGmUmUmCfUmUfGfGmUmGmCmUf CmUfUmGmGmCmUm-3' (SEQ ID NO: 13) siAN2-M1 Sense strand: 5'-AmGmCmCmAmAmGfAmGfCfAfCmCmAmAmG mAmAmCmUmAm-3' (SEQ ID NO: 14) Antisense strand: 5'-UmAfGmUmUmCfUmUfGfGmUmGmCmUf CmUfUmGmGmCmUmUmGm-3' (SEQ ID NO: 15) siAN1-M2 Sense strand: 5'-CmCmAmAmGfAmGfCfAfCmCmAmAmGmAmA mCmUmAm-3' (SEQ ID NO: 12) Antisense strand: 5'-UmAfGmUmUmCfUmUmGmGmUmGmCmUf CmUfUmGmGmCmUm-3' (SEQ ID NO: 16) siAN2-M2 Sense strand: 5'-AmGmCmCmAmAmGfAmGfCfAfCmCmAmAmG mAmAmCmUmAm-3' (SEQ ID NO: 14) Antisense strand: 5'-UmAfGmUmUmCfUmUmGmGmUmGmCmUf CmUfUmGmGmCmUmUmGm-3' (SEQ ID NO: 17) siAN1-M3 Sense strand: 5'-CmCmAmAmGmAmGfCfAfCmCmAmAmGmAmA mCmUmAm-3' (SEQ ID NO: 18) Antisense strand: 5'-UmAfGmUmUmCfUmUmGmGmUmGmCmUf CmUfUmGmGmCmUm-3' (SEQ ID NO: 16) siAN2-M3 Sense strand: 5'-AmGmCmCmAmAmGmAmGfCfAfCmCmAmAmG mAmAmCmUmAm-3' (SEQ ID NO: 19) Antisense strand: 5'-UmAfGmUmUmCfUmUmGmGmUmGmCmUf CmUfUmGmGmCmUmUmGm-3' (SEQ ID NO: 17)

[0091] siRNA with the above modifications is not only inexpensive but also resistant to ribonucleases in the blood. It can make nucleic acids less susceptible to cleavage, thereby improving the stability of the nucleic acid and It has the property of being highly resistant to nuclease hydrolysis.

[0092] In some embodiments, the sense and anti-strands of the siRNA provided by the present disclosure In the sense strand, at least one single strand has a small number of phosphate ester groups in its phosphate-sugar backbone. At least some of the phosphate groups have a modifying group. The phosphate ester group having the modifying group is a phosphodiester bond in the phosphate ester group. At least one oxygen atom of the thiophosphate group is replaced with a sulfur atom. In some embodiments, the phosphate ester group having the modifying group is represented by formula (1): It is a thiophosphate ester group having the structure:

[0093] [ka]

[0094] These modifications stabilize the double-stranded structure of siRNA and enhance the specificity of base pairing. and maintain a high affinity.

[0095] In some embodiments, in the siRNA provided by the present disclosure, thiophosphoryl The acid ester group is located between the first and second nucleotides at either end of the sense or antisense strand. between the second nucleotide and the second nucleotide at either end of the sense or antisense strand between the nucleotide and the third nucleotide, or any combination thereof In some embodiments, the thiophosphate ester is present in a hydroxyl group. Terminator groups are present attached at all of the above positions except the 5' end of the sense strand. In embodiments, the thiophosphate group is located at all of the above positions except the 3' end of the sense strand. In some embodiments, the thiophosphate group is attached to It is present bound at at least one of the positions. Between the first and second nucleotides from the 5' end of the sense strand, Between the second and third nucleotides from the 5' end of the sense strand, Between the first and second nucleotides from the 3' end of the sense strand, Between the second and third nucleotides from the 3' end of the sense strand, The first and second nucleotides from the 5' end of the antisense strand Between, The second and third nucleotides from the 5' end of the antisense strand Between, The first and second nucleotides from the 3' end of the antisense strand Between, and The second and third nucleotides from the 3' end of the antisense strand Between.

[0096] In some embodiments, the siRNA provided by the present disclosure is siAN1-M 1S, siAN2-M1S, siAN1-M2S, siAN2-M2S, siAN1-M 3S, siAN2-M3S, or one of the following: siAN1-M1S Sense strand: 5'-CmsCmsAmAmGfAmGfCfAfCmCmAmAmGmA mAmCmUmAm-3' (SEQ ID NO: 20) Antisense strand: 5'-UmsAfsGmUmUmCfUmUfGfGmUmGmCm UfCmUfUmGmGmsCmsUm-3' (SEQ ID NO: 21) siAN2-M1S Sense strand: 5'-AmsGmsCmCmAmAmGfAmGfCfAfCmCmAmA mGmAmAmCmUmAm-3' (SEQ ID NO: 22) Antisense strand: 5'-UmsAfsGmUmUmCfUmUfGfGmUmGmCm UfCmUfUmGmGmCmUmsUmsGm-3' (SEQ ID NO: 23) siAN1-M2S Sense strand: 5'-CmsCmsAmAmGfAmGfCfAfCmCmAmAmGmA mAmCmUmAm-3' (SEQ ID NO: 20) Antisense strand: 5'-UmsAfsGmUmUmCfUmUmGmGmUmGmCm UfCmUfUmGmGmsCmsUm-3' (SEQ ID NO: 24) siAN2-M2S Sense strand: 5'-AmsGmsCmCmAmAmGfAmGfCfAfCmCmAmA mGmAmAmCmUmAm-3' (SEQ ID NO: 22) Antisense strand: 5'-UmsAfsGmUmUmCfUmUmGmGmUmGmCm UfCmUfUmGmGmCmUmsUmsGm-3' (SEQ ID NO: 25) siAN1-M3S Sense strand: 5'-CmsCmsAmAmGmAmGfCfAfCmCmAmAmGmA mAmCmUmAm-3' (SEQ ID NO: 26) Antisense strand: 5'-UmsAfsGmUmUmCfUmUmGmGmUmGmCm UfCmUfUmGmGmsCmsUm-3' (SEQ ID NO: 24) siAN2-M3S Sense strand: 5'-AmsGmsCmCmAmAmGmAmGfCfAfCmCmAmA mGmAmAmCmUmAm-3' (SEQ ID NO: 27) Antisense strand: 5'-UmsAfsGmUmUmCfUmUmGmGmUmGmCm UfCmUfUmGmGmCmUmsUmsGm-3' (SEQ ID NO: 25)

[0097] In some embodiments, the 5'-terminal nucleotide of the antisense strand of the siRNA The nucleotide is a 5'-phosphate nucleotide or a 5'-phosphate analog-modified nucleotide.

[0098] The conventional 5'-phosphate nucleotide or 5'-phosphate analog modified nucleotide is It is known to those skilled in the art that, for example, a 5'-phosphate nucleotide may have the following structure: .

[0099] [ka]

[0100] Also, for example, Anastasia Khvorova and Jonathan K. Watts,The chemical evolution of oligo nucleotide therapies of clinical utility . Nature Biotechnology,2017,35(3):238~48 discloses the following four types of 5'-phosphate analog modified nucleotides:

[0101] [ka] where R is selected from H, OH, methoxy, and fluorine; Base represents a base; A, It is selected from U, C, G or T.

[0102] In some embodiments, the 5'-phosphate nucleotide is a 5'-phosphate nucleotide, as shown in formula (2): 5'-phosphate analog modified nucleotides are nucleotides containing Vinyl phosphate ester (5'-(E)-vinylphospho) represented by formula (3) nucleotide containing a thiol ester (E-VP) modification, or a thiol ester (E-VP) modification, as shown in formula (5). It is a phosphate-modified nucleotide.

[0103] In some embodiments, the siRNA provided by the present disclosure is siAN1-M 1P1, siAN2-M1P1, siAN1-M2P1, siAN2-M2P1, siA N1-M3P1, siAN2-M3P1, siAN1-M1SP1, siAN2-M1S P1, siAN1-M2SP1, siAN2-M2SP1, siAN1-M3SP1, s It is one of iAN2-M3SP1. siAN1-M1P1 Sense strand: 5'-CmCmAmAmGfAmGfCfAfCmCmAmAmGmAmA mCmUmAm-3' (SEQ ID NO: 12) Antisense strand: 5'-P1-UmAfGmUmUmCfUmUfGfGmUmGmC mUfCmUfUmGmGmCmUm-3' (SEQ ID NO: 28) siAN2-M1P1 Sense strand: 5'-AmGmCmCmAmAmGfAmGfCfAfCmCmAmAmG mAmAmCmUmAm-3' (SEQ ID NO: 14) Antisense strand: 5'-P1-UmAfGmUmUmCfUmUfGfGmUmGmC mUfCmUfUmGmGmCmUmUmGm-3' (SEQ ID NO: 29) siAN1-M2P1 Sense strand: 5'-CmCmAmAmGfAmGfCfAfCmCmAmAmGmAmA mCmUmAm-3' (SEQ ID NO: 12) Antisense strand: 5'-P1-UmAfGmUmUmCfUmUmGmGmUmGmC mUfCmUfUmGmGmCmUm-3' (SEQ ID NO: 30) siAN2-M2P1 Sense strand: 5'-AmGmCmCmAmAmGfAmGfCfAfCmCmAmAmG mAmAmCmUmAm-3' (SEQ ID NO: 14) Antisense strand: 5'-P1-UmAfGmUmUmCfUmUmGmGmUmGmC mUfCmUfUmGmGmCmUmUmGm-3' (SEQ ID NO: 31) siAN1-M3P1 Sense strand: 5'-CmCmAmAmGmAmGfCfAfCmCmAmAmGmAmA mCmUmAm-3' (SEQ ID NO: 18) Antisense strand: 5'-P1-UmAfGmUmUmCfUmUmGmGmUmGmC mUfCmUfUmGmGmCmUm-3' (SEQ ID NO: 30) siAN2-M3P1 Sense strand: 5'-AmGmCmCmAmAmGmAmGfCfAfCmCmAmAmG mAmAmCmUmAm-3' (SEQ ID NO: 19) Antisense strand: 5'-P1-UmAfGmUmUmCfUmUmGmGmUmGmC mUfCmUfUmGmGmCmUmUmGm-3' (SEQ ID NO: 31) siAN1-M1SP1 Sense strand: 5'-CmsCmsAmAmGfAmGfCfAfCmCmAmAmGmA mAmCmUmAm-3' (SEQ ID NO: 20) Antisense strand: 5'-P1-UmsAfsGmUmUmCfUmUfGfGmUmG mCmUfCmUfUmGmGmsCmsUm-3' (SEQ ID NO: 32) siAN2-M1SP1 Sense strand: 5'-AmsGmsCmCmAmAmGfAmGfCfAfCmCmAmA mGmAmAmCmUmAm-3' (SEQ ID NO: 22) Antisense strand: 5'-P1-UmsAfsGmUmUmCfUmUfGfGmUmG mCmUfCmUfUmGmGmCmUmsUmsGm-3' (SEQ ID NO: 33) siAN1-M2SP1 Sense strand: 5'-CmsCmsAmAmGfAmGfCfAfCmCmAmAmGmA mAmCmUmAm-3' (SEQ ID NO: 20) Antisense strand: 5'-P1-UmsAfsGmUmUmCfUmUmGmGmUmG mCmUfCmUfUmGmGmsCmsUm-3' (SEQ ID NO: 34) siAN2-M2SP1 Sense strand: 5'-AmsGmsCmCmAmAmGfAmGfCfAfCmCmAmA mGmAmAmCmUmAm-3' (SEQ ID NO: 22) Antisense strand: 5'-P1-UmsAfsGmUmUmCfUmUmGmGmUmG mCmUfCmUfUmGmGmCmUmsUmsGm-3' (SEQ ID NO: 35) siAN1-M3SP1 Sense strand: 5'-CmsCmsAmAmGmAmGfCfAfCmCmAmAmGmA mAmCmUmAm-3' (SEQ ID NO: 26) Antisense strand: 5'-P1-UmsAfsGmUmUmCfUmUmGmGmUmG mCmUfCmUfUmGmGmsCmsUm-3' (SEQ ID NO: 34) siAN2-M3SP1 Sense strand: 5'-AmsGmsCmCmAmAmGmAmGfCfAfCmCmAmA mGmAmAmCmUmAm-3' (SEQ ID NO: 27) Antisense strand: 5'-P1-UmsAfsGmUmUmCfUmUmGmGmUmGm CmUfCmUfUmGmGmCmUmsUmsGm-3' (SEQ ID NO: 35)

[0104] The inventors of the present disclosure have demonstrated that the siRNA provided by the present disclosure significantly improves plasma and lysosomal activity. Surprisingly, it not only possesses chromatin stability but also maintains extremely high gene silencing activity. Found it.

[0105] The siRNAs provided by the present disclosure can be prepared by conventional siRNA preparation methods in the art (e.g., For example, solid phase synthesis and liquid phase synthesis are used. Commercial customization services are already available. Modified nucleotide groups are introduced into the siRNAs described in this disclosure by using nucleotide-mers. and methods for preparing nucleoside monomers having the corresponding modifications, and modified nucleoside monomers. Methods for introducing leutide groups into siRNA are also well known to those skilled in the art.

[0106] <Drug Composition> The present disclosure provides a drug comprising the above-described siRNA as an active ingredient and a pharmaceutically acceptable carrier. A composition is provided.

[0107] The pharmaceutically acceptable carrier may be a carrier commonly used in the field of siRNA administration. Often, for example, magnetic nanoparticles (e.g., , nanoparticles based on Fe3O4 or Fe2O3), carbon nanotubes (carbon nanotubes, mesoporous silicon n), calcium phosphate nanoparticles rticles), polyethyleneimine (PEI) , polyamidoamine dendrimer (polyamidoamine (PAMAM) d endrimer), poly(L-lysine, PLL), chitosan (chitosan), 1,2-dioleoyl-3-trimethylammonium propane ( 1,2-dioleoyl-3-trimethylammonium-propane , DOTAP), poly(D- or L-lactic acid / glycolic acid copolymers) (poly(D&L-l (actic / glycolic acid) copolymer, PLGA), poly(acetylacetonate) Poly(2-aminoethyl ethylene phosphate ester) Poly(N,N-dimethylaminoethylene phosphate), PPEEA) and poly(N,N-dimethylaminoethylene phosphate) Poly(2-dimethylaminoethyl methacrylate) acrylate), PDMAEMA) and one or more of their derivatives. Not limited to these.

[0108] In some embodiments, the siRNA and pharmaceutically acceptable salts in the pharmaceutical composition Although there are no particular requirements for the content of the soluble carrier, in some embodiments, siR The weight ratio of NA to the pharmaceutically acceptable carrier may be 1:(1 to 500). In some embodiments, the weight ratio is 1:(1 to 50).

[0109] In some embodiments, the drug composition contains other pharmaceutically acceptable additives. The additive may be one or more of various agents or compounds commonly used in the art. For example, the pharmaceutically acceptable additive may be a pH buffer, At least one of a protective agent and an osmolality adjusting agent may be included.

[0110] The pH buffer solution is trishydroxymethylaminomethane hydrochloride at pH 7.5 to 8.5. Buffer solution (tris(hydroxymethyl) aminomethane hyd chloride buffer) and / or phosphate buffer of pH 5.5 to 8.5 For example, it may be a phosphate buffer solution having a pH of 5.5 to 8.5.

[0111] The protective agent may be inositol, sorbitol, sucrose, trehalose, or mannose. The drug composition may be at least one of maltose, lactose, and glucose. The content of the protective agent may be 0.01 to 30% by weight based on the total weight of the composition.

[0112] The osmolality adjusting agent may be sodium chloride and / or potassium chloride. The content of the osmolality adjusting agent is such that the osmolality of the drug composition is 200 to 700 mOsm / kg. Depending on the desired osmolality, one skilled in the art can determine the osmolality of the solution. The content of the osmolality adjusting agent can be easily determined.

[0113] In some embodiments, the pharmaceutical composition may be a liquid formulation, such as an injectable solution. Alternatively, the lyophilized powder may be mixed with a liquid additive at the time of administration to form a liquid preparation. The liquid formulation may be administered by subcutaneous, intramuscular or intravenous injection, but is not limited thereto. The drug is first administered to the lungs by spraying, or is then delivered to other organ tissues (e.g., In some embodiments, the administration may be to the liver, but is not limited to the liver. The pharmaceutical composition is used for intravenous administration.

[0114] In some embodiments, the drug composition may be in the form of a liposomal formulation. In some embodiments, the pharmaceutically acceptable carrier used in the liposome formulation The carrier is an amine-containing transfection compound (hereinafter also referred to as organic amine), auxiliary lipids, The organic amino acid may be a PEG- or PEG-modified lipid. The lipids, co-lipids, and PEGylated lipids were prepared according to CN103380113A (the entire contents of which are incorporated herein by reference). amine-containing transfection compounds described in and one or more selected from the group consisting of a salt or derivative thereof which is acceptable to the present invention, a co-lipid, and a PEGylated lipid. There may be multiple types.

[0115] In some embodiments, the organic amine is a compound described in CN103380113A. The compound may be a compound represented by formula (201) or a pharmaceutically acceptable salt thereof.

[0116] [ka] During the ceremony, X 101 and X 102 are each independently O, S, NA or CA, and A is hydrogen. or C1-C 20 is a hydrocarbon chain, Y and Z are each independently C=O, C=S, S=O, CH—OH, or SO; R 101 , R 102 , R 103 , R 104 , R 105 , R 106 and R 107 Each independently hydrogen, cyclic or acyclic, substituted or unsubstituted, branched or straight chain aliphatic group, cyclic or acyclic, substituted or unsubstituted, branched or straight chain heteroaliphatic groups, substituted or unsubstituted, Branched or straight chain acyl groups, substituted or unsubstituted, branched or straight chain aryl groups, substituted or unsubstituted a substituted, branched or straight chain heteroaryl group; x is an integer from 1 to 10, n is an integer of 1 to 3, m is an integer of 0 to 20, and p is 0 or 1, where m If p=0, then R 102 is hydrogen, When at least one of n or m is 2, R 103 and nitrogen in formula (201) , forming a structure shown in formula (202) or formula (203).

[0117] [ka] In the formula, g, e, and f are each independently an integer of 1 to 6, and "HCC" represents a hydrocarbon chain. represents each * N represents a nitrogen atom in formula (201).

[0118] In some embodiments, R 103 is a polyamine. R 103 is a ketal. In some embodiments, R in formula (201) 10 1 and R 102 each independently being an optionally substituted or unsubstituted branched or linear alkyl group; alkyl or alkenyl, wherein the alkyl or alkenyl has from 3 to about 20 carbon atoms, e.g. For example, 8 to about 18 carbon atoms and 0 to 4 double bonds, for example, 0 to 2 double bonds. It has.

[0119] In some embodiments, when n and m each independently have a value of 1 or 3, R 103 may be any one of the following formulas (204) to (213).

[0120] [ka] In formulas (204) to (213), g, e, and f each independently represent an integer of 1 to 6. , each "HCC" represents a hydrocarbon chain, and each * is R 103 and the nitrogen atom in formula (201) indicates possible points of attachment of * Each H on the position is bonded to the nitrogen atom in formula (201). may be substituted to

[0121] The compound of formula (201) was prepared according to the method described in CN103380113A. Good too.

[0122] In some embodiments, the organic amine is an organic amine represented by formula (214): and / or an organic amine represented by formula (215).

[0123] [ka]

[0124] The co-lipid may be cholesterol, a cholesterol analogue, and / or a cholesterol-lowering agent. It is a derivative of The PEGylated lipid is 1,2-dipalmitamido-sn-glycero-3-phosphatidylinositol. Triethanolamine-N-[methoxy(polyethylene glycol)]-2000(1,2 -dipalmitoyl-sn-glycero-3-phosphatidylet hanolamine-N-[methoxy(polyethylene glyco l)-2000]).

[0125] In some embodiments, the drug composition further comprises the organic amine, the auxiliary lipid, and The molar ratio of the PEGylated lipid to the PEGylated lipid was (19.7-80):(19.7-80):(0.3 (50-70):(20-40):(3-20), for example. stomach.

[0126] In some embodiments, the siRNA of the present disclosure and the amine-containing transfection The drug composition particles formed by the injection reagent have an average diameter of about 30 nm to about 200 nm. , generally about 40 nm to about 135 nm, and more generally, the average diameter of the liposome particles The diameter is about 50 nm to about 120 nm, about 50 nm to about 100 nm, about 60 nm to about 90 nm, or The average diameter of the liposome particles is about 70 nm to about 90 nm, for example, about 30,4 0, 50, 60, 70, 75, 80, 85, 90, 100, 110, 120, 130, 1 40, 150 or 160 nm.

[0127] In some embodiments, the siRNA of the present disclosure and the amine-containing transfection In the drug composition formed by the coupling reagent, siRNA and total lipid (e.g., organic amine , co-lipid and / or PEGylated lipid) is about 1:1 to about 1:5 0, approx. 1:1 to approx. 1:30, approx. 1:3 to approx. 1:20, approx. 1:4 to approx. 1:18, approx. 1:5 about 1:17, about 1:5 to about 1:15, about 1:5 to about 1:12, about 1:6 to about 1:12, or The weight ratio of siRNA to total lipids of the present disclosure is in the range of about 1:6 to about 1:10. Approximately 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11,1:12, 1:13 , 1:14, 1:15, 1:16, 1:17 or 1:18.

[0128] In some embodiments, the pharmaceutical composition is marketed with each component present independently. In some embodiments, the present disclosure provides a method for preparing a pharmaceutical composition comprising administering a pharmaceutical composition containing 100 mg of 10 ... and a pharmaceutically acceptable carrier. may be prepared according to various known methods, and may be used in accordance with the present disclosure in place of conventional siRNA. In some embodiments, the siRNA provided by the following method may be used. It may also be prepared in a similar manner.

[0129] The organic amine, co-lipid, and PEGylated lipid were suspended in alcohol at the above molar ratio and mixed uniformly. The amount of alcohol is adjusted so that the total mass concentration of the obtained lipid solution is 2 to 25%. The alcohol is determined to have a concentration of 8 to 18 mg / mL, for example, 8 to 18 mg / mL. Pharmaceutically acceptable alcohols, such as ethanol, propylene glycol, benzyl Alcohol, glycerin, polyethylene glycol 200, polyethylene glycol 30 0, polyethylene glycol 400, etc., are selected from alcohols that are liquid at around room temperature. The solvent may be one or more of the above, for example, ethanol.

[0130] The siRNA provided by the present disclosure is dissolved in a buffered salt solution to obtain an aqueous siRNA solution. The concentration of the buffer salt solution is 0.05 to 0.5 M, and may be, for example, 0.1 to 0.2 M. The pH of the buffer salt solution is adjusted to 4.0 to 5.5, and may be, for example, 5.0 to 5.2. The amount of the buffered salt solution is such that the siRNA concentration is 0.6 mg / mL or less, for example, 0.2 to 100 mg / mL. The buffer salts are determined to be 0.4 mg / mL. One or more selected from the group consisting of sodium acetate and / or potassium acetate. It can be Um.

[0131] After mixing the lipid solution and the siRNA aqueous solution, the resulting product was heated at 40-60°C for at least Incubate for another 2 minutes, e.g., 5 to 30 minutes, to obtain the incubated liposome preparation. The volume ratio of the iRNA aqueous solution may be 1:(2 to 5), for example, 1:4.

[0132] The cultured liposome formulation is concentrated or diluted, impurities are removed, sterilized, and the drug composition provided by the present disclosure is obtained. Its physicochemical parameters include a pH of 6.5 to 8, an encapsulation efficiency of 80% or more, a particle size of 40 to 200 nm, a polydispersity index of 0.30 or less, and an osmotic pressure of 250 to 400 mOsm / kg. For example, the physicochemical parameters may include a pH of 7.2 to 7.6, an encapsulation efficiency of 90% or more, a particle size of 60 to 100 nm, a polydispersity index of 0.20 or less, and an osmotic pressure of 300 to 400 mOsm / kg. Here, the concentration or dilution may be performed before removing impurities, after removing impurities, or simultaneously. As a method for removing impurities, various conventional methods may be employed. For example, , a tangential flow system and a hollow fiber column are used for ultrafiltration under the condition of 100KDa, and the ultrafiltration exchange solution may be phosphate buffered saline (PBS) with a pH of 7.4. As a sterilization method, various conventional methods may be employed. For example, it may be sterilized by filtration with a 0.22 μm filter.

[0133] [[ID=1�]]

[0134] <siRNA complex> The present disclosure provides an siRNA complex comprising the above siRNA and a complexing group complexed and bound to the siRNA.

[0135] Generally, the complexing group includes at least one targeting group and an optional linker that are pharmaceutically acceptable, and the siRNA, the linker, and the targeting group are sequentially bound. In some embodiments, the number of the targeting groups is 1 to 6. In some embodiments, the number of the targeting groups is 2 to 4. The siRNA molecule is bound to the complexing group. ​​​​​​​​​​​ It may be non-covalently or covalently conjugated, e.g., covalently conjugated to the conjugating group. The conjugation site between the siRNA and the conjugation group may be at the 3' end or the 3' end of the sense strand of the siRNA. may be at the 5' end of the antisense strand, or at the 5' end of the antisense strand, or may be at an internal sequence of the siRNA. In some embodiments, the conjugation site between the siRNA and the conjugation group may be in a row. is at the 3' end of the sense strand of the siRNA.

[0136] In some embodiments, the conjugated group is a phosphate group of a nucleotide, a hydroxyl group at the 2'-position, In some embodiments, the conjugated group may be attached to a hydroxy group or a base. It may be attached to the 3'-hydroxy group, in which case the internucleotide is 2'-5' The conjugated group is attached to the end of the siRNA strand via a phosphate diester bond. When attached to the internal sequence of siRNA, it is usually attached to the phosphate group of a nucleotide. , usually attached to the ribose sugar ring or the base. For various attachment methods, see the literature: Mut hiah Manoharan et.al. siRNA conjugates c arrying sequentially assembled trivalent N-acetylgalactosamine linked through nu cleosides elicit robust gene silencing i n vivo in hepatocytes. ACS Chemical biol ogy,2015,10 (5):1181~7.

[0137] In some embodiments, the bond between the siRNA and the conjugation group is acid-labile or reducible. These chemical bonds can be attached to the nucleosomes in the acidic environment of the cellular endosomes. In this case, the siRNA can be released by decomposition. In this case, the conjugation group is attached to the sense strand of siRNA, and the effect of conjugation on siRNA activity is investigated. can be reduced as much as possible.

[0138] In some embodiments, the pharmaceutically acceptable targeting group is a targeting group that is suitable for use in the field of siRNA administration. Ligands commonly used in the art, such as the various ligands described in WO2009082607A2 The disclosure of which is incorporated herein by reference in its entirety. .

[0139] In some embodiments, the pharmaceutically acceptable targeting group is a target of cholesterol, bile lipophilic molecules such as fatty acids, vitamins (e.g., tocopherol), and lipid molecules of different chain lengths; Polymers such as polyethylene glycol, polypeptides such as membrane-permeable peptides, and aptamers mers, antibodies, quantum dots, lactose, polylactose, mannose, galactose sugars such as galactosamine (GalNAc) and folic acid (folate) , asialoglycoprotein, asialoglycoconjugates, lipoproteins (e.g., high-density lipoproteins) , low-density lipoproteins, glucagon, neurotransmitters (e.g., adrenaline), growth target molecules such as receptor ligands expressed in hepatocytes, such as factors and transferrin, or The ligand may be one or more selected from the ligands formed by the derivatives.

[0140] In some embodiments, each of the ligands is a ligand capable of binding to a receptor on a cell surface. In some embodiments, at least one ligand is independently selected from the group consisting of: is a ligand that can bind to a receptor on the surface of liver cells. At least one ligand is a ligand capable of binding to a receptor on the surface of a mammalian cell. In some embodiments, the at least one ligand binds to a receptor on the surface of human hepatocytes. In some embodiments, at least one ligand is It is a ligand that can bind to the asialoglycoprotein receptor (ASGPR) on the liver surface. These types of ligands are known to those skilled in the art, and their action is generally to bind to the surface of target cells. It binds to specific receptors and mediates the delivery of siRNA bound to the ligand to target cells. do.

[0141] In some embodiments, the pharmaceutically acceptable targeting group is a target of a mammalian hepatocyte surface. Any one of the ligands that bind to the asialoglycoprotein receptor (ASGPR) on the surface In some embodiments, each ligand may independently be an asialoglycoprotein. Proteins, e.g., asialoorosomucoid (A SOR or asialofetuin (ASF). In some embodiments, the ligand is a sugar or a sugar derivative.

[0142] In some embodiments, at least one ligand is a sugar. In some embodiments, each ligand is a sugar. Another ligand is a monosaccharide, polysaccharide, modified monosaccharide, modified polysaccharide, or sugar derivative. In embodiments, at least one of the ligands may be a monosaccharide, a disaccharide, or a trisaccharide. In some embodiments, at least one ligand is a modified sugar. In some embodiments, each ligand is a modified sugar. Each ligand may independently be a polysaccharide, a modified polysaccharide, a monosaccharide, a modified monosaccharide, a polysaccharide derivative, or In some embodiments, each or at least one of the ligands is selected from monosaccharide derivatives. At least one of glucose and its derivatives, mannan and its derivatives, galactose and its derivatives, xylose and its derivatives, ribose and its derivatives, fucose and its derivatives derivatives, lactose and its derivatives, maltose and its derivatives, arabinose and its derivatives The sugar is selected from the group consisting of saccharides, fructose and its derivatives, and sialic acid.

[0143] In some embodiments, each of the ligands is a D-mannopyranose, an L-mannopyranose, or an Pyranose, D-arabinose, D-xylofuranose, L-xylofuranose, D-glucose glucose, L-glucose, D-galactose, L-galactose, α-D-mannofuranoside D-mannofuranose, β-D-mannofuranose, α-D-mannopyranose, β-D-mannopyranolate α-D-glucopyranose, β-D-glucopyranose, α-D-glucofurano β-D-glucofuranose, α-D-fructofuranose, α-D-fructopyranolate α-D-galactopyranose, β-D-galactopyranose, α-D-galactofuranose Ranose, β-D-galactofuranose, glucosamine, sialic acid, galactosamine, N -acetylgalactosamine, N-trifluoroacetylgalactosamine, N-propionyl Galactosamine, Nn-butyrylgalactosamine, N-isobutyrylgalactosamine , 2-amino-3-O-[(R)-1-carboxyethyl]-2-deoxy-β-D-glucan Glucopyranose, 2-deoxy-2-methylamino-L-glucopyranose, 4,6-di Deoxy-4-formamido-2,3-di-O-methyl-D-mannopyranose, 2-de Oxy-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 (methyl 2,3,4-t ris-O-acetyl-1-thio-6-O-trityl-α-D-gluco pyranoside), 4-thio-β-D-galactopyranose, ethyl 3,4,6, 7-Tetra-O-acetyl-2-deoxy-1,5-dithio-α-D-glucoheptopyril D-ribose, 2,5-anhydro-D-alanine, ribose, D-ribose, D-4-thiazolinone olivosulfate, L-ribose, or L-4-thioribose. For other options for the ligand, see, for example, CN105378082A. , the disclosure of which is incorporated herein by reference in its entirety.

[0144] In some embodiments, the pharmaceutically acceptable target in the siRNA complex The group may be galactose or N-acetylgalactosamine, and may be galactose or The N-acetylgalactosamine molecule may be monovalent, divalent, trivalent, or tetravalent. The monovalent, divalent, trivalent, and tetravalent antibodies each contain an siRNA molecule and galactose as the targeting group. or in siRNA complexes formed from conjugated groups containing N-acetylgalactosamine molecules. The molar ratio of siRNA molecules to galactose or N-acetylgalactosamine molecules in the It should be understood that this refers to a ratio of 1:1, 1:2, 1:3 or 1:4. In one embodiment, the pharmaceutically acceptable targeting group is N-acetylgalactosamine. In some embodiments, the siRNA described in the present disclosure comprises N-acetylgalactosamine. When conjugated to a conjugated group containing an N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent or tetravalent. In some embodiments, the siRNA described in the present disclosure is N-acetylgalactosidase. When conjugated to a conjugation group containing galactosamine, the N-acetylgalactosamine molecule is trivalent.

[0145] The targeting group may be attached to the siRNA molecule via a suitable linker, and those skilled in the art will appreciate that the targeting group may be attached to the siRNA molecule via a suitable linker. An appropriate linker can be selected depending on the specific type of target group. For the types of targeting groups and methods of binding to siRNA, see WO2015006740A2 Reference may be made to the disclosure of which the contents are incorporated herein by reference in their entirety.

[0146] In some embodiments, when the targeting group is N-acetylgalactosamine, A suitable linker may be the structure shown in formula (301).

[0147] [ka] During the ceremony, k is an integer from 1 to 3, L A is a chain portion containing an amide bond having a structure represented by formula (302), L A has one of the target groups and one of the L C Bonded to the part by an ether bond will be done.

[0148] [ka] L B is a chain containing N-acylpyrrolidine having the structure shown in formula (303). The chain portion has a carbonyl group at one end thereof, and the L C Bonded to the part by an amide bond The other end has an oxygen group, and is bound to the siRNA via a phosphate ester bond.

[0149] [ka] L C is hydroxymethylaminomethane, dihydroxymethylaminomethane or trihydroxymethylaminomethane is a divalent to tetravalent linker group based on hydroxymethylaminomethane, C is an oxygen source Each of the L's is bonded to an ether bond via a A and is bonded to the a group via a nitrogen atom. The L B The part is connected to the

[0150] In some embodiments, n=3 and L C Trihydroxymethylaminomethyl When the linker is a tetravalent linker group based on an alkyl group, -(L A )3 Trihydroxy Methylaminomethane-L B - N-acetylgalactosamine molecules and siRNA molecules The structure of the siRNA complex formed by binding is shown in the following formula (304).

[0151] [ka] In the formula, the double helix structure represents the siRNA.

[0152] Similarly, the conjugation site between the siRNA and the conjugation group is at the 3' or 5' end of the sense strand of the siRNA. It may be at the 5' end of the antisense strand, at the 5' end of the antisense strand, or in the internal sequence of the siRNA. It is also possible.

[0153] In some embodiments, the 3' end of the sense strand of the siRNA described in this disclosure is , linker-(L A )3-trihydroxymethylaminomethane-L B -Three N-A Covalently conjugated to a cetylgalactosamine (GalNAc) molecule, The molar ratio of GalNAc molecules to si is 1:3, and the structure is shown in the following formula (305): An RNA complex (hereinafter also referred to as (GalNAc)3-siRNA) is obtained.

[0154] [ka] In the formula, the double helix structure represents the siRNA, and the linker represents the sense domain of the siRNA. It is attached to the 3' end of the strand.

[0155] In some embodiments, when the targeting group is N-acetylgalactosamine, A suitable linker may be the structure shown in formula (306).

[0156] [ka] During the ceremony, l is an integer from 0 to 3, * represents the site on the linker that is attached to the targeting group via an ether bond, # represents the site in the linker that is bound to the siRNA via a phosphate bond. .

[0157] In some embodiments, when l=2, the siRNA complex has the formula (30 7) has the structure shown in

[0158] [ka] In the formula, the double helix structure represents the siRNA, and the linker represents the sense domain of the siRNA. It is attached to the 3' end of the strand.

[0159] The conjugates may be synthesized by methods already well described in the prior art. For example, WO2015006740A2 describes in detail a method for preparing multiple types of complexes. The siRNA complexes of the present disclosure can be obtained by methods well known to those skilled in the art. For example, WO2014025805A1 describes a method for preparing the structure shown in formula (305). Rajeev et al., ChemBioChem 2015,16,903-90 8 describes the preparation method of the structure shown in formula (307).

[0160] In some embodiments, the siRNA complex has the structure shown in formula (308): It has.

[0161] [ka] During the ceremony, n1 is an integer selected from 1 to 3, and n3 is an integer selected from 0 to 4. , m1, m2, and m3 are independently integers selected from 2 to 10, R 10 , R 11 , R 12 , R 13 , R 14 and R 15 are each independently H , or C1-C 10 Alkyl groups, C1-C 10 Halogenated alkyl groups and C1-C 10 a alkoxy groups; R3 is a group having the structure shown in formula A59.

[0162] [ka] wherein E1 is OH, SH, or BH2, and Nu is an siRNA of the disclosure.

[0163] R2 is a linear alkylene group of 1 to 20 carbon atoms in length, where one or more of the carbon atoms is , 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-C 18 Heterocyclylene group and C5-C 10 and optionally substituted with one or more selected from the group consisting of 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 Fluorinated alkyl groups, -SC1-C 10 Alkyl group, -SC1-C 10 Alkylphenyl group , -C1-C 10 Alkyl-SH, -SC1-C 10 Halogenated alkyl groups, halogen groups 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), sia -C(O)O(C1-C 10 alkyl group), -CON(C 1-C 10 Alkyl group) (C1-C 10 alkyl group), -CONH(C1-C 10 Archi ), -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 Alkylphenyl group, -C(O)C1-C 10 Haloalkyl Group, -OC(O)C1-C 10 Alkyl group, -SO2(C1-C 10 alkyl group), -S O2 (phenyl group), -SO2 (C1-C 10 halogenated alkyl group), -SO2NH2 , -SO2NH(C1-C 10 alkyl group), -SO2NH (phenyl group), -NHSO 2(C1-C 10 alkyl group), -NHSO2 (phenyl group) and -NHSO2 (C1- C 10 and optionally having one or more substituents selected from the group consisting of halogenated alkyl groups. That's fine.

[0164] Each L1 is a straight chain alkylene group of 1 to 70 carbon atoms in length, is C(O), NH, O, S, CH=N, S(O)2, C2-C 10 Alkenylene group, C 2-C 10 Alkynylene group, C6-C 10 Arylene group, C3-C 18 Heterocyclylene Groups and C5-C 10 and optionally substituted with one or more selected from the group consisting of heteroarylene groups. L1 is converted into C1-C 10 Alkyl groups, C6-C 10 Aryl groups, C5-C 10 Haitai Aryl groups, C1-C 10 Halogenated alkyl groups, -OC1-C 10 Alkyl group, -O C1-C 10 Alkylphenyl group, -C1-C 10 Alkyl-OH, -OC1-C 10 Ha Halogenated alkyl group, -SC1-C 10 Alkyl group, -SC1-C 10 Alkylphenyl group, -C1-C 10 Alkyl-SH, -SC1-C 10 Halogenated alkyl groups, halogens Substituent, -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), Ano 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 Al (kill 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 Archi group, -C(O)C1-C 10 Alkylphenyl group, -C(O)C1-C 10 Hello Aruki group, -OC(O)C1-C 10 Alkyl group, -SO2(C1-C 10 alkyl group), - SO2 (phenyl group), -SO2 (C1-C 10 halogenated alkyl group), -SO2NH 2. -SO2NH(C1-C 10 alkyl group), -SO2NH (phenyl group), -NHS O2(C1-C 10 alkyl group), -NHSO2 (phenyl group) and -NHSO2 (C1 -C 10 and optionally having one or more substituents from the group consisting of halogenated alkyl groups. You may do so.

[0165] In some embodiments, L1 consists of groups A1 through A26 or any combination thereof. The structures and definitions of A1 to A26 are as follows:

[0166] [ka] TIFF0007672163000022.tif32170 where j1 is an integer between 1 and 20, j2 is an integer between 1 and 20, R' is C1-C 10 is an alkyl group of the formula Ra is selected from the group consisting of groups of formulae A27 to A45 or any combination thereof.

[0167] [ka] Rb is C1-C 10 is an alkyl group of the formula TIFF0007672163000024.tif19170 represents the site at which the group is attached to the rest of the molecule.

[0168] For convenience, L1 is defined as a linear alkylene group, although one of skill in the art will recognize that amines and alkenyl groups resulting from the above-described replacement and / or substitution may not be linear or may be named differently. 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, a ring (e.g., heterocyclylene or heteroarylene) obtained by substituting a carbon atom of the linear alkylene is considered to be one atom.

[0169] M1 represents a targeting group, and its definition and selectable range are the same as those of the targeting group described above. In some embodiments, each M1 is an asialoglycoprotein on the surface of a mammalian liver cell. The ligand is one independently selected from ligands having affinity for the protein receptor.

[0170] M1 has affinity for the asialoglycoprotein receptor on the surface of mammalian liver cells. In some embodiments, n1 is an integer from 1 to 3. n3 may be an integer from 0 to 4, and n3 may be the number of M1 targeting groups in the complex. is at least 2. In some embodiments, n1+n3≧2. This indicates that the number of M1 targeting groups is at least 3, and that the M1 targeting groups and the liver surface It allows alloglycoprotein receptors to bind more readily, and the complex is then endocytosed. This action can promote cellular uptake. When the number of targeting groups is three or more, the M1 targeting group and the asialoglycoprotein receptor on the liver surface Since the improvement in ease of binding with the body is not clear, the ease of synthesis, structure / process cost In some embodiments, n1 is 1, taking into consideration various aspects such as the amount of hydroxybenzoates and delivery efficiency. n1 is an integer between 0 and 1, and n1+n3=2 or 3.

[0171] In some embodiments, m1, m2, and m3 are independently selected from integers from 2 to 10. When selected, the spatial position between multiple M1 targeting groups is determined by the relationship between the M1 targeting group and the asialoglycoprotein on the liver surface. The complexes provided by the present disclosure can be adapted to bind to protein receptors. In order to simplify, make synthesis easier, and / or reduce costs, some embodiments In the formula, m1, m2, and m3 are each independently an integer of 2 to 5, and In the embodiment, m1=m2=m3.

[0172] R 10 , R 11 , R 12 , R 13 , R 14 and R 15 But, H, C1-C 10 Alkyl group , C1-C 10 Halogenated alkyl groups and C1-C 10 Independently from each alkoxy If one of them is selected, it is possible to obtain the desired compound without changing the properties of the complex of the present disclosure. It will be understood by those skilled in the art that the objectives of the present invention can be achieved in some embodiments. Hey, R 10 , R 11 , R 12 , R 13 , R 14 and R 15 are each independently H, In some embodiments, R 10 , R 11 , R 12 , R 13 , R 14 and R 15 are both H.

[0173] R3 is a group having the structure shown in formula A59, in which E1 is OH, SH, or BH2 In consideration of the availability of preparation raw materials, in some embodiments, E1 is OH Or SH.

[0174] R2 is selected to achieve a bond between N on the nitrogen-containing backbone and A59. In this context, a "nitrogen-containing backbone" is R 10 , R 11 , R 12 , R 13 , R 14 and R 15 It refers to a chain structure in which the carbon atom to which R2 is attached and the N are bonded to each other. is any linker capable of connecting the A59 group to an N on the nitrogen-containing backbone in a suitable manner. In some embodiments, a compound of formula (308) can be prepared by a solid phase synthesis process. When preparing the siRNA complex shown in Both the binding site bound to P in R1 and the binding site bound to P in R2 must be included. In some embodiments, the site of R2 bonded to N in the nitrogen-containing backbone is The site on R3 that is bonded to P forms an amide bond with N, and the site on R3 that is bonded to P forms a phosphate ester bond with P. In some embodiments, R2 is B5, B6, B5', or B6'. It is also possible.

[0175] [ka] however, TIFF0007672163000026.tif14129 represents the site to which the group is covalently attached.

[0176] The value of q2 may range from 1 to 10, and in some embodiments, q 2 is an integer from 1 to 5.

[0177] L1 connects the M1 targeting group to the N on the nitrogen-containing backbone, forming the siRNP shown in formula (308). In some embodiments, L 1 is selected from one or more bond combinations of groups of formulae A1 to A26. In the formula, L1 is one of A1, A4, A5, A6, A8, A10, A11 and A13, or In some embodiments, L is selected from the group consisting of A, A, At least two of A8, A10 and A11 are selected from the combination of A8, A10 and A11. In the embodiment, L1 is selected from at least two bond combinations of A1, A8, and A10. do.

[0178] In some embodiments, the length of L1 is 3 to 25 atoms, 3 to 20 atoms, It may be 4 to 15 atoms or 5 to 12 atoms. The length of L1 is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 pieces, 14 pieces, 15 pieces, 16 pieces, 17 pieces, 18 pieces, 19 pieces, 20 pieces, 21 pieces, 22 pieces, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60 It is an atom.

[0179] For convenience, L1 is defined as a linear alkylene group, although one of skill in the art will recognize that amines and alkenyl groups resulting from the above-described replacement and / or substitution may not be linear or may be named differently. 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, a ring (e.g., heterocyclylene or heteroarylene) obtained by substituting a carbon atom of the linear alkylene is considered to be one atom.

[0180] In some embodiments, 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 In some embodiments, R' is an alkyl group, and in some embodiments, R' is a methyl group, an ethyl group, and an isopropyl group. Ra is one of A27, A28, A29, A30, and A31. In some embodiments, Ra is A27 or A28. Rb is C1- In some embodiments, Rb is a methyl group, an ethyl group, In some embodiments, the aryl group represented by Formula A1-A2 is one of an isopropyl group and a butyl group. By selecting j1, j2, R', Ra, and Rb in 6, the M1 target group and The binding to N on the nitrogen-containing backbone is realized, and the spatial position between the M1 targeting group and the liver surface is adjusted. It is further adapted for binding to the asialoglycoprotein receptor.

[0181] In some embodiments, the complex has the formula (403), (404), (405): , (406), (407), (408), (409), (410), (411), (41 2), (413), (414), (415), (416), (417), (418), ( 419), (420), (421) or (422).

[0182] [ka] TIFF0007672163000028.tif231170TIFF0007672163000029.tif226170TIFF0007672163 000030.tif245170TIFF0007672163000031.tif179170TIFF0007672163000032.tif92170

[0183] In some embodiments, P in formula A59 is any group in the siRNA sequence. For example, P in formula A59 is a nucleotide in the sense strand of the siRNA. In some embodiments, the nucleotide sequence may be linked to either one of the nucleotides in the antisense or antisense strand. In the above embodiment, P in formula A59 represents any one nucleoside in the sense strand of the siRNA. In some embodiments, P in formula A59 is linked to a nucleotide of the siRNA. attached to the end of the sense strand or antisense strand, and in some embodiments, represented by the formula A59 The P in the formula (I) is bound to the end of the sense strand of the siRNA. Or, in the antisense strand, it refers to the four nucleotides from one end to the front. In this embodiment, P in formula A59 is the sense strand or the antisense strand of the siRNA. In some embodiments, P in formula A59 is linked to the end of the siRNA. When it is bound to the above position of the sense strand of siRNA, , the siRNA complex shown in formula (308) enters the cell and then unfolds. The antisense strand of the siRNA was released, and ANGPTL3 mRNA was expressed as a protein. Blocking the process of translating proteins and the angiopoietin-like protein 3 (ANGPTL3) gene Expression can be suppressed.

[0184] In some embodiments, P in Formula A59 represents a nucleotide in the siRNA. Any possible position on the nucleotide, for example, the 5' position of the nucleotide, the 2' position of the nucleotide, It may be attached to the 3' position of the nucleotide or the base of the nucleotide. In formula A59, P is a phosphodiester bond that binds the siRNP to the ribonucleotide. A may be attached to the 2', 3' or 5' position of the nucleotide in A. In some embodiments, P in formula A59 represents the 3'-terminal nucleotide of the sense strand of the siRNA. The P in formula A59 is bonded to the oxygen atom obtained by dehydrogenating the 3' hydroxy group of the , replacing the hydrogen in the 2'-hydroxy group of one nucleotide in the sense strand of the siRNA. Alternatively, P in formula A59 is an siRNA. by replacing the hydrogen in the 5' hydroxy group of the 5' terminal nucleotide of the sense strand of Attached to a nucleotide.

[0185] The inventors of the present disclosure have unexpectedly discovered that the siRNA complexes of the present disclosure have significantly improved plasma stability, low off-target effects, and further exhibit significantly undiminished ANGPTL3 mRNA silencing activity, and further have high lipid-lowering effects. Thus, in some embodiments, the siRNA in the siRNA complex of the present disclosure may be one of siAN1, siAN2, siAN1-M1, siAN2-M1, siAN1-M2, siAN2-M2, siAN1-M3, siAN2-M3, siAN1-M1S, siAN2-M1S, siAN1-M2S, siAN2-M2S, siAN1-M3S, siAN2-M3S, siAN1-M1P1, siAN2-M1P1, siAN1-M2P1, siAN2-M2P1, siAN1-M3P1, siAN2-M3P1, siAN1-M1SP1, siAN2-M1SP1, siAN1-M2SP1, siAN2-M2SP1, siAN1-M3SP1, siAN2-M3SP1 shown in Table 1.

[0186] [Table 1] JPEG0007672163000034.jpg89164 siRNA sequence in the complex of the present disclosure

[0187] In the siRNA or siRNA complexes described in this disclosure, each adjacent nucleotide The bonds between the diesters are phosphate diester bonds or thiophosphate diester bonds. The non-bridging oxygen or sulfur atoms in the ester or thiophosphate bond are negatively charged. It is charged with a hydroxyl or sulfhydryl group. The hydrogen ions in the hydroxyl or sulfhydryl groups may be partially or completely separated. may be substituted with a cation. The cation may be any cation, for example, a metal Cation, ammonium ion NH4 + , may be one of the organic ammonium cations In consideration of improving solubility, in one embodiment, the cation is an alkali metal ions, ammonium cations formed by tertiary amines, and quaternary ammonium cations The alkali metal ions are one or more selected from the group consisting of K + and / or Na + and the cation formed by the tertiary amine may be The formed ammonium ions and / or N,N-diisopropylethylamine The ammonium ion formed may be an ammonium ion. The NA or siRNA complex may be present at least in part as a salt. In the case of The sulfur atoms are at least partially bonded to sodium ions, and the si The RNA or siRNA complexes are present as sodium salts or partial sodium salts.

[0188] As will be apparent to those skilled in the art, nucleoside monomers with corresponding modifications can be used. Modified nucleotide groups are introduced into the siRNAs described in this disclosure by using Methods for preparing nucleoside monomers with corresponding modifications and modified nucleoside monomers are also described. Methods for introducing nucleotide groups into siRNA are also well known to those skilled in the art. The cleoside monomers may be purchased commercially or may be prepared by known methods.

[0189] <Preparation of siRNA complex represented by formula (308)> The siRNA complex represented by formula (308) can be prepared by any reasonable synthetic route. good.

[0190] In some embodiments, the siRNA complex represented by formula (308) can be prepared by the following method: This method can be carried out under the conditions of phosphoramidite solid phase synthesis. Depending on the nucleotide types and procedures of the sense and antisense strands of the siRNA, Nucleoside monomers are sequentially linked from 1 to 5', and the linkage of each nucleoside monomer is involves four reactions: deprotection, coupling, capping, and oxidation or sulfurization, and and isolating and annealing the sense and antisense strands of the siRNA. , the siRNA of the present disclosure.

[0191] The method also includes reacting a compound of formula (32) under coupling reaction conditions and in the presence of a coupling reagent. 1) The compound shown in 1) is combined with a nucleoside monomer or a nucleotide bound to a solid support. and contacting the compound represented by formula (321) with the sequence, and subjecting the compound represented by formula (321) to a coupling reaction to form a nucleotide. The compound of formula (321) is a conjugated molecule. It is also called.

[0192] [ka] During the ceremony, R4 is a moiety capable of binding to siRNA, as shown in Nu. wherein R4 is a moiety capable of covalently binding to the siRNA shown in Nu. In some embodiments, R4 is linked to N through a phosphodiester bond via a reaction. is a moiety that can be conjugated to any functional group of siRNA shown in u, Each S1 was independently prepared by substituting all active hydroxy groups in M1 with YCOO- groups. each Y is a methyl group, a trifluoromethyl group, a difluoromethyl group, a fluoromethyl group, methyl group, trichloromethyl group, dichloromethyl group, chloromethyl group, ethyl group, n-propyl group Independent of propyl, isopropyl, phenyl, halophenyl and alkylphenyl groups In some embodiments, Y is a methyl group.

[0193] 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 M1 are as described above.

[0194] R4 provides a bond with N on the nitrogen-containing backbone, forming the siRNA complex shown in formula (308). In some embodiments, the hydroxyl group is selected to provide suitable reactive sites for the synthesis of the hydroxyl group. R4 is an R2 linker group or a protected R2 linker group, and by reacting It contains a functional group that can form the structure shown in siRNA and A59.

[0195] In some embodiments, R4 is an siRNA or nucleoside molecule designated Nu. a first functional group capable of forming a phosphite ester with a group on the monomer; and a hydroxy group. or a second functional group capable of reacting with an amino group to form a covalent bond or and a solid support attached by a bond. In some embodiments, the first functional The group is a phosphoramidite, a hydroxy group, or a protected hydroxy group. In one embodiment, the second functional group is a phosphoramidite, a carboxylic acid, or a carbo In some embodiments, the second functional group is a phosphate. a solid support that is bound to another moiety of the molecule, said covalent bond being via a hydroxy or amino group In some embodiments, the solid support is formed of a phosphate ester The bond is a carboxylic acid ester bond or an amide bond. In some embodiments, the solid support is a resin.

[0196] In some embodiments, the first functional group is a hydroxy group, —OR k or the formula ( The second functional group includes a group represented by formula (C1), (C2), (C3), ( It includes the structure shown in (C1') or (C3').

[0197] [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, TIFF0007672163000037.tif15150 represents the site at which the group is attached to the rest of the molecule.

[0198] In some embodiments, the first functional group is a phosphine, as shown in formula (C3): The phosphoramidite group may be at any position on the nucleotide. Coupling with a hydroxy group, for example, a hydroxy group at the 2' position or a hydroxy group at the 3' position This reacts to form a phosphite ester, which is then oxidized or sulfurized to form a diphosphate represented by formula A59. The conjugated molecule is conjugated to the siRNA by forming a ester or phosphorothioate bond. In this case, even if the second functional group is not present, the compound of formula (321) can be to obtain an siRNA complex represented by formula (308) which can be conjugated to a nucleotide. In this case, siR is synthesized by a method such as phosphoramidite solid phase synthesis. After obtaining the sense strand or antisense strand of NA, the compound of formula (321) and the nucleotide sequence In the sequence, the hydroxyl group on the terminal nucleotide is reacted, and in a subsequent oxidation or sulfurization step, to form a phosphodiester bond or a thiophosphate bond, and the compound of formula (321) The substance is conjugated to the siRNA.

[0199] In some embodiments, the first functional group comprises a protected hydroxy group. In some embodiments, the second functional group comprises a group capable of reacting with a solid support. The reaction provides a conjugated molecule comprising the solid support. The functional group of 2 is a carboxyl group, a carboxy group, or a carboxy group as shown in formula (C1), (C2), or (C3). The second functional group may be a carboxyl group or a phosphoramidite. When the compound of formula (321) contains a hydroxyl group on a solid support, e.g., a resin, The group or amino group is subjected to an esterification reaction or an amidation reaction, and the resulting compound is formed into a carboxylic acid ester bond. The second functional group is a phosphoramidite, forming a conjugated molecule containing the solid support. When the compound of formula (321) contains a hydroxyl group, it is possible to combine the compound of formula (321) with a conventional solid support, e.g., a hydroxyl group in a resin. The hydroxyl group was then oxidized and bonded via a phosphodiester bond. Then, the solid support-bound product is used as a starting material to form a complex molecule containing the solid support. Then, nucleoside monomers are sequentially linked according to the phosphoramidite solid phase synthesis method to form a complex. The sense or antisense strand of the siRNA is obtained with the group attached. During the phase synthesis, the first functional group is deprotected and then subjected to coupling reaction conditions. The nucleoside is coupled to a phosphoramidite group in a nucleoside monomer below.

[0200] In some embodiments, the first functional group is a hydroxy group or a protected hydroxy group. The second functional group contains an oxy group, as shown in formula (C1') or (C3'): Carboxylic acid ester bonded solid support or amide bonded solid support The starting material includes a carrier or a solid phase carrier bound by a phosphate ester bond. The compound of formula (321) was used in place of the solid support and the phosphoramidite solid phase synthesis method was followed. The sense strand or the amino acid sequence of the siRNA is formed by sequentially binding the nucleoside monomers and attaching the complex group. The antisense strand is obtained.

[0201] In some embodiments, the carboxylate is —COO - M + This may be expressed as So, M + is a cation, for example, a metal cation, an ammonium cation NH + , In one embodiment, the gold cation is one selected from organic ammonium cations. The group ion is one selected from alkali metal ions, for example, K + or Na + in In order to improve solubility and facilitate the reaction, some embodiments are In this case, the organic ammonium ion is the ammonium cation formed by a tertiary amine. or quaternary ammonium cations, e.g., ammonia formed with triethylamine Ammonium ions or ammonium ions formed by N,N-diisopropylethylamine In some embodiments, the carboxylate is triethylamine carboxylic acid. salt or N,N-diisopropylethylamine carboxylate.

[0202] In some embodiments, R4 is selected from the group consisting of formula (B9), (B10), (B9′), (B1 0'), (B11), (B12), (B11') or (B12') .

[0203] [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, TIFF0007672163000039.tif12169 represents the site at which the group is attached to the remainder of the molecule. is 1 or 2. In some embodiments, q2 is an integer from 1 to 5. In this embodiment, R4 comprises a structure shown in formula (B9) or (B10). In some embodiments, R4 comprises a structure shown in formula (B11) or (B12).

[0204] In some embodiments, Rk is one or more of Tr (trityl group), MMTr (4-methoxytrityl group), DMTr (4,4'-bismethoxytrityl group), TMTr (4,4',4'-trimethoxytrityl group). In some embodiments, Rk can be DMTr, i.e., 4,4'-dimethoxytrityl.

[0205] The definition of L1 is as described above.

[0206] In some embodiments, L1 attaches the M1 targeting group to an N atom on the nitrogen-containing backbone. , is used to provide liver targeting functionality to the siRNA complex shown in formula (308). In some embodiments, L1 includes any one of A1 to A26 or a combination thereof. nothing.

[0207] From the above description, it will be readily apparent to those skilled in the art that phosphorylation is possible using phosphorylation methods known in the art. Compared with the midite solid phase synthesis method, the first functional group and the optional second functional group allow for complex The nucleotide sequence may be inserted at any possible position in the nucleotide sequence, for example, at the end of the nucleotide sequence, to obtain an siRNA complex represented by formula (308) linked to the end of the nucleotide sequence. Accordingly, unless otherwise specified, in the following description of the preparation of the complex, This allows for reactions such as "deprotection," "coupling," "capping," "oxidation," and "sulfurization." Where reference is made, reaction conditions for solid phase synthesis of phosphoramidite nucleic acids known in the art are used. It should be understood that the same applies to these reactions. The drugs are described in detail below.

[0208] In some embodiments, each S1 is independently M1. wherein each S1 independently substituted at least one active hydroxy group in M1 with a hydroxyl group. In some embodiments, each S is independently a group protected by a protecting group. All of the active hydroxy groups present in the compound are protected with hydroxy protecting groups. In embodiments, any hydroxy protecting group known to one skilled in the art can be used to protect the active hydroxy in M1. In some embodiments, the protected hydroxyl group can be used to protect the hydroxyl group. The hydroxy group may be represented by the formula YCOO-, where each Y is independently C-C 10 Al alkyl groups and C6-C 10 aryl groups, wherein the C1-C 10 Alkyl Groups and C6-C 10 The aryl group is optionally substituted with one or more substituents, said substituents being , halogen, and a C1-C6 alkyl group. wherein each Y is independently a methyl group, a trifluoromethyl group, a difluoromethyl group, a monomethyl group, Monofluoromethyl group, trichloromethyl group, dichloromethyl group, chloromethyl group, ethyl group, n-propyl group, isopropyl group, phenyl group, halophenyl group and C1-C6 alkoxy group alkylphenyl groups.

[0209] In some embodiments, each S1 independently consists of formula A46-A54 is selected from the group.

[0210] [ka]

[0211] In some embodiments, S1 is of formula A49 or A50.

[0212] In some embodiments, each Y is a methyl group, a trifluoromethyl group, a difluoromethyl group, Methyl group, monofluoromethyl group, trichloromethyl group, dichloromethyl group, chloromethyl phenyl, halophenyl, and aryl groups. In some embodiments, Y is one independently selected from alkylphenyl groups. is a methyl group.

[0213] As described above, the method for preparing the siRNA complex represented by formula (308) is The other strand of the siRNA is synthesized (for example, the sense strand of the siRNA to which the conjugated molecule is bound in the above step). When the antisense strand of siRNA is synthesized, it is further recommended to synthesize the antisense strand of siRNA according to the solid phase synthesis method. and vice versa), isolating and annealing the sense and antisense strands. Specifically, in the isolation step, the nucleotide sequence and / or The solid support bound to the conjugated molecule is cleaved and the necessary protecting groups are removed (in this case In this case, each S1 group in the compound of formula (321) is converted to the corresponding M1 target group), The sense strand (or antisense strand) of the siRNA to which the conjugated molecule is bound and the corresponding antisense strand are The sense strand and antisense strand are annealed to form a double-stranded RNA. The A structure is formed to obtain the siRNA complex shown in formula (308).

[0214] In some embodiments, the method for preparing the siRNA complex of formula (308) comprises: Under coupling reaction conditions and in the presence of a coupling reagent, a compound of formula (321) contacting the first nucleoside monomer at the 3' end of the sense strand or the antisense strand; The compound represented by formula (321) is attached to the first nucleotide in the sequence, and phospho The desired sense or antisense nucleotide species are synthesized under the conditions of thiamidite solid phase synthesis. Nucleoside monomers are sequentially linked from 3' to 5' according to the method and procedure, and siRNP is synthesized. A step of synthesizing a sense strand or an antisense strand of A, wherein the compound (321) is R4 a first functional group containing a protected hydroxy group, and a compound represented by formula (C1') or (C3'): A compound represented by formula (321) containing a second functional group having a structure represented by formula (321): Before coupling with the second nucleoside monomer, the compound of formula (321) is deprotected and each nucleoside is The bond between the thiol and thiol monomers can be achieved through four steps: deprotection, coupling, capping, oxidation, or sulfurization. a step of reacting a nucleic acid with a conjugated group to obtain a sense strand or an antisense strand of the nucleic acid; The conditions for solid-phase thiamidite synthesis depend on the type and procedure of nucleotides in the antisense or sense strand. By this sequence, nucleoside monomers are linked in order from 3' to 5' to form the antisense strand of the nucleic acid. The reverse or sense strand is synthesized, and the bonds of each nucleoside monomer are deprotected, coupled, and It involves four reactions: capping, oxidation, or sulfurization, which remove the protecting groups and cleave the peptide from the solid support. The method includes steps of isolating and purifying the sense and antisense strands of the nucleic acid, and annealing them.

[0215] In some embodiments, the method for preparing the siRNA complex of formula (308) comprises: The type and method of nucleotides in the sense strand or antisense strand of the double-stranded siRNA Nucleoside monomers are sequentially linked from 3' to 5', and the sense strand and the The antisense strand is synthesized, and the bonds of each nucleoside monomer are deprotected, coupled, and capped. The four reactions include cleaving, oxidation, or sulfurization, resulting in a sense strand bound to a solid support, and Obtaining the antisense strand bound to the substrate; coupling reaction conditions and coupling reagents In the presence of The antisense strand is contacted with a carrier-bound antisense strand, and a first phosphoramidite group is attached to R4. A step of binding a compound of formula (321) containing a functional group to a sense strand or an antisense strand. The protecting groups are removed, the solid support is cleaved, and the si- The method includes the steps of obtaining a sense strand or an antisense strand of RNA and annealing them.

[0216] In some embodiments, P in formula A59 is the nucleotide of the sense strand in the siRNA. A method for preparing an siRNA complex bound to the 3' end and represented by formula (308) is as follows: (1) A compound of formula (321) (the compound of formula (321) has a protected hydroxyl group at R4) Shi-base OR k and a first functional group having a structure represented by formula (C1') or (C3'): a hydroxy-protecting group R in k Remove the The deprotected product is converted to a nucleoside under coupling reaction conditions and in the presence of a coupling reagent. and contacting the monomer to obtain a nucleoside monomer bound to the solid support by a conjugation molecule. thing, (2) Starting with the nucleoside monomer that is bound to the solid support by the conjugated molecule, Synthesize the sense strand of siRNA by phosphoramidite solid-phase synthesis in the '-5' direction. To do, (3) Synthesize the antisense strand of siRNA by phosphoramidite solid-phase synthesis. To do so, (4) The sense and antisense strands of the siRNA are isolated and annealed to form a nucleotide sequence represented by the formula (308): and obtaining an siRNA complex as shown in

[0217] In step (1), the protecting group R in the compound of formula (321) k The method to remove The method comprises contacting a compound of formula (321) with a deprotecting reagent under deprotecting conditions. In some embodiments, the temperature is 0 to 50°C, and in some embodiments, 15 to 35°C. The reaction time is 30 to 300 seconds, and in some embodiments, 50 to 150 seconds. The deprotection reagent is selected from trifluoroacetic acid, trichloroacetic acid, dichloroacetic acid, and chloroacetic acid. In some embodiments, the compound may be dichloroacetic acid. The molar ratio of the deprotection reagent to the compound of formula (321) is 10:1 to 1000:1, In some embodiments, it is from 50:1 to 500:1.

[0218] The coupling reaction conditions and coupling reagents are those suitable for the above coupling reaction. In some embodiments, solid phase synthesis is employed. The same conditions and reagents as in the coupling reaction in the method may be used.

[0219] In some embodiments, the coupling reaction conditions include a reaction temperature of 0 to 5 0° C., and in some embodiments, 15 to 35° C. Compound of Formula (321) and the nucleoside monomer in a molar ratio of 1:1 to 1:50. The molar ratio of the compound of formula (321) to the coupling reagent is 1:2 to 1:5. :1 to 1:50, and in some embodiments, 1:3 to 1:10. In some embodiments, the reaction time is 200 to 3000 seconds, and in some embodiments, 500 to 150 The coupling reagents are 1H-tetrazole, 5-ethylthio 1H-tetrazole, 1H-tetrazole, 5-benzylthio-1H-tetrazole, In some embodiments, the coupling is 5-ethylthio-1H-tetrazole. The reaction may be carried out in an organic solvent, such as anhydrous acetonitrile, anhydrous DMF, or the like. and anhydrous dichloromethane, and in some embodiments, The amount of the organic solvent used for the compound of formula (321) is The concentration is 3 to 50 L / mol, and in some embodiments, 5 to 20 L / mol.

[0220] 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 that is bound to a solid support by a conjugated molecule, the 3'-5' In this case, the sense strand S of the second type of siRNA complex is synthesized in the direction of The nucleotide sequence is attached to the 3' end of the sense strand.

[0221] 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 type and dosage of the oxidation reagent, the sulfurization reaction conditions, the sulfurization reagent and dosage, and the method of the present invention are generally used in the art. Various reagents, dosages and conditions are used.

[0222] For example, in some embodiments, in steps (2) and (3), the solid phase synthesis Then, the following conditions may be used:

[0223] The deprotection conditions for the nucleoside monomer are a temperature of 0 to 50°C, and several experiments have been carried out. In some embodiments, the temperature is 15 to 35°C, and the reaction time is 30 to 300 seconds. In an embodiment, the deprotection time is 50 to 150 seconds, and the deprotection reagent is trifluoroacetic acid, trichloroacetic acid, The compound may be one or more selected from acetic acid, dichloroacetic acid, and chloroacetic acid. In some embodiments, the deprotection reagent is dichloroacetic acid. The molar ratio of the dimethoxytrityl protecting group to the methyltrityl protecting group may be 2:1 to 100:1. In this embodiment, the ratio is 3:1 to 50:1.

[0224] 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 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 support to the coupling reagent is 1:1 to 1: 100, and in some embodiments, 1:50 to 1:80, and the reaction time and the The selection of coupling reagents is the same as above.

[0225] 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 is 1:100 to 100:1. In some embodiments, the ratio is 1:10 to 10:1. When molar amounts of acetic anhydride and N-methylimidazole are used, The molar ratio of imidazole 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.

[0226] The oxidation reaction conditions include 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. In some embodiments, the reaction time is 5 to 5 0 seconds, and the oxidizing reagent is, in some embodiments, iodine (in some embodiments (In some cases, it is provided as iodine water.) The oxidation reagent and the solid phase in the coupling step The molar ratio of the nucleic acid sequence bound to the carrier may be 1:1 to 100:1, and 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 include 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 50 to 2000 seconds. In some embodiments, the sulfurization reagent is xanthan gum. A sulfurization reagent and a nucleic acid sequence that is bound to a solid support in a coupling step. The molar ratio of is 10:1 to 1000:1, and in some embodiments, is 10:1 to 5 In some embodiments, the sulfurization reaction is carried out in a solvent mixture of acetonitrile:pyridinyl ... It is carried out in a mixed solvent of 1:3 to 3:1 ethanol.

[0227] After all nucleoside monomers have been attached, but before annealing, the method comprises: The method further comprises isolating the sense and antisense strands of the nucleic acid sequence. Isolation methods are well known to those skilled in the art. Generally, the synthesized nucleotide sequence is cleaved from the solid support and the bases are phosphate- This includes removing the protecting groups on the base and on the ligand, purifying and desalting.

[0228] 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 the usual cleavage and deprotection methods used in siRNA synthesis. For example, the obtained solid support-bound nucleotide sequence may be treated with concentrated aqueous ammonia. In the deprotection process, the protecting group YCOO- of A46 to A54 is converted to a hydroxy group and converting the S1 group to the corresponding M1 group to form the complex shown in formula (308). Here, the concentrated ammonia water may be 25 to 30% by weight of ammonia water. The dose of concentrated ammonia water is 0.2 ml / μmol to the target siRNA sequence. It may be 0.8 ml / μmol.

[0229] If the synthesized nucleotide sequence has at least one 2'-TBDMS protection, The method involves removing the solid support from the nucleotide sequence by triethylamine trihydrofluoride. This further comprises removing the 2'-TBDMS protection by contacting the In this case, the resulting desired siRNA sequence contains the corresponding nucleic acid with a free 2'-hydroxy group. The dose of pure triethylamine trihydrofluoride is The amount may be 0.4 ml / μmol to 1.0 ml / μmol for the iRNA sequence. As a result, the siRNA complex represented by formula (308) can be obtained.

[0230] Methods of purification and desalting are well known to those skilled in the art. For example, preparative ion chromatography Purification of nucleic acids by gradient elution of NaBr or NaCl using a hydrochromatographic purification column The product was collected and combined, and then desalted using a reversed phase chromatography purification column. It is possible.

[0231] In the siRNA complex thus obtained, represented by formula (308), the nucleotide A non-bridging oxygen atom or sulfur atom in the phosphodiester or thiophosphodiester bond between The yellow atom is essentially bonded to a sodium ion, and the siR shown in formula (308) The NA complex exists primarily as a sodium salt. and replacing the sodium ions with hydrogen ions and / or other cations to form other forms of the formula The cation can be used in the same manner as described above. That's right.

[0232] During the synthesis process, the purity and molecular weight of nucleic acid sequences can be constantly monitored to better control the synthesis quality. Such detection methods are known to those skilled in the art. For example, ion exchange chromatography Nucleic acid purity was detected by liquid chromatography and purified by liquid chromatography tandem mass spectrometry. The molecular weight can be measured by this method.

[0233] Annealing methods are also well known to those skilled in the art. The sense strand (S strand) and antisense strand (AS strand) were mixed in an equal molar ratio with water for injection and incubated for 70-9 min. It is heated to 5°C and then cooled to room temperature, allowing the formation of a double-stranded structure through hydrogen bonding. In this way, the siRNA complex shown in formula (308) can be obtained.

[0234] After obtaining the complex, in some embodiments, the complex may be purified by, for example, liquid chromatography. Using methods such as tandem mass spectrometry, the synthesized compound shown in formula (308) was analyzed by molecular weight detection. We have clarified the characteristics of the siRNA complexes synthesized and confirmed that the synthesized siRNA complexes are The siRNA complex represented by formula (308) and the sequence of the synthesized siRNA is the desired siRNA sequence, for example one of the sequences shown in Table 1. can.

[0235] The compound of formula (321) can be prepared in an organic solvent under esterification reaction conditions and a base. and contacting the compound represented by formula (313) with a cyclic acid anhydride in the presence of an esterification catalyst. ion exchange and isolation to obtain the compound of formula (321). can be obtained by

[0236] [ka] In the formula, 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. R6 is a group that provides R4 in formula (321). In some embodiments, , R6 has the structure shown in formula (A61).

[0237] [ka] In the formula, R i can achieve bonding with N on the nitrogen-containing backbone, R k O to form one free R is any group to which a hydroxy group is attached. k is a hydroxy protecting group. In this case, R4 includes a first functional group as a hydroxy protecting group and a second functional group, and the second functional group is A compound of formula (321) containing a structure represented by formula (C1) or (C2) is obtained. do.

[0238] The esterification reaction conditions are a reaction temperature of 0 to 100°C and a reaction time of 8 to 4 hours. In some embodiments, the esterification reaction conditions include a reaction temperature of The temperature is 10 to 40°C, and the reaction time is 20 to 30 hours.

[0239] In some embodiments, the organic solvent is an epoxy solvent, an ether solvent, or a halogen-based solvent. Halogenated alkyl solvents, dimethyl sulfoxide, N,N-dimethylformamide and N , N-diisopropylethylamine. The epoxy solvent is dioxane and / or tetrahydrofuran, and the ether The terephthalic solvent is ethyl ether and / or methyl tert-butyl ether, Halogenated alkyl solvents include dichloromethane, trichloromethane and 1,2-dichloromethane. In some embodiments, the organic solvent is one or more of dichloromethane. The amount of the organic solvent is 3 to 100% by weight of the compound represented by the formula (313). In some embodiments, the concentration is 5 to 20 L / mol.

[0240] In some embodiments, the cyclic acid anhydride is succinic anhydride, glutaric anhydride adipic anhydride or pimelic anhydride, The compound represented by the formula (313) is a compound represented by the formula (314). The ratio of the fluorine-containing polymer to the fluorine-containing polymer is 1:1 to 10:1, and in some embodiments, 2:1 to 5:1.

[0241] The esterification catalyst may be any catalyst that catalyzes the esterification reaction, for example For example, the catalyst may be 4-dimethylaminopyridine. ) is 1:1 to 10:1 in some embodiments, , 2:1 to 5:1.

[0242] In some embodiments, the base is any inorganic base, organic base, or combination thereof. In consideration of solubility and stability of the product, the base may be, for example, The organic base may be a tertiary amine. In some embodiments, the tertiary amine The organic base is triethylamine or N,N-diisopropylethylamine. The molar ratio of the tertiary amine organic base to the compound represented by formula (313) is 1:1 to 20:1. and in some embodiments, 3:1 to 10:1.

[0243] The ion exchange reaction is carried out by converting the compound of formula (321) into the desired carboxylic acid or carboxylic acid salt. The ion exchange method is known to those skilled in the art, and a suitable ion exchange Using the solution and exchange conditions, the aforementioned cations were + A composite molecule can be obtained, where In some embodiments, the ion exchange reaction is carried out by trimethylsilylsilane. The reaction is carried out using an ethylamine phosphate solution, and the concentration of the triethylamine phosphate solution is In some embodiments, the triethylamine phosphate The concentration of the salt solution is 0.4 to 0.6 M, and the triethylamine is added to the compound of formula (313). The dosage of the diaminomethane phosphate solution is 3 to 6 L / mol, and in a further embodiment 4 ~5L / mol.

[0244] Isolating the compound of formula (321) from the reaction mixture by any suitable isolation method. In some embodiments, after the solvent is evaporated, the resulting mixture is subjected to a chromatographic method. The compound of formula (321) can be isolated by, for example, (1) normal phase purification silica gel Silica gel packing of 200 to 300 mesh is mixed with a diluent containing 1% by weight of triethylamine. Gradient elution with chloromethane:methanol = 100:18 to 100:20, or 2) Reverse-phase purification: C 18 C8 reversed-phase packing material was used, and methanol:acetonitrile=0.1:1 The isolation was achieved using two chromatographic conditions: a gradient elution at a ratio of 1:0.1; In some embodiments, the solvent can be directly removed to provide a crude product of the compound of Formula (321). The crude product can be used as is in the subsequent reaction.

[0245] In some embodiments, the method for preparing a compound of Formula (321) comprises the steps of: In an organic solvent, the above ion exchange reaction is carried out in the presence of a condensing agent and a tertiary amine organic base. The product obtained by the above step is further contacted with a solid phase carrier containing an amino group or a hydroxy group. In this case, R4 includes a first functional group and a second functional group, and the first functional group wherein the functional group comprises a hydroxy protecting group and the second functional group comprises a structure shown in formula (C1'): 321) compound is obtained.

[0246] The solid support is one of the supports used in solid-phase synthesis of siRNA, and Some are known to those skilled in the art. For example, the solid support may have an active hydroxy or amino group. In some embodiments, the solid support may be selected from a solid support comprising a functional group. The resin is an amino resin or a hydroxy resin. The resin or hydroxy resin has a particle size of 100 to 400 mesh, and the surface The parameter of the amount of amino or hydroxyl groups carried is 0.2 to 0.5 mmol / g. The dosage ratio of the compound represented by the formula (321) to the solid phase carrier is 10 to 400 μmol. In some embodiments, the compound is present in an amount of 1000 mg / g of solid support. The dosage ratio of the compound of formula (321) to the solid support is 50 to 200 μmol / g. .

[0247] 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 Solvents, halogenated alkyl solvents, dimethyl sulfoxide, N,N-dimethylformamide In some embodiments, the amine is one or more of: methylaminomethylpropional; In this embodiment, the epoxy solvent is dioxane and / or tetrahydrofuran. The ether solvent is ethyl ether and / or methyl tert-butyl ether. The halogenated alkyl solvent is selected from the group consisting of dichloromethane, trichloromethane, and 1,2-chloromethane. In some embodiments, the organic solvent is one or more of: is acetonitrile. For the compound of formula (321), the amount of the organic solvent is 20 In some embodiments, the ion exchange rate is 50 to 100 L / mol. do.

[0248] In some embodiments, the condensing agent is (benzotriazol-1-yloxy ) Tripyrrolidinophosphonium hexafluorophosphate, 3-diethoxyphosphoryl -1,2,3-benzoxazol-4(3H)-one and / or O-benzotriazole -tetramethyluronium hexafluorophosphate, In one embodiment, the condensing agent is O-benzotriazole-tetramethyluronium hexahydrate. The molar ratio of the condensing agent to the compound represented by formula (321) is In a further embodiment, it is from 1:1 to 20:1, and in a further embodiment from 1:1 to 5:1.

[0249] In some embodiments, the tertiary amine organic base is triethylamine and and / or N,N-diisopropylethylamine, and in some embodiments, N, N-diisopropylethylamine, and the tertiary amine organic base and the formula (321) The molar ratio of the compound shown is 1:1 to 20:1, and in some embodiments, 1 :1~5:1.

[0250] In some embodiments, the method for preparing a compound of Formula (321) comprises reacting the resulting condensation product The substance is subjected to capping reaction under conditions of an organic solvent, a capping reagent and an acylation catalyst and isolating to obtain a compound of formula (321). The effect of the capping reaction is to avoid the generation of unwanted by-products in subsequent reactions. The first step is to remove any active reactive functional groups that have not yet fully reacted in order to achieve the desired result. The capping reaction is carried out under the conditions of a reaction 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 1 to 10 hours. The capping reagent is a siRNA solid-phase capping reagent known to those skilled in the art. A capping reagent used in the synthesis may also be used.

[0251] In some embodiments, the capping reagent consists of capping reagent 1 (cap1) and capping reagent 2 (cap2). Capping reagent 1 is N-methylimidazole, and in some embodiments, N-methylimidazole is provided as a pyridine / acetonitrile mixed solution, with the volume ratio of pyridine to acetonitrile being 1:10 to 1:1, and in some embodiments, 1:3 to 1:1. The ratio of the total volume of pyridine and acetonitrile to the volume of N-methylimidazole is 1:1 to 10:1, and in some embodiments, 3:1 to 7:1. Capping reagent 2 is acetic anhydride. In some embodiments, the capping reagent 2 is provided as an acetonitrile solution of acetic anhydride, with the volume ratio of acetic anhydride to acetonitrile being 1:1 to 1:10, and in further embodiments, 1:2 to 1:6.

[0252] In some embodiments, the N-methylimidazole is The ratio of the volume of the mixed solution to the mass of the compound of formula (321) 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 (321) is 0.5 ml / g. 0 ml / g, and in some embodiments, 1 ml / g to 5 ml / g.

[0253] In some embodiments, the capping reagent comprises equimolar amounts of acetic anhydride and N In some embodiments, the organic solvent is acetonitrile. Nitriles, epoxy solvents, ether solvents, halogenated alkyl solvents, dimethyl sulfonates 1 of phenoxide, N,N-dimethylformamide and N,N-diisopropylethylamine In some embodiments, the organic solvent is acetonitrile. The amount of the organic solvent is 10 to 50 L / mol relative to the compound of formula (321). In some embodiments, the concentration is 5 to 30 L / mol.

[0254] In some embodiments, the acylation catalyst is an esterification condensation or an amidation condensation catalyst. The catalyst may be selected from any catalyst that can be used in the reaction of the present invention, such as an alkali heterocyclic compound. In one embodiment, the acylation catalyst is 4-dimethylaminopyridine. The mass ratio of the solvent to the compound represented by formula (321) is 0.001:1 to 1:1, and several In some embodiments, it is 0.01:1 to 0.1:1.

[0255] In some embodiments, the compound of formula (321) can be isolated from the reaction mixture by any suitable isolation method. In some embodiments, the compound can be isolated by thorough washing with an organic solvent. purifying and filtering to remove unreacted reactants, excess capping reagent, and other impurities; The compound of formula (321) can be obtained by the above-mentioned method. The organic solvent is acetonitrile, dichloromethane, methanol, and in some embodiments, acetonitrile It is.

[0256] In some embodiments, the method for preparing the conjugated molecule of formula (321) comprises the step of: In the reaction, under coupling reaction conditions and in the presence of a coupling reagent, The compound shown is contacted with a phosphorodiamidite and isolated to give the compound shown in formula (321). In this case, R4 includes a first functional group and a second functional group, and the first functional group is wherein the functional group comprises a hydroxy protecting group and the second functionality comprises a structure shown in formula (C3): 321) compound is obtained.

[0257] In some embodiments, the coupling reaction conditions include a temperature of 0 to 50°C. For example, the reaction temperature may be 15 to 35°C, and the compound of formula (313) and phosphorodiamidite may be The molar ratio of the compound represented by the formula (3 The molar ratio of the compound 13) to the coupling reagent may be 1:1 to 1:100, e.g. For example, the ratio may be 1:50 to 1:80, and the reaction time may be 200 to 3000 seconds. The phosphorodiamidite is, for example, bis(diisopropyl (amino)(2-cyanoethoxy)phosphine may be used, or a commercially available product may be purchased. Alternatively, it may be synthesized by methods known in the art. 1H-tetrazole, 5-ethylthio-1H-tetrazole, 5-benzylthio-1H-tetrazole The compound is one or more selected from the group consisting of 5-ethylthio-1H-tetrazole, for example. The coupling reaction may be carried out in an organic solvent, and the organic solvent may be anhydrous acetonitrile. anhydrous dichloromethane, anhydrous dichloromethane, anhydrous dichloromethane, In some embodiments, for the compound of Formula (313), The amount of the organic solvent is 3 to 50 L / mol, for example, 5 to 20 L / mol. By carrying out the coupling reaction, the hydrochloride in the compound of formula (313) can be obtained. The oxy group reacts with a phosphoramidite to form a phosphoramidite group. In this embodiment, the solvent can be removed directly to give the crude compound of formula (321). The crude product can be used as is in the subsequent reaction.

[0258] In some embodiments, the method for preparing a compound of Formula (321) comprises a coupling reaction The resulting product was isolated by reacting the product in the presence of a coupling agent in an organic solvent under the conditions and then contacting the solid support with a hydroxy group-containing solid support. The compound of formula (321) is obtained by carrying out a cycloaddition reaction, an oxidation reaction, and isolating the compound. and a second functional group, the first functional group comprising a hydroxy protecting group and the second functional group comprising a A compound of formula (321) is obtained in which the functional group has the structure shown in formula (C3').

[0259] In some embodiments, the solid support is amenable to solid phase nucleic acid synthesis as known in the art. For example, a commercially available general-purpose solid phase support (NittoPhase) that has been deprotected is used. ase(R)HL UnyLinker TM 300 Oligonucleotides ide Synthesis Support, Kinovate Life Science nces, Inc., the structure of which is shown in formula B80.

[0260] [ka]

[0261] Deprotection reactions are known to those skilled in the art. In some embodiments, deprotection conditions include the temperature is 0 to 50°C, for example 15 to 35°C, and the reaction time is 30 to 300 seconds; For example, 50 to 150 seconds. The deprotection reagent is trifluoroacetic acid, trichloroacetic acid, dichloroacetic acid, In some embodiments, the acetic acid may be one or more selected from the group consisting of acetic acid, acetic acid, and chloroacetic acid. In this embodiment, the deprotection reagent is dichloroacetic acid. The molar ratio of the r(4,4'-dimethoxytrityl) protecting group is 2:1 to 100:1, e.g. For example, the ratio is 3:1 to 50:1. By carrying out the deprotection, a reaction is formed on the surface of the solid phase support. This provides a free active hydroxy group, making it amenable to subsequent coupling reactions.

[0262] The coupling reaction conditions and the selection of coupling reagents are as described above. By carrying out a coupling reaction, the free hydroxy group formed in the deprotection reaction is converted into a phosphoryl group. The midite group is reacted to form a phosphite ester bond.

[0263] In some embodiments, the capping reaction conditions include a temperature of 0 to 50°C. For example, the temperature is 15 to 35°C, and the reaction time is 5 to 500 seconds, for example, 10 to 100 seconds. and the capping reaction is carried out in the presence of a capping reagent. The selection and dosage are as above.

[0264] The oxidation reaction conditions are a temperature of 0 to 50°C, for example, 15 to 35°C. The reaction time may be 1 to 100 seconds, for example, 5 to 50 seconds, and the oxidation reagent may be, for example, Alternatively, it may be iodine (provided as iodine water in some embodiments). In some embodiments, the molar ratio of the oxidizing reagent to the phosphite ester groups is 1:1-100. :1, and may be, for example, 5:1 to 50:1. In some embodiments, The oxidation reaction was carried out in a mixed solvent of tetrahydrofuran, water, and pyridine in a ratio of 3:1:1 to 1:1:3. This is done with a drug.

[0265] In some embodiments, R6 is one of the groups of formula B7 or B8.

[0266] [ka] In the formula, the definition of q2 is as described above. In this case, the compound represented by formula (313) can be reacted in an organic solvent under amidation reaction conditions: and amidation reaction in the presence of a condensing agent and a tertiary amine organic base, a compound represented by formula (A-1) or a compound represented by formula (A-2), It can be obtained by a preparation method in which the compound is isolated.

[0267] [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 selectable ranges of each are as described above. be.

[0268] 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 a reaction time. The reaction temperature is 10 to 40°C, and the reaction time is 2 to 16 hours.

[0269] In some embodiments, the organic solvent is an alcohol solvent, an epoxy solvent, Ether solvents, halogenated alkyl solvents, dimethyl sulfoxide, N,N-dimethyl The solvent may be one or more of formamide and N,N-diisopropylethylamine. In some embodiments, the alcohol solvent is methanol, ethanol, propanol, or the like. In some embodiments, the epoxy is one or more of the epoxy groups. The alkoxy solvent, in some embodiments, is dioxane and / or tetrahydrofuran. In some embodiments, the ether solvent is ethyl ether and / or is methyl tert-butyl ether. The halogenated alkyl solvents include several In the embodiment, dichloromethane, trichloromethane and 1,2-dichloroethane In some embodiments, the organic solvent is dichloromethane. The amount of the organic solvent is 3 to 50 L / mol relative to the compound of formula (314), In a further embodiment, it is 3 to 20 L / mol.

[0270] In some embodiments, the amidation reaction condensing agent is (benzotriazole-1 -yloxy)tripyrrolidinophosphonium hexafluorophosphate, 3-diethoxy Diphosphoryl-1,2,3-benzoxazol-4(3H)-one, 4-(4,6-dimethyl Trimethyltriazin-2-yl)-4-methylmorpholine hydrochloride (4-(4,6-dimethicone) thoxytriazin-2-yl)-4-methylmorpholine hy drochloride), 2-ethoxy-1-ethoxycarbonyl-1,2-dihydro Quinoline (EEDQ) or O-benzotriazole-tetramethyluronium hexafluoro In a further embodiment, 3-diethoxyphosphoryl-1, 2,3-benzoxazol-4(3H)-one. 14) may be 1:1 to 10:1 in molar ratio, and in some embodiments In terms of morphology, it is 2.5:1 to 5:1.

[0271] In some embodiments, the tertiary amine organic base is triethylamine or In a further embodiment, N,N-diisopropylethylamine is N,N-diisopropylethylamine. The tertiary amine organic base and the formula (314) The molar ratio of the compound to the hydroxy group is 3:1 to 20:1, and in some embodiments, 5:1 to 1 The ratio is 0:1.

[0272] 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, glycerol 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. The cyclic acid anhydride can be reacted with HCl to prepare a compound of formula (A-2), The cyclic acid anhydride may have 4 to 13 carbon atoms, and in some embodiments, 4 to 8 carbon atoms. As can be easily understood by those skilled in the art, the selection of the cyclic acid anhydride can be determined by the following: A to A2) correspond to different values ​​of q2 in the compounds, for example, When the cyclic acid anhydride is succinic anhydride, q2=1, and the cyclic acid anhydride is glutaric anhydride. In this case, q2=2, which can be inferred.

[0273] In some variations, the compound of formula (314) can be prepared by reacting the cyclic acid 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 This can be easily achieved by the vendor using the above method.

[0274] Similarly to 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 chromatographic Compounds of formula (313) can be isolated by the following two methods: It can be isolated under chromatographic conditions. (1) Normal phase purified silica gel: 200 300 mesh silica gel packing was packed in petroleum ether:ethyl acetate:dichloromethane:N Gradient elution with N-dimethylformamide = 1:1:1:0.5 to 1:1:1:0.6 (2) Reverse phase purification: C 18 C8 reversed-phase packing material was used, methanol:acetonitrile=0.1: In some embodiments, the solvent is removed directly to obtain a compound of formula ( 313) can be obtained as a crude product, which can be used directly in the subsequent reaction. It is possible.

[0275] In some embodiments, the compound of Formula (314) can be prepared by reacting the compound of Formula (314) in an organic solvent with Compound (315) is contacted with haloacetic acid under deprotection reaction conditions and then isolated. The compound can be obtained by a preparation method including the steps of:

[0276] [ka] In the formula, R7 is a group represented by the formula (330), (331), (332) or (333). In some embodiments, the structure of R7 is shown in formula (330).

[0277] [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.

[0278] The haloacetic acid may be selected from dichloroacetic acid, trichloroacetic acid, chloroacetic acid, and trifluoroacetic acid. In some embodiments, dichloroacetic acid is one or more selected from the group consisting of: .

[0279] The deprotection reaction conditions are a reaction temperature of 0 to 100°C and a reaction time of 0.1 to 2 In some embodiments, the reaction temperature is 10 to 40°C, and the reaction time is 4 hours. is 0.5 to 16 hours.

[0280] In some embodiments, the organic solvent is an epoxy solvent, an ether solvent, or a halogen-based solvent. Halogenated alkyl solvents, dimethyl sulfoxide, N,N-dimethylformamide and N , N-diisopropylethylamine. In some embodiments, the ether is dioxane and / or tetrahydrofuran. The tertiary solvent may, in some embodiments, be ethyl ether and / or methyl tert In some embodiments, the halogenated alkyl solvent is butyl ether. and one or more of dichloromethane, trichloromethane, and 1,2-dichloroethane. In some embodiments, the organic solvent is dichloromethane. 5) For the compound, the amount of the organic solvent is 3 to 50 L / mol; In this case, the concentration is 5 to 20 L / mol.

[0281] The molar ratio of the haloacetic acid to the compound represented by the formula (315) is 5:1 to 100:1. and in some embodiments, from 10:1 to 50:1.

[0282] Similarly to 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 chromatographic Compounds of formula (314) can be isolated by a fluorochemical method, for example by (1) Normal phase purified silica gel: 200 300 mesh silica gel packing was dissolved in dichloromethane:methanol = 100:30 Gradient elution: 100:40. (2) Reverse phase purification: C 18 , C8 reversed-phase packing, methanol Gradient elution is performed using acetonitrile: acetonitrile = 0.1:1 to 1:0.1. In some embodiments, The solvent can then be directly removed to give the crude compound of formula (314), which can be The product can be used as it is in the subsequent reaction.

[0283] The compound represented by formula (315) can be prepared by reacting a condensing agent for amidation reaction with a tertiary amide in an organic solvent. In the presence of an organic base such as amines, a compound represented by formula (317) is converted into a compound represented by formula (3 16), and then isolating the compound. can.

[0284] [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 and S1 are as described above.

[0285] The compound of formula (316) is, for example, the compound described in J. Am. Chem. Soc. 20 14,136,16958-16961 may be used, or the compounds of the formula Compounds of formula (316) can be prepared by a person skilled in the art by a variety of methods, for example, as described in US Pat. With reference to the method disclosed in Example 1 of US Pat. No. 8,106,022 B2, several formulas Compounds of formula (316) can be prepared, the entire contents of which are incorporated herein by reference. and is incorporated herein by reference.

[0286] In some embodiments, the condensation reaction conditions include a reaction temperature of 0 to 100°C. and the reaction time is 0.1 to 24 hours. 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.

[0287] 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, 2.5:1 to 5:1. be.

[0288] In some embodiments, the organic solvent is acetonitrile, an epoxy solvent, or an ethoxylated Ether solvents, halogenated alkyl solvents, dimethyl sulfoxide, N,N-dimethyl ether and N,N-diisopropylethylamine, The alkoxy solvent, in some embodiments, is dioxane and / or tetrahydrofuran. In some embodiments, the ether solvent is ethyl ether and / or is methyl tert-butyl ether, and the halogenated alkyl solvent is In the embodiment, dichloromethane, trichloromethane and 1,2-dichloroethane In some embodiments, the organic solvent is one or more of acetonitrile. For the compound of formula (317), the amount of the organic solvent is 3 to 50 L / mol. In some embodiments, the concentration is 5 to 20 L / mol.

[0289] In some embodiments, the amidation reaction condensing agent is (benzotriazole-1 -yloxy)tripyrrolidinophosphonium hexafluorophosphate, 3-diethoxy Diphosphoryl-1,2,3-benzoxazol-4(3H)-one (DEPBT), O- Benzotriazole-tetramethyluronium hexafluorophosphate or 4-(4 ,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride, and further In some embodiments, 4-(4,6-dimethoxytriazin-2-yl)-4-methylmono The compound represented by formula (317) is a compound represented by formula (317) and the condensing agent for the amidation reaction is methyl ... The ratio may be from 2:1 to 10:1, and in some embodiments, from 2.5:1 to 5: :1.

[0290] The tertiary amine organic base is N-methylmorpholine, triethylamine or N,N In some embodiments, N-methyl- and the tertiary amine organic base and the compound represented by formula (317) are The molar ratio may be from 3:1 to 20:1, and in some embodiments, from 5:1 to 10 :1.

[0291] Similarly to 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 chromatographic Compounds of formula (315) can be isolated by a method similar to that described below. It can be isolated under chromatographic conditions. (1) Normal phase purified silica gel: 200 300 mesh silica gel packing was dissolved in dichloromethane:methanol=100:5-10 Gradient elution: 0:7. (2) Reverse phase purification: C 18 C8 reversed phase packing was added to methanol:acetone In some embodiments, the solvent is eluted with a gradient of 0.1:1 to 1:0.1 ammonia. The reagent can be directly removed to give the crude compound of formula (315), which can be prepared by the following steps: It can be used as is in the subsequent reaction.

[0292] 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 obtained by reacting the compounds in a single reaction. In some embodiments, the L1 moieties are the same. The compound of formula (316) is converted into a compound of formula (31) having different L1 and / or S1. 6), the compound of formula (315) was reacted with the compound of formula (316) in a batch reaction. For example, the formula (317) of 1eq can be expressed as the mixture is contacted with 2 eq of a first compound of formula (316) and After attaching the first S1-L1 moiety to the two terminal primary amine groups of the compound, (n3+n1-1)eq (the definitions of n3 and n1) are added to a second compound of formula (316) and the range of values ​​are as described above), (n3 + n1 - 1) A second S1-L1 moiety can be attached to the secondary amine group.

[0293] In some embodiments, the compound of Formula (317) can be prepared in the presence of an organic solvent and and deprotection reaction conditions, the compound of formula (318) is contacted with an aqueous methylamine solution. The compound can be obtained by a preparative method comprising the steps of:

[0294] [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 each are as described above.

[0295] 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 and the reaction time is 1 0 to 30 hours.

[0296] The organic solvent may be selected from alcohols, and in some embodiments, ethanol, and isopropanol, and in some embodiments , methanol, and the amount of the organic solvent is 1 to 20 L relative to the compound of formula (318). / mol, and in some embodiments, 1.5 to 10 L / mol.

[0297] 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, it is 50:1 to 200:1.

[0298] Similarly to 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 chromatographic Compounds of formula (317) can be isolated by the following two methods: It can be isolated under chromatographic conditions. (1) Normal phase purified silica gel: 200 300 mesh silica gel packing was dissolved in dichloromethane:methanol:ammonia water (2 5% by weight) = 1:1:0.05 to 1:1:0.25. (2) Reverse-phase purification: C 18 C8 reversed-phase packing was used, and a gradient of methanol:acetonitrile was used from 0.1:1 to 1:0.1. In some embodiments, the solvent is directly removed to obtain a crude compound of Formula (317). A crude product can be obtained, and the crude product can be used directly in the subsequent reaction.

[0299] In some embodiments, the compound of Formula (318) can be reacted in the presence of an organic solvent and and substitution reaction conditions, the compound of formula (319) is converted to triphenylchloromethane (T rCl), diphenylethylphenylchloromethane, phenyldiethylphenylchloromethane ethane or triethylphenylchloromethane, in some embodiments triphenylchloromethane and then isolating the resulting mixture. can.

[0300] [ka] In the formula, n1, n3, m1, m2, m3, R 10 , R 11 , R 12 , R 13 , R 14 , R 15 The definitions and selectable ranges of each are as described above.

[0301] 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 conditions include a reaction temperature of 10 to 4 The temperature is 0°C and the reaction time is 10 to 30 hours.

[0302] Triphenylchloromethane (TrCl), diphenylethylphenylchloromethane, Phenyldiethylphenylchloromethane or triethylphenylchloromethane is a commercially available product. Triphenylchloromethane (TrCl), diphenylethylphenyl chloromethane, phenyldiethylphenylchloromethane or triethylphenylchloromethane The molar ratio of methane to the compound represented by formula (319) may be 1:1 to 10:1; In some embodiments, it is 1:1 to 3:1.

[0303] The organic solvent may be an epoxy solvent, an ether solvent, an alkyl halide solvent, a dimethicone solvent, or a dimethylsilyl alcohol. Dimethyl sulfoxide, N,N-dimethylformamide and N,N-diisopropylethyl ether In some embodiments, the epoxy solvent may be one or more of the following: The ether solvent may be dioxane and / or tetrahydrofuran. In some embodiments, ethyl ether and / or methyl tert-butyl ether In some embodiments, the halogenated alkyl solvent may be a terephthalate. One or more of dichloromethane, trichloromethane and 1,2-dichloroethane In some embodiments, the organic solvent is dichloromethane. 319) The amount of the organic solvent may be 3 to 50 L / mol relative to the compound; In some embodiments, it is 5 to 20 L / mol.

[0304] Similarly to 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 chromatographic Compounds of formula (318) can be isolated by the following two methods: It can be isolated under chromatographic conditions. (1) Normal phase purified silica gel: 200 300 mesh silica gel packing was dissolved in methanol:dichloromethane=0.01:1-0 0.5:1, or methanol:dichloromethane:ethyl acetate:petroleum ether = 0.1:1 Gradient elution: 1:1 to 1:1:1:1. (2) Reverse-phase purification: C 18 , C8 reversed phase packing Gradient elution was performed using methanol:acetonitrile = 0.1:1 to 1:0.1. In an embodiment, the solvent can be directly removed to provide a crude compound of Formula (318); The crude product can be used as is in the subsequent reaction.

[0305] In some embodiments, the compound of Formula (319) can be prepared by reacting the compound of Formula (319) in an organic solvent with Compound of formula (320) is contacted with ethyl trifluoroacetate under substitution reaction conditions. The compound can be obtained by a preparative method comprising the steps of:

[0306] [ka] In the formula, n1, n3, m1, m2, m3, R 10 , R 11 , R 12 , R 13 , R 14 , R 15 The definitions and selectable ranges of each are as described above.

[0307] In some embodiments, the organic solvent is acetonitrile, an epoxy solvent, or an ethoxylated Ether solvents, halogenated alkyl solvents, dimethyl sulfoxide, N,N-dimethyl ether The most commonly used compounds are one or more of: methyl amine, methyl amine, and N,N-diisopropylethylamine. In the embodiment, the epoxy solvent is dioxane and / or tetrahydrofuran. In some embodiments, the ether solvent is ethyl ether and / or is methyl tert-butyl ether, and in some embodiments, the halogen The alkyl chlorinated solvents are dichloromethane, trichloromethane and 1,2-dichloroethane. In some embodiments, the organic solvent is one or more of acetonitrile. The amount of the organic solvent is 1 to 50 L / mol relative to the compound of formula (320). In some embodiments, the concentration is 1 to 20 L / mol.

[0308] 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 substitution reaction conditions include a reaction temperature of The temperature is 10 to 40°C and the reaction time is 10 to 30 hours.

[0309] Compounds of formula (320) can be obtained commercially or by methods known to those skilled in the art. For example, m1 = m2 = m3 = 3, n1 = 1, n3 = 2, and R 10 , R 11 , R 12 , R 13 , R 14 , R 15 When both are H, the chemical formula of formula (320) The compound is commercially available from Alfa Aesar.

[0310] The molar ratio of the ethyl trifluoroacetate to the compound represented by formula (320) is 2:1 to 1 0:1, and in some embodiments, 3:1 to 5:1.

[0311] Similarly to 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 chromatographic Compounds of formula (319) can be isolated by the following two methods: It can be isolated under chromatographic conditions. (1) Normal phase purified silica gel: 200 300 mesh silica gel packing was dissolved in methanol:dichloromethane=0.01:1-0 0.5:1, or methanol:dichloromethane:ethyl acetate:petroleum ether = 0.1:1 Gradient elution: 1:1 to 1:1:1:1. (2) Reverse-phase purification: C 18 , C8 reversed phase packing Gradient elution was performed using methanol:acetonitrile = 0.1:1 to 1:0.1. In an embodiment, the solvent can be directly removed to provide the crude compound of Formula (319); The crude product can be used as is in the subsequent reaction.

[0312] The siRNA complexes of the present disclosure may be used in combination with other pharmaceutically acceptable additives, The additive may be one or more of various agents or compounds commonly used in the art. For details, see the description of the pharmaceutical compositions of the present disclosure above.

[0313] <Use of the siRNA of the present disclosure, and the drug composition and complex containing the siRNA> In some embodiments, the present disclosure provides siRNA and / or drug compositions of the present disclosure. and / or siRNA complexes in the preparation of a medicament for the treatment and / or prevention of dyslipidemia. Provides for use in the

[0314] In some embodiments, the present disclosure provides siRNA and / or drug compositions of the present disclosure. and / or administering an effective amount of the siRNA complex to a subject in need thereof. A method for preventing and / or treating dyslipidemia is provided.

[0315] RNase inhibitors can be administered to a subject in need thereof using siRNA active ingredients of the present disclosure. The purpose of preventing and / or treating dyslipidemia can be achieved through the A-interference mechanism. Therefore, the siRNA, and / or drug composition and / or siRNA complex of the present disclosure may be Drugs used for the prevention and / or treatment of lipid disorders or for the prevention and / or treatment of lipid disorders It can be used to prepare

[0316] The lipid abnormality is caused by overexpression of the ANGPTL3 gene in hepatocytes. It usually refers to the level of lipids such as triglycerides and cholesterol in the blood and / or lipoproteins. High levels of lipids are associated with high blood pressure, cardiovascular Hypertriglyceridemia is highly associated with atherosclerosis, diabetes, and other pathological disorders. The dyslipidemia described in this disclosure is associated with high cholesterol and arteriosclerosis and can also lead to pancreatitis. including, but not limited to, hypertriglyceridemia, hyperlipidemia, or atherosclerosis It will not be done.

[0317] As used herein, the term "drug administration / administration" refers to the administration of siRNA, drug compositions of the present disclosure. and / or localizing at least a portion of the siRNA complex to a desired site to produce a desired effect. The siRNA, drug composition and / or siRNA complex of the present disclosure can be administered by a method or route that allows the siRNA to be administered. Suitable routes of administration for the methods of the present disclosure include topical administration and Local administration generally results in greater siRNA complexation than systemic administration in a subject. Although the siRNA, drug compositions and and / or the siRNA complex is delivered to the subject's entire body. Considering that the intended use is to provide a therapeutic means, in some embodiments This is an administration method that can deliver drugs to the liver.

[0318] Administration to a subject can be by any suitable route known in the art, including: Oral administration or parenteral routes, such as intravenous, intramuscular, subcutaneous, transdermal, or endotracheal administration. Oral (aerosol), pulmonary, nasal, rectal and topical (buccal and sublingual) administration The frequency of administration may be daily, weekly, or biweekly. The period may be once or more than once a year, every three weeks, every month or every year.

[0319] The dosage of the siRNA, drug composition or siRNA complex described in this disclosure is determined by methods known in the art. The dose may be a normal dose for the patient, said dose being determined depending on various parameters, in particular the age of the subject. The dose may be determined by standard pharmaceutical procedures in cell cultures or experimental animals. Toxicity and therapeutic effects are measured, e.g., LD50 (the dose that kills 50% of the colony) and ED 50 (In quantitative responses, this refers to the dose that can induce 50% of the maximum response intensity. (In qualitative reactions, this refers to the dose at which 50% of test subjects have a positive reaction) A range of human dosages may be determined based on data obtained from cell culture assays and animal studies. You can get the range.

[0320] administering the siRNA, drug composition, and / or siRNA complex described in this disclosure In this case, for example, male or female C57BL / 6J, 6 to 12 weeks old, weighing 18 to 25 g, or For ob / ob mice weighing 30–45 g, the following amounts of siRNA were administered: (i) siRNA complex; For the combination, the siRNA dose may be 0.001 to 100 mg / kg body weight. In a further embodiment, it is 0.01 to 50 mg / kg body weight, and in an even further embodiment, it is 0.01 to 50 mg / kg body weight. In one embodiment, the dose is 0.05 to 20 mg / kg body weight, and in a further embodiment, 0. (ii) 1 to 10 mg / kg body weight of siRNA and a pharmaceutically acceptable carrier; The resulting pharmaceutical composition has an siRNA dose of 0.001 to 50 mg / kg body weight. In a further embodiment, the dose is 0.01 to 10 mg / kg body weight, more preferably 0.01 to 10 mg / kg body weight. In yet a further embodiment, 0.05 to 5 mg / kg body weight, and in yet a further embodiment, In this case, the dose is 0.1 to 3 mg / kg body weight.

[0321] In some embodiments, the present disclosure provides siRNA and / or drug compositions of the present disclosure. and / or contacting hepatocytes with an effective amount of the siRNA complex of the present disclosure, and / or Alternatively, a drug composition and / or a siRNA complex is introduced into the liver cells, and the cells are stimulated by the RNA interference mechanism. and suppressing the expression of the ANGPTL3 gene in hepatocytes. The present invention provides a method for inhibiting the expression of the ANGPTL3 gene in hepatocytes. 3B, HepG2, Huh7, or other hepatoma cell lines or isolated primary hepatocytes. In some embodiments, the cells are Huh7 hepatoma cells.

[0322] The method provided by the present disclosure inhibits the expression of the ANGPTL3 gene in cells. The siRNA in the provided modified siRNA, drug composition and / or siRNA complex The NA dose can generally reduce the expression of target genes and is between 1 pM and 1 μM on the target cell surface. , 0.01 nM to 100 nM, 0.05 nM to 50 nM, or 0.05 nM to about 5 nM The amount required to achieve this local concentration will depend on the delivery method, delivery site, and other factors. These include the location of the delivery site, the number of cell layers between the delivery site and the target cell or tissue, and whether delivery is local or systemic. The concentration at the delivery site varies depending on various factors, including the amount of The concentration may be significantly higher than that in the

[0323] <Kit> The present disclosure provides a method for the preparation of at least one of the modified siRNAs, drug compositions, and siRNA complexes of the present disclosure. A kit containing an effective amount of the species is provided.

[0324] In some embodiments, the kits described herein include a container containing the modified siRNA. In some embodiments, the kits described herein can provide In some embodiments, the composition may include a container providing a pharmaceutically acceptable excipient. Therefore, the kit may also contain other ingredients, such as stabilizers or preservatives. In some embodiments, the kits described herein include the modified s At least one other therapeutic agent may be contained in a container separate from the container providing the iRNA. In some embodiments, the kit comprises a modified siRNA and a pharmaceutically acceptable carrier and and / or instructions for mixing the additives or other ingredients, if any.

[0325] In the kit of the present disclosure, the modified siRNA and a pharmaceutically acceptable carrier and / or Additives and the modified siRNA, drug composition and / or siRNA complex and / or The complex, and / or pharmaceutically acceptable excipient may be in any form, for example, a liquid form, a dry form, or the like. In some embodiments, the modified form may be provided in a dry or lyophilized form. a modified siRNA and a pharmaceutically acceptable carrier and / or excipient, and the pharmaceutical composition; and / or the complex and any pharmaceutically acceptable additives are essentially clean and / or Sterile. In some embodiments, sterile water can be provided in the kits of the present disclosure. Cut.

[0326] The present disclosure will be further described below with reference to examples, but the present disclosure is not limited thereto. do not have. [Example]

[0327] 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 Borough Press (1989) This is done by referring to the method described above.

[0328] HEK293A cells were provided by the Nucleic Acid Technology Laboratory, Institute of Molecular Medicine, Peking University, and were collected from 20 % fetal bovine serum (FBS, Hyclone) and 0.2% penicillin-stress Penicillin-Streptomycin (Gibco, Inv The cells were cultured in DMEM complete medium (Hyclone) containing 5% ethanol at 37°C. The cells were cultured in an incubator containing CO2 / 95% air.

[0329] 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), D containing 1% non-essential amino acids (NEAA, Corning) Cells were cultured in MEM complete medium (Hyclone) at 37°C in 5% CO2 / 95% air. The cells were cultured in an incubator.

[0330] siRNA and siRNA complexes against the ANGPTL3 gene synthesized according to the present disclosure Cells were transfected with siRNA or siRNA complexes as negative controls. If transfection is performed using Lipofectamine as the transfection reagent, TM 2000 (Invitrogen) was used, and the specific procedures were provided by the manufacturer. Please refer to the instructions.

[0331] Unless otherwise stated, all reagent proportions provided below are by volume (v / v). It is calculated.

[0332] The animal models used are as follows: C57BL / 6N mice: 6-8 weeks old, purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd. Hereafter, these mice will be simply referred to as c57 mice. 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 7Bres / J, purchased from Jackson Laboratory, USA. Monkeys with metabolic syndrome: Nonhuman Primate Research Center, Institute of Molecular Medicine, Peking University Provided by.

[0333] The experimental data are all

number

[0334] (Preparation Example 1) Preparation of Complexes 1 to 4 In this preparation example, complex 1, complex 2, and complex 4 (hereinafter referred to as L10-siAN1M 3SVP, also known as L10-siAN1M3SP and L10-siAN1M3S complexes) We were also able to synthesize complex 3 (called the L10-siAN1M3SPs complex). The aforementioned complexes were expected to be able to be synthesized by the L-9 complex molecules, each of which was identified by the number siAN1M3SV P, siAN1M3SP, siAN1M3S, or siAN1M3SPs siRNA The siRNA sequence in the complex was as shown in Table 1. Shown in Figure 3.

[0335] (1-1) Synthesis of Compound L-10 Compound L-10 was synthesized according to the following method.

[0336] [ka]

[0337] (1-1-1) Synthesis of GAL-5 Complex Terminal Segments

[0338] [ka] (1-1-1a) 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) was added under ice-water bath. 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 acetone was added until completely dissolved. Acetonitrile / toluene mixed solvent (acetonitrile:toluene volume ratio = 1:1) was added, The solvent was evaporated to dryness to give 130.0 g of a white solid product, GAL-2.

[0339] (1-1-1b) Synthesis of GAL-3 The GAL-2 (35.1 g, 90.0 mmol) obtained in step (1-1-1a) was Dissolve in 3 ml of anhydrous 1,2-dichloroethane and simmer in an ice-water bath under nitrogen protection for 24 hours. 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.

[0340] The reaction mixture was diluted with 400 ml of dichloromethane, filtered through diatomaceous earth, and added to 1 L of saturated charcoal. An aqueous solution of sodium hydrogen carbonate was added, and the mixture was stirred uniformly. The organic phase was separated, and the aqueous phase was diluted with dichloroethane. Extract twice with 300 ml each time, combine the organic phases, and add 300 ml of saturated Wash with aqueous sodium bicarbonate solution and 300 ml of saturated saline, separate the organic phase, and The mixture was dried over sodium sulfate, and the solvent was evaporated to dryness under reduced pressure to give 26.9 g of a pale yellow, viscous starch syrup. The product GAL-3 was obtained.

[0341] (1-1-1c) Synthesis of GAL-4 The GAL-3 (26.9 g, 81.7 mmol) obtained in step (1-1-1b) was Dissolve in 6 ml of anhydrous 1,2-dichloroethane, add 30 g of dried 4 Å molecular sieve powder, 9.0 g of 5-hexen-1-ol (CAS number: 821-41-0, Adamas- Beta Co., Ltd., 89.9 mmol) was added, and the mixture was stirred at room temperature for 30 minutes. 9.08 g of TMSOTf (40.9 mmol) was added under protection, and the reaction was stirred at room temperature overnight. The 4Å molecular sieve powder was filtered off, and the filtrate was diluted with 300 ml of dichloromethane. Filter through diatomaceous earth, add 500 ml of saturated aqueous sodium bicarbonate solution, stir for 10 minutes, and wash. The organic phase was separated, the aqueous phase was extracted once with 300 ml of dichloroethane, and the organic phase was evaporated. Then, 300 ml of saturated sodium bicarbonate solution and 300 ml of saturated saline solution were used. The organic phase was separated, dried over anhydrous sodium sulfate, and the solvent was evaporated to dryness under reduced pressure. 0.3g of yellow starch syrup-like product GAL-4 was obtained, which was used in the next oxidation reaction without further purification. It was.

[0342] (1-1-1d) Synthesis of GAL-5 GAL-4 (14.9 g, 34%) obtained by the method described in step (1-1-1c) Dissolve 0.7 mmol) in a mixed solvent of 77 ml of dichloromethane and 77 ml of acetonitrile. Each solution was dissolved in 103 ml of deionized water and 29.7 g of sodium periodate (CA S number: 7790-28-5, purchased from Aladdin, 138.8 mmol) The mixture was stirred in an ice-water bath for 10 minutes, and ruthenium(III) chloride (CAS number: 14898-6 7-0, purchased from Energy Co., 238 mg, 1.145 mmol) was added and the mixture was left at room temperature. 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 by adding 100 ml of HCl, the organic phase was separated and discarded, and the aqueous phase was washed once with dichloromethane and once with HCl. The aqueous phase was adjusted to pH 3 with solid citric acid. The mixture was extracted three times with 200 ml of dichloromethane, and the organic phases were combined and diluted with sodium sulfate anhydride. The mixture was dried over sodium hydroxide, and the solvent was evaporated to dryness under reduced pressure to give 6.85 g of a white foamy solid product, GAL-5 obtained. 1 H NMR (400 MHz,DMSO) δ 12.01 (br,1H ),7.83 (d,J = 9.2 Hz,1H),5.21 (d,J = 3.2 Hz,1H),4.96 (dd,J = 11.2,3.2 Hz,1H),4.4 9 (d,J = 8.4 Hz,1H),4.07 - 3.95 (m,3H),3 .92 - 3.85 (m,1H),3.74 - 3.67 (m,1H),3.4 8 - 3.39 (m,1H),2.20 (t,J = 6.8 Hz,2H),2 .11 (s,3H),2.00 (s,3H),1.90 (s,3H),1.77 (s,3H), 1.55 - 1.45 (m,4H).

[0343] (1-1-2) Synthesis of M-11-T3:

[0344] [ka] J-0 (1.883 g, 10 mmol, purchased from Alfa Aesar) 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 mixture was foam-dried for 18 hours using a vacuum oil pump. 5.342 g of a solid crude product M-11-T3 was obtained, which was directly used in subsequent steps without further purification. It was used in the reaction. MS m / z:C 15 H 22 F9N4O3, [M+H] + , theoretical value: 4 77.35, Actual value: 477.65.

[0345] (1-1-3) Synthesis of M-11-T3-Tr:

[0346] [ka] M-11-T3 crude product (5.342 g, 10 mmol) in 50 ml of dichloromethane The reaction mixture was dissolved in TrCl (3.345 g, 12 mmol) and triethylamine (1 The mixture was stirred at room temperature for 20 hours and saturated sodium bicarbonate was added. The reaction mixture was washed twice with 20 ml of water and once with 20 ml of saturated saline. The organic phase was dried over anhydrous sodium sulfate, filtered, and then the organic solvent was evaporated to dryness under reduced pressure. After pumping overnight, 7.763 g of the crude solid product M-11-T3-Tr was obtained. S m / z:C 34 H 36 F9N4O3,[M+Na] + ,Theoretical value: 741.25,Actual measurement Value: 741.53. The solid crude product M-11-T3-Tr was used in the next step without further purification. It was used in the synthesis of M-18-Tr.

[0347] (1-1-4) Synthesis of M-18-Tr:

[0348] [ka] The crude M-11-T3-Tr product (7.763 g, 10 mm ol) was dissolved in 100 ml of methanol, and 100 ml of methylamine aqueous solution (40 mass%) was added. % by volume, and the mixture was stirred at 50°C for 23 hours. Insoluble particles were removed by filtration, and the solvent was evaporated under reduced pressure. The mixture was dried and 200 ml of a 1:1 volume ratio dichloromethane:methanol mixed solvent was added, and the mixture was diluted with water for 50 minutes. The aqueous phase was washed with dichloromethane (DCM) in one portion. Extract with 50 ml three times, combine the organic phases, dry with anhydrous sodium sulfate, and filter. The solvent was evaporated to dryness under reduced pressure, and the mixture was foam-dried overnight using a vacuum oil pump. The mixture was purified on a silica gel column, and petroleum ether was added to the column, followed by 1% by weight of triethylamine. The acidity of the silica gel was neutralized with dichloromethane:methanol:ammonia water (25 wt.%). Gradient elution was performed with a chromatogram of 1:1:0.05 to 1:1:0.25, and the product eluate was collected. The mixture was evaporated to dryness under reduced pressure and then foam dried using 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 ,6H),7.32 -7.24 (m,6H),7.19 - 7.12 (m,3H ),2.60 - 2.47 (m,4H),2.46 - 2.19 (m,13H) ,1.70 - 1.55 (m,4H),1.40 (p, J = 6.8 Hz,2 H). MS m / z:C 28 H 39 N4, [M+H] + ,Theoretical value: 431.65,Actual measurement Value: 432.61.

[0349] Synthesis of (1-1-5)L-5-Tr:

[0350] [ka] M-18-Tr (2.02 g, 4.69 mmol) obtained in step (1-1-4) and Mixed with GAL-5 (6.93 g, 15.48 mmol) obtained in step (1-1-1). The resulting solution was dissolved in 47 ml of acetonitrile and N-methylmorpholine (3.13 g, 30.9 ml) was added. 6mmol) and 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpho Add phosphate hydrochloride (DMTMM, 4.28 g, 15.48 mmol) and stir at room temperature for 2 h. The reaction mixture was diluted with 200 ml of dichloromethane and added with 100 ml of saturated sodium bicarbonate. The organic phase was washed with thorium solution, and then with 100 ml of saturated saline solution. After drying over aqueous sodium sulfate and filtration, the solvent was evaporated to dryness under reduced pressure to obtain the crude product. Purify the mixture on a 0-300 mesh normal phase silica gel column, add petroleum ether to the column, and The acidity of the silica gel was neutralized with wt% triethylamine, and the mixture was diluted with dichloromethane:methanol = 1 Gradient elution was performed at 00:5 to 100:7, and the product eluate was collected and evaporated to dryness under reduced pressure to give 7.49 g of pure 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 - 3.83 (m,3H),3.77 - 3.6 4 (m,3H),3.45 - 3.35 (m,3H),3.12 - 2.87 (m,8H),2.30 - 2.15 (m,3H),2.11 - 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 11 9N7O 30 ,[M+H] + , theoretical value: 1718.81, actual value: 1718.03.

[0351] Synthesis of (1-1-6)L-8:

[0352] [ka] The L-5-Tr (5.94 g, 3.456 mmol) obtained in step (1-1-5) was Dissolved in 9 ml of dichloromethane, dichloroacetic acid (13.367 g, 103.67 mm ol) was added, and the mixture was allowed to react at room temperature for 2 hours. 100 ml of dichloromethane was added to dilute the reaction mixture. Then, add saturated sodium bicarbonate solution to wash and adjust the pH to 7-8. The extract was extracted six times with dichloromethane, each time with 30 ml of dichloromethane. The organic phases were combined and washed with anhydrous sodium sulfate. After drying over HCl and filtration, the solvent was evaporated to dryness under reduced pressure to obtain the crude product. Using ~300 mesh normal phase silica gel, the silica gel was purified with 10 wt% triethylamine. The acidity was neutralized, the column was equilibrated with 1 wt% triethylamine, and dichloromethane:methanol was added. Gradient elution was performed with a ratio of 100:30 to 100:40, and the product eluate was collected. The solvent was evaporated under reduced pressure. After drying, 4.26 g of pure L-8 was obtained. 1 H NMR (400 MHz, DMS O) 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 - 2.08 (m,9H),2.07 - 1.98 (m,13 H),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 N7O3 0,[M+H] + , theoretical value: 1477.59, measured value: 1477.23.

[0353] (1-1-7a) Synthesis of A-1

[0354] [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 was added to the flask (12.88 g, 45.0 mmol) and reacted at 45°C for 22 hours. The reaction mixture was filtered. The cake was rinsed with 200 ml of DCM, the filtrate was concentrated under reduced pressure to dryness, and the residue was It was redissolved in 500 ml of dichloromethane and diluted with 0.5 M triethylamine phosphate (p Wash twice with 200 ml of HCl (H=7-8) each time, and then wash the aqueous phase with 200 ml of dichloromethane each time. Extract twice with 200 ml of water, combine the organic phases, dry with anhydrous sodium sulfate, filter, and dissolve. The agent was evaporated to dryness under reduced pressure, purified with a 200-300 mesh normal phase silica gel column, and extracted with petroleum ether. Ether:ethyl acetate:dichloromethane:methanol=1:1:1:0.35 to 1:1:1 Gradient elution was performed with a gradient of 0.55, and the product eluate was collected. The solvent was evaporated to dryness under reduced pressure. Redissolve in dichloromethane and dilute with 200 ml of 0.5 M triethylamine phosphate. The aqueous phase was extracted twice with 200 ml of dichloromethane, and the organic phases were combined. Dry over anhydrous sodium sulfate, filter, evaporate the solvent to dryness under reduced pressure, and then vacuum the mixture overnight in an oil pump. After decompression, 20.7 g of a white solid product A-1 was obtained. 1 H NMR (400 MHz, D MSO-d6) δ 7.46 (ddd,J = 6.5,2.3,1.1 Hz,1 H),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,1 H),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] - , Theoretical value: 407.15, Actual value: 40 6.92.

[0355] (1-1-7b) Synthesis of L-7:

[0356] [ka] L-8 (2.262 g, 1.532 mmol) obtained in step (1-1-6) and step ( A-1 (2.342 g, 4.596 mmol) obtained in 1-1-7a) was mixed with 1 Dissolve in 6 ml of dichloromethane and add 3-diethoxyphosphoryl-1,2,3-benzoxanthate. Add 1.375 g (4.596 mmol) of 4(3H)-dioxazolone (DEPBT) Then, diisopropylethylamine (1.188 g, 9.191 mmol) was added. The reaction was stirred at 25°C for 2 hours, and the organic phase was washed with 10 ml of saturated sodium bicarbonate. The extract was extracted three times with dichloromethane, each time with 10 ml of dichloromethane, and the organic phase was extracted with 10 ml of saturated saline. The aqueous phase was extracted twice with 10 ml of dichloromethane, and the organic phases were combined and washed with water. After drying with aqueous sodium sulfate and filtering, the solvent was evaporated to dryness under reduced pressure and then vacuumed overnight in an oil pump. After foam drying, 4,900 g of crude product was obtained. 0 mesh normal phase silica gel was used, and the silica gel was acidified with 20 ml of triethylamine. The column was neutralized and equilibrated with petroleum ether containing 1 wt% triethylamine. ethanol:ethyl acetate:dichloromethane:N,N-dimethylformamide=1:1:1:0.5 Gradient elution was performed with a ratio of 1:1:1:0.6, and the product eluate was collected. The solvent was evaporated to dryness under reduced pressure to obtain 2 0.336g of pure L-7 was obtained. 1 H NMR (400 MHz, DMSO) δ7 .90 - 7.78 (m,4H),7.75 - 7.64 (m,1H),7.3 8 - 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,3 H),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 - 1.4 0 (m, 28H). MS m / z: C 90 H 128 N7O 35 ,[M-DMTr] + , theoretical value: 1564.65, measured value: 1564.88.

[0357] Synthesis of (1-1-8)L-9:

[0358] [ka] L-7 (2.300 g, 1.26 mmol) obtained in step (1-1-7b), amber Acid anhydride (0.378 g, 3.78 mmol) and 4-dimethylaminopyridine (DMAC) P, 0.462 g, 3.78 mmol) were mixed and dissolved in 13 ml of dichloromethane. , and diisopropylethylamine (DIEA, 0.814 g, 6.30 mmol) The mixture was stirred at 25°C for 24 hours, and the reaction mixture was washed with 5 ml of 0.5 M triethylamine phosphate. The aqueous phase was extracted three times with 5 ml of dichloromethane, and the combined organic phases were evaporated under reduced pressure. After drying, 2.774 g of crude product was obtained. For column purification, 60 g of 200-300 mesh The acidity of the silica gel was neutralized with 1% by weight of triethylamine. The column was equilibrated with dichloromethane and diluted with dichloromethane:methylene chloride containing 1 wt% triethylamine. The product eluate was collected and the solvent was reduced. Evaporation to dryness under reduced pressure gave 1.874 g of pure L-9 complex molecule. 1 H NMR (400 MHz,DMSO) δ 8.58 (d,J = 4.2 Hz,1H),7.94 - 7.82 (m,3H),7.41 - 7.29 (m,5H),7.22 (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. 4Hz,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,2 H),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: 1 664.72, Actual value: 1665.03.

[0359] Synthesis of (1-1-9)L-10 compound:

[0360] [ka] In this step, the L-9 conjugated molecule is bound to a solid support, thereby forming the L-10 compound. was prepared.

[0361] The L-9 complex molecule obtained in step (1-1-8) (0.233 g, 0.1126 mmol) ), O-benzotriazole-tetramethyluronium hexafluorophosphate (H BTU, 0.064 g, 0.1689 mmol) and diisopropylethylamine (DI EA, 0.029 g, 0.2252 mmol) and dissolved in 19 ml of acetonitrile. The reaction mixture was stirred at room temperature for 5 minutes, and then aminomethyl resin (0.901 g, 100-20 0 mesh, amino group loading 400 μmol / g, purchased from Nankai Wasei Co., Ltd.) was added and the mixture was stirred at 25°C. The reaction was carried out in a shaker at 220 rpm for 15 hours, after which the mixture was filtered and Rinse the plate twice with 30 ml of DCM and twice with 30 ml of acetonitrile. Rinse three times with 1 ml of ethyl ether, rinse once with 30 ml of ethyl ether, and dry for 2 h using a vacuum oil pump. After that, the raw materials (CapA, CapB, 4-dimethylamine) were mixed in the proportions shown in Table 2. The capping reaction was carried out by adding 25 hydroxybenzoates (DMAP and acetonitrile). The mixture was left in a shaker at 200 rpm for 5 hours, and the reaction mixture was filtered. Rinse the filter with acetonitrile three times, 30 ml each time, and suction filter until dry. , dried under reduced pressure overnight using a vacuum oil pump, and the resulting 1.100 g, loading amount 90.8 μmol / g L- Ten compounds (ie, L-9 conjugate molecules bound to a solid support) were obtained.

[0362] [Table 2] where CapA and CapB are capping reagent solutions, and CapA is 20 volumes % N-methylimidazole in a pyridine / acetonitrile mixture. The volume ratio of CapB to acetonitrile is 3:5, and CapC is 20% by volume of acetic anhydride in acetonitrile. It was a tolyl solution.

[0363] (1-2) Synthesis of the sense strand of complexes 1 to 4 Since the sense strand sequences of conjugates 1 to 4 were the same, the preparation methods were also the same. Using the solid-phase phosphoramidite method, the L-10 compound prepared in the above step was used as a starting material, and nucleoside monomers were linked one by one in the 3'-5' direction according to the nucleotide sequence of the sense strand. After each nucleoside monomer was linked, four reactions were performed: deprotection, coupling, capping, oxidation, or sulfurization. When two nucleotides were linked via a phosphate ester, four reactions were performed when linking the next nucleoside monomer: deprotection, coupling, capping, and oxidation. When two nucleotides were linked via a thiophosphate ester, four reactions were performed when linking the next nucleoside monomer: deprotection, coupling, capping, and sulfurization. The synthesis conditions were specified as follows:

[0364] Nucleoside monomers were provided in acetonitrile at a concentration of 0.1 M, and each deprotection reaction The conditions were the same, that is, the temperature was 25°C, the reaction time was 70 seconds, and the deprotection reagent was , a solution of dichloroacetic acid in dichloromethane (3% v / v), The molar ratio of the 4,4'-dimethoxytrityl protecting group in the methyl group to the 4,4'-dimethoxytrityl protecting group in the methyl group was 5:1.

[0365] The coupling reaction conditions were the same for all of them: the temperature was 25°C, and the solid support was bound to the The molar ratio of the nucleic acid sequence to the nucleoside monomers is 1:10, and the nucleic acid sequence 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 0.5 M acetonite. The solution was a distillate solution.

[0366] The capping conditions were the same for all samples: temperature 25°C, reaction time 15 seconds. The capping reagent solution was a mixed solution of CapA and CapB 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 The ratio of N-methylimidazole to the nucleic acid sequence bound to the solid support was 1:1:1.

[0367] The oxidation reaction conditions were the same for each reaction, with the temperature at 25°C and the reaction time at 15 seconds. The drug was iodine solution with a concentration of 0.05M. Iodine and the solid support in the coupling step The molar ratio of tetrahydrofuran:water:to the nucleic acid sequence to be bound was 30:1. The reaction was carried out using a mixed solvent of pyridine (3:1:1).

[0368] The conditions for each sulfurization reaction were the same: temperature 25°C, reaction time 300 seconds, and sulfurization. The sulfurization reagent was xanthan hydride. The molar ratio of acetonitrile:pyridin to the nucleic acid sequence to be bound was 120:1. The reaction was carried out using a mixed solvent of 1:1.

[0369] The cleavage and deprotection conditions are as follows: The column was added to ammonia water with a concentration of 25% by weight, and the dose of ammonia water was 0.5 ml / μmol. The mixture was reacted at 55°C for 16 hours, and the liquid was removed and concentrated to dryness in vacuo.

[0370] Purification and desalting: Preparative ion chromatography purification column (Source 15Q) The purification of nucleic acids was completed by gradient elution with NaCl. mM sodium phosphate (pH 8.1), solvent: water / acetonitrile = 9:1 (volume ratio) 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: eluent A: eluent B Gradient elution was performed with B = 100:0 to 50:50. The product eluates were collected and combined, and the reversed-phase column was The product was desalted using a chromatography purification column. The column was desalted by centrifugation, the packing material was dextran gel G25, and the column was eluted with deionized water.

[0371] Detection: Purity was detected using ion exchange chromatography (IEX-HPLC) and the liquid The molecular weight was analyzed by liquid chromatography-mass spectrometry (LC-MS). The theoretical value was 7584. The actual value was 7584.0, and the actual value was consistent with the theoretical value. It was demonstrated that the sense strand S conjugated with the conjugated molecule was synthesized.

[0372] (1-3) Synthesis of antisense strand (1-3A) Preparation of the antisense strand of complex 1 The solid-phase phosphoramidite method was used to prepare a general-purpose solid support (UnyLinker TM loa NittoPhase® HL Solid Supports, Ki The starting material was circulated as a starting material (novate Life Sciences), and the enzyme complex 1 was annealed. The cis-sense chain AS was synthesized. Deprotection, coupling, and capping in solid-phase synthesis. The reaction conditions for oxidation or sulfurization, cleavage and deprotection, purification and desalting conditions are the same as those for the synthesis of the sense strand. there were.

[0373] Detection: Purity was determined by ion exchange chromatography (IEX-HPLC) and molecular The amount was analyzed by liquid chromatography-mass spectrometry (LC-MS). The theoretical value was 7007. 46, the actual measured value was 7006.2. The actual measured value was consistent with the theoretical value, and the target sequence was obtained. This indicates that the antisense strand AS was synthesized.

[0374] Here, 2'-methoxy-modified uracil nucleosides modified with vinyl phosphate esters Monomer (VP-Um) (2'-methoxy modified uracil n The ucleoside monomer (VP-Um) was synthesized according to the following method. Ta.

[0375] [ka]

[0376] (1-3-1) Synthesis of VP-U-2 The VP-U-2 molecule was synthesized according to the following method.

[0377] [ka]

[0378] 2'-methoxy modified uracil nucleoside uracil nucleoside)(2'-OMe-U, 51.30g, 91.6 mmol), tert-butyldiphenylchlorosilane (TBDPSCl, 50.35 g , 183.2 mmol) and imidazole (12.47 g, 183.2 mmol) were mixed. The mixture was dissolved in 450 ml of N,N-dimethylformamide (DMF) and stirred at room temperature for 20 hours. The DMF was distilled off, and the residue was dissolved in 600 ml of dichloromethane and then added to 300 ml of saturated The aqueous phase was washed with dichloromethane (DCM) in 3 portions. Extract three times with 00 ml of water, combine the organic phases, and wash the aqueous phase with 5% oxalic acid until the pH is <5. After evaporating the solvent until dry, crude VP-U-1 was obtained, which was then used as VP-U- It was used in the synthesis of 2.

[0379] The crude VP-U-1 product was dissolved in 100 ml of dichloromethane, and then the solution was left in an ice bath for 10 minutes. The mixture was stirred for 1 minute, and then 450 ml of 2% p-toluenesulfonyl ether, which had been previously chilled in a refrigerator at 4°C, was added. 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, and 200 ml of saturated sodium bicarbonate was added to quench the reaction. The organic phase was washed with saturated aqueous 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 mixture was washed once with 200 ml of saturated saline, and the solvent was evaporated to dryness. The mixture was purified using a 0-300 mesh normal phase silica gel column, and petroleum ether was added to the column. Oil ether: ethyl acetate: dichloromethane: methanol = 1:1:1:0.05 to 1:1 The product eluate was collected, the solvent was evaporated to dryness under reduced pressure, and the column was filtered with a vacuum oil pump. The mixture was dried in a foam pump to give a total of 40.00 g of pure VP-U-2. 1 H NMR (4 00 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 = 4.7 Hz,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),4.05 (dd,J = 4.8, 4.0 Hz,1H),3.96 (t,J = 4.7 Hz,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, Measured value: 4 97.45.

[0380] (1-3-2) Synthesis of VP-U-4:

[0381] [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 dimethylformamide (DMF) and trifluoroacetic acid (6.61 g, 53.2 mmol) in 200 ml of dimethylformamide (DMF). The mixture was dissolved in dimethyl sulfoxide (DMSO) and stirred at room temperature for 20 hours. Tetraethyl phenyldiphosphonate (21.44 g, 74.4 mmol) in 120 mL of THF The solution was dissolved in t-BuOK (11.36 g, 101.2 mmHg) and cooled in an ice bath. After adding 10 ml of the mixture at ice bath temperature, the mixture was allowed to react for 10 minutes, then warmed to room temperature and allowed to react for 0.5 hours. Then, add the above-mentioned reaction mixture over about 1 hour, react at ice bath temperature for 1 hour, and then cool to room temperature. The temperature was raised and the reaction was continued for 18 hours. Water was added to quench the reaction, and the aqueous phase was washed with dichloromethane. The organic phases were combined and extracted once with 200 ml of saturated saline. After washing, the solvent was evaporated until dry. Normal phase silica gel (200-300 mesh) Purify with a column, put petroleum ether into the column, and petroleum ether: ethyl acetate = 1:1-1 Gradient elution was performed with 4:1000 ethanol, and the product eluate was collected. The solvent was evaporated to dryness under reduced pressure and foamed with a vacuum oil pump. Drying gave a total of 14.00 g of pure VP-U-4. 1 H NMR (400 MH z,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). MS m / z:C 31 H 42 N2O8PSi,[M+H] + , Theoretical value: 629.24, Measured value: 6 29.51.

[0382] (1-3-3) Synthesis of VP-U-5:

[0383] [ka] VP-U-4 (14.00 g, 22.29 mmol) was dissolved in 100 ml of tetrahydrofuran. Dissolve triethylamine trihydrofluoride (17.96 g, 111.45 mmol) in water. ) was added and stirred at room temperature for 20 hours to allow the reaction to complete. The solvent was then evaporated to dryness. The crude product was dissolved in 50 ml of dichloromethane and then evaporated to dryness. The product was purified on a 200-300 mesh normal phase silica gel column, and petroleum ether was added to the column. The mixture was placed in a mixture of petroleum ether, ethyl acetate, dichloromethane, and methanol in a ratio of 1:1:1:0. Gradient elution was performed with a ratio of 0.5 to 1:1:1:0.25, and the product eluate was collected. The solvent was evaporated to dryness under reduced pressure. The mixture was then foam-dried using a vacuum oil pump to obtain a total of 6.70 g of pure VP-U-5. 1 HN MR (400 MHz,DMSO-d6) δ 7.96 (d,J = 7.8 H z,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),5.2 7 (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.

[0384] (1-3-4) Synthesis of VP-U-6:

[0385] [ka] VP-U-5 (391 mg, 1.5 mL) was dissolved in 10 mL of anhydrous dichloromethane under argon protection. 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 evaporated until dry, and the residue was purified by column chromatography. Purify (200-300 mesh normal phase silica gel) with dichloromethane:acetonitrile (containing 0.5 wt% triethylamine) = 3:1 to 1:3 gradient elution), product solution The exudate was collected and the solvent was removed by concentration to obtain a total of 508 mg of the target product VP-U-6. 31 P 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, Measured value: 591.55. VP-U-6 is the target product VP-Um. It was shown to be involved in the synthesis of RNA chains as a nucleoside monomer.

[0386] (1-3B) Preparation of the antisense strand of complex 2 The antisense strand of complex 2 is distinguished from the antisense strand of complex 1 by the first position at the 5'-end. The only difference was the nucleotide modification of the nucleotides. When preparing the sense strand, the last nucleoside monomer attached is 2'-methoxy-modified. Uracil nucleoside monomer (Um) is a nucleoside that can be deprotected, coupled, capped, and Four oxidation reactions were carried out to obtain CPR-I monomer (Suzhou Jiji, Cat#13-2601 -XX) was attached to the 5' end of the antisense strand to form a 5'-phosphate modification.

[0387] [ka]

[0388] General purpose solid support used in synthesis, deprotection, coupling, capping, oxidation The conditions for the sulfurization reaction, cleavage and deprotection, purification and desalting were the same as those for the synthesis of the sense strand. .

[0389] Purity was detected by ion exchange chromatography (IEX-HPLC) and liquid chromatography The molecular weight was analyzed by liquid chromatography-mass spectrometry (LC-MS), and the theoretical value was 7011. 47, the actual measured value was 7011.3. The actual measured value was consistent with the theoretical value, and the target sequence was obtained. This indicates that the antisense strand AS was synthesized.

[0390] (1-3C) Preparation of the antisense strand of complex 3 When CPR-I monomers were bound, sulfurization reaction conditions were used instead of the oxidation reaction conditions described above. The synthesis process was the same as that for the antisense strand of Complex 2, except that 5'-thiophosphoryl We anticipated that we could prepare the antisense strand of complex 3 with acid ester modifications.

[0391] (1-3D) Preparation of the antisense strand of complex 4 The distinction between the antisense strand of complex 4 and that of complex 1 is based on the first position at the 5'-end. The only difference was the nucleotide modification of the nucleotides. When preparing the sense strand, the last nucleoside monomer attached is 2'-methoxy-modified. It was a uracil nucleoside monomer (Um).

[0392] Purity was detected by ion exchange chromatography (IEX-HPLC) and liquid chromatography The molecular weight was analyzed by liquid chromatography-mass spectrometry (LC-MS), and the theoretical value was 6931. 47, the actual measured value was 6930.9. The actual measured value was consistent with the theoretical value, and the target sequence was obtained. This indicates that the antisense strand AS was synthesized.

[0393] (1-4) Synthesis of Complexes 1 to 4 For complex 1, the S chain and AS chain were each dissolved in water for injection to give a solution of 40 mg / mL. The resulting solutions were mixed in an equimolar ratio, heated at 50°C for 15 minutes, cooled to room temperature, and then added to water. The double-stranded structure was formed by the ion bond. Dilute the complex using a pressure of 18.2 MΩ*cm (25°C) until the concentration reaches 0.2 mg / mL. After dilution, the sample was analyzed by liquid chromatography mass spectrometry (LC-MS). Matography-Mass Spectrometry, purchased from Waters The molecular weight was detected using a chromatograph (model number: LCT Premier). The measured value was consistent with the theoretical value. The synthesized complex 1 is a purpose-designed double-stranded nucleic acid sequence containing the L-9 complex molecule. It was shown that:

[0394] The same method was used to prepare complexes 2 to 4, and the molecular weights were measured. The values ​​were consistent with those of the synthesized complex, which was a purpose-designed double-stranded nucleic acid sequence containing the L-9 complex molecule. The structures of complexes 1 to 4 are shown in formula (403).

[0395] (Preparation Example 2) Preparation of Complexes 5 to 21 and Comparative Complex 1 1) The siRNA was compared with Comparative Complex 1 and Complexes 5 to 21 shown in Table 3. 2) if the target sequence contains unmodified nucleotides, the cleavage and deprotection conditions are After treatment with aqueous ammonia, the concentration of single-stranded nucleic acid was 0.4 ml / μmol. After dissolving the product in N-methylpyrrolidone, 0.3 ml / μmol of triethyl Add amine and 0.6 ml / μmol triethylamine trihydrofluoride to the ribose The complex was prepared in the same manner as in Preparation Example 1, except that the 2'-TBDMS protection in We synthesized 5, 6, 7, and comparative conjugate 1. We anticipated that conjugates 8-21 could be produced.

[0396] [Table 3] JPEG0007672163000072.jpg191144JPEG0007672163000073.jpg115143

[0397] (Preparation Example 3) Preparation of P10-siAN1M3SVP complex (complex 22) (3-1) Synthesis of P-10 Compound Compound P-10 was synthesized according to the following method.

[0398] [ka]

[0399] (3-1-1) Synthesis of GAL5-C4-1 In 40 ml of N,N-dimethylformamide, The resulting GAL-5 (13.43 g, 30.0 mmol), 4-amino acid tert- ethyl ester hydrochloride (5.87 g, 30.0 mmol), O-benzotriazole-tet Tetramethyluronium hexafluorophosphate (13.65 g, 36.0 mmol) and Add diisopropylethylamine (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 mixture. Add aqueous solution, extract with ethyl acetate three times with 200 ml each time, combine the organic phases, The organic phase was separated and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure until dry, and 30.3 g of oily crude product GAL5-C4-1 was obtained. The reaction mixture was then directly subjected to the next reaction.

[0400] (3-1-2) Synthesis of GAL5-C4-2 The crude GAL5-C4-1 product obtained in step (3-1-1) (30.3 g, 30 mmol) ) was dissolved in 180 ml of formic acid and the mixture was stirred at room temperature for 16 hours. Evaporated and purified by column chromatography (200-300 mesh normal phase silica The gel was eluted with a gradient of dichloromethane:methanol = 100:18 to 100:20. The reaction eluate was collected, and the solvent was removed by concentration to obtain a total of 14.84 g of the target product GAL5-C4- I got 2.

[0401] Synthesis of (3-1-3)P-6: M-18-Tr (2.02 g, 4 0.69 mmol) and GAL5-C4-2 (8.24 g, 1 (5.48 mmol, two batches of product combined) was mixed and added to 47 ml of acetonite. The mixture was dissolved in ethanol and N-methylmorpholine (3.13 g, 30.96 mmol) was added. Finally, 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine salt The acid salt (DMTMM, 4.28 g, 15.48 mmol) was added and the reaction was carried out at room temperature with stirring for 2 hours. The reaction mixture was diluted with 20 ml of dichloromethane and added to 10 ml of saturated sodium bicarbonate solution. The organic phase was washed with 10 ml of saturated saline solution, and the organic phase was combined and washed with anhydrous sulfuric acid. After drying with sodium hydroxide and filtering, the solvent was evaporated to dryness under reduced pressure to obtain a crude product. The mixture was purified on a 00 mesh normal phase silica gel column, and petroleum ether was added to the column, and 1 wt.% Neutralize the acidity of the silica gel with triethylamine and dissolve it in dichloromethane:methanol=100: Gradient elution was performed with a ratio of 5 to 100:7, and the product eluate was collected and evaporated to dryness under reduced pressure to give a total of 8.27 g. Pure P-6 was obtained.

[0402] Synthesis of (3-1-4)P-7: P-6 (6.82 g, 3.456 mmol) obtained in (3-1-3) above was added to 69 ml of dichloromethane and dichloroacetic acid (13.367 g, 103.67 mmol) The reaction mixture was diluted with 100 ml of dichloromethane and allowed to react at room temperature for 2 hours. Further, add saturated sodium bicarbonate solution to wash and adjust the pH to 7-8. The organic phases were combined and extracted with dichloromethane six times, each time with 30 ml of dichloromethane. After drying over sodium and filtering, the solvent was evaporated to dryness under reduced pressure to obtain the crude product. The mixture was purified with mesh normal phase silica gel, and the silica gel was acidified with 10 wt% triethylamine. The column was neutralized and equilibrated with 1 wt% triethylamine, and the mixture was diluted with dichloromethane:methanol = Gradient elution was performed at 100:30 to 100:40, and the product eluate was collected. The solvent was evaporated to dryness under reduced pressure. A total of 4.82 g of P-7 was obtained. MS m / z:C 78 H 127 N 10 O 33 ,[M+ H] + , theoretical value: 1732.91, actual value: 1735.73.

[0403] Synthesis of (3-1-5)P-8:

[0404] [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-Benzoxazol-4(3H)-one (DEPBT) (1.375 g, 4. 596 mmol) was added, and diisopropylethylamine (1.188 g, 9.19 1 mmol) 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 phase was washed with saturated brine, and the aqueous phase was extracted twice with dichloromethane, each time with 10 ml of dichloromethane. 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 obtained by foam drying overnight using a vacuum oil pump. Using 0-300 mesh normal phase silica gel, 20 ml of triethylamine was added to the silica gel. The acidity of the column was neutralized, and the column was equilibrated with petroleum ether containing 1 wt% triethylamine. Oil ether: ethyl acetate: dichloromethane: N,N-dimethylformamide = 1:1:1 Gradient elution was performed with a ratio of 0.5 to 1:1:1:0.6, and the product eluate was collected and the solvent was evaporated to dryness under reduced pressure. Solidification gave a total of 2.793 g of pure P-8.

[0405] Synthesis of (3-1-6)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 hours. The reaction mixture was diluted with 50 ml of dichloromethane and then added 100 ml of 0.5 M triethyl ether. Add dimethylamine phosphate to wash the reaction mixture, and then add 10 ml of dichloromethane to the aqueous phase. The organic phases were combined and evaporated to dryness under reduced pressure to obtain the crude product. g of 200-300 mesh normal phase silica gel was used, and silica was purified with 1 wt% triethylamine. The acidity of the gel was neutralized, and the column was equilibrated with dichloromethane and 1 wt% triethylamine. The product was eluted with a gradient of dichloromethane:methanol=100:18 to 100:20. The eluate was collected, and the solvent was evaporated to dryness under reduced pressure to obtain a total of 200 mg of pure P-9 conjugate molecule. MS m / z:C 106 H 153 N 10 O 41 ,[M-DMTr] + , theoretical value: 1921 .05, Actual value: 1920.97.

[0406] Synthesis of (3-1-7)P-10: Instead of the L-9 conjugate molecule, a P-9 conjugate molecule was used, and the P-9 conjugate was bound to a solid support. P-10 was obtained by the same method as in step (1-1-9) in Preparation Example 1, except that the following compound was obtained: Prepared.

[0407] (3-2) Synthesis of P10-siAN1M3SVP complex The sense strand was synthesized using the P-10 compound instead of the L-10 compound as the starting point. Except for the above, the same procedures as in steps (1-2), (1-3A), and (1-4) of Preparation Example 1 were carried out. Complex 22 was prepared. The P10-siAN1M3SVP complex, whose structure is shown in formula (404), Amalgamation was achieved.

[0408] (Preparation Example 4) Preparation of R5-siAN1M3SVP complex (complex 23) (4-1) Synthesis of R-5 Compound The R-5 compound was synthesized according to the following method.

[0409] [ka]

[0410] (4-1-1) Synthesis of GAL-C7-1 GAL-3 (26.4 g, 80%) obtained by the method described in step (1-1-1b) 0.2 mmol) was dissolved in 134 ml of anhydrous 1,2-dichloroethane and 60 g of 4 Å Add sieve powder and then add 7-octen-1-ol (11.3 g, 88.2 mmol). The reaction was stirred at room temperature for 10 minutes, and then the mixture was cooled to room temperature and cooled in an ice bath under nitrogen protection with trifluoromethanesulfonic acid. Trimethylsilyl (8.9 g, 40.1 mmol) was added, and the reaction was allowed to proceed with stirring at room temperature for 24 hours. The 4Å molecular sieve powder was removed by filtration, and 500 ml of saturated aqueous sodium bicarbonate solution was added to the filtrate. The organic phase was separated and the aqueous phase was extracted once with 100 ml of dichloromethane. The mixture was combined and washed once with 250 ml of saturated saline, and the organic phase was separated and washed with anhydrous sodium sulfate. The solvent was distilled off under reduced pressure until dry, and 33.3 g of a yellow starch syrup-like product, GAL-C 7-1 was obtained and used in the next oxidation reaction without further purification.

[0411] (4-1-2) Synthesis of GAL-C7-2 GAL-C7-1 (33.3 g, 72.8 mmol) obtained in step (4-1-1) Dissolve it in a mixed solvent of 160 ml of dichloromethane and 160 ml of acetonitrile, 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 chloride (III) (498 mg The reaction mixture was stirred for 23 hours after the temperature was naturally raised to room temperature. Add 00ml of water, dilute and stir, then add saturated sodium bicarbonate to adjust the pH to 7.5. The organic phase was separated, and the aqueous phase was extracted three times with dichloromethane. The organic phase was discarded, and the aqueous phase was extracted with solid crystals. The pH was adjusted to about 3 with acetic acid, and the extract was then extracted three times with 200 ml of dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed by distillation under reduced pressure. Chromatography (200-300 mesh normal phase silica gel in dichloromethane:methanol The resulting product was purified by gradient elution (100:18 to 100:20) to give 22.4 g of a white foamy solid. The product GAL-C7-2 was obtained. MS m / z:C 21 H 32 NO 11 ,[M+H] + , Theoretical value: 476.50, measured value: 475.94.

[0412] (4-1-3) Synthesis of R-1: M-18-Tr (2.02 g, 4 4.69 mmol) and GAL-C7-2 (7.36 g, 15.48 mmol) were mixed. Dissolved in 7 ml of acetonitrile and further added N-methylmorpholine (3.13 g, 30. 96 mmol) was added, and finally 4-(4,6-dimethoxytriazin-2-yl)-4- Methylmorpholine hydrochloride (DMTMM, 4.28 g, 15.48 mmol) was added, and the mixture was stirred at room temperature. The reaction mixture was stirred for 2 hours at RT. The reaction mixture was diluted with 200 ml of dichloromethane and added to 100 ml of saturated Wash the organic phase with saturated sodium bicarbonate solution, then wash the organic phase with 100 ml of saturated brine. 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 purified on a 200-300 mesh normal phase silica gel column and then dissolved in petroleum ether. The acidity of the silica gel was neutralized with 1% by weight of triethylamine, and dichloromethane was added. Gradient elution was performed with ethanol:methanol = 100:5 to 100:7, and the product eluate was collected and distilled under reduced pressure. After drying, 7.82 g of pure R-1 was obtained.

[0413] (4-1-4) 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 hours. Add 100 ml of dichloromethane to dilute the reaction mixture, and add saturated sodium bicarbonate solution. Wash the mixture with dichloromethane to adjust the pH to 7-8. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and then the solvent was removed. The residue was evaporated to dryness under reduced pressure to obtain a crude product. The acidity of the silica gel was neutralized with 10% by weight triethylamine, and then 1% by weight triethylamine was added. The column was equilibrated and gradient elution was performed with dichloromethane:methanol = 100:30 to 100:40. The solvent was evaporated to dryness under reduced pressure to give 4.49 g of pure R-2.

[0414] (4-1-5) 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- 1,2,3-Benzoxazol-4(3H)-one (DEPBT) (1.375 g, 4. 596 mmol) was added, and diisopropylethylamine (1.188 g, 9.19 1 mmol) 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 phase was washed with saturated brine, and the aqueous phase was extracted twice with dichloromethane, each time with 10 ml of dichloromethane. 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 obtained by foam drying overnight using a vacuum oil pump. Using 0-300 mesh normal phase silica gel, 20 ml of triethylamine was added to the silica gel. The acidity of the column was neutralized, and the column was equilibrated with petroleum ether containing 1 wt% triethylamine. Oil ether: ethyl acetate: dichloromethane: N,N-dimethylformamide = 1:1:1 Gradient elution was performed with a gradient of 0.5 to 1:1:1:0.6, and the solvent was evaporated to dryness under reduced pressure to obtain 2.642 g of pure PEG-40. I got a stylish R-3.

[0415] (4-1-6) Synthesis of R-4: R-3 (795 mg, 0.4074 mmol), succinic anhydride (82 mg, 0.81 48 mmol) and 4-dimethylaminopyridine (DMAP, 100 mg, 0.8148 (mmol) were mixed and dissolved in 4 ml of dichloromethane, and then diisopropyl ethyl Amine (DIEA, 100 mg, 0.8148 mmol) was added and the mixture was stirred at 25°C for 18 h. The reaction mixture was washed with 5 ml of 0.5 M triethylamine phosphate, and the aqueous phase was The mixture was extracted three times with 5 ml of methyl ether, and the organic phases were combined and evaporated to dryness under reduced pressure to obtain the crude product. For column purification, 30 g of 200-300 mesh normal phase silica gel was used, and 1 wt. The acidity of the silica gel was neutralized with % triethylamine, and the column was equilibrated with dichloromethane. Dichloromethane containing 1% by weight of triethylamine:methanol=100:18-100: The product eluate was collected and the solvent was evaporated to dryness under reduced pressure to give 505 mg of pure 1,2-dimethyl-2,2-dichloro ... The R-4 conjugate molecule was obtained.

[0416] (4-1-7) Synthesis of R-5 conjugate molecules: Instead of the L-9 conjugate molecule, an R-4 conjugate molecule was used, and the R-4 conjugate was bound to a solid support. R-5 was prepared in the same manner as in step (1-1-9) in Preparation Example 1, except that the compound was obtained. Made.

[0417] (4-2) Synthesis of R5-siAN1M3SVP complex The sense strand was synthesized using the R-5 compound instead of the L-10 compound as the starting material. In addition, multiple compounds can be prepared in the same manner as in steps (1-2), (1-3A), and (1-4) in Preparation Example 1. Complex 23 was prepared. The R5-siAN1M3SVP complex, whose structure is shown in formula (407), I expected to be able to get it.

[0418] (Preparation Example 5) Preparation of LA5-siAN1M3SVP complex (complex 24) It was anticipated that the LA-5 compound could be synthesized by the following process route.

[0419] [ka]

[0420] The sense strand was synthesized using the LA-5 compound instead of the L-10 compound as the starting point. Except for the above, the same procedures as in steps (1-2), (1-3A), and (1-4) of Preparation Example 1 were carried out. Complex 24 was prepared. The LA5-siAN1M3SVP complex, whose structure is shown in formula (412), I expected to get a merger.

[0421] (Preparation Example 6) Preparation of LB5-siAN1M3SVP complex (complex 25) (6-1) Synthesis of LB-5 Compound The LB-5 compound was synthesized according to the following method.

[0422] [ka]

[0423] Synthesis of (6-1-1)LB-1: L-8 (5.0 g, 3.386 m) obtained by the method described in step (1-1-6) mol), adipic anhydride (870 mg, 6.772 mmol) and 4-dimethylamine Mix 130 ml of HCl with 100 mg of HCl (DMAP, 827 mg, 6.772 mmol) of HCl in a 130 ml flask. The mixture was dissolved in 160 ml of diisopropylethylamine (DIEA, 2.2 g, 16 0.931 mmol) was added and the mixture was stirred at 25°C for 4 hours. The reaction mixture was diluted with 0.5M triethylamine phosphate, and the aqueous phase was washed with 0.5M triethylamine phosphate. Extract with chloromethane four times, 10 ml each time, and combine the organic phases. Evaporate to dryness under reduced pressure to obtain a crude product. For column purification, 120 g of 200-300 mesh normal phase silica gel was used. The acidity of the silica gel was neutralized with 1 wt% triethylamine, and the column was flattened with dichloromethane. Equilibrated, petroleum ether: ethyl acetate: dichloromethane: methanol = 1:1:1:0.2 Gradient elution at ~1:1:1:1 and evaporation of the solvent to dryness yielded 4.267 g of pure LB-1 obtained.

[0424] Synthesis of (6-1-2)LB-2: LB-1 (4.697 g, 2.7 53 mmol, two batches of product combined), 3-amino-1,2-propanedioic acid ol (313 mg, 3.442 mmol), 4-(4,6-dimethoxytriazine-2- (methyl)-4-methylmorpholine hydrochloride (DMTMM, 953 mg, 3.442 mmol) and N-methylmorpholine (700 mg, 6.884 mmol) in 30 ml of acetone. The mixture was added to a mixture of nitrile and 3 ml of methanol, and the mixture was stirred overnight at room temperature. The solvent was evaporated until the eluate was completely dissolved, and the eluate was purified by column chromatography (200-300 mesh normal phase silica gel). The product was purified by gradient elution with dichloromethane:methanol = 1:0.07 to 1:0.5. The product eluate was collected and the solvent was removed by concentration to obtain 3.27 g of the target product LB-2. Ta.

[0425] Synthesis of (6-1-3)LB-3: LB-2 (2.27 g, 1.353 mmol) was dissolved in 14 ml of anhydrous pyridine. Furthermore, 4,4'-bismethoxytrityl chloride (688 mg, 2.03 mmol) The mixture was stirred at room temperature overnight, and the reaction was quenched by adding 150 ml of methanol. The solvent was evaporated until dry. The silica gel was eluted with a gradient of dichloromethane:methanol = 1:0.05 to 1:0.2. The product eluate was collected, and the solvent was removed by concentration to obtain 1.647 g of the desired product. Got LB-3.

[0426] Synthesis of (6-1-4)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 DIEA (270 mg, 2.07 5mmol) was added and the reaction was stirred overnight at 25°C. The reaction mixture was washed three times with 1 ml of dichloromethane, and the aqueous phase was extracted three times with 2 ml of dichloromethane. The mixture was combined and evaporated to dryness under reduced pressure to obtain a crude product. The acidity of silica gel was neutralized with 5% by weight of triethylamine, and petroleum ether was added. The column was equilibrated with ether and diluted with dichloromethane:methanol containing 1 wt% triethylamine. Gradient elution was performed with a ratio of 100:5 to 100:20, and the solvent was evaporated to dryness under reduced pressure to obtain 787 mg of pure PEG. The LB-4 conjugate molecule was obtained.

[0427] Synthesis of (6-1-5)LB-5: The LB-4 conjugate molecule was used instead of the L-9 conjugate molecule, and the LB-4 conjugate bound to a solid support was used. LB- was prepared in the same manner as in step (1-1-9) of Preparation Example 1, except that the synthetic molecule was obtained. 5 was prepared.

[0428] (6-2) Synthesis of LB5-siAN1M3SVP complex The sense strand was synthesized using the LB-5 compound instead of the L-10 compound as the starting material. Except for the above, the same procedures as in steps (1-2), (1-3A), and (1-4) of Preparation Example 1 were carried out. Complex 25 was prepared using LB5-siAN1M3SVP, whose structure is shown in formula (413). I expected to get it.

[0429] (Preparation Example 7) Synthesis of V8-siAN1M3SVP conjugate (conjugate 26) It was anticipated that compound V-8 could be synthesized via the following process route.

[0430] [ka]

[0431] The sense strand was synthesized using the V-8 compound instead of the L-10 compound as the starting material. In addition, multiple compounds can be prepared in the same manner as in steps (1-2), (1-3A), and (1-4) in Preparation Example 1. Compound 26 was prepared to give V8-siAN1M3SVP, the structure of which is shown in formula (414). I expected it would be possible.

[0432] (Preparation Example 8) Preparation of W8-siAN1M3SVP complex (complex 27) (8-1) Synthesis of W-8 Compound Compound W-8 was synthesized according to the following method.

[0433] [ka]

[0434] Synthesis of (8-1-1)W-1: W-0 (2.024 g, 10 mmol) was dissolved in 25 ml of acetonitrile. Add triethylamine (4.048 g, 40 mmol) and cool to about 0°C in an ice-water bath. Ethyl trifluoroacetate (5.683 g, 40 mmol) was added and the mixture was allowed to react at room temperature for 22 hours. The solvent was evaporated to dryness under reduced pressure, and the mixture was foam-dried using a vacuum oil pump for 18 hours to obtain 5.835 g of solid. A crude product W-1 was obtained.

[0435] (8-1-2) 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 reaction mixture was washed twice with sodium chloride and once with 20 ml of saturated saline. The organic phases were combined and After drying with sodium sulfate and filtering, the organic solvent was evaporated to dryness under reduced pressure and then extracted with a vacuum oil pump. After overnight foam drying, 8.012 g of solid crude product W-2 was obtained. A deprotection reaction was carried out.

[0436] Synthesis of (8-1-3)W-3: W-2 crude product (8.012 g, 10 mmol) was dissolved in 100 ml of methanol. Further, 100 ml of methylamine aqueous solution (40 wt%) 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 DCM-methanol mixed solvent and wash the organic phase with 50 ml of saturated sodium bicarbonate. The aqueous phase was extracted three times with dichloromethane, each time with 50 ml of dichloromethane. The organic phases were combined and extracted with anhydrous sulfuric acid. After drying with sodium hydroxide and filtering, the solvent was evaporated to dryness under reduced pressure and the mixture was foamed overnight with a vacuum oil pump. The mixture was dried and purified on a 200-300 mesh normal phase silica gel column, and petroleum ether was added to the column. The silica gel was placed in a ram, and the acidity of the silica gel was neutralized with 1% by weight of triethylamine. Methanol:ammonia water (25 wt%) = 1:1:0.05 to 1:1:0.25 gradient The product eluate was collected, the solvent was evaporated to dryness under reduced pressure, and the mixture was dried with a vacuum oil pump. 3.062 g of pure W-3 was obtained.

[0437] (8-1-4) Synthesis of W-4: W-3 (0.675 g, 1.517 mmol) and GAL-C7-2 (2.60 g, 5. 46 mmol) were mixed and dissolved in 47 ml of acetonitrile, and then diisopropyl Ethylamine (1.57 g, 12.14 mmol) was added, and finally 3-diethoxyphosphoric acid was added. 1.816 g of aryl-1,2,3-benzoxazol-4(3H)-one (DEPBT, 6.04 mmol) was added and the mixture was stirred at room temperature for 2.5 hours. The reaction mixture was diluted with ethanol, and the organic phase was washed with 80 ml of saturated sodium bicarbonate solution. The organic phase was washed with 1 liter of saturated brine, 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. The column was purified with petroleum ether, and the silica gel was treated with 1% by weight 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 give 1.610 g of pure W-4.

[0438] Synthesis of (8-1-5)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 further added and the mixture was allowed to react at room temperature for 1 hour. The reaction mixture was neutralized with 150 ml of pyridine, and the solvent was evaporated to dryness under reduced pressure to obtain a crude product. Normal phase silica gel of 200-300 mesh was used, and the mixture was washed with 10% by weight of triethylamine. The acidity of the silica gel was neutralized, and the column was equilibrated with 1% by weight of triethylamine. 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 give 1.26 g of pure W-5.

[0439] Synthesis of (8-1-6)W-6: W-5 (1.25 g, 0.793 mmol) and the method described in step (1-1-7a) A-1 (1.21 g, 2.38 mmol) obtained by the above procedure was mixed with 12 ml of dichloromethane. Dissolved in methane, 3-diethoxyphosphoryl-1,2,3-benzoxazole 4(3 H)-one (DEPBT, 0.712 g, 2.38 mmol) was added, and diisopropyl Pyroethylamine (0.615 g, 4.76 mmol) was added, and the mixture was stirred at 25°C for 3 hours. The organic phase was washed with 80 ml of saturated sodium bicarbonate, and the aqueous phase was diluted with dichloromethane. Extract three times with 10 ml each time, combine the organic phases, wash with 10 ml of saturated saline, and The phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated to dryness under reduced pressure. The crude product was obtained by drying overnight in an oil pump. 0 mesh normal phase silica gel was used, and the silica gel was acidified with 20 ml of triethylamine. The column was neutralized and equilibrated with petroleum ether containing 1 wt% triethylamine. ethanol:ethyl acetate:dichloromethane:N,N-dimethylformamide=1:1:1:0.1 Gradient elution was performed with a ratio of 1:1:0.7 to collect the product eluate, and the solvent was evaporated to dryness under reduced pressure to obtain a 1.5 7 g of pure W-6 was obtained.

[0440] (8-1-7) 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 mmol) ol) were mixed and dissolved in 7 ml of dichloromethane, and DIEA (0.407 g, 3.15 mmol) was added and the mixture was stirred at 25°C for 24 hours. The reaction mixture was washed with dimethylamine phosphate, and the aqueous phase was extracted three times with 5 ml of dichloromethane. The organic phases were combined and evaporated to dryness under reduced pressure to obtain a crude product. Using ~300 mesh normal phase silica gel, acidify the silica gel with 1 wt% triethylamine. The column was neutralized with dichloromethane and equilibrated with dichloromethane containing 1% by weight of triethylamine. Gradient elution was performed with chloromethane and methanol (100:18 to 100:20), and the product eluate was collected. The mixture was collected and the solvent was evaporated to dryness under reduced pressure to give 1.033 g of pure W-7 complex molecule. z:C 101 H 146 N7O 38 ,[M-DMTr] + ,Theoretical value: 1763.92,Actual measurement Value: 1763.21.

[0441] Synthesis of (8-1-8)W-8: The W-7 conjugate molecule was used instead of the L-9 conjugate molecule, and the W-7 conjugate was bound to a solid support. W-8 was prepared in the same manner as in step (1-1-9) in Preparation Example 1, except that the following compound was obtained: Made.

[0442] (8-2) Synthesis of W8-siAN1M3SVP complex The sense strand was synthesized using compound W-8 instead of compound L-10 as the starting material. In addition, multiple compounds can be prepared in the same manner as in steps (1-2), (1-3A), and (1-4) in Preparation Example 1. Compound 27 was prepared. W8-siAN1M3SVP, whose structure is shown in formula (415), was obtained. .

[0443] (Preparation Example 9) Preparation of X8-siAN1M3SVP complex (complex 28) It was anticipated that compound X-8 could be synthesized by the following process route.

[0444] [ka]

[0445] The sense strand was synthesized using the X-8 compound instead of the L-10 compound as the starting material. In addition, multiple compounds can be prepared in the same manner as in steps (1-2), (1-3A), and (1-4) in Preparation Example 1. Compound 28 was prepared to give X8-siAN1M3SVP, the structure of which is shown in formula (421). I expected it would be possible.

[0446] (Preparation Example 10) Preparation of Z5-siAN1M3SVP complex (complex 29) (10-1) Synthesis of Z-5 Compound Compound Z-5 was synthesized according to the following method.

[0447] [ka]

[0448] Synthesis of (10-1-1)Z-1: W-3 (1.50 g, 3.37 m) obtained by the method described in step (8-1-3) mol) and GAL5-C4-2 (7 mol) obtained by the method described in step (3-1-2). 0.18g, 13.48mmol) were mixed and dissolved in 34ml of dichloromethane. Diisopropylethylamine (3.48 g, 26.96 mmol) was added to the 3- Diethoxyphosphoryl-1,2,3-benzoxazol-4(3H)-one (DEPBT 4.04 g, 13.48 mmol) was added and the reaction was stirred at room temperature for 4.5 hours. Dilute the reaction mixture with 1 ml of dichloromethane and add 80 ml of saturated sodium bicarbonate solution to the organic phase. The organic phase was washed with 80 ml of saturated saline solution, and the organic phases were combined and washed with anhydrous sodium sulfate. After drying in a tumbler and filtering, the solvent was evaporated to dryness under reduced pressure to obtain a crude product, which was then filtered to a thickness of 200-300 mesh. The column was purified with a normal phase silica gel column containing 1% by weight of petroleum ether. Neutralize the acidity of silica gel with amine, and use dichloromethane:methanol = 30:1 to 15:1 The product eluate was collected and evaporated to dryness under reduced pressure to give 3.97 g of pure Z-1. MS m / z:C 98 H 143 N 10 O 33 ,[M+H] + , theoretical value: 1987.9 8. Actual value: 1987.90.

[0449] (10-1-2) 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 the mixture was reacted at room temperature for 1 hour. The reaction mixture was neutralized by adding pyridine, and the solvent was evaporated to dryness under reduced pressure to obtain a crude product. 220 g of 200-300 mesh normal phase silica gel was placed in a column and eluted with 10% pyridine. The acidity of the silica gel was neutralized, and the column was equilibrated with 1‰ pyridine and then diluted with dichloromethane:methanol. Gradient elution was performed with a ratio of 10:1 to 2:1, and the product eluate was collected. The solvent was evaporated to dryness under reduced pressure. 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.

[0450] Synthesis of (10-1-3)Z-3: Z-2 (3.49 g, 2.0 mmol) and the method described in step (1-1-7a) The resulting A-1 (3.06 g, 6.0 mmol) was mixed with 30 ml of dichloromethane. 3-diethoxyphosphoryl-1,2,3-benzoxazole 4(3H)- Add diisopropyl ether (DEPBT, 1.80 g, 6.0 mmol) and Amine (1.55 g, 12.0 mmol) was added, and the reaction was stirred at 25°C for 3 hours. Dilute the reaction mixture with ml of dichloromethane and add saturated sodium bicarbonate to the organic phase in one portion. Wash twice with 30 ml of dichloromethane, extract the aqueous phase with 10 ml of dichloromethane, combine the organic phases, and add 50 ml of The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and then dissolved in water. The agent was evaporated to dryness under reduced pressure and then dried overnight with a vacuum oil pump to obtain a crude product. 200g of 200-300 mesh normal phase silica gel was used, and 20ml of triethylamine was added. The acidity of the silica gel was neutralized with amine, and the silica gel was then washed with petroleum ether containing 1% by weight of triethylamine. The column was equilibrated and eluted with a gradient of dichloromethane:methanol = 25:1 to 15:1. The eluate was collected and the solvent was evaporated to dryness under reduced pressure to give 2.2 g of pure Z-3. MS m / z :C 103 H 151 N 10 O 38 ,[M+H] + , Theoretical value: 2136.02, Actual value: 2 136.20.

[0451] Synthesis of (10-1-4)Z-4: Z-3 (2.10 g, 0.983 mmol) was dissolved in DIEA (0.635 g, 4.915 mmol) mol) in 14.8 ml of dichloromethane and (DMAP, 240 mg, 1.966 mmol) was added and stirred to clarify the solution, and then succinic acid Anhydride (197 mg, 1.966 mmol) was added and the reaction was stirred at 25°C for 18 hours. Add 0 ml of dichloromethane to dilute the reaction mixture, and add 80 ml of 0.5 M triethylamine. The organic phase is washed with phosphate and the aqueous phase is extracted twice with dichloromethane, each time with 50 ml. The organic phases were combined and evaporated to dryness under reduced pressure to obtain a crude product. 00 mesh normal phase silica gel was used, and the silica gel was acidified with 1 wt% triethylamine. The column was neutralized and equilibrated with dichloromethane, and then diluted with dichloromethane containing 1% by weight of triethylamine. Gradient elution was performed with methane:methanol = 10:1 to 3:1, and the product eluate was collected and the solvent was reduced. Evaporation to dryness under reduced pressure gave 1.95 g of pure Z-4 ​​conjugate molecule. MS m / z:C 107 H1 55 N 10 O 41 ,[M+H] + , theoretical value: 1935.07, actual value: 1935.29.

[0452] Synthesis of (10-1-5)Z-5 The Z-4 ​​conjugate molecule was used instead of the L-9 conjugate molecule, and the Z-4 ​​conjugate molecule was bound to a solid support. Z-5 was prepared in the same manner as in step (1-1-9) in Preparation Example 1, except that the following compound was obtained: Made.

[0453] (10-2) Synthesis of Z5-siAN1M3SVP conjugate The sense strand was synthesized using the Z-5 compound instead of the L-10 compound as the starting material. In addition, multiple compounds can be prepared in the same manner as in steps (1-2), (1-3A), and (1-4) in Preparation Example 1. Complex 29 was prepared. Z5-siAN1M3SVP complex, the structure of which is shown in formula (422). obtained.

[0454] (Preparation Example 11) Preparation of Complexes F1 to F13 In this preparation example, complexes F1 to F13 were synthesized, and the sequences of the siRNAs conjugated to the complexes were as follows: , as shown in Table 3.

[0455] (11-1) Synthesis of FIN-2 conjugate molecules Rajeev et al., ChemBioChem 2015, 16, 903-908 The FIN-2 conjugate molecule was synthesized by the following process route, referring to the preparation method described in the previous paper.

[0456] Synthesis of (11-1-1)PRO-10

[0457] [ka] (11-1-1a) Synthesis of PRO-7 2.93 g of PRO-6 (L-hydroxyproline, CAS number: 51-35-4, E 22.4 mmol) in 22.5 ml of 1,4-dioxane ( 1,4-dioxane (CAS number: 123-91-1) and dissolved in 34 ml of 10% ( An aqueous solution of 100% Na2CO3 (w / w) was added to form a suspension, and 6.95 g of Fmoc-Cl( 9-Fluorenylmethyl chloroformate, CAS number: 28920-43-6, Energ (purchased from Company Y, 26.8 mmol) was dissolved in 34 ml of 1,4-dioxane and placed on ice. The reaction mixture was added to the suspension in a bath, allowed to warm to room temperature, and reacted overnight. The mixture was poured into ice water and extracted three times with methyl tert-butyl ether, each time at 100 ml. The organic phase was discarded, and the aqueous phase was adjusted to pH 5 or less with concentrated HCl, and 100 ml of ethyl acetate was added. The organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was evaporated to dryness under reduced pressure. This gave 7.83 g of a white foamy solid product, PRO-7. 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),7.3 8 - 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). HRM S (ESI) m / z theoretical value C 20 H 19 NO5[MH] - 352.1190 , measured value 352.1033.

[0458] (11-1-1b) Synthesis of PRO-8 7.83 g of PRO-7 (22.2 mmol) was dissolved in 80 ml of THF (CAS number: 10 9-99-9), heated to 65°C in an oil bath, and refluxed. / L of BH3-Me2S in THF (CAS No. 13292-87-0, J&K Sc (Purchased from Ientific, 73.2 mmol) was added, and the reaction was continued under reflux for 3 hours. The reaction solution is drained, the remaining solids are dissolved in methanol, and the reaction solution is stirred until gas disappears. Methanol was added to the mixture, and stirring was continued for 30 minutes. The solvent was evaporated under reduced pressure, and then petroleum ether was added. The mixture was purified three times by the above procedure to give 7.1 g of a white solid product, PRO-8. 1 H NMR (400 MHz,DMSO-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 C 20 H2 1NO4[M+Na] + 362.1368, actual value 362.1012.

[0459] (11-1-1c) Synthesis of PRO-9 7.1 g of PRO-8 (21 mmol) was dissolved in 100 ml of pyridine. Add 1 g of DMTr-Cl (4,4'-bismethoxytrityl chloride, 42 mmol) The reaction was stirred at room temperature for 5 hours. The solvent was removed by distillation under reduced pressure, and the crude product was dissolved in ethyl acetate. Salt impurities were removed by filtration, the solvent was distilled off under reduced pressure, and the mixture was purified using a silica gel column. The column was pre-alkalined with pyridine, and then the crude product was dissolved in DCM and loaded. DMTr-Cl was eluted with DCM containing pyridine (v / v), and the product was then extracted with ethyl acetate. The product eluate was collected, and the solvent was evaporated to dryness under reduced pressure to obtain 8.2 g of a white solid product P RO-9 was obtained. HRMS (ESI) m / z theoretical value C 41 H 39 NO6[M+ Na] + 664.2675, Actual value 664.2348, C18 RP-HPLC (Lot No. JJS160324-1) Purity 94.20%.

[0460] (11-1-1d) Synthesis of PRO-10 8.2 g of PRO-9 (12.8 mmol) was dissolved in 64 ml of DMF (N,N-dimethylformamide). 40 ml of piperidine (384 mmol) was added and the mixture was stirred at room temperature for 30 minutes. The reaction mixture was stirred for 1 minute, and then poured into 300 ml of ice water. The organic phases were combined and washed with 200 ml of saturated saline solution. After drying with anhydrous sodium sulfate and distilling off the solvent under reduced pressure, the residue was purified with a silica gel column. The silica gel column was pre-alkalined with pyridine, and then the crude product was dissolved in DCM and loaded. Fmoc was eluted with DCM containing 1% (v / v) pyridine, and the product was then extracted with ethyl acetate. The product eluate was collected, and the solvent was evaporated to dryness under reduced pressure to obtain 4.65 g of a white solid product. Got PRO-10. 1 H NMR (400 MHz,DMSO-d6) δ 7.4 0 (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 C 26 H 29 NO4[M+Na] + 442.1994, actual value 442 .1999, C18 RP-HPLC (Lot No. JJS160329-1) Purity 97. 07%.

[0461] (11-1-2) Synthesis of FIN-1 [ka]

[0462] GAL-5 (4.5 g, 10 mmol) obtained by the method described in (1-1-1) ) was dissolved in 40 ml of DMF, and then 3.9 g of DIEA (N,N-diisopropyl ether) was added. Thiolamine, CAS number: 7087-68-5, purchased from Aladdin, 30mmol l) and 3.8 g of HBTU (benzotriazole-N,N,N',N'-tetramethyl Uronium hexafluorophosphate, CAS number: 94790-37-2, Alad din Co., Ltd., 11 mmol), and stirred at room temperature for 10 minutes. d) PRO-10 (4.2 g, 10 mmol) obtained in step d) was dissolved in 40 ml of DMF. After that, it was added to the reaction solution, and anhydrous sodium sulfate was added to the reaction solution to dry it, and the mixture was then left at room temperature for 2 hours. The reaction mixture was poured into 120 ml of ice water and washed with ethyl acetate three times at 60 ml each time. The organic phases were combined and washed with 20 ml of water and 20 ml of saturated saline solution. The phases were separated and dried over anhydrous sodium sulfate, the solvent was removed by distillation under reduced pressure, and the residue was purified using a silica gel column. The silica gel column was preliminarily alkalized with pyridine, and the sample was loaded onto it. Elution with a dichloromethane (DCM) solution containing 1% triethylamine and 1% methanol by volume The product 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 F. I got IN-1. 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 (td,J = 8.9,3.5 Hz,5H),6.93 - 6.84 (m,4H),5.21 (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 (dq,J = 9.3,5.3,4.3 Hz,1H),2.22 (s,2H),2.07 (s,3H),1.99 (s,3 H),1.90 (s,4H),1.74 (s,3H),1.50 (s,3H),1 .36 (s,1H). C18 RP-HPLC (Lot No. LJ160422) Purity 9 5.45%.

[0463] (11-1-3) Synthesis of FIN-2

[0464] [ka] FIN-1 (3.0 g, 3.53 mmol) obtained in step (11-1-2) was dissolved in acetone. The water was removed by azeotropy with the nitrile, and the mixture was dried by vacuum suction, dissolved in 10 ml of DMF, and then dried with nitrogen. 2.13 g of PA (bis(diisopropylamino)(2-cyanoethoxy) phosphate) was prepared under the protection of Sphingol, purchased from Adamas, product number 11356B, 7.06 mmol), 34 6 mg of tetrazole (CAS number: 288-94-8, purchased from Aladdin, 4 0.94mmol) was added, and the mixture was stirred at room temperature. 10ml of DMF was added, and the mixture was stirred for 1 hour. The reaction was continued. After the solvent was distilled off under reduced pressure, the residue was purified by silica gel column chromatography. The silica gel column was alkalized with pyridine beforehand, and the crude product was dissolved in DCM. The product eluate was collected, and the solvent was removed under reduced pressure to give 4.5 g of free The crude product was dissolved completely in 50% acetonitrile aqueous solution. The sample was dissolved in HCl and purified on a C-18, 330 g, 300 Å medium pressure purification column. The gel was first alkalized with a 1% by volume solution of pyridine in acetonitrile, and then gradient elution was performed to identify the product. The peaks were collected and the solvent was removed under reduced pressure to obtain 2.2 g of the white powder product FIN-2 conjugate molecule. . 31 P NMR (162 MHz,CDCl3) δ 148.04,147.94 ,147.62,147.19, Phosphorus spectral purity 92%, C18 RP-HPLC pure Alcohol content: 90.54%.

[0465] (11-2) Binding of FIN-2 conjugate molecules to solid supports The FIN-2 conjugate molecule obtained in step (11-1-3) was synthesized three times by solid-phase nucleic acid synthesis. By circulating the solution, the general-purpose solid support (UnyLinker TM Loaded Ni ttoPhase® HL Solid Supports) and conjugated groups Binding of (FIN_FIN_FIN) to the 3' end of the RNA sense strand was achieved.

[0466] Rajeev et al., ChemBioChem 2015, 16, 903-908 The above-mentioned binding is carried out by referring to the preparation method described above. Specifically, first, the above-mentioned general-purpose solid phase carrier is used. That is, the hydroxy protecting group on the solid support is removed, and the coupling reaction conditions and coupling The FIN-2 complex is contacted with the FIN-2 complex molecule in the presence of a capping agent, followed by coupling and capping. After reaction and oxidation, a FIN conjugate molecule bound to the solid support was obtained. The hydroxy protecting group DMTr in the FIN complex molecule bound to the FIN-2 complex is removed. The resulting product is brought into contact with a synthetic molecule to couple the product, followed by a capping reaction and an oxidation reaction. The deprotection-coupling-capping-oxidation process was repeated once to obtain the third FIN-2 complex. The resulting molecules were coupled to obtain a composite group (FIN_FIN_FIN) bound to a solid support.

[0467] In the above reaction, the above-mentioned deprotection, coupling, capping and oxidation are carried out under the reaction conditions The solvent and reagent amounts were the same as those in the solid phase nucleic acid synthesis method described in the above step (1-2). It was.

[0468] (11-3) Synthesis of Complexes F1 to F13 1) synthesizing a sense strand starting from the compound obtained in step (11-2); Preparation Example 1 except that the RNA has a sequence corresponding to complexes F1 to F13 shown in Table 3 The title complex was prepared by the same method as in steps (1-2), (1-3), and (1-4) in did.

[0469] Liquid Chromatography Mass Spectrometer (LC-MS) raphy-Mass Spectrometry, purchased from Waters, model number: L The molecular weight was detected by CT Premier. As a result, the measured value was consistent with the theoretical value. The synthesized complex is a compound of the intended design, whose structure is shown in formula (307). was confirmed.

[0470] (Preparation Example 12) Preparation of Comparative Complex 2 In this preparation example, comparative complex 2 was synthesized, and the sequence of the siRNA conjugated to the complex is shown in Table 3. The complex is shown in WO2016168286A1 as compound AD-656. The structure was the same as 95.

[0471] (12-1) Synthesis of (GalNAc)3 conjugated molecules Compound 30, i.e., the above, was prepared by the preparation method described in WO2014025805A1. Such a linker - (L A )3-trihydroxymethylaminomethane-L B - and the target group N-acetylgalactosamine molecules (where each L A One N-acetylgalactosamine Since three N-acetylgalactosamine molecules can be bound to one linker, (GalNAc)3 conjugate molecules (also called (GalNAc)3 conjugate molecules) containing the The structure of compound 30 is shown below.

[0472] [ka]

[0473] (12-2)(GalNAc)3 conjugation to solid support (GalNAc)3 conjugated molecule by the same method as in step (1-1-9) in Preparation Example 1. was bound to a solid support to obtain a (GalNAc)3 conjugated molecule bound to the solid support.

[0474] (12-3) Synthesis of comparative complex 2 1) synthesizing a sense strand starting from the compound obtained in step (12-2); 2) synthesizing a conjugated si Preparation Example 1, except that the RNA has the sequence shown in Table 3 under number AD-65695. Comparative complex 2 was obtained by the same method as steps (1-2), (1-3D), and (1-4) in Prepared.

[0475] Liquid Chromatography Mass Spectrometer (LC-MS) raphy-Mass Spectrometry, purchased from Waters, model number: L The molecular weight was detected by CT Premier. The theoretical value of the sense strand was 8625.32. The actual value was 8623.7, the theoretical value for the antisense strand was 7726.15, and the actual value was 7725. 2. The measured value was consistent with the theoretical value, and the synthesized complex had the structure of formula (305) It was confirmed that the compound was the compound of the intended design shown in

[0476] (Preparation Example 13) Synthesis of siRNA sequence The siRNA sequences provided by the present disclosure shown in Table 4 were obtained by conventional solid phase synthesis methods. After dissolving an equimolar mixture of sense and antisense strands in DEPC water, were annealed to form siRNA duplexes.

[0477] [Table 4]

[0478] After the preparation of the siRNA or complex of the present disclosure described above is completed, the siRNA or complex is stored in a standard container until use. The solution was freeze-dried as a solid powder and stored. Alternatively, the compound may be dissolved first and used as a solution of a desired concentration.

[0479] (Experimental Example 1) Inhibition of activity and off-target activity of siRNA in the in vitro psiCHECK system Detection of the 'B' effect

[0480] In this experiment, the activity of siRNAs 1, 4, 5, 7, and 8 in the in vitro psiCHECK system was The on-target activity and off-target effects of each of the five siRNAs were investigated. Measure activity targeting a perfectly matched target sequence or a target sequence that matches the seed region did.

[0481] 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. Plasmids were constructed and co-transfected with test siRNAs into HEK293A cells. The expression level of the dual luciferase reporter gene was used to determine the effect of siRNA on the expression of the dual luciferase reporter gene. The target activity and off-target effects were reflected. The specific steps are as follows: .

[0482] [1] Construction of detection plasmid psiCHECK TM -2(Promega TM ) plasmid to generate four recombinant plants The plasmids were constructed, of which GSCM represents the on-target plasmid and PSCM , GSSM, and PSSM represent off-target plasmids. (1) GSCM is a 21-nucleotide sequence of the antisense strand of the test siRNA. The target sequence is completely complementary to all of the sequences. (2) PSCM is a 21-nucleotide sequence of the antisense strand of the test siRNA. Contains the target sequence, which matches all of the columns exactly. (3) GSSM is a nucleotide sequence of the 1st to 8th positions from the 5' end of the antisense strand of the test siRNA. The nucleotide sequence is completely complementary to the antisense strand of the test siRNA. It corresponds to the nucleotide sequence of positions 9 to 21 from the 5' end, and the sequence is not completely complementary, i.e. , any one of nucleotides 9 to 21 from the 5' end of the antisense strand in the test siRNA If the nucleotide is G, C, A, or U, the nucleotide at the corresponding position in the target sequence is Each of the sequences contains a sequence of interest that is T, A, C, or G. (4) PSSM is a sequence of nucleotides 1 to 8 from the 5' end of the sense strand of the test siRNA. The remaining 9' end of the sense strand in the test siRNA is completely complementary to the 5' end of the sense strand. nucleotide sequence at position 19, which is not complementary to the nucleotide sequence at position 19, i.e., the test siR In NA, any nucleotide from positions 9 to 19 from the 5' end of the sense strand is G, C, A, or is U, the nucleotide at the corresponding position in the target sequence is T, A, C, or contains a target sequence that is G. To make the length of the target sequence of GSSM equal, Nucleotides T and C were added in order to the 3' end.

[0483] psiCHECK the target sequence TM Cloned into the XhoI / NotI site of the -2 plasmid I trained.

[0484] [2] Transfection Lipofectamine in a 96-well plate TM 2000(Invitro According to the instruction manual of gen, 11 groups were identified for each siRNA and plasmid. The above-mentioned plasmids were co-transfected with the corresponding siRNA at a concentration of 0.05. Transfect 10 ng of plasmid per well and add 0.2 μL of Lipof ectamine TM 2000 was used. siRNA1, siRNA4, siRNA5 The concentration of siRNA8 was diluted from 5 nM to 0.00008 nM in 3-fold increments, for a total of 11 The concentration of siRNA7 was doubled from 0.5 nM to 0.0005 nM. There were 11 concentrations in total. There were 3 replicate wells per group.

[0485] [3] Detection After 24 hours of co-transfection, dual luciferase reporter gene assays were performed. 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. For each specific concentration of siRNA test group, an siRNA-untreated group was used as a control (con.). The effect of Renilla lucifera on firefly luciferase (Fir) protein levels The results were normalized to Renase (Ren) protein levels.

[0486] The suppression of expression of the four recombinant plasmids by siRNA 8 is shown in Figures 1A-1D. For the suppression of expression of the four recombinant plasmids by siRNA 1, see Figure 2A. As can be seen from the figure, unmodified siRNA 8 at 5 nM inhibited GSS The expression of M and PSCM was suppressed by approximately 20%, and the antisense strand seed region was While the target effect and off-target effect of the sense strand were only slightly demonstrated, the present disclosure No off-target effects were observed with the modified siRNA 1 provided by siRNAs 4, 5, and 7, like siRNA 1, did not have any off-target effects. It was not accepted.

[0487] The activity results measured at different siRNA concentrations were analyzed using Graphpad 5.0 software. warelog(inhibitor) vs. response-Variable s The dose-effect curve was fitted using the lope function, and the following calculations were made from the dose-effect curve: IC of the tested siRNA targeting GSCM by the method 50 The values ​​were calculated and the results are shown in Table 5. did.

[0488]

number

[0489] [Table 5]

[0490] This experiment demonstrates that the modified siRNA provided by the present disclosure is effective in the in vitro psiCHECK system. It has extremely high inhibitory activity against 50 At 3-30 pM, and at 5 nM, It was shown that no off-target effects were detected in the modified siRNAs tested.

[0491] (Experimental Example 2) Detection of the suppressive activity of siRNA and siRNA complexes in an in vitro cell system (Experimental Example 2-1) ANGPTL3 mRNA expression by siRNA in Huh7 cells Detection of suppression efficiency relative to the amount of present. Lipofectamine TM 2000 to test siRNA (siRNA1, 2, 4, 5, 6, 7) were transfected into the human hepatoma cell line Huh7, and siRN The final concentrations of A were 5 nM, 0.25 nM, and 0.05 nM, respectively. There were two replicate wells. Cells not treated with any siRNA served as blank controls. (marked as "blank" in Figure 3A).

[0492] Fluorescent quantitative real-time PCR CR) to Huh7 cells transfected with siRNA at various concentrations. The expression level of ANGPTL3 mRNA was detected in the transfected cells. The transfected cells were cultured for 24 hours and then treated with Trizol (Thermo Fisher Scientific) Total RNA was extracted from the cells using a standard operating procedure for total RNA extraction using a ELISA kit (manufactured by r). 1 μg of total RNA was taken from each sample and transfected using a reverse transcription kit (Promega, product code A3500). cDNA was obtained by reverse transcription according to the instructions in the manufacturer's manual. Mixture(with ROX)(Beijing Kangwei Century Biological Technology Co., Ltd., No. CW09 56) Using the kit, cDNA was used as a template and ANGPTL3 was isolated according to the instructions. The mRNA expression levels of ANGPTL3 and the endogenous reference gene GAPDH were detected. The PCR primers for amplification are shown in Table 6.

[0493] [Table 6]

[0494] The expression level of ANGPTL3 mRNA was calculated as follows: ANGPTL3 mRNA expression level = (test group AN GPTL3 mRNA expression level / GAPDH mRNA expression level in test group / (control group AN GPTL3 mRNA expression level / GAPDH mRNA expression level in the control group) × 100% It was calculated using the following equation.

[0495] The suppression rate of ANGPTL3 mRNA expression by siRNA was (1-ANGP TL3 mRNA expression level) × 100%. Each test group was treated with siRNA at each concentration. The control group was Huh7 cells treated with siRNA. The results are shown in Figure 3A.

[0496] As can be seen from FIG. 3A, the modified siRNAs provided by the present disclosure inhibited the expression of the Huh7 cell line. It has high inhibitory activity in the SI65695 gene, comparable to the activity of the positive control si65695.

[0497] (Experimental Example 2-2) ANGPTL3 mRNA expression in Huh7 cells by siRNA complex Detection of suppression efficiency relative to NA expression level. The test samples were complexes F1 to F11, and the final concentration of the complex (as the amount of siRNA) ) were 50 nM and 5 nM, respectively. The in vitro inhibitory activity of each complex is shown in Figure 3B.

[0498] As can be seen from FIG. 3B, the siRNA complexes provided by the present disclosure inhibited Huh7 cells. The complex exhibited high inhibitory activity in the system, and suppressed ANGPTL3 mRNA expression by 5 nM. The inhibition rate reached 60-80%.

[0499] (Experimental Example 2-3) ANGPTL3 mRNA in siRNA complexes in Huh7 cells IC for 50 Measurement of. The test samples were complexes F3, F4, F8, and F9, and the final concentration of the complex ( (as the amount of ion) was diluted in 5-fold increments from 50 nM to 0.016 nM, with the lowest concentration being 0.00 001 nM, for a total of seven concentrations, with three replicate wells per group. Detection was carried out in the same manner as in Experimental Example 2-1.

[0500] In other experiments, the test sample was Complex 2, and the final concentration of the complex ( The concentration was doubled from 2 nM to 0.0078 nM, for a total of nine concentrations. There were two replicate wells per well.

[0501] In other experiments, the test samples were conjugates 1, 4, 5, and 7, and the final concentrations of the conjugates ( The siRNA concentration was doubled from 0.5 nM to 0.03125 nM, and the highest concentration was was 5 nM for a total of 6 concentrations with 2 replicate wells per group.

[0502] Effect of siRNA complex on ANGPTL3 mRNA expression levels measured at different concentrations From the inhibition rate, IC50 was calculated in the same manner as in Experimental Example 1, and the inhibitory effect of the test complex on Huh7 cells in vitro was calculated. ICs in cells 50 The results are shown in Table 7.

[0503] [Table 7]

[0504] As can be seen from Table 7, the siRNA complexes provided by the present disclosure exhibited excellent activity in an in vitro cell system. It has extremely high inhibitory activity against 50 was in the range of 0.085 to 0.462 nM.

[0505] (Experimental Example 3) Stability of siRNA and siRNA complexes in plasma and lysosomes detection (Experimental Example 3-1) Detection of siRNA stability in lysosomes. In this experiment, lysosomal lysates derived from mice containing siRNA 1, 2, 4, 5, 6, and 7 were The stability at

[0506] Preparation of lysosomal lysate-treated test samples: 6 μl of each siRNA (20 μM ) were added to 27.2 μL of sodium citrate solution (pH 5.0), 4.08 μL of Deionized water and 2.72 μL of mouse-derived lysosomal lysate (Rat Liver T ritosomes, Xenotech, product number R0610.LT, lot number 1610 069) and mixed uniformly, and the final concentration of acid phosphatase was 0.2 mU / μL. The mixture was incubated at 37°C. 5 μL of the mixture was added at 0, 1, 2, 4, 6, and 24 hours. Remove the sample and add 15 μL of 9 M urea solution to denature it, then add 4 μL of 6x sample loading buffer. Add the solution (Solarbio, product code 20160830) and immediately place in a refrigerator at -80°C. The reaction was stopped by freezing. At time 0, the test sample and the lysosomal lysate were mixed uniformly. This represents the point in time immediately after the event.

[0507] Preparation of a reference sample not treated with lysosomal lysate: Equimolar amounts of siRNA (2 1.5 μL of each solution was taken and added to 7.5 μL of sodium citrate solution (pH 5 0), mix evenly with 1 μL of deionized water, and then add 30 μL of 9 M urea solution to denature. After that, add 8 μL of 6× sample loading buffer, mix evenly, and immediately refrigerate in a refrigerator at -80°C. The reaction was stopped by freezing. The reference sample for each siRNA was marked with M in the electropherogram. can be.

[0508] 16% by weight of non-denatured polyacrylamide gel was mixed, and the test samples and reference samples were 20 μl of each sample was loaded onto the gel and electrophoresed at a constant current of 20 mA for 10 minutes. After the electrophoresis, the gel was electrophoresed for 30 minutes at a constant current of 40 mA. The mixture was placed on a shaker and stained with Gelred dye (BioTium, product no. 13G1203). Staining was performed for 10 minutes. Gel imaging was performed, and the results were photographed and shown in Figure 4A.

[0509] As can be seen from FIG. 4A, the modified siRNA provided by the present disclosure is derived from a mouse. It was able to remain stable in the endothelial cells for at least 24 hours.

[0510] (Experimental Example 3-2) Detection of stability of siRNA complex in lysosomes. In this experiment, the stability of complexes 1, 4, 5, and 7 in mouse lysosomal lysates was investigated. I looked into it.

[0511] The test samples were complexes 1, 4, 5, and 7, and the concentration of the complexes was calculated as the amount of siRNA. The detection time points are 0 hours, 5 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, and 6 hours. The detection was carried out in the same manner as in Experimental Example 3-1, except that the time was 100 s. Shown in 4B.

[0512] As can be seen from FIG. 4B, the siRNA complexes provided by the present disclosure were derived from mouse It remains in the lysosome for at least 1 hour without being degraded, and then the main barrier The band was only slightly shifted downward, and the corresponding siRNA was Considering the high stability of α-glucan in the presence of α-glucan, the band shift may be due to the cleavage of the monosaccharide at the complex group. These results suggest that the siRNA complexes of the present disclosure exhibit satisfactory stability.

[0513] (Experimental Example 3-3) Detection of stability of siRNA complex in plasma. In this example, the stability of conjugates 1, 4, 5, and 7 in human plasma was investigated.

[0514] Complexes 1, 4, 5, 7 and control siRNA 8 (concentration of siRNA or siRNA complex) The concentrations were both 20 μM and 12 μl, calculated based on the amount of siRNA in the complex. Each was diluted with 108 μL of 90% human plasma (PBS) and The mixture was mixed and incubated at 37°C. A 10 μL sample was taken at each time point, immediately fl...

Claims

1. 1. A siRNA conjugate comprising an siRNA and a conjugated group conjugated to the siRNA, the siRNA comprises a sense strand and an antisense strand, each nucleotide of the siRNA being independently modified or unmodified; The sense strand comprises a nucleotide sequence I, and the antisense strand comprises a nucleotide sequence II, and the nucleotide sequence I and the nucleotide sequence II form a double-stranded region in a reverse-complementary manner at least in a portion thereof, and the nucleotide sequence I comprises a nucleotide sequence A of the same length as the nucleotide sequence shown in SEQ ID NO: 1, which has one or less nucleotide difference, and the nucleotide sequence II comprises a nucleotide sequence B of the same length as the nucleotide sequence shown in SEQ ID NO: 2, which has one or less nucleotide difference; 5'-CCAAGAGCACCAAGAACUZ-3' (SEQ ID NO: 1), 5'-Z'AGUUCUUGGUGCUCUUGG-3' (SEQ ID NO: 2), Z is A and Z' is U; Nucleotide sequence A comprises nucleotide Z A at the corresponding position of Z, and nucleotide sequence B comprises nucleotide Z′ B at the corresponding position of Z′; Z' Ｂ is the first nucleotide at the 5' end of the antisense strand, and the corresponding positions are at the same position in the nucleotide sequence from the same end of the nucleotide sequence; Z A is the last nucleotide at the 3' end of nucleotide sequence A; the sense and antisense strands are the same or different in length, the sense strand is 19-23 nucleotides in length and the antisense strand is 20-26 nucleotides in length; The siRNA conjugate has the structure shown in formula (308): 【Chemistry 1】 During the ceremony, n1 is an integer selected from 1 to 3, and n3 is an integer selected from 0 to 4; m1, m2, and m3 are independently an integer selected from 2 to 10; R 10 , R 11 , R 12 , R 13 , R 14 and R 15 are each independently H or selected from the group consisting of C 1 -C 10 alkyl groups, C 1 -C 10 halogenated alkyl groups and C 1 -C 10 alkoxy groups; R 3 is a group having the structure shown in formula A59; 【Chemistry 2】 In the formula, E 1 is OH, SH or BH 2 , and Nu is siRNA; R 2 is a straight chain alkylene group of from 1 to 20 carbon atoms in length, one or more of which are optionally substituted with one or more selected from the group consisting of C(O), NH, O, S, CH═N, S(O) 2 , a C 2 -C 10 alkenylene group, a C 2 -C 10 alkynylene group, a C 6 -C 10 arylene group, a C 3 -C 18 heterocyclylene group, and a C 5 -C 10 heteroarylene group; R 2 is a C 1 -C 10 alkyl group, a C 6 -C 10 aryl group, a C 5 -C 10 heteroaryl group, a C 1 -C 10 halogenated alkyl group, a —OC 1 -C 10 alkyl group, a —OC 1 -C 10 alkylphenyl group, a —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 group, halogen substituent, -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), 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 alkyl group), -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 haloalkyl group, -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 NH 2 , -SO 2 NH(C 1 -C 10 alkyl group), -SO 2 NH (phenyl group), -NHSO 2 (C 1 -C 10optionally bearing one or more substituents selected from the group consisting of -NHSO 2 (alkyl group), -NHSO 2 (phenyl group), and -NHSO 2 (C 1 -C 10 halogenated alkyl group); Each L 1 is a straight chain alkylene group of 1 to 70 carbon atoms in length, one or more of which are optionally substituted with one or more selected from the group consisting of C(O), NH, O, S, CH═N, S(O) 2 , a C 2 -C 10 alkenylene group, a C 2 -C 10 alkynylene group, a C 6 -C 10 arylene group, a C 3 -C 18 heterocyclylene group, and a C 5 -C 10 heteroarylene group; L 1 is a C 1 -C 10 alkyl group, a C 6 -C 10 aryl group, a C 5 -C 10 heteroaryl group, a C 1 -C 10 halogenated alkyl group, a —OC 1 -C 10 alkyl group, a —OC 1 -C 10 alkylphenyl group, a —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 group, halogen substituent, -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), 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 alkyl group), -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 haloalkyl group, -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 NH 2 , -SO 2 NH(C 1 -C 10 alkyl group), -SO 2 NH (phenyl group), -NHSO 2 (C 1 -C 10optionally bearing one or more substituents selected from the group consisting of -NHSO 2 (alkyl group), -NHSO 2 (phenyl group), and -NHSO 2 (C 1 -C 10 halogenated alkyl group); 【change】 represents the site at which the group is attached to the remainder of the molecule, M 1 represents a targeting group in the siRNA conjugate.

2. 2. The siRNA complex of claim 1, wherein nucleotide sequence I further comprises nucleotide sequence III, nucleotide sequence II further comprises nucleotide sequence IV, nucleotide sequence III and nucleotide sequence IV each independently have a length of 1 to 4 nucleotides, nucleotide sequence III is linked to the 5' end of nucleotide sequence A, nucleotide sequence IV is linked to the 3' end of nucleotide sequence B, and nucleotide sequence III and nucleotide sequence IV are equal in length and reverse complementary.

3. the nucleotide sequences III and IV are each one nucleotide in length, and the base of the nucleotide sequence III is G; Alternatively, the nucleotide sequences III and IV are both 2 nucleotides in length, and from the 5' end to the 3' end, the base sequence of nucleotide sequence III is AG; Alternatively, the nucleotide sequences III and IV are each 3 nucleotides in length, and from the 5' end to the 3' end, the base sequence of nucleotide sequence III is AAG; Alternatively, the nucleotide sequences III and IV are each 4 nucleotides in length, and the base sequence of nucleotide sequence III from the 5' end to the 3' end is CAAG.

4. The siRNA complex of claim 1, wherein the nucleotide sequence II further comprises a nucleotide sequence V, the nucleotide sequence V 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.

5. the nucleotide sequence V is 2 nucleotides in length; and The siRNA complex of claim 4, wherein the nucleotide sequence V is two consecutive thymine deoxyribonucleotides or two consecutive uracil ribonucleotides, or the nucleotide sequence V is complementary to a nucleotide at a corresponding position of a target mRNA.

6. the sense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO:3, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:5; 5'-CCAAGAGCACCAAAGAACUZ Ａ -3' (SEQ ID NO:3), 5'-Z' Ｂ AGUUCUUGGUGCUCUUGGCU-3' (SEQ ID NO:5), Alternatively, the sense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO:6, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO:7; 5'-AGCCAAGAGCACCAAGAACUZ Ａ -3' (SEQ ID NO:6), 5'-Z' Ｂ AGUUCUUGGUGCUCUUGGCUUGGCUUG-3' (SEQ ID NO: 7), However, Z' Ｂ is the first nucleotide at the 5' end of the antisense strand, and Z Ａ is selected from A, U, G or C; Z' Ｂ Is Z Ａ The siRNA complex of claim 1 , wherein the nucleotide is complementary to:

7. 2. The siRNA complex of claim 1, wherein the siRNA is siAN1 or siAN2. siAN1 Sense strand: 5'-CCAAGAGCACCAAGAACUA-3' (SEQ ID NO: 8) and antisense strand: 5'-UAGUUCUUGGUGCUCUUGGCU-3' (SEQ ID NO: 9); siAN2 Sense strand: 5'-AGCCAAGAGCACCAAGAACUA-3' (SEQ ID NO: 10) and antisense strand: 5'-UAGUUCUUGGUGCUCUUGGCUUG-3' (SEQ ID NO: 11).

8. 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 nucleotides are located at nucleotide sequence A and nucleotide sequence B; From the 5' to the 3' end, the 7th, 8th and 9th nucleotides of the nucleotide sequence A are fluoro-modified nucleotides; and The siRNA complex of claim 1, wherein, from the 5' end to the 3' end, the 2nd, 6th, 14th, and 16th nucleotides of the nucleotide sequence B are fluoro-modified nucleotides, and each non-fluoro-modified nucleotide is a methoxy-modified nucleotide, and the methoxy-modified nucleotide refers to a nucleotide in which the 2'-hydroxy group of the ribose group is substituted with methoxy.

9. From the 5' to the 3' end, the nucleotides at positions 5, 7, 8, and 9 of nucleotide sequence A in the sense strand of the siRNA are fluoro-modified nucleotides and the remaining nucleotides in the sense strand of the siRNA are methoxy-modified nucleotides; from the 5' to the 3' end, the nucleotides at positions 2, 6, 8, 9, 14, and 16 of nucleotide sequence B in the antisense strand of the siRNA are fluoro-modified nucleotides and the remaining nucleotides in the antisense strand of the siRNA are methoxy-modified nucleotides; or From the 5' to the 3' end, the nucleotides at positions 5, 7, 8, and 9 of nucleotide sequence A in the sense strand of the siRNA are fluoro-modified nucleotides and the nucleotides at the remaining positions of the sense strand of the siRNA are methoxy-modified nucleotides; from the 5' to the 3' end, the nucleotides at positions 2, 6, 14, and 16 of nucleotide sequence B in the antisense strand of the siRNA are fluoro-modified nucleotides and the nucleotides at the remaining positions of the antisense strand of the siRNA are methoxy-modified nucleotides. Alternatively, from the 5' to the 3' end, the nucleotides at positions 7, 8, and 9 of nucleotide sequence A in the sense strand of the siRNA are fluoro-modified nucleotides, and the nucleotides at the remaining positions of the sense strand of the siRNA are methoxy-modified nucleotides, and from the 5' to the 3' end, the nucleotides at positions 2, 6, 14, and 16 of nucleotide sequence B in the antisense strand of the siRNA are fluoro-modified nucleotides, and the nucleotides at the remaining positions of the antisense strand of the siRNA are methoxy-modified nucleotides. The siRNA complex of claim 8.

10. the siRNA is any one of siAN1-M1, siAN2-M1, siAN1-M2, siAN2-M2, siAN1-M3, or siAN2-M3; siAN1-M1: Sense strand: 5'-CmCmAmAmGfAmGfCfAfCmCmAmAmGmAmAmCmUmAm-3' (SEQ ID NO: 12); Antisense strand: 5'-UmAfGmUmUmCfUmUfGfGmUmGmCmUfCmUfUmGmGmCmUm-3' (SEQ ID NO: 13); siAN2-M1: Sense strand: 5'-AmGmCmCmAmAmGfAmGfCfAfCmCmAmAmGmAmAmCmUmAm-3' (SEQ ID NO: 14); Antisense strand: 5'-UmAfGmUmUmCfUmUfGfGmUmGmCmUfCmUfUmGmGmCmUmUmGm-3' (SEQ ID NO: 15); siAN1-M2: Sense strand: 5'-CmCmAmAmGfAmGfCfAfCmCmAmAmGmAmAmCmUmAm-3' (SEQ ID NO: 12); Antisense strand: 5'-UmAfGmUmUmCfUmUmGmGmUmGmCmUfCmUfUmGmGmCmUm-3' (SEQ ID NO: 16); siAN2-M2: Sense strand: 5'-AmGmCmCmAmAmGfAmGfCfAfCmCmAmAmGmAmAmCmUmAm-3' (SEQ ID NO: 14); Antisense strand: 5'-UmAfGmUmUmCfUmUmGmGmUmGmCmUfCmUfUmGmGmCmUmUmGm-3' (SEQ ID NO: 17); siAN1-M3: Sense strand: 5'-CmCmAmAmGmAmGfCfAfCmCmAmAmGmAmAmCmUmAm-3' (SEQ ID NO: 18); Antisense strand: 5'-UmAfGmUmUmCfUmUmGmGmUmGmCmUfCmUfUmGmGmCmUm-3' (SEQ ID NO: 16); siAN2-M3: Sense strand: 5'-AmGmCmCmAmAmGmAmGfCfAfCmCmAmAmGmAmAmCmUmAm-3' (SEQ ID NO: 19); Antisense strand: 5'-UmAfGmUmUmCfUmUmGmGmUmGmCmUfCmUfUmGmGmCmUmUmGm-3' (SEQ ID NO: 17) The siRNA complex described in claim 1, wherein the capital letters C, G, U, and A represent the base sequence of a nucleotide, the lower case letter m represents that one nucleotide adjacent to the left of the letter m is a methoxy-modified nucleotide, and the lower case letter f represents that one nucleotide adjacent to the left of the letter f is a fluoro-modified nucleotide.

11. In the siRNA, at least one phosphate group is a thiophosphate group, the thiophosphate group being Between the first and second nucleotides from the 5' end of the sense strand, Between the second and third nucleotides from the 5' end of the sense strand, Between the first and second nucleotides from the 3' end of the sense strand, Between the second and third nucleotides from the 3' end of the sense strand, Between the first and second nucleotides from the 5' end of the antisense strand, Between the second and third nucleotides from the 5' end of the antisense strand, Between the first and second nucleotides from the 3' end of the antisense strand, and The siRNA complex according to claim 1, which is bound to at least one nucleotide selected from the group consisting of the second nucleotide and the third nucleotide from the 3' end of the antisense strand.

12. the siRNA is any one of siAN1-M1S, siAN2-M1S, siAN1-M2S, siAN2-M2S, siAN1-M3S, and siAN2-M3S; siAN1-M1S: Sense strand: 5'-CmsCmsAmAmGfAmGfCfAfCmCmAmAmGmAmAmCmUmAm-3' (SEQ ID NO: 20); Antisense strand: 5'-UmsAfsGmUmUmCfUmUfGfGmUmGmCmUfCmUfUmGmGmsCmsUm-3' (SEQ ID NO: 21); siAN2-M1S: Sense strand: 5'-AmsGmsCmCmAmAmGfAmGfCfAfCmCmAmAmGmAmAmCmUmAm-3' (SEQ ID NO: 22); Antisense strand: 5'-UmsAfsGmUmUmCfUmUfGfGmUmGmCmUfCmUfUmGmGmCmUmsUmsGm-3' (SEQ ID NO: 23); siAN1-M2S: Sense strand: 5'-CmsCmsAmAmGfAmGfCfAfCmCmAmAmGmAmAmCmUmAm-3' (SEQ ID NO: 20); Antisense strand: 5'-UmsAfsGmUmUmCfUmUmGmGmUmGmCmUfCmUfUmGmGmsCmsUm-3' (SEQ ID NO: 24); siAN2-M2S: Sense strand: 5'-AmsGmsCmCmAmAmGfAmGfCfAfCmCmAmAmGmAmAmCmUmAm-3' (SEQ ID NO: 22); Antisense strand: 5'-UmsAfsGmUmUmCfUmUmGmGmUmGmCmUfCmUfUmGmGmCmUmsUmsGm-3' (SEQ ID NO:25); siAN1-M3S: Sense strand: 5'-CmsCmsAmAmGmAmGfCfAfCmCmAmAmGmAmAmCmUmAm-3' (SEQ ID NO:26); Antisense strand: 5'-UmsAfsGmUmUmCfUmUmGmGmUmGmCmUfCmUfUmGmGmsCmsUm-3' (SEQ ID NO: 24); siAN2-M3S: Sense strand: 5'-AmsGmsCmCmAmAmGmAmGfCfAfCmCmAmAmGmAmAmCmUmAm-3' (SEQ ID NO:27); Antisense strand: 5'-UmsAfsGmUmUmCfUmUmGmGmUmGmCmUfCmUfUmGmGmCmUmsUmsGm-3' (SEQ ID NO: 25) The siRNA complex described in claim 1, wherein the capital letters C, G, U, and A represent the base sequence of a nucleotide, the lower case letter m represents that one nucleotide adjacent to the left of the letter m is a methoxy-modified nucleotide, the lower case letter f represents that one nucleotide adjacent to the left of the letter f is a fluoro-modified nucleotide, and the lower case letter s represents that the two nucleotides on the left and right of the letter are bonded via a thiophosphate group.

13. the 5'-terminal nucleotide of the antisense strand is a 5'-phosphate nucleotide or a 5'-phosphate analog-modified nucleotide; and The 5'-phosphate nucleotide is a nucleotide having a structure represented by formula (2), and the 5'-phosphate analog modified nucleotide is selected from nucleotides having a structure represented by any one of formulas (3) to (6), 【Chemistry 3】 The siRNA complex of claim 1 , wherein R is selected from H, OH, a methoxy group, or fluorine, and Base represents a base selected from A, U, C, G, or T.

14. the siRNA is any one of siAN1-M1P1, siAN2-M1P1, siAN1-M2P1, siAN2-M2P1, siAN1-M3P1, siAN2-M3P1, siAN1-M1SP1, siAN2-M1SP1, siAN1-M2SP1, siAN2-M2SP1, siAN1-M3SP1, and siAN2-M3SP1; siAN1-M1P1: Sense strand: 5'-CmCmAmAmGfAmGfCfAfCmCmAmAmGmAmAmCmUmAm-3' (SEQ ID NO: 12); Antisense strand: 5'-P1-UmAfGmUmUmCfUmUfGfGmUmGmCmUfCmUfUmGmGmCmUm-3' (SEQ ID NO:28); siAN2-M1P1: Sense strand: 5'-AmGmCmCmAmAmGfAmGfCfAfCmCmAmAmGmAmAmCmUmAm-3' (SEQ ID NO: 14); Antisense strand: 5'-P1-UmAfGmUmUmCfUmUfGfGmUmGmCmUfCmUfUmGmGmCmUmUmGm-3' (SEQ ID NO:29); siAN1-M2P1: Sense strand: 5'-CmCmAmAmGfAmGfCfAfCmCmAmAmGmAmAmCmUmAm-3' (SEQ ID NO: 12); Antisense strand: 5'-P1-UmAfGmUmUmCfUmUmGmGmUmGmCmUfCmUfUmGmGmCmUm-3' (SEQ ID NO: 30); siAN2-M2P1: Sense strand: 5'-AmGmCmCmAmAmGfAmGfCfAfCmCmAmAmGmAmAmCmUmAm-3' (SEQ ID NO: 14); Antisense strand: 5'-P1-UmAfGmUmUmCfUmUmGmGmUmGmCmUfCmUfUmGmGmCmUmUmGm-3' (SEQ ID NO: 31); siAN1-M3P1: Sense strand: 5'-CmCmAmAmGmAmGfCfAfCmCmAmAmGmAmAmCmUmAm-3' (SEQ ID NO: 18); Antisense strand: 5'-P1-UmAfGmUmUmCfUmUmGmGmUmGmCmUfCmUfUmGmGmCmUm-3' (SEQ ID NO: 30); siAN2-M3P1: Sense strand: 5'-AmGmCmCmAmAmGmAmGfCfAfCmCmAmAmGmAmAmCmUmAm-3' (SEQ ID NO: 19); Antisense strand: 5'-P1-UmAfGmUmUmCfUmUmGmGmUmGmCmUfCmUfUmGmGmCmUmUmGm-3' (SEQ ID NO: 31); siAN1-M1SP1: Sense strand: 5'-CmsCmsAmAmGfAmGfCfAfCmCmAmAmGmAmAmCmUmAm-3' (SEQ ID NO: 20); Antisense strand: 5'-P1-UmsAfsGmUmUmCfUmUfGfGmUmGmCmUfCmUfUmGmGmsCmsUm-3' (SEQ ID NO: 32); siAN2-M1SP1: Sense strand: 5'-AmsGmsCmCmAmAmGfAmGfCfAfCmCmAmAmGmAmAmCmUmAm-3' (SEQ ID NO: 22); Antisense strand: 5'-P1-UmsAfsGmUmUmCfUmUfGfGmUmGmCmUfCmUfUmGmGmCmUmsUmsGm-3' (SEQ ID NO: 33); siAN1-M2SP1: Sense strand: 5'-CmsCmsAmAmGfAmGfCfAfCmCmAmAmGmAmAmCmUmAm-3' (SEQ ID NO: 20); Antisense strand: 5'-P1-UmsAfsGmUmUmCfUmUmGmGmUmGmCmUfCmUfUmGmGmsCmsUm-3' (SEQ ID NO: 34); siAN2-M2SP1: Sense strand: 5'-AmsGmsCmCmAmAmGfAmGfCfAfCmCmAmAmGmAmAmCmUmAm-3' (SEQ ID NO: 22); Antisense strand: 5'-P1-UmsAfsGmUmUmCfUmUmGmGmUmGmCmUfCmUfUmGmGmCmUmsUmsGm-3' (SEQ ID NO: 35); siAN1-M3SP1: Sense strand: 5'-CmsCmsAmAmGmAmGfCfAfCmCmAmAmGmAmAmCmUmAm-3' (SEQ ID NO:26); Antisense strand: 5'-P1-UmsAfsGmUmUmCfUmUmGmGmUmGmCmUfCmUfUmGmGmsCmsUm-3' (SEQ ID NO: 34); siAN2-M3SP1: Sense strand: 5'-AmsGmsCmCmAmAmGmAmGfCfAfCmCmAmAmGmAmAmCmUmAm-3' (SEQ ID NO:27); Antisense strand: 5'-P1-UmsAfsGmUmUmCfUmUmGmGmUmGmCmUfCmUfUmGmGmCmUmsUmsGm-3' (SEQ ID NO: 35) The siRNA conjugate of claim 1, wherein the capital letters C, G, U, and A represent a base sequence of a nucleotide, the lower case letter m represents a nucleotide adjacent to the left side of the letter m that is a methoxy-modified nucleotide, the lower case letter f represents a nucleotide adjacent to the left side of the letter f that is a fluoro-modified nucleotide, the lower case letter s represents that the two nucleotides on the left and right of the letter are bonded via a thiophosphate group, and P1 represents a nucleotide adjacent to the right side of the letter that is a 5'-phosphate nucleotide or a 5'-phosphate analog-modified nucleotide.

15. Each L １ is a combination of one or more bonds independently selected from the groups of formulae A1 to A19, and A21 to A26: 【Chemistry 4】 In the formula, j1 is an integer from 1 to 20, and j2 is an integer from 1 to 20. R' is C １ -C １０ is an alkyl group of the formula Ra is selected from the group consisting of groups of formulae A27 to A31, A34 to A36, A40, A43, A45, and any combination thereof; 【Chemistry 5】 Rb is C １ -C １０ is an alkyl group.

16. L １ is a combination of one or more bonds selected from A1, A4, A5, A6, A8, A10, A11, and A13, or L １ The siRNA complex of claim 15, wherein A is a combination of at least two bonds selected from A1, A4, A8, A10 and A11.

17. L １ 2. The siRNA conjugate of claim 1, wherein the length of

18. The siRNA complex of claim 1, wherein n1 is an integer from 1 to 2, n3 is an integer from 0 to 1, and n1 + n3 = 2 to 3.

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

20. each said targeting group is independently a ligand that has affinity for the asialoglycoprotein receptor (ASGPR) on the surface of mammalian hepatocytes; or Each of the targeting groups may be 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-glucofuranose, Furanose, α-D-fructofuranose, α-D-fructopyranose, α-D-galactopyranose, β-D-galactopyranose, α-D-galactofuranose, β-D-galactofuranose, glucosamine, sialic acid, galactosamine, N-acetylgalactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-n-butyrylgalactosamine, N-isobutyrylgalactosamine, 2- Amino-3-O-[(R)-1-carboxyethyl]-2-deoxy-β-D-glucopyranose, 2-deoxy-2-methylamino-L-glucopyranose, 4,6-dideoxy-4-formamido-2,3-di-O-methyl-D-mannopyranose, 2-deoxy-2-sulfoamino-D-glucopyranose, N-glycolyl-α-neuraminic acid, 5-thio-β-D-glucopyranose, methyl 2,3,4-tris-O-acetylacetate 2. The siRNA conjugate of claim 1, wherein the siRNA conjugate is independently selected from the group consisting of ethyl 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-allonitrile, ribose, D-ribose, D-4-thioribose, L-ribose, and L-4-thioribose.

21. R ２ The nitrogen-containing backbone is bonded to the N atom and R ３ and R ２ The site bonded to N in the nitrogen-containing skeleton above forms an amide bond with N, and the R ３ The site bound to P in R ２ is selected from B5, B6, B5' or B6'; 【Chemistry 6】 During the ceremony, 【change】 represents the site at which the group is covalently attached; q ２ The siRNA complex of claim 1, wherein is an integer from 1 to 10.

22. The siRNA conjugate of claim 1, having a structure as shown in formula (403), (404), (405), (406), (407), (408), (409), (410), (411), (412), (413), (414), (415), (416), (417), (418), (419), (420), (421) or (422). 【Chemistry 7】 【change】 【change】 【change】 【change】

23. The siRNA conjugate of claim 1 , wherein the P atom in formula A59 is attached to the 3′ end of the sense strand of the siRNA.

24. A composition for treating dyslipidemia, comprising the siRNA complex according to any one of claims 1 to 23.

25. 25. The composition of claim 24, wherein the lipid abnormality is hypercholesterolemia, hypertriglyceridemia or atherosclerosis.

26. A composition for use in suppressing expression of the ANGPTL3 gene in hepatocytes, comprising an siRNA complex according to any one of claims 1 to 23.

27. A kit comprising the siRNA complex according to any one of claims 1 to 23.

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