Double-stranded oligonucleotides, compositions and complexes containing double-stranded oligonucleotides, and methods of preparation and use

Abstract

The present disclosure provides a modified double-stranded oligonucleotide, wherein the sense strand comprises nucleotide sequence 1 and the antisense strand comprises nucleotide sequence 2, wherein nucleotide sequences 1 and 2 are both 19 nucleotides in length, and from the 5' end to the 3' end, the nucleotides at positions 7, 8, and 9 of nucleotide sequence 1 and the nucleotides at positions 2, 6, 14, and 16 of nucleotide sequence 2 are all fluoro-modified nucleotides, and the nucleotides at the other positions are independently one of non-fluoro-modified nucleotides. The present disclosure further provides a drug composition comprising the oligonucleotide, a conjugate, and its use in preparing a drug. [Selection diagram] Figure 15

Description

[Technical Field]

[0001] The present disclosure relates to double-stranded oligonucleotides, compositions and complexes comprising double-stranded oligonucleotides, and methods of preparation and use. [Background technology]

[0002] Double-stranded oligonucleotides are well known as active ingredients of drugs. In the development of small molecule nucleic acid drugs, delivery systems are one of the key technologies. Summary of the Invention [Problem to be solved by the invention]

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

[0004] In some embodiments, the present disclosure provides a double-stranded oligonucleotide. the first nucleotide sequence fragment is a nucleotide sequence fragment in a target mRNA; from the 5' end to the 3' end, the nucleotides at positions 7, 8, and 9 of nucleotide sequence 1 are fluoro-modified nucleotides; each of the nucleotides at positions 2, 6, 14, and 16 of nucleotide sequence 2 is a fluoro-modified nucleotide; and each of the nucleotides at positions 2, 6, 14, and 16 of nucleotide sequence 2 is a fluoro-modified nucleotide; and each of the nucleotides at positions 2, 6, 14, and 16 of nucleotide sequence 2 is a fluoro-modified nucleotide;

[0005] In some embodiments, the present disclosure further provides a pharmaceutical composition comprising the double-stranded oligonucleotide of the present disclosure, the pharmaceutical composition comprising the double-stranded oligonucleotide of the present disclosure and a pharmaceutically acceptable carrier.

[0006] In some embodiments, the present disclosure further provides a complex comprising the double-stranded oligonucleotide of the present disclosure, the complex comprising the double-stranded oligonucleotide of the present disclosure and a ligand conjugated to the double-stranded oligonucleotide.

[0007] In some embodiments, the present disclosure provides use of the double-stranded oligonucleotide, drug composition, or complex of the present disclosure in the preparation of a drug for the treatment and / or prevention of a pathological condition or disease caused by the expression of a specific gene in liver cells.

[0008] In some embodiments, the present disclosure provides a method for treating a pathological condition or disease caused by the expression of a particular gene in liver cells, the method comprising administering to a subject suffering from the disease a double-stranded oligonucleotide, drug composition, or complex of the present disclosure.

[0009] In some embodiments, the present disclosure provides a method for suppressing expression of a specific gene in a hepatocyte, comprising contacting the hepatocyte with a double-stranded oligonucleotide, drug composition, or complex of the present disclosure.

[0010] In some embodiments, the present disclosure provides a kit comprising the double-stranded oligonucleotide, drug composition, or conjugate of the present disclosure.

[0011] Other features and advantages of the present invention are described in detail in the detailed description section that follows.

[0012] [Incorporated by reference] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. [Brief explanation of the drawings]

[0013] [Figure 1-2] 1 shows semi-quantitative results of an in vitro tritosome stability test of siRNA complexes. [Figure 3-4] 1 shows semi-quantitative results of an in vitro stability test of siRNA complexes in human plasma. [Figure 5-6] 1 shows semi-quantitative results of an in vitro monkey plasma stability test of siRNA complexes. [Figure 7-10] The time course metabolism curves of PK / TK plasma concentration or tissue concentration of complex A1 in rat plasma at a dose of 10 mg / kg (FIG. 7), complex A1 in rat liver and kidney at a dose of 10 mg / kg (FIG. 8), complex A1 in rat plasma at a dose of 50 mg / kg (FIG. 9), and complex A1 in rat liver and kidney at a dose of 50 mg / kg (FIG. 10) are shown. [Figure 11-14] Each shows the inhibitory effect of the complex of the present disclosure on HBV mRNA in the 44BriHBV model. [Figure 15-16] 1 shows the time-dependent inhibitory effects of the complex of the present disclosure on serum HBsAg and HBV DNA in an AAV-HBV model, respectively. [Figure 17] 1 shows the time-dependent inhibitory effect of the complex of the present disclosure on serum HBsAg in an M-Tg model. [Figure 18-19] 1 shows the time-dependent inhibitory effect of the complex of the present disclosure on serum HBsAg and HBV DNA in an HBV-Tg model with 1.28 copies each. [Figures 20A-20D] 1 shows the inhibitory effects of different concentrations of complex A1 on the expression of GSCM, GSSM, PSCM, and PSSM. [Figure 21-22] 1 shows the inhibitory effects of the complex of the present disclosure on target mRNA and off-target mRNA in vitro, respectively. [Figure 23-25] Each shows the results of an in vitro stability test of the conjugate of the present disclosure. [Figure 26-28] 1 shows the inhibitory effect of the complex of the present disclosure on HBV mRNA in vivo. [Figure 29-31] 1 shows the time-dependent inhibitory effect of the complex of the present disclosure on the expression of HBsAg and HBV DNA in the serum of different HBV transgenic mice. [Figure 32-34] 1 shows the results of an in vitro stability test of a conjugate of the present disclosure. [Figure 35-36] 1 shows the inhibitory effect of the complex of the present disclosure on target mRNA and off-target mRNA in vitro. [Figure 37]4 shows the in vivo mRNA suppression effect of the complex of the present disclosure in the 44BriHBV model. [Figure 38] 1 shows the time-dependent inhibitory effect of the complex of the present disclosure on HBsAg expression in mice. [Figure 39] 1 shows the in vivo inhibitory effect of the complex of the present disclosure on mRNA in M-Tg model mice. [Figure 40-42] 1 shows the results of an in vitro stability test of a conjugate of the present disclosure. [Figure 43-44] 1 shows the inhibitory effect of the complex of the present disclosure on target mRNA and off-target mRNA in vitro. [Figure 45] The complex of the present disclosure exhibits an inhibitory effect on HBV mRNA in the body. [Figure 46] 1 shows the time-dependent inhibitory effect of the complex of the present disclosure on HBsAg expression in the serum of HBV transgenic mice. [Figure 47] 1 shows the inhibitory effect of the complex of the present disclosure on HBV mRNA in M-Tg model mice. [Figures 48A-48D] 1 shows the inhibitory effect of comparative siRNA3 on target mRNA and off-target mRNA in vitro. [Figures 49A-49D] 1 shows the inhibitory effect of siRNA E1 of the present disclosure on target mRNA and off-target mRNA in vitro. [Figure 50A-50B] 1 shows the inhibitory effect of siRNA and siRNA complexes of the present disclosure on ANGPTL3 mRNA in vitro. [Figures 51A-51D] Each shows the results of an in vitro stability experiment of the complex of the present disclosure. [Figures 52A-52D] The inhibition rate of lipids (blood lipids) by the complex of the present disclosure is shown, and is expressed as total cholesterol (CHO) and triglycerides (TG) in serum. [Figures 53A-53D] 1 shows the suppression rate of ANGPTL3 mRNA expression in vivo by the complex of the present disclosure. [Fig. 54A-54D]The inhibition rates of lipids by the complexes of the present disclosure are shown in terms of serum total cholesterol (CHO) and triglycerides (TG). [Figure 55A-55B] 1 shows the inhibition rate of lipids over time by the complex of the present disclosure, expressed as serum total cholesterol (CHO) and triglycerides (TG). [Figure 55C] 1 shows the inhibition rate of ANGPTL3 mRNA expression by the complex of the present disclosure. [Figures 56A-56B] 1 shows the inhibition rate of lipids over time by the complex of the present disclosure, expressed as serum total cholesterol (CHO) and triglycerides (TG). [Figures 57A-57D] 1 shows the inhibition rate of lipids over time by the complex of the present disclosure, expressed as serum total cholesterol (CHO) and triglycerides (TG). [Figure 58A-58B] 1 shows the inhibition rate of lipids over time by the complex of the present disclosure, expressed as serum total cholesterol (CHO) and triglycerides (TG). [Figure 58C] 1 shows the inhibition rate of ANGPTL3 mRNA expression by the complex of the present disclosure. [Figure 59] 1 shows the in vitro inhibition rate of APOC3 expression by the complex of the present disclosure. [Figure 60] The inhibition rate of APOC3 expression in liver tissue by the complex of the present disclosure on day 14 is shown. [Figure 61A-61B] 1 shows the inhibition rate of lipids over time by the complex of the present disclosure, expressed as serum total cholesterol (CHO) and triglycerides (TG). [Figure 62A-62B] 1 shows the inhibition rate of lipids over time by the complex of the present disclosure, expressed as serum total cholesterol (CHO) and triglycerides (TG). [Figure 63A-63D] 1 shows the inhibition rate of lipids over time by different doses of the conjugate of the present disclosure, expressed as serum total cholesterol (CHO) and triglycerides (TG). [Figure 64-65] 1 shows the results of an in vitro stability test of a conjugate of the present disclosure. [Figure 66-68]1 shows the time-dependent inhibition rates of serum surface antigen, serum e antigen, and HBV DNA by different doses of the conjugate of the present disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0014] The following describes in detail the embodiments of the present disclosure. It should be understood that the embodiments described herein are merely for the purpose of explaining or interpreting the present disclosure, and are not intended to limit the present disclosure.

[0015] (definition) Unless otherwise specified in the context, capital letters C, G, U, and A represent the base sequence of nucleotides, lowercase letter m represents that one nucleotide adjacent to the left of the letter m is a 2'-methoxy-modified nucleotide, lowercase letter f represents that one nucleotide adjacent to the left of the letter f is a 2'-fluoro-modified nucleotide, lowercase letter s represents that two nucleotides adjacent to the left and right of the letter s are linked by a thiophosphate group, and P1 represents that one nucleotide adjacent to the right of P1 is a 5'-phosphate nucleotide or a 5'-phosphate analog-modified nucleotide, and particularly refers to a vinyl phosphate-modified nucleotide (represented by VP in the following examples), a 5'-phosphate nucleotide (represented by P in the following examples), or a 5'-thiophosphate-modified nucleotide (represented by Ps in the following examples).

[0016] In the context of this specification, the terms "complementary" and "reverse complementary" may be used interchangeably and have the meaning well known to those skilled in the art, i.e., complementary pairing of bases on one strand with bases on the other strand in a double-stranded nucleic acid molecule. In DNA, the purine base adenine (A) always pairs with the pyrimidine base thymine (T) (or uracil (U) in RNA), and the purine base guanine (G) always pairs with the pyrimidine base cytosine (C). Each base pair contains one purine and one pyrimidine. When adenine on one strand always pairs with thymine (or uracil) on the other strand and guanine always pairs with cytosine, the strands are considered complementary and the sequence of the strands can be deduced from the sequence of the complementary strand. Correspondingly, "mismatch" as used in the art refers to the absence of complementary pairing of bases at corresponding positions in a double-stranded nucleic acid.

[0017] Unless otherwise specified in context, "essentially reverse complementary" refers to the presence of no more than three base mismatches between two related nucleotide sequences, "substantially reverse complementary" refers to the presence of no more than one base mismatch between two nucleotide sequences, and "fully complementary" refers to the absence of any base mismatches between two nucleotide sequences.

[0018] In this context, the presence of a "nucleotide difference" between one nucleotide sequence and another nucleotide sequence refers to a change in the type of nucleotide at the same position in the former compared to the latter. For example, if one nucleotide base in the latter is A and the corresponding nucleotide base at the same position in the former is U, C, G, or T, it is recognized that a nucleotide difference exists between the two nucleotide sequences at that position. In some embodiments, a nucleotide difference at a position is also considered to have occurred when an abasic nucleotide or a nucleotide analog is used instead of the nucleotide at the original position.

[0019] In the context of the present disclosure, particularly in describing the methods for preparing the double-stranded oligonucleotides, drug compositions, and / or oligonucleotide conjugates, unless otherwise specified, the term "nucleoside monomer" refers to modified or unmodified nucleoside monomers (unmodified or modified RNA phosphoramidites; RNA phosphoramidites are sometimes referred to as nucleoside phosphoramidites) used in phosphoramidite solid-phase synthesis, depending on the type and order of nucleotides in the double-stranded oligonucleotides, drug compositions, and / or oligonucleotide conjugates being prepared. Phosphoramidite solid-phase synthesis is a method used for RNA synthesis known to those skilled in the art. All of the nucleoside monomers used in the present disclosure are commercially available.

[0020] As used herein, a dash ("-") that is not between two alphabetic letters or symbols is used to indicate the location of the point of attachment of a substituent. For example, -C1-C 10 Alkyl-NH2 is C1-C 10 It is attached via an alkyl group.

[0021] As used herein, "optional" or "optionally" refers to the fact that the subsequently described event or circumstance may or may not occur, and that the description includes both cases where the event or circumstance occurs and cases where it does not. For example, "optionally substituted alkyl" includes "alkyl" and "substituted alkyl" as defined in the following sentence. With respect to any groups that contain one or more substituents, it will be understood by those of skill in the art that it is not intended that these groups introduce any substitutions or patterns that are sterically impractical, synthetically impractical, and / or inherently unstable.

[0022] As used herein, "alkyl" refers to straight and branched chain alkyls having a specified number of carbon atoms, typically 1 to 20 carbon atoms, e.g., 1 to 10 carbon atoms, 1 to 8, or 1 to 6 carbon atoms. For example, C1-C6 alkyl includes straight and branched chain alkyls of 1 to 6 carbon atoms. When naming an alkyl residue having a specific number of carbon atoms, it is intended to include all branched and straight chain forms having that number of carbon atoms. Thus, for example, "butyl" is meant to include n-butyl, sec-butyl, isobutyl, and tert-butyl, and "propyl" includes n-propyl and isopropyl. Alkylene is a subset of alkyl and refers to a residue similar to alkyl but having two points of attachment.

[0023] As used herein, "alkenyl" refers to an unsaturated branched or straight-chain alkyl group having at least one carbon-carbon double bond, obtained by removing one hydrogen molecule from adjacent carbon atoms of a parent alkyl group. The group may be in the cis or trans configuration of the double bond. Exemplary alkenyl groups include, but are not limited to, vinyl, propenyl groups such as prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl (allyl), and prop-2-en-2-yl, and butenyl groups such as but-1-en-1-yl, but-1-en-2-yl, 2-methylprop-1-en-1-yl, but-2-en-1-yl, but-2-en-2-yl, buta-1,3-dien-1-yl, and buta-1,3-dien-2-yl. In some embodiments, alkenyl has from 2 to 20 carbon atoms, and in other embodiments, from 2 to 10, 2 to 8, or 2 to 6 carbon atoms. Alkenylene is a subset of alkenyl, referring to the same residues as alkenyl but with two points of attachment.

[0024] As used herein, "alkynyl" refers to an unsaturated branched or straight-chain alkyl group having at least one carbon-carbon triple bond, where the carbon-carbon triple bond is obtained by removing two hydrogen molecules from adjacent carbon atoms of a parent alkyl group. Exemplary alkynyl groups include, but are not limited to, ethynyl, propynyl groups such as prop-1-yn-1-yl and prop-2-yn-1-yl, and butynyl groups such as but-1-yn-1-yl, but-1-yn-3-yl, and but-3-yn-1-yl. In some embodiments, alkynyl groups have 2 to 20 carbon atoms, and in other embodiments, 2 to 10, 2 to 8, or 2 to 6 carbon atoms. Alkynylene is a subset of alkynyl and refers to the same residue as alkynyl but with two points of attachment.

[0025] As used herein, "alkoxy" refers to an alkyl of the specified number of carbon atoms attached through an oxygen bridge, e.g., methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, pentyloxy, 2-pentyloxy, isopentyloxy, neopentyloxy, hexyloxy, 2-hexyloxy, 3-hexyloxy, 3-methylpentyloxy, etc. Alkoxy typically has 1 to 10, 1 to 8, 1 to 6, or 1 to 4 carbon atoms attached through the oxygen bridge.

[0026] As used herein, "aryl" refers to a group formed by removing a hydrogen atom from a ring carbon atom derived from an aromatic monocyclic or polycyclic hydrocarbon ring system. The aromatic monocyclic or polycyclic hydrocarbon ring system contains only hydrogen and carbon atoms from 6 to 18 carbon atoms, and at least one ring in the ring system is fully unsaturated, i.e., it contains a cyclic, delocalized (4n+2) π-electron system according to the Hückel theory. Aryl includes, but is not limited to, groups such as phenyl, fluorenyl, and naphthyl. Arylene is a subset of aryl and refers to a residue similar to aryl but with two points of attachment.

[0027] As used herein, "cycloalkyl" refers to a non-aromatic carbocyclic ring, typically having 3 to 7 ring carbon atoms. The ring may be saturated or may contain one or more carbon-carbon double bonds. Illustrative examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, and cyclohexenyl, as well as bridged and cage ring groups such as norbornane.

[0028] As used herein, "halogen substituent" or "halo" refers to fluoro, chloro, bromo, and iodo, and the term "halogen" includes fluorine, chlorine, bromine, and iodine.

[0029] As used herein, "halogenated alkyl" refers to an alkyl group, as defined above, in which a specified number of carbon atoms are substituted with one or more halogen atoms, up to the maximum permitted number. Illustrative examples of halogenated alkyl groups include, but are not limited to, trifluoromethyl, difluoromethyl, 2-fluoroethyl, and pentafluoroethyl.

[0030] A "heterocyclic group" refers to a stable 3- to 18-membered non-aromatic cyclic group containing 2 to 12 carbon atoms and 1 to 6 heteroatoms selected from nitrogen, oxygen, and sulfur. Unless otherwise specified in the specification, a heterocyclic group is a monocyclic, bicyclic, tricyclic, or tetracyclic ring system and may include fused or bridged ring systems. The heteroatoms in a heterocyclic group may be optionally oxidized. One or more nitrogen atoms (if present) are optionally quaternized. A heterocyclic group may be partially saturated or fully saturated. A heterocyclic group can be attached to the rest of the molecule through any atom of the ring. Illustrative examples of such heterocyclic groups include, but are not limited to, dioxanyl, thiophenyl[1,3]disulfonyl, decahydroisoquinolinyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindole, octahydroisoindole, 2-oxapiperazinyl, 2-oxapiperidyl, 2-oxapyrimidinyl, oxazolidinyl, piperidyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trisulfonyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxothiomorpholinyl, and 1,1-dioxothiomorpholinyl.

[0031] "Heteroaryl" refers to a group derived from a 3- to 18-membered aromatic ring containing 2 to 17 carbon atoms and 1 to 6 heteroatoms selected from nitrogen, oxygen, and sulfur. As used herein, heteroaryl may be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, in which at least one ring in the ring system is fully unsaturated, i.e., it contains a cyclic delocalized (4n+2) π-electron system according to Hückel theory. Heteroaryl includes fused or bridged ring systems. Heteroatoms in heteroaryl are optionally oxidized. One or more nitrogen atoms (if present) are optionally quaternized. Heteroaryl is attached to the remainder of the molecule through any atom in the ring. Illustrative examples of heteroaryl include 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, benzodioxaphenyl, benzopyranyl, benzopyranonyl, benzofuryl, benzofuranonyl, benzothiophenyl, benzothieno[3,2-d]pyrimidinyl, benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridyl, carbazolyl, cinnolyl, cyclopenta[d]pyrimidinyl, 6,7-dihydrobenzoyl, and the like. dro-5H-cyclopenta[4,5]thieno[2,3-d]pyrimidinyl, 5,6-dihydrobenzo[h]quinazolinyl, 5,6-dihydrobenzo[h]cinnolinyl, 6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-c]pyridazinyl, dibenzofuryl, dibenzothiophenyl, furyl, furanonyl, furo[3,2-c]pyridyl, 5,6,7,8,9,10-hexaphenyl 5,6,7,8,9,10-Hexahydrocycloocta[d]pyridazinyl, 5,6,7,8,9,10-Hexahydrocycloocta[d]pyridyl, isothiazolyl, indazolyl, imidazolyl, indole, isoindole, indolinyl, isoindolinyl, isoquinolyl, indolizinyl, isoxazolyl, 5,8-methano-5,6,7,8-Tetrahydroquinazolinyl, naphthyridinonyl, 1,6-naphthyridinonyl, 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]pyrimidinyl, pyrazinyl, pyrimidinyl, pyridazinyl, pyrrolyl, quinazolinyl, quinox These include, but are not limited to, salinyl, quinolyl, isoquinolyl, tetrahydroquinolyl, 5,6,7,8-tetrahydroquinazolinyl, 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidinyl, 6,7,8,9-tetrahydro-5H-cyclohepta[4,5]thieno[2,3-d]pyrimidinyl, 5,6,7,8-tetrahydropyrido[4,5-c]pyridazinyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl, triazinyl, thieno[2,3-d]pyrimidinyl, thieno[3,2-d]pyrimidinyl, thieno[2,3-c]pridinyl, and thiophenyl.

[0032] A variety of hydroxy protecting groups can be used in the present disclosure. Generally, a protecting group can render a chemical functionality insensitive to certain reaction conditions and can be added to and removed from that functionality in a molecule without substantially damaging the remainder of the molecule. Representative hydroxy protecting groups are disclosed in Beaucage et al., Tetrahedron 1992, 48, 2223-2311, and Greene and Wuts, Protective Groups in Organic Synthesis, Chapter 2, 2d ed., John Wiley & Sons, New York, 1991, which are incorporated herein by reference in their entireties. In some embodiments, the protecting group is stable under basic conditions but can be removed under acidic conditions. In some embodiments, non-exclusive examples of hydroxy protecting groups that can be used herein include dimethoxytrityl (DMT), monomethoxytrityl, 9-phenylxanthen-9-yl (Pixyl) "9-phenylxanthen-9-yl(Pixyl)" and 9-(p-methoxyphenyl)xanthen-9-yl (Mox) "9-(p-methoxyphenyl)xanthen-9-yl(Mox)". In some embodiments, non-exclusive examples of hydroxy protecting groups that can be used herein include Tr (trityl group), MMTr (4-methoxytrityl), DMTr (4,4'-dimethoxytrityl) and TMTr (4,4',4''-trimethoxytrityl).

[0033] The term "subject," as used herein, refers to any animal, e.g., a mammal or marsupial. The subject of this disclosure includes, but is not limited to, humans, non-human primates (e.g., rhesus monkeys or other types of macaques), mice, pigs, horses, donkeys, cows, sheep, rats, and any type of poultry.

[0034] As used herein, the terms "method of treatment," "treatment," "alleviation," or "amelioration" may be used interchangeably herein. These terms refer to a method of obtaining a beneficial or desired result, including, but not limited to, a therapeutic effect. A "therapeutic effect" means eradicating or ameliorating the underlying disorder being treated. A therapeutic effect may also be achieved by observing an improvement in the patient by eradicating or ameliorating one or more physiological symptoms associated with the underlying disorder, even though the patient may still be afflicted by the underlying disorder.

[0035] As used herein, "prevention" and "prophylaxis" may be used interchangeably. These terms refer to a method of obtaining a beneficial or desired result, including, but not limited to, a prophylactic effect. To obtain a "prophylactic effect," a complex or composition can be administered to a patient at risk of contracting a particular disease, or to a patient who has reported one or more pathological symptoms of a disease, although the patient may not have been diagnosed with the disease.

[0036] <Modified double-stranded oligonucleotide> In one aspect, the present disclosure provides double-stranded oligonucleotides capable of modulating gene expression.

[0037] The double-stranded oligonucleotide of the present disclosure contains a nucleotide group as a basic structural unit, and the nucleotide group contains a phosphate group, a ribose group, and a base, which is known to those skilled in the art, and therefore, a detailed description thereof will be omitted here.

[0038] CN102140458B discloses an siRNA that specifically inhibits HBV genes, and has investigated several chemical modification strategies for the siRNA. The study found that different modification strategies have completely different effects on indicators such as siRNA stability, biological activity, and cytotoxicity. Seven effective modification methods were demonstrated in the study, and compared with unmodified siRNA, siRNA obtained by one of these modification methods had improved blood stability while maintaining essentially the same inhibitory activity as unmodified siRNA.

[0039] The double-stranded oligonucleotide of the present disclosure comprises a sense strand and an antisense strand, each nucleotide of the sense strand and the antisense strand is a modified nucleotide, the sense strand comprises nucleotide sequence 1, and the antisense strand comprises nucleotide sequence 2, the length of both nucleotide sequence 1 and nucleotide sequence 2 is 19 nucleotides, and nucleotide sequence 1 and nucleotide sequence 2 form a double-stranded region in a reverse complementary manner at least in part, and nucleotide sequence 2 is at least partially reverse complementary to a first nucleotide sequence fragment, and the first nucleotide sequence fragment is a target mRNA. A nucleotide sequence fragment in A, wherein, from the 5' end to the 3' end, the nucleotides at positions 7, 8, and 9 of nucleotide sequence 1 are fluoro-modified nucleotides, and each of the other nucleotides at positions 1 are independently one of the non-fluoro-modified nucleotides, the first nucleotide at the 5' end of nucleotide sequence 2 is the first nucleotide at the 5' end of the antisense strand, the nucleotides at positions 2, 6, 14, and 16 of nucleotide sequence 2 are fluoro-modified nucleotides, and each of the other nucleotides at positions 2, 6, 14, and 16 of nucleotide sequence 2 are independently one of the non-fluoro-modified nucleotides. In the context of the present disclosure, a "fluoro-modified nucleotide" refers to a nucleotide in which the hydroxy group at the 2' position of the ribose group of the nucleotide is substituted with fluorine, and a "non-fluoro-modified nucleotide" refers to a nucleotide or nucleotide analog in which the hydroxy group at the 2' position of the ribose group of the nucleotide is substituted with a non-fluorinated group.

[0040] "Nucleotide analog" refers to a group that can substitute for a nucleotide in a nucleic acid but that differs in structure from adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, or thymine deoxyribonucleotide, for example, an isonucleotide, bridged nucleic acid (BNA), or acyclic nucleotide.

[0041] In some embodiments, nucleotide sequence 2 is essentially the reverse complement, essentially the complete reverse complement, or the complete reverse complement of the first nucleotide sequence fragment.

[0042] In some embodiments, from the 5' to the 3' end, at least nucleotides 2 to 19 of nucleotide sequence 2 are complementary to the first nucleotide sequence fragment. In some specific embodiments, from the 5' to the 3' end, the first nucleotide of nucleotide sequence 2 is A or U.

[0043] In some embodiments, the nucleotide sequence 1 and the nucleotide sequence 2 are essentially reverse complementary, essentially completely reverse complementary, or completely reverse complementary.

[0044] In some embodiments, the sense strand further comprises nucleotide sequence 3, and the antisense strand further comprises nucleotide sequence 4, wherein each nucleotide in nucleotide sequence 3 and nucleotide sequence 4 is independently a non-fluoro-modified nucleotide, the length of nucleotide sequence 3 and nucleotide sequence 4 is 1 to 4 nucleotides, respectively, the length of nucleotide sequence 3 and nucleotide sequence 4 is equal, and they are essentially completely reverse complementary or completely reverse complementary, nucleotide sequence 3 is attached to the 5' end of nucleotide sequence 1, and nucleotide sequence 4 is attached to the 3' end of nucleotide sequence 2, and nucleotide sequence 4 is essentially completely reverse complementary or completely reverse complementary to a second nucleotide sequence fragment, and the second nucleotide sequence fragment refers to a nucleotide sequence adjacent to the first nucleotide sequence fragment in the target mRNA and having the same length as nucleotide sequence 4.

[0045] In some embodiments, nucleotide sequence 3 and nucleotide sequence 4 are fully complementary, each nucleotide sequence 3 and nucleotide sequence 4 is 1 nucleotide in length, and nucleotide sequence 4 is fully reverse complementary to a second nucleotide sequence fragment; or nucleotide sequence 3 and nucleotide sequence 4 are fully complementary, each nucleotide sequence 3 and nucleotide sequence 4 is 2 nucleotides in length, and nucleotide sequence 4 is fully reverse complementary to a second nucleotide sequence fragment; or nucleotide sequence 3 and nucleotide sequence 4 are fully complementary, each nucleotide sequence 3 and nucleotide sequence 4 is 3 nucleotides in length, and nucleotide sequence 4 is fully reverse complementary to a second nucleotide sequence fragment; or nucleotide sequence 3 and nucleotide sequence 4 are fully complementary, each nucleotide sequence 3 and nucleotide sequence 4 is 4 nucleotides in length, and nucleotide sequence 4 is fully reverse complementary to a second nucleotide sequence fragment.

[0046] The nucleotide sequence 3 and the nucleotide sequence 4 are completely reverse complementary, and the nucleotide sequence 4 is completely reverse complementary to a second nucleotide sequence fragment, and when the nucleotide sequence associated with the target mRNA is determined, the nucleotide sequence 3 and the nucleotide sequence 4 are also determined.

[0047] Thus, the length of the sense strand or antisense strand may independently be 19 to 23 nucleotides.

[0048] In some embodiments, the double-stranded oligonucleotide further comprises nucleotide sequence 5, wherein each nucleotide of nucleotide sequence 5 is independently one of the non-fluoro-modified nucleotides, and nucleotide sequence 5 is 1 to 3 nucleotides in length and is attached to the 3' end of the antisense strand, constituting a 3' overhang end of the antisense strand.

[0049] Thus, the ratio of the lengths of the sense strand to the antisense strand in the double-stranded oligonucleotides provided by the present disclosure may be 19 / 19, 19 / 20, 19 / 21, 19 / 22, 20 / 20, 20 / 21, 20 / 22, 20 / 23, 21 / 21, 21 / 22, 21 / 23, 21 / 24, 22 / 22, 22 / 23, 22 / 24, 22 / 25, 23 / 23, 23 / 24, 23 / 25, or 23 / 26.

[0050] In some embodiments, the length of nucleotide sequence 5 is 2 nucleotides, and from the 5' end to the 3' end, nucleotide sequence 5 is two consecutive thymine deoxyribonucleotides, two consecutive uracil ribonucleotides, or is completely reverse complementary to a third nucleotide sequence fragment, where the third sequence fragment refers to a nucleotide sequence adjacent to the first nucleotide sequence fragment or the second nucleotide sequence fragment in the target mRNA and having a length equal to that of nucleotide sequence 5.

[0051] Therefore, in some embodiments, the length ratio of the sense strand to the antisense strand of the double-stranded oligonucleotide provided by the present disclosure is 19 / 21 or 21 / 23, and when this is the case, the double-stranded oligonucleotide provided by the present disclosure has better target mRNA silencing activity.

[0052] In this context, a fluoro-modified nucleotide refers to a nucleotide in which the hydroxy group at the 2' position of the ribose group of the nucleotide is substituted with fluorine, as shown in formula (101), wherein Base represents a base selected from C, G, A, or U.

[0053] A non-fluoro-modified nucleotide refers to a nucleotide or nucleotide analog in which the 2'-hydroxy group of the ribose group of the nucleotide is replaced with a non-fluorinated group. In some embodiments, each non-fluoro-modified nucleotide is independently selected from nucleotides or nucleotide analogs in which the 2'-hydroxy group of the ribose group of the nucleotide is replaced with a non-fluorinated group.

[0054] Nucleotides in which the hydroxy group at the 2'-position of these ribose groups is substituted with a non-fluorinated group are known to those skilled in the art, and these nucleotides may be, for example, one selected from 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, and 2'-deoxynucleotides.

[0055] In some embodiments, the 2'-alkoxy modified nucleotide may be a methoxy modified nucleotide (2'-OMe) as shown in formula (102). In some embodiments, the 2'-substituted alkoxy modified nucleotide may be a 2'-O-methoxyethyl modified nucleotide (2'-MOE) as shown in formula (103). In some embodiments, the 2'-amino modified nucleotide (2'-NH2) is shown in formula (104). In some embodiments, the 2'-deoxynucleotide (DNA) is shown in formula (105).

[0056] [ka]

[0057] A nucleotide analog refers to a group that can substitute for a nucleotide in a nucleic acid but that differs in structure from adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, or thymine deoxyribonucleotide. In some embodiments, a nucleotide analog may be an isonucleotide, bridged nucleic acid (BNA), or acyclic nucleotide.

[0058] BNA refers to a constrained or inaccessible nucleotide. BNAs may comprise a five-, six-, or seven-membered bridge structure with a "fixed" C3'-endo sugar pucker. Typically, the bridge is introduced at the 2'-, 4'-position of the ribose to provide a 2',4'-BNA nucleotide, such as an LNA as shown in formula (106), an ENA as shown in formula (107), or a cET BNA as shown in formula (108).

[0059] [ka]

[0060] An acyclic nucleotide refers to an "open ring" nucleotide in which the sugar ring of the nucleotide is opened, for example, an unlocked nucleic acid (UNA) shown in formula (109) or a glycerol nucleic acid (GNA) shown in formula (110).

[0061] [ka]

[0062] In the above formula (109) and formula (110), R is selected from H, OH, or alkoxy (O-alkyl).

[0063] An isonucleotide refers to a compound in which the position of the base on the ribose ring in a nucleotide has been changed, and may be, for example, a compound represented by formula (111) or (112) in which the base has been shifted from the 1'-position to the 2'-position or the 3'-position on the ribose ring.

[0064] [ka]

[0065] In the compounds of the above formulae (111) to (112), Base represents a base such as A, U, G, C, or T, and R is selected from H, OH, F, or the non-fluorinated groups described above.

[0066] In some embodiments, the nucleotide analog is one selected from an isonucleotide, LNA, ENA, cET, UNA, and GNA. In some embodiments, each non-fluoro-modified nucleotide is a methoxy-modified nucleotide, which in this context refers to a nucleotide in which the 2'-hydroxy group of the ribose group is replaced with a methoxy group.

[0067] In this context, the terms "fluoro-modified nucleotide," "2'-fluoro-modified nucleotide," "nucleotide in which the 2'-hydroxy group of the ribose group is substituted with fluorine," and "2'-fluororibose group" have the same meaning and all refer to a compound having the structure shown in formula (101) in which the 2'-hydroxy group of the nucleotide is substituted with fluorine, and the terms "methoxy-modified nucleotide," "2'-methoxy-modified nucleotide," "nucleotide in which the 2'-hydroxy group of the ribose group is substituted with a methoxy group," and "2'-methoxyribose group" have the same meaning and all refer to a compound in which the 2'-hydroxy group of the nucleotide ribose group is substituted with a methoxy group to form the structure shown in formula (102).

[0068] In some embodiments, the double-stranded oligonucleotides of the present disclosure can improve the blood stability of nucleic acids by resisting cleavage by ribonucleases in the blood, making the nucleic acids more resistant to nuclease hydrolysis while maintaining high target gene-modulating activity.

[0069] In some embodiments, the double-stranded oligonucleotides described in the present disclosure have a high balance of plasma stability and gene expression regulation efficiency in animal experiments, and some also have the advantages of being simpler and less expensive. Some examples are shown below.

[0070] From the 5' end to the 3' end, in the sense strand, the nucleotides at positions 7, 8, and 9 of nucleotide sequence 1 are fluoro-modified nucleotides, and the nucleotides at other positions in the sense strand are methoxy-modified nucleotides; and in the antisense strand, the nucleotides at positions 2, 6, 14, and 16 of nucleotide sequence 2 are fluoro-modified nucleotides, and the nucleotides at other positions in the antisense strand are methoxy-modified nucleotides.

[0071] In some embodiments, the double-stranded oligonucleotides of the present disclosure further comprise other modified nucleotide groups, which do not appreciably weaken or eliminate the function of the double-stranded oligonucleotide to modulate target gene expression.

[0072] Currently, in the art, there are several methods available for modifying double-stranded oligonucleotides, including not only the ribose group modifications mentioned above, but also backbone modifications (e.g., phosphate group modifications) and base modifications (see, for example, Watts, JK, G.F. Deleavy and M.J. Damha, Chemically modified siRNA: tools and applications. Drug Discovery Today, 2008.13(19-20):pp.842-55, the contents of which are incorporated herein by reference in their entirety).

[0073] In some embodiments, at least one of the phosphate groups in the phosphate-sugar backbone of at least one single strand in the sense strand and the antisense strand is a phosphate group having a modified group. The phosphate group having a modified group may be a phosphorothioate group in which at least one oxygen atom in the phosphodiester bond of the phosphate group is replaced with a sulfur atom. By substituting one sulfur atom for a non-bridging oxygen atom in the phosphodiester bond, the phosphodiester bond is replaced with a phosphorothioate bond, i.e., two nucleotides are linked by a phosphorothioate group, which may be a phosphorothioate structure as shown in formula (121). This modification stabilizes the structure of the double-stranded oligonucleotide and maintains high specificity and high affinity for base pairing.

[0074] [ka]

[0075] In some embodiments, in the double-stranded oligonucleotide, a phosphorothioate group is present bound to at least one of the following: between the first and second nucleotides at any end of the sense strand or the antisense strand; between the second and third nucleotides at any end of the sense strand or the antisense strand; or any combination thereof. In some embodiments, a phosphorothioate group is present bound to all of the above positions except the 5'-end of the sense strand. In some embodiments, a phosphorothioate group is present bound to all of the above positions except the 3'-end of the sense strand. In some embodiments, a phosphorothioate group is present bound to at least one of the following 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, 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 Between the second and third nucleotides from the 3' end of the antisense strand.

[0076] In some embodiments, the 5'-terminal nucleotide of the antisense strand sequence of the double-stranded oligonucleotide molecule is a 5'-phosphate nucleotide or a 5'-phosphate analog-modified nucleotide.

[0077] In some embodiments, the 5'-phosphate nucleotide has the structure shown in formula (122):

[0078] [ka]

[0079] At the same time, the types of commonly used 5'-phosphate analog-modified nucleotides are known to those skilled in the art. For example, Anastasia Khvorova and Jonathan K. Watts, "The chemical evolution of oligonucleotide therapies of clinical utility," Nature Biotechnology, 2017, 35(3):238-48, discloses nucleotides represented by formulas (123) to (126).

[0080] [ka] where R represents a group selected from the group consisting of H, OH, F and a methoxy group, and Base represents a base selected from A, U, C, G or T.

[0081] In some embodiments, the 5'-phosphate analog modified nucleotide is a nucleotide containing a vinyl phosphate ester (E-vinylphosphonate, E-VP) as shown in formula (123) or a nucleotide containing a thiophosphate ester as shown in formula (125).

[0082] The modification means disclosed herein can be applied to various double-stranded oligonucleotides that regulate gene expression. In some embodiments, the double-stranded oligonucleotides may be those that suppress or down-regulate gene expression, such as siRNA, and in some embodiments, the double-stranded oligonucleotides may be those that activate or up-regulate gene expression, such as saRNA.

[0083] The double-stranded oligonucleotides modified by the present disclosure unexpectedly exhibit improved stability in blood, improved stability in lysosomes, reduced off-target effects, and / or improved activity of the double-stranded oligonucleotides, while exhibiting excellent in vivo inhibitory effects without significant reduction in target gene expression regulation activity.

[0084] The modified double-stranded oligonucleotides, pharmaceutical compositions, and conjugates provided by the present disclosure can be used to regulate the abnormal expression of various genes and treat various pathological conditions or diseases caused by abnormal gene expression. These genes may be various endogenous genes in the human body or animal body, or pathogen genes that reproduce in the human body or animal body. Double-stranded oligonucleotides having specific nucleotide sequences and the above-mentioned modification means can be designed and prepared according to the desired target mRNA.

[0085] According to some embodiments of the present disclosure, the double-stranded oligonucleotide of the present disclosure may be, for example, the following siRNA:

[0086] said nucleotide sequence 1 is the sequence shown in SEQ ID NO: 1 and said nucleotide sequence 2 is the sequence shown in SEQ ID NO: 2; or said nucleotide sequence 1 is the sequence shown in SEQ ID NO:3 and said nucleotide sequence 2 is the sequence shown in SEQ ID NO:4; or said nucleotide sequence 1 is the sequence shown in SEQ ID NO:5 and said nucleotide sequence 2 is the sequence shown in SEQ ID NO:6; or said nucleotide sequence 1 is the sequence shown in SEQ ID NO: 7 and said nucleotide sequence 2 is the sequence shown in SEQ ID NO: 8; or said nucleotide sequence 1 is the sequence shown in SEQ ID NO: 9 and said nucleotide sequence 2 is the sequence shown in SEQ ID NO: 10; or said nucleotide sequence 1 is the sequence shown in SEQ ID NO: 11 and said nucleotide sequence 2 is the sequence shown in SEQ ID NO: 12; or The nucleotide sequence 1 is the sequence shown in SEQ ID NO:13, and the nucleotide sequence 2 is the sequence shown in SEQ ID NO:14.

[0087] 5'-CmCmUmUmGmAmGfGfCfAmUmAmCmUmUmCmAmAmAm-3' (SEQ ID NO: 1) 5'-UmUfUmGmAmAfGmUmAmUmGmCmCmUfCmAfAmGmGm-3' (SEQ ID NO: 2) 5'-UmGmCmUmAmUmGfCfCfUmCmAmUmCmUmUmCmUmAm-3' (SEQ ID NO: 3) 5'-UmAfGmAmAmGfAmUmGmAmGmGmCmAfUmAfGmCmAm-3' (SEQ ID NO: 4) 5'-UmCmUmGmUmGmCfCfUfUmCmUmCmAmUmCmUmGmAm-3' (SEQ ID NO: 5) 5'-UmCfAmGmAmUfGmAmGmAmAmGmGmCfAmCfAmGmAm-3' (SEQ ID NO: 6) 5'-CmGmUmGmUmGmCfAfCfUmUmCmGmCmUmUmCmAmAm-3' (SEQ ID NO: 7) 5'-UmUfGmAmAmGfCmGmAmAmGmUmGmCfAmCfAmCmGm-3' (SEQ ID NO: 8) 5'-GmAmAmAmGmUmAfUfGfUmCmAmAmCmGmAmAmUmAm-3' (SEQ ID NO: 9) 5'-UmAfUmUmCmGfUmUmGmAmCmAmUmAfCmUfUmUmCm-3' (SEQ ID NO: 10) 5'-CmCmAmAmGmAmGfCfAfCmCmAmAmGmAmAmCmUmAm-3' (SEQ ID NO: 11) 5'-UmAfGmUmUmCfUmUmGmGmUmGmCmUfCmUfUmGmGm-3' (SEQ ID NO: 12) 5'-CmAmAmUmAmAmAfGfCfUmGmGmAmCmAmAmGmAmAm-3' (SEQ ID NO: 13) 5'-UmUfCmUmUmGfUmCmCmAmGmCmUmUfUmAfUmUmGm-3' (SEQ ID NO: 14)

[0088] Here, the capital letters C, G, U, and A represent the base sequence of nucleotides, the lowercase letter m indicates that one nucleotide adjacent to the left of the letter m is a 2'-methoxy-modified nucleotide, and the lowercase letter f indicates that one nucleotide adjacent to the left of the letter f is a 2'-fluoro-modified nucleotide.

[0089] According to some embodiments of the present disclosure, the double-stranded oligonucleotides of the present disclosure may be, for example, siRNAs shown in Tables 1A-1F.

[0090] [Table 1] siRNA Sequences in Some Embodiments [Table 1A]

[0091] [Table 1B]

[0092] [Table 1C]

[0093] [Table 1D]

[0094] [Table 1E]

[0095] [Table 1F]

[0096] [Table 1G] TIFF0007727308000015.tif193170 * S: sense strand, AS: antisense strand

[0097] wherein capital letters C, G, U, and A represent the base sequence of nucleotides, lowercase letter m represents that one nucleotide adjacent to the left of the letter m is a 2'-methoxy-modified nucleotide, lowercase letter f represents that one nucleotide adjacent to the left of the letter f is a 2'-fluoro-modified nucleotide, lowercase letter s represents that two nucleotides adjacent to the left and right of the letter s are linked by a thiophosphate group, and P1 represents that one nucleotide adjacent to the right of P1 is a 5'-phosphate nucleotide or a 5'-phosphate analog-modified nucleotide, which in some embodiments is a vinyl phosphate-modified nucleotide (represented by VP in the examples below), a 5'-phosphate-modified nucleotide (represented by P in the examples below), or a thiophosphate-modified nucleotide (represented by Ps in the examples below).

[0098] As those skilled in the art will readily appreciate, the double-stranded oligonucleotides described herein can be obtained by conventional methods for preparing double-stranded oligonucleotides (e.g., solid-phase synthesis and liquid-phase synthesis). Commercial customization services are already available for solid-phase synthesis. Modified nucleotide groups can be introduced into the double-stranded oligonucleotides described herein by using nucleotide monomers with corresponding modifications. Methods for preparing nucleotide monomers with corresponding modifications and methods for introducing modified nucleotide groups into double-stranded oligonucleotides are also well known to those skilled in the art.

[0099] The modified double-stranded oligonucleotide provided by the present disclosure may be used alone, or may form a pharmaceutical composition with a pharmaceutically acceptable carrier, or may be combined with a conjugate molecule to form a complex, or may be in other forms. An effective amount of the double-stranded oligonucleotide, pharmaceutical composition, or complex is contacted with a cell to regulate the expression of a target gene, or the double-stranded oligonucleotide, pharmaceutical composition, or complex is administered to a subject to regulate the expression of a target gene, thereby achieving treatment of a pathological condition or disease caused by abnormal expression of the target gene.

[0100] Forming a drug composition with an appropriate carrier or complexing it with an appropriate conjugate molecule can further improve the blood stability of the double-stranded oligonucleotide of the present disclosure, enhance its targeting, and solve problems such as the in vivo delivery of the double-stranded oligonucleotide of the present disclosure. For double-stranded oligonucleotides, carriers or complexes that can impart or enhance targeting are highly advantageous because they significantly improve the efficiency of double-stranded oligonucleotides in regulating target gene expression and reduce potential side effects. Furthermore, after delivery of the targeting carrier or complex, the double-stranded oligonucleotide must still be able to act at the target site; that is, the encapsulation / complexation of the carrier or complex must not affect the activity of the double-stranded oligonucleotide itself (e.g., if the double-stranded oligonucleotide is an siRNA, it must not affect the RNAi mechanism that internalizes the siRNA, i.e., the RISC complex). Furthermore, these targeting carriers or complexes must also have good biocompatibility and as low toxicity as possible.

[0101] The pharmaceutical composition can be distributed systematically to various parts of the body, or can be specifically enriched in a specific part of the body. The conjugate generally has targeting properties, and the type of conjugate molecule can be changed according to the expression distribution of the target gene in the human or animal body to achieve the purpose of delivering the double-stranded oligonucleotide to the relevant part, for example, the conjugate molecule can be a conjugate molecule targeting the liver, lung, kidney, or cancer cell.

[0102] In some embodiments, the present disclosure further provides a pharmaceutical composition comprising the modified double-stranded oligonucleotide and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be any available carrier.

[0103] In some embodiments, the present disclosure further provides an oligonucleotide conjugate comprising the modified double-stranded oligonucleotide and a ligand conjugated to the double-stranded oligonucleotide. Depending on the expression distribution of the target gene, different conjugate molecules can be used to deliver the double-stranded oligonucleotide to different organs or cells. For example, the conjugate molecule described below is suitable for delivering the double-stranded oligonucleotide to the liver to regulate the expression of a target endogenous gene expressed in the liver or a gene of a pathogen that reproduces in the liver, thereby achieving the goal of treating a pathological condition or disease caused by the abnormal expression of an endogenous gene expressed in the liver or a gene of a pathogen that reproduces in the liver.

[0104] In some embodiments, the present disclosure provides use of the double-stranded oligonucleotide, a pharmaceutical composition comprising the above-described double-stranded oligonucleotide, or the above-described oligonucleotide conjugate in the preparation of a medicament for the treatment and / or prevention of a pathological condition or disease caused by overexpression of a gene.

[0105] In some embodiments, the present disclosure provides a method for treating a pathological condition or disease caused by abnormal gene expression, comprising administering to a subject an effective amount of the double-stranded oligonucleotide, the pharmaceutical composition, or the oligonucleotide conjugate.

[0106] In some embodiments, the present disclosure provides a method for modulating gene expression, comprising contacting an effective amount of the double-stranded oligonucleotide, the pharmaceutical composition, or the oligonucleotide conjugate with a cell expressing the gene of interest. In some embodiments, the abnormal expression is overexpression, and accordingly, the modulation is suppression of the overexpression.

[0107] In some embodiments, the double-stranded oligonucleotide, pharmaceutical composition, or oligonucleotide conjugate exhibits unexpected stability and activity in regulating genes expressed in the liver or treating pathological conditions or diseases caused by abnormal gene expression in hepatocytes. Examples of genes expressed in the liver include, but are not limited to, ApoB, ApoC, ANGPTL3, PCSK9, SCD1, TIMP-1, Col1A1, FVII, STAT3, p53, HBV, and HCV. In some embodiments, the specific gene is selected from the group consisting of hepatitis B virus genes, angiopoietin-like protein 3 genes, and apolipoprotein C3 genes. Accordingly, the disease is selected from chronic liver disease, hepatitis, hepatic fibrosis, hepatic hyperplasia, and dyslipidemia. In some embodiments, the dyslipidemia is hypercholesterolemia, hypertriglyceridemia, or atherosclerosis.

[0108] In some embodiments, the double-stranded oligonucleotide, the pharmaceutical composition, or the oligonucleotide complex can also be used to treat other liver diseases, including diseases characterized by unwanted cell proliferation, hematological diseases, metabolic diseases, and diseases characterized by inflammation. Liver proliferative diseases can be benign or malignant diseases, such as cancer, hepatocellular carcinoma (HCC), liver metastasis, or hepatoblastoma. Liver hematological or inflammatory diseases can be diseases related to blood coagulation factors, complement-mediated inflammation, or fibrosis. Liver metabolic diseases include lipid abnormalities and irregular glucose regulation.

[0109] The present disclosure further provides a kit comprising the double-stranded oligonucleotide, the pharmaceutical composition, or the oligonucleotide conjugate.

[0110] The following description of the drug composition and oligonucleotide conjugate is based on the aforementioned double-stranded oligonucleotide suitable for regulating gene expression. However, the descriptions of the pharmaceutically acceptable carrier and the ligand in the drug conjugate are also suitable for systemic administration of the modified double-stranded oligonucleotide and delivery of the double-stranded oligonucleotide to a target organ or tissue, particularly the liver, to regulate the expression of endogenous genes expressed in the target organ or tissue or genes of pathogens propagating in the target organ or tissue.

[0111] <Drug Composition> In one aspect, the present disclosure provides a pharmaceutical composition comprising the above-described double-stranded oligonucleotide as an active ingredient and a pharmaceutically acceptable carrier.

[0112] The pharmaceutically acceptable carrier may be a carrier commonly used in the field of double-stranded oligonucleotide administration, such as magnetic nanoparticles (e.g., nanoparticles based on Fe3O4 or Fe2O3), carbon nanotubes, mesoporous silicon, calcium phosphate nanoparticles, polyethylenimine (PEI), polyamidoamine (PAMAM) dendrimer, poly(L-lysine) (PLL), chitosan, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), poly(D&L-lactic / glycolic acid) copolymer (PLGA), poly(aminoethyl ethylene phosphate ester ... The polymerizable monomers include, but are not limited to, one or more of poly(N,N-dimethylaminoethyl methacrylate) (PPEEA) and poly(2-dimethylaminoethyl methacrylate) (PDMAEMA) and derivatives thereof.

[0113] In some embodiments, there are no particular requirements for the contents of the double-stranded oligonucleotide and the pharmaceutically acceptable carrier in the pharmaceutical composition, but in some embodiments, the weight ratio of the double-stranded oligonucleotide to the pharmaceutically acceptable carrier may be 1:(1 to 500). In some specific embodiments, the weight ratio is 1:(1 to 50).

[0114] In some embodiments, the pharmaceutical composition may contain other pharmaceutically acceptable additives, which may be one or more of various agents or compounds commonly used in the art. For example, the other pharmaceutically acceptable additives may include at least one of a pH buffer, a protective agent, and an osmotic pressure adjusting agent.

[0115] The pH buffer solution may be a tris(hydroxymethyl) aminomethane hydrochloride buffer solution of pH 7.5 to 8.5 and / or a phosphate buffer solution of pH 5.5 to 8.5, for example, a phosphate buffer solution of pH 5.5 to 8.5.

[0116] The protective agent may be at least one of inositol, sorbitol, sucrose, trehalose, mannose, maltose, lactose, and glucose, and the content of the protective agent may be 0.01 to 30% by weight based on the total weight of the pharmaceutical composition.

[0117] The osmotic pressure adjusting agent may be sodium chloride and / or potassium chloride. The content of the osmotic pressure adjusting agent is determined so that the osmotic pressure of the drug composition is 200 to 700 mOsmol / kilogram. Those skilled in the art can easily determine the content of the osmotic pressure adjusting agent depending on the desired osmotic pressure.

[0118] In some embodiments, the pharmaceutical composition may be a liquid formulation such as an injection solution, or may be a lyophilized powder injection that is mixed with a liquid additive at the time of administration to form a liquid formulation. The liquid formulation may be used for, but is not limited to, subcutaneous, intramuscular, or intravenous administration, and may also be administered to the lungs by aerosolization, or may be administered to other organ tissues (e.g., the liver) through the lungs by aerosolization. In some specific embodiments, the pharmaceutical composition is used for intravenous administration.

[0119] In some embodiments, the pharmaceutical composition may be in the form of a liposome formulation. In some embodiments, the pharmaceutically acceptable carrier used in the liposome formulation comprises an amine-containing transfection compound (hereinafter also referred to as an organic amine), a co-lipid, and / or a polyethylene glycol (PEG)-modified lipid. Here, the organic amine, co-lipid, and PEG-modified lipid may be one or more selected from the amine-containing transfection compounds or pharmaceutically acceptable salts or derivatives thereof, co-lipids, and PEG-modified lipids described in CN1033113A (incorporated herein by reference in its entirety).

[0120] In some embodiments, the organic amine may be a compound represented by formula (201) described in CN1033113A or a pharmaceutically acceptable salt thereof.

[0121] [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 represents hydrogen, a cyclic or acyclic, substituted or unsubstituted, branched or straight-chain aliphatic group, a cyclic or acyclic, substituted or unsubstituted, branched or straight-chain heteroaliphatic group, a substituted or unsubstituted, branched or straight-chain acyl group, a substituted or unsubstituted, branched or straight-chain aryl group, or a substituted or unsubstituted, branched or straight-chain heteroaryl group; X is an integer from 1 to 10, When n is an integer of 1 to 3, m is an integer of 0 to 20, and p is 0 or 1, and both m and p are 0, R 102 is hydrogen, When at least one of n or m is 2, R 103 and the nitrogen in formula (201) form a structure shown in formula (202) or formula (203).

[0122] [ka] In the formula, g, e, and f are each independently an integer of 1 to 6, "HCC" represents a hydrocarbon chain, and each * N represents a nitrogen atom shown in formula (201).

[0123] In some embodiments, R 103 is a polyamine. In other embodiments, R 103 is a ketal. In some embodiments, R in formula (201) 101 and R 102 are each independently an optionally substituted or unsubstituted, branched or straight chain alkyl or alkenyl, said alkyl or alkenyl having 3 to about 20 carbon atoms, e.g., 8 to about 18 carbon atoms, and 0 to 4 double bonds, e.g., 0 to 2 double bonds.

[0124] 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).

[0125] [ka] In formulas (204) to (213), each "HCC" represents a hydrocarbon chain, and each * is R 103 and the nitrogen atom in formula (201), and * Each H on position may be substituted to provide a bond with the nitrogen atom in formula (201).

[0126] Here, the compound of formula (201) may be prepared according to the description in CN1033113A.

[0127] In some specific embodiments, the organic amine is an organic amine represented by formula (214) and / or an organic amine represented by formula (215).

[0128] [ka]

[0129] the co-lipid is cholesterol, a cholesterol analogue and / or a cholesterol derivative; The PEGylated lipid is 1,2-dipalmitamido-sn-glycero-3-phosphatidylethanolamine-N-[methoxy(polyethylene glycol)]-2000.

[0130] In some embodiments, the molar ratio of the organic amine, the colipid, and the PEGylated lipid in the drug composition is (19.7-80):(19.7-80):(0.3-50), and may be, for example, (50-70):(20-40):(3-20).

[0131] In some embodiments, the drug composition particles formed by the double-stranded oligonucleotide of the present disclosure and the amine-containing transfection reagent have an average diameter of about 30 nm to about 200 nm, typically about 40 nm to about 135 nm; more typically, the average diameter of the liposome particles is about 50 nm to about 120 nm, about 50 nm to about 100 nm, about 60 nm to about 90 nm, or about 70 nm to about 90 nm; for example, the average diameter of the liposome particles is about 30, 40, 50, 60, 70, 75, 80, 85, 90, 100, 110, 120, 130, 140, 150, or 160 nm.

[0132] In some embodiments, in a drug composition formed from a double-stranded oligonucleotide of the present disclosure and the amine-containing transfection reagent, the weight ratio (weight / weight ratio) of the double-stranded oligonucleotide to total lipids (e.g., organic amine, auxiliary lipid, and / or PEGylated lipid) is within the range of about 1:1 to about 1:50, about 1:1 to about 1:30, about 1:3 to about 1:20, about 1:4 to about 1:18, about 1:5 to 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 about 1:6 to about 1:10, for example, the weight ratio of the double-stranded oligonucleotide of the present disclosure to total lipids is about 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.

[0133] In some embodiments, the pharmaceutical composition may be marketed with each component being present independently, or may be present as a liquid formulation at the time of use. In some embodiments, the pharmaceutical composition formed from the double-stranded oligonucleotide provided by the present disclosure and the pharmaceutically acceptable carrier may be prepared according to various known methods, and the double-stranded oligonucleotide provided by the present disclosure may be used instead of a conventional double-stranded oligonucleotide. In some specific embodiments, the pharmaceutical composition may be prepared according to the following method.

[0134] The organic amine, co-lipid, and PEGylated lipid are suspended in alcohol at the above molar ratio and mixed uniformly to obtain a lipid solution. The amount of alcohol is determined so that the total mass concentration of the resulting lipid solution is 2 to 25 mg / mL, for example, 8 to 18 mg / mL. The alcohol may be one or more selected from pharmaceutically acceptable alcohols that are liquid at or near room temperature, such as ethanol, propylene glycol, benzyl alcohol, glycerin, polyethylene glycol 200, polyethylene glycol 300, and polyethylene glycol 400, and may be, for example, ethanol.

[0135] The double-stranded oligonucleotide provided by the present disclosure is dissolved in a buffer salt solution to obtain an aqueous double-stranded oligonucleotide solution. The concentration of the buffer salt solution is 0.05 to 0.5 M, for example, 0.1 to 0.2 M. The pH of the buffer salt solution is adjusted to 4.0 to 5.5, for example, 5.0 to 5.2. The volume of the buffer salt solution is determined so that the concentration of the double-stranded oligonucleotide is 0.6 mg / mL or less, for example, 0.2 to 0.4 mg / mL. The buffer salt is one or more selected from soluble acetates and soluble citrates, for example, sodium acetate and / or potassium acetate.

[0136] After mixing the lipid solution and the double-stranded oligonucleotide aqueous solution, the resulting product is incubated at 40-60°C for at least 2 minutes, for example, 5-30 minutes, to obtain an incubated liposome preparation. The volume ratio of the lipid solution to the double-stranded oligonucleotide aqueous solution may be 1:(2-5), for example, 1:4.

[0137] The cultured liposome preparation is concentrated or diluted, impurities are removed, and bacteria are sterilized to obtain a pharmaceutical composition provided by the present disclosure, whose physicochemical parameters are pH 6.5 to 8, encapsulation efficiency of 80% or more, particle size of 40 to 200 nm, polydispersity index of 0.30 or less, and osmotic pressure of 250 to 400 mOsm / kg; for example, the physicochemical parameters may be pH 7.2 to 7.6, encapsulation efficiency of 90% or more, particle size of 60 to 100 nm, polydispersity index of 0.20 or less, and osmotic pressure of 300 to 400 mOsm / kg.

[0138] Here, concentration or dilution may be performed before, after, or simultaneously with the removal of impurities. Various conventional methods may be used to remove impurities, such as ultrafiltration at 100 kDa using a tangential flow system or a hollow fiber column, and the ultrafiltration exchange solution may be converted into a phosphate buffer solution (PBS) at pH 7.4. Various conventional methods may be used to sterilize the solution, such as filtration through a 0.22 μm filter.

[0139] <Oligonucleotide conjugate> In one aspect, the present disclosure provides an oligonucleotide conjugate comprising the double-stranded oligonucleotide described above and a conjugation group bound to the double-stranded oligonucleotide.

[0140] In the context of the present disclosure, unless otherwise specified, the term "conjugate" refers to the covalent binding of two or more chemical moieties, each having a specific function, to each other. Accordingly, the term "conjugate" refers to a compound formed by the covalent binding of the respective chemical moieties. Furthermore, the term "oligonucleotide conjugate" refers to a compound formed by covalently binding one or more chemical moieties having a specific function to a double-stranded oligonucleotide. Hereinafter, the oligonucleotide conjugate of the present disclosure may also be referred to simply as a "conjugate." More specifically, in the context of the present disclosure, a "conjugate molecule" should be understood to be a specific compound that can be conjugated to a double-stranded oligonucleotide by reaction to ultimately form the oligonucleotide conjugate of the present disclosure. The types and binding methods of the ligands are known to those skilled in the art. Their function generally involves binding to a specific receptor on the surface of a target cell and mediating the delivery of the double-stranded oligonucleotide bound to the ligand to the target cell.

[0141] Typically, the conjugated group comprises at least one pharmaceutically acceptable targeting group and an optional linker, with the double-stranded oligonucleotide, the linker, and the targeting group being sequentially linked. In one embodiment, the number of targeting groups is 1 to 6. In one embodiment, the number of targeting groups is 2 to 4. The double-stranded oligonucleotide molecule may be conjugated to the conjugated group non-covalently or covalently, for example, covalently. The conjugation site between the double-stranded oligonucleotide and the conjugated group may be at the 3'-end or 5'-end of the sense strand of the double-stranded oligonucleotide, at the 5'-end of the antisense strand, or within an internal sequence of the double-stranded oligonucleotide. In some specific embodiments, the conjugation site between the double-stranded oligonucleotide and the conjugated group is at the 3'-end of the sense strand of the double-stranded oligonucleotide.

[0142] In some embodiments, the conjugated group may be attached to the phosphate group, 2'-hydroxy group, or base of a nucleotide. In some embodiments, the conjugated group may be attached to the 3'-hydroxy group, in which case the nucleotides are linked via a 2'-5' phosphodiester bond. When attached to the end of a double-stranded oligonucleotide, the conjugated group is typically attached to the phosphate group of a nucleotide. When attached to an internal sequence of a double-stranded oligonucleotide, the conjugated group is typically attached to the ribose sugar ring or base. For various attachment methods, see Muthiah Manoharan et al., "siRNA conjugates carrying sequentially assembled trivalent N-acetylgalactosamine linked through nucleosides elicit robust gene silencing in vivo in hepatocytes." ACS Chemical Biology, 2015, 10 (5): 1181-7.

[0143] In some embodiments, the double-stranded oligonucleotide and the conjugation group may be linked by an acid-labile or reducible chemical bond, and these chemical bonds can be degraded in the acidic environment of cell endosomes, and the double-stranded oligonucleotide can be released.For non-degradable conjugation methods, the conjugation group is bound to the sense strand of the double-stranded oligonucleotide, so that the influence of conjugation on the activity of the double-stranded oligonucleotide can be minimized.

[0144] The targeting group may be linked to the double-stranded oligonucleotide molecule via a suitable linker, and those skilled in the art can select a suitable linker according to the specific type of targeting group. For these linkers, types of targeting groups and methods of linking to double-stranded oligonucleotides, reference may be made to the disclosure of WO2015006740A2, the contents of which are incorporated herein by reference in their entirety.

[0145] In some embodiments, when the targeting group is N-acetylgalactosamine, a suitable linker may be the structure shown in formula (301):

[0146] [ka] In the formula, k is an integer of 1 to 3, L A is a chain portion containing an amide bond having a structure represented by formula (302), and each of the L A has one of the target groups and one of the L C It is bonded to the moiety by an ether bond.

[0147] [ka] L B is a chain portion containing an N-acylpyrrolidine having a structure represented by formula (303), the chain portion having a carbonyl group at one end thereof, CThe oligonucleotide has an oxygen group at the other end and is bonded to the double-stranded oligonucleotide by a phosphate ester bond.

[0148] [ka] L C is a divalent to tetravalent linker group based on hydroxymethylaminomethane, dihydroxymethylaminomethane or trihydroxymethylaminomethane, and said L C is bonded to each of the L A moieties by an ether bond via an oxygen atom, and is bonded to each of the L A moieties by an amide bond via a nitrogen atom. B The part is connected to the

[0149] In some embodiments, n=3 and L C When -(L) is a tetravalent linker group based on trihydroxymethylaminomethane, -(L) as a linker A )3-trihydroxymethylaminomethane-L B The structure of the oligonucleotide conjugate formed by linking an N-acetylgalactosamine molecule and a double-stranded oligonucleotide molecule by - is shown in formula (304) below.

[0150] [ka] In the formula, the double helix structure represents a double-stranded oligonucleotide.

[0151] Similarly, the conjugation site between the double-stranded oligonucleotide and the conjugation group may be at the 3'-end or 5'-end of the sense strand of the double-stranded oligonucleotide, or at the 5'-end of the antisense strand, or in an internal sequence of the double-stranded oligonucleotide.

[0152] In some specific embodiments, the 3' end of the sense strand of the double-stranded oligonucleotide described in the present disclosure is linked to a linker-(L A )3-trihydroxymethylaminomethane-L B- to three N-acetylgalactosamine (GalNAc) molecules, thereby obtaining an oligonucleotide conjugate having the structure shown in the following formula (305) (hereinafter also referred to as (GalNAc)3-Nu), in which the molar ratio of the double-stranded oligonucleotide molecule to the GalNAc molecule is 1:3.

[0153] [ka] wherein the double helix structure represents the double-stranded oligonucleotide; The linker is attached to the 3' end of the sense strand of the double-stranded oligonucleotide.

[0154] In some embodiments, when the targeting group is N-acetylgalactosamine, a suitable linker may be the structure shown in formula (306):

[0155] [ka] In the formula, l is an integer of 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 double-stranded oligonucleotide via a phosphate ester bond.

[0156] In some specific embodiments, when l=2, the oligonucleotide conjugate has the structure shown in formula (307).

[0157] [ka] wherein the double helix structure represents the double-stranded oligonucleotide; The linker is attached to the 3' end of the sense strand of the double-stranded oligonucleotide.

[0158] The above conjugates may be synthesized by methods already described in detail in the prior art. For example, WO2015006740A2 describes in detail the preparation of several types of conjugates. WO20140255A1 also describes a method for preparing the structure shown in formula (305). Furthermore, Rajeev et al., ChemBioChem 2015, 16, 903-908, described a method for preparing the structure shown in formula (307).

[0159] In some embodiments, the complex has the structure shown in formula (308).

[0160] [ka] In the formula, 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 C-C 10 Alkyl groups, C1-C 10 Halogenated alkyl groups and C1-C 10 alkoxy groups; R3 is a group having the structure shown in formula A59.

[0161] [ka] wherein E1 is OH, SH or BH2, and Nu is a double-stranded oligonucleotide.

[0162] R2 is a straight chain alkylene group of 1 to 20 carbon atoms in length, where one or more of the carbon atoms is selected from the group consisting of C(O), NH, O, S, CH=N, S(O)2, C2-C 10 Alkenylene group, C2-C 10 Alkynylene group, C6-C 10Arylene group, C3-C 18 Heterocyclylene groups and C5-C 10 heteroarylene groups, and R2 is C1-C 10 Alkyl groups, C6-C 10 Aryl groups, C5-C 10 Heteroaryl groups, C1-C 10 Halogenated alkyl groups, -OC1-C 10 Alkyl group, -OC1-C 10 Alkylphenyl group, -C1-C 10 Alkyl-OH, -OC1-C 10 Halogenated alkyl groups, -SC1-C 10 Alkyl group, -SC1-C 10 Alkylphenyl group, -C1-C 10 Alkyl-SH, -SC1-C 10 Halogenated alkyl groups, halogen substituents, -OH, -SH, -NH2, -C1-C 10 Alkyl-NH2, -N(C1-C 10 Alkyl group) (C1-C 10 alkyl group), -NH(C1-C 10 alkyl group), cyano group, nitro group, -CO2H, -C(O)O(C1-C 10 alkyl group), -CON(C1-C 10 Alkyl group) (C1-C 10 alkyl group), -CONH(C1-C 10 alkyl group), -CONH2, -NHC(O)(C1-C 10 alkyl group), -NHC(O)(phenyl group), -N(C1-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), -SO2 (phenyl group), -SO2 (C1-C 10Halogenated alkyl group), -SO2NH2, -SO2NH(C1-C 10 alkyl group), -SO2NH (phenyl group), -NHSO2 (C1-C 10 alkyl group), -NHSO2 (phenyl group) and -NHSO2 (C1-C 10 The alkyl group may optionally have one or more substituents selected from the group consisting of halogenated alkyl groups.

[0163] Each L1 is a straight chain alkylene group of 1 to 70 carbon atoms in length, where one or more of the carbon atoms is selected from the group consisting of 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 groups and C5-C 10 heteroarylene groups, and L is C-C 10 Alkyl groups, C6-C 10 Aryl groups, C5-C 10 Heteroaryl groups, C1-C 10 Halogenated alkyl groups, -OC1-C 10 Alkyl group, -OC1-C 10 Alkylphenyl group, -C1-C 10 Alkyl-OH, -OC1-C 10 Halogenated alkyl groups, -SC1-C 10 Alkyl group, -SC1-C 10 Alkylphenyl group, -C1-C 10 Alkyl-SH, -SC1-C 10 Halogenated alkyl groups, halogen substituents, -OH, -SH, -NH2, -C1-C 10 Alkyl-NH2, -N(C1-C 10 Alkyl group) (C1-C 10 alkyl group), -NH(C1-C 10 alkyl group), cyano group, nitro group, -CO2H, -C(O)O(C1-C 10 alkyl group), -CON(C1-C 10 Alkyl group) (C1-C 10 alkyl group), -CONH(C1-C10 alkyl group), -CONH2, -NHC(O)(C1-C 10 alkyl group), -NHC(O)(phenyl group), -N(C1-C 10 alkyl)C(O)(C1-C 10 alkyl group), -N(C1-C 10 alkyl)C(O)(phenyl 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), -SO2 (phenyl group), -SO2 (C1-C 10 Halogenated alkyl group), -SO2NH2, -SO2NH(C1-C 10 alkyl group), -SO2NH (phenyl group), -NHSO2 (C1-C 10 alkyl group), -NHSO2 (phenyl group) and -NHSO2 (C1-C 10 M1 may optionally have one or more substituents from the group consisting of halogenated alkyl groups. M1 represents a targeting group.

[0164] In some embodiments, L1 may be selected from the group consisting of groups A1 to A26 or any combination thereof, where the structures and definitions of A1 to A26 are as follows:

[0165] [ka] where j1 is an integer from 1 to 20, and j2 is an integer from 1 to 20, R' is C1-C 10 is an alkyl group of the formula Ra is one selected from the groups of formulae A27 to A45.

[0166] [ka] Rb is C1-C 10 is an alkyl group of the formula TIFF0007727308000031.tif5163 represents the site at which the group is attached to the rest of the molecule.

[0167] For convenience, L1 is the linear Alkylene group However, it will be understood by those skilled in the art that, for example, amines and alkenyl groups resulting from the above-described replacement and / or substitution may not be linear groups or may have different names. For purposes of this disclosure, the length of L1 is the number of atoms in the chain connecting the two attachment points. For this purpose, a ring (e.g., heterocyclylene or heteroarylene) obtained by substituting a carbon atom of the linear alkylene is considered to be one atom.

[0168] In some embodiments, the pharmaceutically acceptable targeting group may be a ligand commonly used in the field of double-stranded oligonucleotide administration, such as various ligands described in WO2009082607A2, the disclosure of which is incorporated herein by reference in its entirety.

[0169] In some embodiments, each of the ligands is independently selected from ligands capable of binding to a cell surface receptor. In some embodiments, at least one ligand is capable of binding to a receptor on the surface of hepatocytes. In some embodiments, at least one ligand is capable of binding to a receptor on the surface of mammalian hepatocytes. In some embodiments, at least one ligand is capable of binding to a receptor on the surface of human hepatocytes. In some embodiments, at least one ligand is capable of binding to an asialoglycoprotein receptor (ASGPR) on the surface of liver. These types of ligands are known to those skilled in the art, and their function generally involves binding to a specific receptor on the surface of target cells and mediating the delivery of a double-stranded oligonucleotide bound to the ligand to the target cell.

[0170] In some embodiments, the pharmaceutically acceptable targeting group can be any one of a number of ligands that bind to the asialoglycoprotein receptor (ASGPR) on the surface of mammalian liver cells. In one embodiment, each ligand is independently an asialoglycoprotein, such as asialoorosomucoid (ASOR) or asialofetuin (ASF).

[0171] In some embodiments, the pharmaceutically acceptable targeting group may be one or more selected from the group consisting of lipophilic molecules such as cholesterol, bile acids, vitamins (e.g., tocopherol), and lipid molecules of different chain lengths; polymers such as polyethylene glycol; polypeptides such as membrane-permeable peptides; aptamers; antibodies; quantum dots; sugars such as lactose, polylactose, mannose, galactose, and N-acetylgalactosamine (GalNAc); folate; and ligands formed by targeting molecules or derivatives thereof, such as asialoglycoproteins, asialoglycores, lipoproteins (e.g., high-density lipoproteins, low-density lipoproteins, etc.), glucagon, neurotransmitters (e.g., adrenaline), growth factors, and receptor ligands expressed in hepatic parenchymal cells, such as transferrin.

[0172] In one embodiment, the ligand is a sugar or a sugar derivative.

[0173] In some embodiments, at least one ligand is a sugar. In some embodiments, each of the ligands is a sugar. In some embodiments, at least one ligand is a monosaccharide, a polysaccharide, a modified monosaccharide, a modified polysaccharide, or a sugar derivative. In some 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 of the ligands is independently selected from a polysaccharide, a modified polysaccharide, a monosaccharide, a modified monosaccharide, a polysaccharide derivative, or a monosaccharide derivative. In some embodiments, each or at least one of the ligands is selected from the group consisting of glucose and derivatives thereof, mannan and derivatives thereof, galactose and derivatives thereof, xylose and derivatives thereof, ribose and derivatives thereof, fucose and derivatives thereof, lactose and derivatives thereof, maltose and derivatives thereof, arabinose and derivatives thereof, fructose and derivatives thereof, and sialic acid.

[0174] In some embodiments, each of the ligands is 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, 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, Nn-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 The ligands may be independently selected from ethyl 2,3,4-tris-O-acetyl-1-thio-6-O-trityl-α-D-glucopyranoside, 4-thio-β-D-galactopyranose, ethyl 3,4,6,7-tetra-O-acetyl-2-deoxy-1,5-dithio-α-D-glucoheptopyranoside, 2,5-anhydro-D-allonitrile, ribose, D-ribose, D-4-thioribose, L-ribose, and L-4-thioribose. For other options for the ligands, see, for example, CN105378082A, the disclosure of which is incorporated herein by reference in its entirety.

[0175] In some embodiments, the pharmaceutically acceptable targeting group in the oligonucleotide conjugate may be galactose or N-acetylgalactosamine, and the galactose or N-acetylgalactosamine molecule may be monovalent, divalent, trivalent, or tetravalent. It should be understood that monovalent, divalent, trivalent, and tetravalent herein refer to a molar ratio of double-stranded oligonucleotide molecules to galactose or N-acetylgalactosamine molecules of 1:1, 1:2, 1:3, or 1:4, respectively, in an oligonucleotide conjugate formed from a double-stranded oligonucleotide molecule and a conjugated group containing a galactose or N-acetylgalactosamine molecule as a targeting group. In some embodiments, the pharmaceutically acceptable targeting group is N-acetylgalactosamine. In some embodiments, when a double-stranded oligonucleotide described in the present disclosure is conjugated to a conjugated group containing N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent or tetravalent. In some embodiments, when the double-stranded oligonucleotides described in the present disclosure are conjugated to a conjugation group that includes N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent.

[0176] M1 represents a targeting group, and its definition and selectable range are the same as those described above. In some embodiments, each M1 is independently selected from ligands having affinity for the asialoglycoprotein receptor on the surface of mammalian liver cells.

[0177] When M1 is a ligand having affinity for the asialoglycoprotein receptor on the surface of mammalian liver cells, in some embodiments, n1 may be an integer from 1 to 3, and n3 may be an integer from 0 to 4, ensuring that the number of M1 ligands in the conjugate is at least 2. In some embodiments, n1 + n3 ≥ 2, whereby the number of M1 ligands is at least 3, which facilitates binding of the M1 ligand to the asialoglycoprotein receptor on the liver surface and promotes endocytosis of the conjugate. Experiments have shown that using three or more M1 ligands does not significantly improve the ease of binding of the M1 ligand to the asialoglycoprotein receptor on the liver surface. Therefore, taking into consideration various aspects such as ease of synthesis, structure / processing cost, and delivery efficiency, in some embodiments, n1 is an integer from 1 to 2, n3 is an integer from 0 to 1, and n1 + n3 = 2 to 3.

[0178] In some embodiments, when m1, m2, and m3 are independently selected from integers of 2 to 10, the spatial positioning between multiple M1 ligands can be optimized for binding of the M1 ligand to the asialoglycoprotein receptor on the liver surface. To simplify, synthesize, and / or reduce costs of the conjugates provided by the present disclosure, in some embodiments, m1, m2, and m3 are each independently an integer of 2 to 5, and in some embodiments, m1 = m2 = m3.

[0179] R 10 , R 11 , R 12 , R 13 , R 14 and R 15 are independently H, C1-C 10 Alkyl groups, C1-C 10 Halogenated alkyl groups and C1-C 10It will be understood by those skilled in the art that when R is one selected from the group consisting of alkoxy, any of the above will achieve the objectives of the present disclosure without changing the properties of the conjugates disclosed herein. 10 , R 11 , R 12 , R 13 , R 14 and R 15 are each independently selected from H, a methyl group, and an ethyl group. In some embodiments, R 10 , R 11 , R 12 , R 13 , R 14 and R 15 are both H.

[0180] In the oligonucleotide conjugates provided by the present disclosure, R3 is a group having the structure shown in formula A59, in which E1 is OH, SH, or BH2, and in some embodiments, E1 is OH or SH, taking into account the availability of preparation raw materials.

[0181] In some embodiments, R2 is selected to provide a bond between N on the nitrogen-containing backbone and A59. In the context of this disclosure, a "nitrogen-containing backbone" refers to R 10 , R 11 , R 12 , R 13 , R 14 and R 15A59 refers to a chain structure in which the carbon atom to which A59 is attached and the N are bonded to each other. Thus, R2 may be any linker group capable of linking the A59 group to the N on the nitrogen-containing backbone in an appropriate manner. In some embodiments, when preparing the oligonucleotide conjugate of the present disclosure by solid phase synthesis, the R2 group must include both a binding site bound to the N on the nitrogen-containing backbone and a binding site bound to P in R3. In some embodiments, the site bound to N in the nitrogen-containing backbone in R2 forms an amide bond with N, and the site bound to P in R3 forms a phosphate ester bond with P. In some embodiments, R2 is B5, B6, B5', or B6'.

[0182] [ka] however, TIFF0007727308000033.tif4165 represents the site to which the group is covalently attached.

[0183] The value of q2 may range from 1 to 10, and in some embodiments, q2 is an integer from 1 to 5.

[0184] L1 serves to connect the M1 ligand to an N on the nitrogen-containing backbone and provide targeting functionality to the oligonucleotide conjugates of the present disclosure. In some embodiments, L1 is a combination of one or more bonds selected from groups of formulae A1-A26. In some embodiments, L1 is a combination of one or more bonds selected from A1, A4, A5, A6, A8, A10, A11, and A13. In some embodiments, L1 is a combination of at least two bonds selected from A1, A4, A8, A10, and A11. In some embodiments, L1 is a combination of at least two bonds selected from A1, A8, and A10.

[0185] In some embodiments, the length of L1 can be 3 to 25 atoms, 3 to 20 atoms, 4 to 15 atoms, or 5 to 12 atoms. In some embodiments, the length of L1 is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, or 60 atoms.

[0186] In some embodiments, j1 is an integer from 2 to 10, and in some embodiments, j1 is an integer from 3 to 5. In some embodiments, j2 is an integer from 2 to 10, and in some embodiments, j2 is an integer from 3 to 5. R' is a C1-C4 alkyl group, and in some embodiments, R' is one of methyl, ethyl, and isopropyl. Ra is one of A27, A28, A29, A30, and A31, and in some embodiments, Ra is A27 or A28. Rb is a C1-C5 alkyl group, and in some embodiments, Rb is one of methyl, ethyl, isopropyl, and butyl. In some embodiments, j1, j2, R', Ra, and Rb in Formulae A1-A26 are selected, respectively, to achieve binding between the M1 ligand and N on the nitrogen-containing backbone and further optimize the spatial location between the M1 ligands for binding to the asialoglycoprotein receptor on the liver surface.

[0187] In some embodiments, the oligonucleotide conjugate of the present disclosure has 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).

[0188] [ka] TIFF0007727308000035.tif220153TIFF0007727308000036.tif211163TIFF0007727308 000037.tif245153TIFF0007727308000038.tif226159TIFF0007727308000039.tif89145

[0189] In some embodiments, P in Formula A59 may be attached to any possible position in the double-stranded oligonucleotide sequence. For example, P in Formula A59 may be attached to any one nucleotide in the sense strand or antisense strand of the double-stranded oligonucleotide. In some embodiments, P in Formula A59 is attached to any one nucleotide in the sense strand of the double-stranded oligonucleotide. In some embodiments, P in Formula A59 is attached to the end of the sense strand or antisense strand of the double-stranded oligonucleotide. In some embodiments, P in Formula A59 is attached to the end of the sense strand of the double-stranded oligonucleotide. The end refers to the previous 4 nucleotides from one end of the sense strand or the antisense strand. In some embodiments, P in Formula A59 is attached to the end of the sense strand or the antisense strand of the double-stranded oligonucleotide. In some embodiments, P in Formula A59 is attached to the 3' end of the sense strand of the double-stranded oligonucleotide. When bound to the above-described position of the sense strand of a double-stranded oligonucleotide, the conjugate provided by the present disclosure can enter a cell and, upon unwinding, release the single antisense strand of the double-stranded oligonucleotide, thereby modulating target gene expression.

[0190] P in Formula A59 may be attached to any available position on a nucleotide in a double-stranded oligonucleotide, for example, the 5' position of the nucleotide, the 2' position of the nucleotide, the 3' position of the nucleotide, or the base of the nucleotide. In some embodiments, P in Formula A59 may be attached to the 2', 3', or 5' position of a nucleotide in the double-stranded oligonucleotide by forming a phosphodiester bond. In some embodiments, P in Formula A59 is attached to the dehydrogenated oxygen atom of the 3' hydroxy group of the 3'-terminal nucleotide in the sense strand of the double-stranded oligonucleotide, or P in Formula A59 is attached to a nucleotide by replacing the hydrogen in the 2'-hydroxy group of a nucleotide in the sense strand of the double-stranded oligonucleotide, or P in Formula A59 is attached to a nucleotide by replacing the hydrogen in the 5' hydroxy group of the 5'-terminal nucleotide in the sense strand of the double-stranded oligonucleotide.

[0191] In the double-stranded oligonucleotide or oligonucleotide complex described in the present disclosure, adjacent nucleotides are linked by a phosphodiester bond or a phosphorothiodiester bond, and non-bridging oxygen or sulfur atoms in the phosphodiester or phosphorothiodiester bond may be negatively charged and exist as hydroxyl or sulfhydryl groups, and the hydrogen ions in the hydroxyl or sulfhydryl groups may be partially or completely replaced by cations. The cations may be any cation, for example, a metal cation, an ammonium ion (NH4), or the like. + , or one of organic ammonium cations. In one embodiment, in consideration of improving solubility, the cation is one or more selected from alkali metal ions, ammonium cations formed with tertiary amines, and quaternary ammonium cations. The alkali metal ion is K + and / or Na +and the cation formed by the tertiary amine may be an ammonium ion formed by triethylamine and / or an ammonium ion formed by N,N-diisopropylethylamine. Thus, the double-stranded oligonucleotides or oligonucleotide complexes described in the present disclosure may exist at least partially as salts. In one form, at least a portion of the non-bridging oxygen or sulfur atoms in the phosphodiester or thiophosphodiester linkages are bound to sodium ions, and the double-stranded oligonucleotides or oligonucleotide complexes described in the present disclosure exist as sodium salts or partial sodium salts.

[0192] As those skilled in the art will be aware, modified nucleotide groups can be introduced into the double-stranded oligonucleotides described in the present disclosure by using nucleoside monomers with corresponding modifications.Methods for preparing nucleoside monomers with corresponding modifications and methods for introducing modified nucleotide groups into double-stranded oligonucleotides are also well known to those skilled in the art.All modified nucleoside monomers can be purchased commercially or prepared by known methods.

[0193] <Preparation of Oligonucleotide Conjugate of Formula (308)> The oligonucleotide conjugates of the present disclosure may be prepared by any reasonable synthetic route.

[0194] In some embodiments, the oligonucleotide conjugate of formula (308) can be prepared by the following method. The method includes sequentially linking nucleoside monomers from 3' to 5' according to the type and order of nucleotides in the sense and antisense strands of the double-stranded oligonucleotide under phosphoramidite solid-phase synthesis conditions, the linking of each nucleoside monomer comprising four reactions: deprotection, coupling, capping, and oxidation or sulfurization, and isolating and annealing the sense and antisense strands of the double-stranded oligonucleotide. The double-stranded oligonucleotide is the double-stranded oligonucleotide disclosed above.

[0195] The method further comprises contacting the compound of formula (321) with a nucleoside monomer or a nucleotide sequence bound to a solid support under coupling reaction conditions and in the presence of a coupling reagent, thereby coupling the compound of formula (321) to the nucleotide sequence. Hereinafter, the compound of formula (321) is also referred to as a conjugated molecule.

[0196] [ka] During the ceremony, R4 is a moiety that can be bound to a double-stranded oligonucleotide of the present disclosure. In some embodiments, R4 is a moiety that can be bound to a double-stranded oligonucleotide of the present disclosure by a covalent bond. In some embodiments, R4 is a moiety that can be reacted to be conjugated to any functional group of a double-stranded oligonucleotide by a phosphodiester bond; Each S1 is independently a group in which all active hydroxy groups in M1 have been replaced with YCOO- groups, and each Y is independently one selected from a methyl group, a trifluoromethyl group, a difluoromethyl group, a fluoromethyl group, a trichloromethyl group, a dichloromethyl group, a chloromethyl group, an ethyl group, an n-propyl group, an isopropyl group, a phenyl group, a halophenyl group, and an alkylphenyl group.

[0197] 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.

[0198] R4 is selected to provide attachment to the N on the nitrogen-containing backbone and to provide a suitable reactive site for synthesis of an oligonucleotide conjugate of formula 308. In some embodiments, R4 includes an R2 linker group or a protected R2 linker group and a functional group that can react with a double-stranded oligonucleotide to form the structure shown in A59.

[0199] In some embodiments, R4 comprises a first functional group capable of forming a phosphite ester with a group on the double-stranded oligonucleotide or nucleoside monomer and a second functional group capable of reacting with a hydroxyl or amino group to form a covalent bond, or comprises a solid support covalently bound thereto. In some embodiments, the first functional group is a phosphoramidite, a hydroxyl group, or a protected hydroxyl group. In some embodiments, the second functional group is a phosphoramidite, a carboxylic acid, or a carboxylate. In some embodiments, the second functional group is a solid support bound to another part of the molecule via a covalent bond, the covalent bond being formed by a hydroxyl or amino group. In some embodiments, the solid support is bound via a phosphate ester bond, a carboxylic ester bond, or an amide bond. In some embodiments, the solid support is a resin.

[0200] In some embodiments, the first functional group is a hydroxy group, —OR k or a group represented by formula (C3), and the second functional group comprises a structure represented by formula (C1), (C2), (C3), (C1') or (C3').

[0201] [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, TIFF0007727308000042.tif4166 represents the site at which the group is covalently attached to the remainder of the molecule.

[0202] In some embodiments, the first functional group comprises a phosphoramidite group, as shown in Formula (C3), which can be coupled with a hydroxy group at any position on a nucleotide, such as the 2'- or 3'-hydroxy group, to form a phosphite ester, which can then be oxidized or sulfurized to form a phosphodiester or thiophosphate bond as shown in Formula A59, allowing the conjugated molecule to be conjugated to a double-stranded oligonucleotide. In this case, even if the second functional group is absent, the compound of Formula (321) can also be conjugated to a nucleotide, and this does not affect the formation of the oligonucleotide conjugate shown in Formula (308). In this case, after obtaining the sense or antisense strand of a double-stranded oligonucleotide by a method such as phosphoramidite solid-phase synthesis, the compound of Formula (321) can be reacted with the hydroxy group on the terminal nucleotide in the nucleotide sequence, and a subsequent oxidation or sulfurization process can form a phosphodiester or thiophosphate bond, allowing the compound of Formula (321) to be conjugated to a double-stranded oligonucleotide.

[0203] 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, thereby providing a conjugated molecule comprising the solid support. In some embodiments, the second functional group comprises a carboxy group, a carboxylate, or a phosphoramidite, as shown in formula (C1), (C2), or (C3). When the second functional group comprises a carboxy group or a carboxylate, the compound of formula (321) is esterified or amidated with a hydroxy group or an amino group on a solid support, e.g., a resin, to form a conjugated molecule comprising the solid support linked by a carboxylic acid ester bond or an amide bond. When the second functional group comprises a phosphoramidite functional group, the compound of formula (321) is coupled with a hydroxy group on a general-purpose solid support, e.g., a resin, and oxidized to form a conjugated molecule comprising the solid support linked by a phosphodiester bond. Then, starting from the solid support-bound product, nucleoside monomers are sequentially coupled according to the phosphoramidite solid-phase synthesis method to obtain the sense or antisense strand of a double-stranded oligonucleotide to which a conjugated group is attached. During the phosphoramidite solid-phase synthesis process, the first functional group is deprotected and then coupled with the phosphoramidite group on the nucleoside monomer under coupling reaction conditions.

[0204] In some embodiments, the first functional group comprises a hydroxy group or a protected hydroxy group, and the second functional group comprises a solid-phase support bound by a carboxylic acid ester bond, a solid-phase support bound by an amide bond, or a solid-phase support bound by a phosphate ester bond, as shown in formula (C1') or (C3'). In this case, a compound of formula (321) is used as a starting material instead of a solid-phase support, and nucleoside monomers are sequentially bound according to a phosphoramidite solid-phase synthesis method to obtain a sense strand or an antisense strand of a double-stranded oligonucleotide bound with a conjugated group.

[0205] In some embodiments, the carboxylate is —COO - M+ where M + is a cation, for example, a metal cation, an ammonium cation NH + In one embodiment, the metal ion is an alkali metal ion, such as K + or Na + In some embodiments, in order to improve solubility and facilitate the reaction, the organic ammonium ion is an ammonium cation formed with a tertiary amine or a quaternary ammonium cation, such as an ammonium ion formed with triethylamine or an ammonium ion formed with N,N-diisopropylethylamine. In some embodiments, the carboxylate is triethylamine carboxylate or N,N-diisopropylethylamine carboxylate.

[0206] In some embodiments, R4 comprises a structure shown in formula (B9), (B10), (B9'), (B10'), (B11), (B12), (B11'), or (B12').

[0207] [ka] In the formula, q1 is an integer of 1 to 4, q2 is an integer of 1 to 10, X is O or NH, and M + is a cation and R k is a hydroxy protecting group, SPS represents a solid phase support, TIFF0007727308000044.tif5167 represents the site at which the group is covalently attached to the remainder of the molecule. In some embodiments, q1 is 1 or 2. In some embodiments, q2 is an integer from 1 to 5. In some embodiments, R4 comprises a structure shown in formula (B9) or (B10). In some embodiments, R4 comprises a structure shown in formula (B11) or (B12).

[0208] In some embodiments, Rk are Tr (trityl group), MMTr (4-methoxytrityl group), DMTr (4,4'-bismethoxytrityl group), TMTr (4,4',4'-trimethoxy Trityl In some embodiments, R k may be DMTr, ie, 4,4'-dimethoxytrityl.

[0209] L1 refers to a linear alkylene group of 1 to 70 carbon atoms in length, in which one or more carbon atoms are selected from the group consisting of 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 groups and C5-C 10 heteroarylene groups, and L is C-C 10 Alkyl groups, C6-C 10 Aryl groups, C5-C 10 Heteroaryl groups, C1-C 10 Halogenated alkyl groups, -OC1-C 10 Alkyl group, -OC1-C 10 Alkylphenyl group, -C1-C 10 Alkyl-OH, -OC1-C 10 Halogenated alkyl groups, -SC1-C 10 Alkyl group, -SC1-C 10 Alkylphenyl group, -C1-C 10 Alkyl-SH, -SC1-C 10 Halogenated alkyl groups, halogen substituents, -OH, -SH, -NH2, -C1-C 10 Alkyl-NH2, -N(C1-C 10 Alkyl group) (C1-C 10 alkyl group), -NH(C1-C 10 alkyl group), cyano group, nitro group, -CO2H, -C(O)OC1-C 10 Alkyl group, -CON(C1-C 10 Alkyl group) (C1-C 10alkyl group), -CONH(C1-C 10 alkyl group), -CONH2, -NHC(O)(C1-C 10 alkyl group), -NHC(O)(phenyl group), -N(C1-C 10 alkyl)C(O)(C1-C 10 alkyl group), -N(C1-C 10 alkyl)C(O)(phenyl 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), -SO2 (phenyl group), -SO2 (C1-C 10 Halogenated alkyl group), -SO2NH2, -SO2NH(C1-C 10 alkyl group), -SO2NH (phenyl group), -NHSO2 (C1-C 10 alkyl group), -NHSO2 (phenyl group) and -NHSO2 (C1-C 10 The alkyl group may optionally have one or more substituents selected from the group consisting of halogenated alkyl groups.

[0210] In some embodiments, L1 is used to attach the M1 ligand to an N atom on the nitrogen-containing backbone and provide liver targeting functionality to the oligonucleotide conjugate. In some embodiments, L1 comprises any one or combination of A1-A26.

[0211] From the above description, those skilled in the art can easily understand that, compared to the phosphoramidite solid-phase synthesis method known in the art, the above-mentioned first functional group and optional second functional group can be used to obtain an oligonucleotide conjugate in which the conjugated molecule is bound to any possible position of the nucleotide sequence, for example, the end of the nucleotide sequence. Accordingly, unless otherwise specified, when reactions such as "deprotection," "coupling," "capping," "oxidation," and "sulfurization" are mentioned in the following description of the preparation of the conjugate, it should be understood that the reaction conditions and reagents related to the phosphoramidite nucleic acid solid-phase synthesis method known in the art also apply to these reactions. Exemplary reaction conditions and reagents are described in detail below.

[0212] In some embodiments, each S1 is independently M1. In some embodiments, each S1 is independently a group in which at least one active hydroxy group in M1 is protected with a hydroxy-protecting group. In some embodiments, each S1 is independently a group in which all active hydroxy groups present in M1 are protected with hydroxy-protecting groups. In some embodiments, any hydroxy-protecting group known to those skilled in the art can be used to protect the active hydroxy group in M1. In some embodiments, the protected hydroxy group may be represented by the formula YCOO-, where each Y is independently C1-C 10 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 selected from the group consisting of halogen and C1-C6 alkyl groups. In some embodiments, each Y is independently selected from the group consisting of methyl, trifluoromethyl, difluoromethyl, monofluoromethyl, trichloromethyl, dichloromethyl, chloromethyl, ethyl, n-propyl, isopropyl, phenyl, halophenyl, and C1-C6 alkylphenyl groups.

[0213] In some embodiments, each S1 is independently selected from the group consisting of formulae A46-A54.

[0214] [ka]

[0215] In some embodiments, S1 is of formula A49 or A50.

[0216] In some embodiments, each Y is independently selected from a methyl group, a trifluoromethyl group, a difluoromethyl group, a fluoromethyl group, a trichloromethyl group, a dichloromethyl group, a chloromethyl group, an ethyl group, an n-propyl group, an isopropyl group, a phenyl group, a halophenyl group, and an alkylphenyl group; in some embodiments, Y is a methyl group.

[0217] As described above, the method for preparing an oligonucleotide conjugate of the present disclosure further includes synthesizing the other strand of the double-stranded oligonucleotide (for example, if the sense strand of the double-stranded oligonucleotide to which a conjugated group is attached is synthesized in the above step, the method further includes synthesizing the antisense strand of the double-stranded oligonucleotide according to a solid-phase synthesis method, or vice versa), isolating the sense strand and the antisense strand, and annealing them. Specifically, in the isolation step, the solid-phase support to which the nucleotide sequence and / or the conjugated group is attached is cleaved, and necessary protecting groups are removed (in this case, each S1 group in the compound of formula (321) is converted to the corresponding M1 ligand), thereby obtaining the sense strand (or antisense strand) of the double-stranded oligonucleotide to which a conjugated group is attached and the corresponding antisense strand (or sense strand). The sense strand and the antisense strand are then annealed to form a double-stranded RNA structure, thereby obtaining the oligonucleotide conjugate represented by formula (308).

[0218] In some embodiments, the method for preparing the oligonucleotide conjugate includes contacting a compound represented by formula (321) with the first nucleoside monomer at the 3' end of a sense strand or an antisense strand under coupling reaction conditions and in the presence of a coupling reagent, thereby binding the compound represented by formula (321) to the first nucleotide in the sequence, and sequentially binding the nucleoside monomers from 3' to 5' according to the type and order of nucleotides in the desired sense strand or antisense strand under phosphoramidite solid phase synthesis conditions, thereby synthesizing the sense strand or antisense strand of a double-stranded oligonucleotide, wherein the compound represented by formula (321) includes a first functional group containing a protected hydroxy group and a second functional group having a structure represented by formula (C1') or (C3') in R4. the compound of formula (321) is deprotected before binding to the first nucleoside monomer, and the binding of each nucleoside monomer involves four reactions, namely, deprotection, coupling, capping, and oxidation or sulfurization, to obtain a sense strand or an antisense strand of nucleic acid bound to a conjugated group; and the nucleoside monomers are sequentially bound from 3' to 5' under phosphoramidite solid-phase synthesis conditions, depending on the type and order of nucleotides in the antisense strand or the sense strand, to synthesize an antisense strand or a sense strand of nucleic acid, and the binding of each nucleoside monomer involves four reactions, namely, deprotection, coupling, capping, oxidation or sulfurization, to remove the protecting groups, cleave the compound from the solid-phase support, isolate and purify the compound, and then anneal the resulting nucleic acid sense strand and antisense strand.

[0219] In some embodiments, the method for preparing the oligonucleotide conjugate includes the steps of synthesizing a sense strand and an antisense strand by sequentially linking nucleoside monomers from 3' to 5' depending on the type and order of nucleotides in the sense strand or antisense strand of the double-stranded oligonucleotide, wherein the linkage of each nucleoside monomer involves four reactions: deprotection, coupling, capping, and oxidation or sulfurization, to obtain a sense strand bound to a solid support and an antisense strand bound to a solid support; contacting a compound represented by formula (321) with the sense strand bound to the solid support or the antisense strand bound to the solid support under coupling reaction conditions and in the presence of a coupling reagent to link the compound represented by formula (321) containing a first functional group that is a phosphoramidite group at R4 to the sense strand or the antisense strand; removing the protecting groups and cleaving the compound from the solid support, isolating and purifying the compound, respectively, to obtain the sense strand or the antisense strand of the double-stranded oligonucleotide, and annealing the resulting compound. A conjugated group is linked to the sense strand or the antisense strand of the double-stranded oligonucleotide.

[0220] In some embodiments, P in formula A59 is attached to the 3' end of the sense strand in the double-stranded oligonucleotide, and the method for preparing the oligonucleotide conjugate of the present disclosure comprises: (1) A compound of formula (321) (the compound of formula (321) has a protected hydroxy group OR at R4) k and a second functional group having a structure represented by formula (C1') or (C3'). k and contacting the deprotected product with a nucleoside monomer under coupling reaction conditions and in the presence of a coupling reagent to obtain a nucleoside monomer attached to a solid support by a conjugated group. (2) Starting from the nucleoside monomer bound to the solid support by the conjugated molecule, synthesizing the sense strand of a double-stranded oligonucleotide by phosphoramidite solid-phase synthesis in the 3'-5' direction; (3) synthesizing the antisense strand of the double-stranded oligonucleotide by phosphoramidite solid phase synthesis; (4) isolating and annealing the sense and antisense strands of the double-stranded oligonucleotide to obtain the oligonucleotide complex of the present disclosure.

[0221] In step (1), the protecting group R in the compound of formula (321) k The method for removing (321) includes contacting the compound of Formula (321) with a deprotecting reagent under deprotecting conditions. The deprotecting conditions include a temperature of 0 to 50°C, and in some embodiments, 15 to 35°C, a reaction time of 30 to 300 seconds, and in some embodiments, 50 to 150 seconds, and the deprotecting reagent may be one or more selected from trifluoroacetic acid, trichloroacetic acid, dichloroacetic acid, and chloroacetic acid, and in some embodiments, is dichloroacetic acid. The molar ratio of the deprotecting reagent to the compound of Formula (321) is 10:1 to 1000:1, and in some embodiments, 50:1 to 500:1.

[0222] The coupling reaction conditions and coupling reagents may be any conditions and reagents suitable for the above-described coupling reaction, and in some embodiments, may be the same as those used for the coupling reaction in the solid-phase synthesis method employed.

[0223] In some embodiments, the coupling reaction conditions include a reaction temperature of 0 to 50°C, and in some embodiments, 15 to 35°C. The molar ratio of the compound of Formula (321) to the nucleoside monomer may be 1:1 to 1:50, and in some embodiments, 1:2 to 1:5. The molar ratio of the compound of Formula (321) to the coupling reagent may be 1:1 to 1:50, and in some embodiments, 1:3 to 1:10. The reaction time is 200 to 3000 seconds, and in some embodiments, 500 to 1500 seconds. The coupling reagent may be one or more selected from 1H-tetrazole, 5-ethylthio-1H-tetrazole, and 5-benzylthio-1H-tetrazole, and in some embodiments, 5-ethylthio-1H-tetrazole. The coupling reaction may be carried out in an organic solvent, and the organic solvent may be one or more selected from anhydrous acetonitrile, anhydrous DMF, and anhydrous dichloromethane, and in some embodiments, anhydrous acetonitrile. With respect to the compound of formula (321), the amount of the organic solvent is 3 to 50 L / mol, and in some embodiments, 5 to 20 L / mol.

[0224] In step (2), the sense strand S of the oligonucleotide conjugate is synthesized in the 3'-5' direction using the nucleoside monomer bound to the solid support by the conjugated molecule prepared in the above step by phosphoramidite nucleic acid solid phase synthesis method, in which the conjugated group is bound to the 3' end of the obtained sense strand.

[0225] Other conditions for the solid-phase synthesis in steps (2) and (3) include deprotection conditions for the nucleoside monomer, the type and dosage of the deprotection reagent, coupling reaction conditions, the type and dosage of the coupling reagent, capping reaction conditions, the type and dosage of the capping reagent, oxidation reaction conditions, the type and dosage of the oxidation reagent, sulfurization reaction conditions, and the sulfurization reagent and dosage, and various reagents, dosages, and conditions commonly used in this field are adopted.

[0226] For example, in some embodiments, in steps (2) and (3), the solid phase synthesis may use the following conditions:

[0227] Deprotection conditions for the nucleoside monomer include a temperature of 0 to 50°C, and in some embodiments, 15 to 35°C, a reaction time of 30 to 300 seconds, and in some embodiments, 50 to 150 seconds, and the deprotection reagent may be one or more selected from trifluoroacetic acid, trichloroacetic acid, dichloroacetic acid, and chloroacetic acid, and in some embodiments, is dichloroacetic acid. The molar ratio of the deprotection reagent to the 4,4'-dimethoxytrityl protecting group on the solid support may be 2:1 to 100:1, and in some embodiments, is 3:1 to 50:1.

[0228] Coupling reaction conditions include a temperature of 0 to 50°C, and in some embodiments, 15 to 35°C; a molar ratio of the nucleic acid sequence bound to the solid phase carrier to the nucleoside monomer may be 1:1 to 1:50, and in some embodiments, 1:5 to 1:15; a molar ratio of the nucleic acid sequence bound to the solid phase carrier to the coupling reagent may be 1:1 to 1:100, and in some embodiments, 1:50 to 1:80; and the reaction time and selection of the coupling reagent are the same as described above.

[0229] Capping reaction conditions include a temperature of 0 to 50°C, and in some embodiments, 15 to 35°C, a reaction time of 5 to 500 seconds, and in some embodiments, 10 to 100 seconds, and the selection of capping reagent is the same as described above. The molar ratio of the total amount of capping reagent to the nucleic acid sequence bound to the solid phase carrier is 1:100 to 100:1, and in some embodiments, 1:10 to 10:1. When equimolar amounts of acetic anhydride and N-methylimidazole are used as capping reagents, the molar ratio of acetic anhydride, N-methylimidazole, and the nucleic acid sequence bound to the solid phase carrier is 1:1:10 to 10:10:1, and in some embodiments, 1:1:2 to 2:2:1.

[0230] The oxidation reaction conditions include a temperature of 0 to 50°C, and in some embodiments, 15 to 35°C, a reaction time of 1 to 100 seconds, and in some embodiments, 5 to 50 seconds. In some embodiments, the oxidation reagent is iodine (provided as iodine water). The molar ratio of the oxidation reagent to the nucleic acid sequence bound to the solid phase carrier in the coupling step may be 1:1 to 100:1, and in some embodiments, 5:1 to 50:1. In some embodiments, the oxidation reaction is 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, 15 to 35°C, a reaction time of 50 to 2000 seconds, and in some embodiments, 100 to 1000 seconds. In some embodiments, the sulfurization reagent is xanthan hydride. The molar ratio of the sulfurization reagent to the nucleic acid sequence bound to the solid phase carrier in the coupling step may be 10:1 to 1000:1, and in some embodiments, is 10:1 to 500:1. In some embodiments, the sulfurization reaction is carried out in a mixed solvent of acetonitrile:pyridine=1:3 to 3:1.

[0231] After all nucleoside monomers have been linked and before annealing, the method further includes isolating the sense and antisense strands of the double-stranded oligonucleotide. Isolation methods are known to those skilled in the art and generally involve cleaving the synthesized nucleotide sequence from the solid support, removing protecting groups on the bases, phosphate groups, and ligands, and purifying and desalting.

[0232] Cleavage of the synthesized nucleotide sequence from the solid support and removal of the protecting groups on the bases, phosphate groups, and ligands can be performed using conventional cleavage and deprotection methods used in double-stranded oligonucleotide synthesis. For example, the resulting solid-support-bound nucleotide sequence can be contacted with concentrated aqueous ammonia. During the deprotection process, the YCOO- protecting groups at A46 to A54 are converted to hydroxy groups, and the S1 group is converted to the corresponding M1 group, producing the conjugate shown in formula (308). The concentrated aqueous ammonia may be 25 to 30 wt. % aqueous ammonia, and the dosage of the concentrated aqueous ammonia may be 0.2 ml / μmol to 0.8 ml / μmol relative to the desired double-stranded oligonucleotide sequence.

[0233] If the synthesized nucleotide sequence contains at least one 2'-TBDMS-protected group, the method further comprises contacting the nucleotide sequence from which the solid support has been removed with triethylamine trihydrofluoride to remove the 2'-TBDMS-protected group. In this case, the resulting double-stranded oligonucleotide sequence has a free 2'-hydroxy group corresponding to the corresponding nucleoside. The amount of pure triethylamine trihydrofluoride may be 0.4 ml / μmol to 1.0 ml / μmol relative to the double-stranded oligonucleotide sequence. Thus, the oligonucleotide conjugate of formula (308) can be obtained.

[0234] Methods for purification and desalting are well known to those skilled in the art. For example, nucleic acid purification can be completed using a preparative ion chromatography purification column with gradient elution of NaBr or NaCl, and the product can be recovered and combined, and then desalted using a reversed-phase chromatography purification column.

[0235] In the oligonucleotide conjugates obtained in this manner, non-bridging oxygen or sulfur atoms in the internucleotide phosphodiester or phosphorothiodiester bonds are essentially bound to sodium ions, and the oligonucleotide conjugates exist essentially as sodium salts. By well-known ion exchange methods, the sodium ions can be replaced with hydrogen ions and / or other cations, as previously described, to obtain other forms of oligonucleotide conjugates.

[0236] During the synthesis process, the purity and molecular weight of the nucleic acid sequence can be constantly monitored to better control the synthesis quality. Such detection methods are well known to those skilled in the art. For example, the purity of the nucleic acid can be detected by ion exchange chromatography, and the molecular weight can be measured by liquid chromatography tandem mass spectrometry.

[0237] Annealing methods are also well known to those skilled in the art. For example, a simply synthesized sense strand (S strand) and an antisense strand (AS strand) can be mixed in an equimolar ratio in water for injection, heated to 70-95°C, and then cooled to room temperature to form a double-stranded structure through hydrogen bonding. In this way, the oligonucleotide complex of the present disclosure can be obtained.

[0238] After obtaining the conjugate of the present disclosure, in some embodiments, the synthesized oligonucleotide conjugate can be characterized by molecular weight detection or the like using a method such as liquid chromatography tandem mass spectrometry, and it can be confirmed that the synthesized oligonucleotide conjugate is the intended oligonucleotide conjugate and that the sequence of the synthesized double-stranded oligonucleotide is the desired double-stranded oligonucleotide sequence, for example, one of the sequences shown in Table 1.

[0239] Compounds of formula (321) can be prepared by a process comprising contacting compounds of formula (313) with a cyclic acid anhydride in an organic solvent under esterification conditions and in the presence of a base and an esterification catalyst, followed by ion exchange and isolation to obtain compounds of formula (321).

[0240] [ka] In the formula, n1, n3, m1, m2, m3, R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , L 1、 The definition and selectable range of each 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).

[0241] [ka] In the formula, R i can achieve bonding with N on the nitrogen-containing backbone, R k Any group having one free hydroxy group bonded to O, R k is a hydroxy-protecting group. In this case, R4 contains a first functional group as a hydroxy-protecting group and a second functional group, and a compound of formula (321) is obtained in which the second functional group contains a structure represented by formula (C1) or (C2).

[0242] The esterification reaction conditions include a reaction temperature of 0 to 100°C and a reaction time of 8 to 48 hours, and in some embodiments, the esterification reaction conditions include a reaction temperature of 10 to 40°C and a reaction time of 20 to 30 hours.

[0243] In some embodiments, the organic solvent includes one or more of an epoxy solvent, an ether solvent, a halogenated alkyl solvent, dimethyl sulfoxide, N,N-dimethylformamide, and N,N-diisopropylethylamine. In some embodiments, the epoxy solvent is dioxane and / or tetrahydrofuran, the ether solvent is ethyl ether and / or methyl tert-butyl ether, and the halogenated alkyl solvent is one or more of dichloromethane, trichloromethane, and 1,2-dichloroethane. In some embodiments, the organic solvent is dichloromethane. The amount of the organic solvent relative to the compound represented by formula (313) is 3 to 50 L / mol, and in some embodiments, 5 to 20 L / mol.

[0244] In some embodiments, the cyclic acid anhydride is one of succinic anhydride, glutaric anhydride, adipic anhydride, or pimelic anhydride, and in some embodiments, succinic anhydride. The molar ratio of the cyclic acid anhydride to the compound represented by formula (313) is 1:1 to 10:1, and in some embodiments, 2:1 to 5:1.

[0245] The esterification catalyst may be any catalyst that catalyzes the esterification reaction, for example, the catalyst may be 4-dimethylaminopyridine, and the molar ratio of the catalyst to the compound represented by Formula (313) is 1:1 to 10:1, and in some embodiments, 2:1 to 5:1.

[0246] In some embodiments, the base may be any inorganic base, organic base, or a combination thereof. Considering solubility and product stability, the base may be, for example, a tertiary amine organic base. In some embodiments, the tertiary amine 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.

[0247] The ion exchange process converts the compound of formula (321) into the desired carboxylic acid or carboxylate form. The ion exchange process is known to those skilled in the art and involves the use of suitable ion exchange solutions and conditions to convert the aforementioned cations into M + In some embodiments, the ion exchange reaction is carried out using a triethylamine phosphate solution, and the concentration of the triethylamine phosphate solution is 0.2 to 0.8 M, and in some embodiments, the concentration of the triethylamine phosphate solution is 0.4 to 0.6 M. The amount of the triethylamine phosphate solution relative to the compound of Formula (313) is 3 to 6 L / mol, and in further embodiments, 4 to 5 L / mol.

[0248] The compound of formula (321) can be isolated from the reaction mixture by any suitable isolation method. In some embodiments, after evaporating and removing the solvent, the compound of formula (321) can be isolated by chromatography. For example, (1) normal-phase purified silica gel: 200-300 mesh silica gel packing is eluted with a gradient of dichloromethane:methanol=100:18 to 100:20 containing 1 wt% triethylamine, or (2) reverse-phase purified silica gel: C18 or C8 reverse-phase packing is eluted with a gradient of methanol:acetonitrile=0.1:1 to 1:0.1. In some embodiments, the solvent can be directly removed to obtain a crude product of the compound of formula (321), which can be used directly in the subsequent reaction.

[0249] In some embodiments, the method for preparing a compound of Formula (321) further comprises contacting the product obtained by the ion exchange reaction with a solid support containing an amino group or a hydroxy group in the presence of a condensing agent and a tertiary amine organic base in an organic solvent under condensation reaction conditions, thereby obtaining a compound of Formula (321), in which R4 contains a first functional group and a second functional group, the first functional group containing a hydroxy-protecting group, and the second functional group containing the structure shown in Formula (C1').

[0250] The solid support is one of the supports used in solid-phase synthesis of double-stranded oligonucleotides, some of which are known to those skilled in the art. For example, the solid support may be selected from solid supports containing an active hydroxyl group or amino functional group. In some embodiments, the solid support is an amino resin or a hydroxy resin. In some embodiments, the amino resin or the hydroxy resin has parameters of a particle size of 100 to 400 mesh and a surface amino or hydroxy group loading of 0.2 to 0.5 mmol / g. The dosage ratio of the compound represented by formula (321) to the solid support is 10 to 400 μmol of compound / gram of solid support (μmol / g). In some embodiments, the dosage ratio of the compound represented by formula (321) to the solid support is 50 to 200 μmol / g.

[0251] The organic solvent may be any suitable solvent or solvent mixture known to those skilled in the art. In some embodiments, the organic solvent is one or more of acetonitrile, epoxy solvents, ether solvents, alkyl halide solvents, dimethyl sulfoxide, N,N-dimethylformamide, and N,N-diisopropylethylamine. In some embodiments, the epoxy solvent is dioxane and / or tetrahydrofuran, the ether solvent is ethyl ether and / or methyl tert-butyl ether, and the alkyl halide solvent is one or more of dichloromethane, trichloromethane, and 1,2-dichloroethane. In some embodiments, the organic solvent is acetonitrile. The dosage of the organic solvent relative to the compound of Formula (321) is 20 to 200 L / mol, and in some embodiments, 50 to 100 L / mol.

[0252] In some embodiments, the condensing agent may be (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate, 3-diethoxyphosphoryl-1,2,3-benzoxazol-4(3H)-one, and / or O-benzotriazole-tetramethyluronium hexafluorophosphate, and in some embodiments, the condensing agent is O-benzotriazole-tetramethyluronium hexafluorophosphate. The molar ratio of the condensing agent to the compound represented by Formula (321) is 1:1 to 20:1, and in further embodiments, 1:1 to 5:1.

[0253] In some embodiments, the tertiary amine organic base is triethylamine and / or N,N-diisopropylethylamine, and in some embodiments, N,N-diisopropylethylamine. The molar ratio of the tertiary amine organic base to the compound represented by Formula (321) is 1:1 to 20:1, and in some embodiments, 1:1 to 5:1.

[0254] In some embodiments, the method for preparing a compound of Formula (321) may further include contacting the resulting condensation product with a capping reagent and an acylation catalyst in an organic solvent under capping reaction conditions, followed by isolation to obtain a compound of Formula (321). The function of the capping reaction is to remove any active reactive functional groups that have not yet fully reacted to avoid the generation of unwanted by-products in subsequent reactions. The capping reaction conditions include a reaction temperature of 0 to 50°C, and in some embodiments, 15 to 35°C, and a reaction time of 1 to 10 hours, and in some embodiments, 3 to 6 hours. The capping reagent may be a capping reagent known to those skilled in the art for use in solid-phase nucleic acid synthesis.

[0255] In some embodiments, the capping reagent comprises capping reagent A (capA) and capping reagent B (capB), and capping reagent A is N-methylimidazole. In some embodiments, N-methylimidazole is provided as a pyridine / acetonitrile mixed solution, and the volume ratio of pyridine to acetonitrile is 1:10 to 1:1, and in some embodiments, 1:3 to 1:1. The total volume of pyridine and acetonitrile and the volume of N-methylimidazole are Volume of with ratio In some embodiments, the ratio of capping reagent B to acetic anhydride is 1:1 to 10:1, and in some embodiments, 3:1 to 7:1. In some embodiments, the capping reagent B is acetic anhydride. In some embodiments, the capping reagent B is provided as a solution of acetic anhydride in acetonitrile, and the volume ratio of acetic anhydride to acetonitrile is ratio is from 1:1 to 1:10, and in a further embodiment from 1:2 to 1:6.

[0256] In some embodiments, the ratio of the volume of the N-methylimidazole pyridine / acetonitrile mixed solution to the mass of the compound of Formula (321) is 5 ml / g to 50 ml / g, and in some embodiments, 15 ml / g to 30 ml / g.The ratio of the volume of the acetic anhydride acetonitrile solution to the mass of the compound of Formula (321) is 0.5 ml / g to 10 ml / g, and in some embodiments, 1 ml / g to 5 ml / g.

[0257] In some embodiments, equimolar amounts of acetic anhydride and N-methylimidazole are used as capping reagents. In some embodiments, the organic solvent is one or more of acetonitrile, epoxy solvents, ether solvents, alkyl halide solvents, dimethyl sulfoxide, N,N-dimethylformamide, and N,N-diisopropylethylamine. In some embodiments, the organic solvent is acetonitrile. In some embodiments, the amount of the organic solvent relative to the compound of Formula (321) is 10 to 50 L / mol, and in some embodiments, 5 to 30 L / mol.

[0258] In some embodiments, the acylation catalyst may be selected from any catalyst that can be used in esterification condensation or amidation condensation, such as an alkali heterocyclic compound. In some embodiments, the acylation catalyst is 4-dimethylaminopyridine. The mass ratio of the catalyst to the compound represented by formula (321) is 0.001:1 to 1:1, and in some embodiments, 0.01:1 to 0.1:1.

[0259] 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 of Formula (321) can be obtained by thoroughly washing with an organic solvent and filtering to remove unreacted reactants, excess capping reagent, and other impurities. The organic solvent is selected from acetonitrile, dichloromethane, and methanol, and in some embodiments, is acetonitrile.

[0260] In some embodiments, a method for preparing a conjugated molecule of Formula 321 includes contacting a compound of Formula 313 with a phosphorodiamidite in an organic solvent under coupling reaction conditions and in the presence of a coupling reagent, and isolating to obtain a compound of Formula 321, wherein R4 comprises a first functional group and a second functional group, the first functional group comprising a hydroxy protecting group, and the second functional group comprising a structure according to Formula C3.

[0261] In some embodiments, the coupling reaction conditions include a temperature of 0 to 50°C, e.g., 15 to 35°C; a molar ratio of the compound of Formula (313) to the phosphorodiamidite of 1:1 to 1:50, e.g., 1:5 to 1:15; a molar ratio of the compound of Formula (313) to the coupling reagent of 1:1 to 1:100, e.g., 1:50 to 1:80; and a reaction time of 200 to 3000 seconds, e.g., 500 to 1500 seconds. The phosphorodiamidite may be, for example, bis(diisopropylamino)(2-cyanoethoxy)phosphine, which may be commercially available or synthesized by methods known in the art. The coupling reagent may be one or more selected from 1H-tetrazole, 5-ethylthio-1H-tetrazole, and 5-benzylthio-1H-tetrazole, e.g., 5-ethylthio-1H-tetrazole. The coupling reaction may be carried out in an organic solvent, and the organic solvent may be one or more selected from anhydrous acetonitrile, anhydrous DMF, and anhydrous dichloromethane, such as anhydrous acetonitrile. In some embodiments, the amount of the organic solvent relative to the compound of Formula (313) may be 3 to 50 L / mol, for example, 5 to 20 L / mol. By carrying out the coupling reaction, the hydroxy group in the compound of Formula (313) reacts with the phosphoramidite to form a phosphoramidite group. In some embodiments, the solvent can be directly removed to obtain a crude product of the compound of Formula (321), which can be used directly in the subsequent reaction.

[0262] In some embodiments, the method for preparing a compound of Formula (321) further comprises contacting the isolated product with a hydroxyl-containing solid support in the presence of a coupling reagent in an organic solvent under coupling reaction conditions, followed by capping and oxidation, followed by isolation to obtain a compound of Formula (321). In this case, a compound of Formula (321) is obtained in which R4 includes a first functional group and a second functional group, the first functional group including a hydroxyl-protecting group, and the second functional group having the structure shown in Formula (C3').

[0263] In some embodiments, the solid support is a solid support that can be used for solid-phase nucleic acid synthesis known in the art, such as a deprotected commercially available general-purpose solid support (NittoPhase® HL UnyLinker TM 300 Oligonucleotide Synthesis Support, Kinovate Life Sciences, the structure of which is shown in formula B80).

[0264] [ka]

[0265] Deprotection reactions are well known to those skilled in the art. In some embodiments, deprotection conditions include a temperature of 0 to 50°C, for example, 15 to 35°C, and a reaction time of 30 to 300 seconds, for example, 50 to 150 seconds. The deprotection reagent may be one or more selected from trifluoroacetic acid, trichloroacetic acid, dichloroacetic acid, and chloroacetic acid, and in some embodiments, the deprotection reagent is dichloroacetic acid. The molar ratio of the deprotection reagent to the -DMTr (4,4'-dimethoxytrityl) protecting groups on the stationary phase is 2:1 to 100:1, for example, 3:1 to 50:1. By performing the deprotection, reactive free hydroxy groups are obtained on the surface of the solid phase support, facilitating the subsequent coupling reaction.

[0266] The coupling reaction conditions and coupling reagents are selected as described above. By carrying out the coupling reaction, the free hydroxy group formed in the deprotection reaction reacts with the phosphoramidite group to form a phosphite bond.

[0267] In some embodiments, the capping reaction conditions include a temperature of 0 to 50°C, for example, 15 to 35°C, a reaction time of 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 of which are as described above.

[0268] The oxidation reaction conditions may include a temperature of 0 to 50°C, for example, 15 to 35°C, a reaction time of 1 to 100 seconds, for example, 5 to 50 seconds, and an oxidation reagent such as iodine (provided as iodine water in some embodiments). In some embodiments, the molar ratio of the oxidation reagent to the phosphite ester group is 1:1 to 100:1, for example, 5:1 to 50:1. In some embodiments, the oxidation reaction is carried out in a mixed solvent of tetrahydrofuran:water:pyridine=3:1:1 to 1:1:3.

[0269] In some embodiments, R6 is one of the groups of formula B7 or B8.

[0270] [ka] In the formula, the definition of q2 is as described above.

[0271] In this case, the compound represented by formula (313) can be obtained by a preparation method in which the compound represented by formula (314) is contacted with the compound represented by formula (A-1) or the compound represented by formula (A-2) in an organic solvent under amidation reaction conditions and in the presence of an amidation reaction condensing agent and a tertiary amine organic base, and then isolated.

[0272] [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.

[0273] The amidation reaction conditions may include a reaction temperature of 0 to 100°C and a reaction time of 1 to 48 hours, and in some embodiments, the amidation reaction conditions include a reaction temperature of 10 to 40°C and a reaction time of 2 to 16 hours.

[0274] In some embodiments, the organic solvent is one or more of an alcohol solvent, an epoxy solvent, an ether solvent, a halogenated alkyl solvent, dimethyl sulfoxide, N,N-dimethylformamide, and N,N-diisopropylethylamine. In some embodiments, the alcohol solvent is one or more of methanol, ethanol, and propanol, and in some embodiments, ethanol. In some embodiments, the epoxy solvent is dioxane and / or tetrahydrofuran. In some embodiments, the ether solvent is ethyl ether and / or methyl tert-butyl ether. In some embodiments, the halogenated alkyl solvent is one or more of dichloromethane, trichloromethane, and 1,2-dichloroethane. In some embodiments, the organic solvent is dichloromethane. The amount of the organic solvent relative to the compound of Formula (314) is 3 to 50 L / mol, and in further embodiments, 3 to 20 L / mol.

[0275] In some embodiments, the amidation reaction condensing agent is (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate, 3-diethoxyphosphoryl-1,2,3-benzoxazol-4(3H)-one, 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride, 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ), or O-benzotriazole-tetramethyluronium hexafluorophosphate, or in further embodiments, 3-diethoxyphosphoryl-1,2,3-benzoxazol-4(3H)-one. The molar ratio of the amidation reaction condensing agent to the compound represented by Formula (314) may be 1:1 to 10:1, and in some embodiments, is 2.5:1 to 5:1.

[0276] In some embodiments, the tertiary amine organic base is triethylamine or N,N-diisopropylethylamine, and in further embodiments, N,N-diisopropylethylamine. The molar ratio of the tertiary amine organic base to the compound represented by Formula (314) is 3:1 to 20:1, and in some embodiments, 5:1 to 10:1.

[0277] In some embodiments, compounds of formula (A-1) and formula (A-2) may be prepared by any suitable method. For example, R kis a DMTr group, a compound of formula (A-1) can be prepared by reacting calcium glycerate with DMTrCl. Similarly, a compound of formula (A-2) can be prepared by contacting 3-amino-1,2-propanediol with a cyclic acid anhydride, followed by reaction with DMTrCl, where the cyclic acid anhydride may be a cyclic acid anhydride having 4 to 13 carbon atoms, and in some embodiments, 4 to 8 carbon atoms. As one skilled in the art will readily appreciate, the choice of cyclic acid anhydride corresponds to different values ​​of q2 in the compound of formula (A-2); for example, if the cyclic acid anhydride is succinic anhydride, q2 = 1; if the cyclic acid anhydride is glutaric anhydride, q2 = 2; and so forth.

[0278] In some modifications, the compound of formula (313) can also be prepared by sequentially reacting the compound of formula (314) with the cyclic acid anhydride, 3-amino-1,2-propanediol, and DMTrCl. As can be readily understood by those skilled in the art, these modifications do not affect the structure and function of the compound of formula (313) and can be easily realized by those skilled in the art using the above method.

[0279] As described above, the compound of formula (313) can be isolated from the reaction mixture by any suitable isolation method. In some embodiments, after evaporating the solvent, the compound of formula (313) can be isolated by chromatography. For example, the following two chromatographic conditions can be used: (1) normal-phase purification: 200-300 mesh silica gel packing, gradient elution with petroleum ether: ethyl acetate: dichloromethane: N,N-dimethylformamide = 1:1:1:0.5 to 1:1:1:0.6; (2) reverse-phase purification: C18, C8 reverse-phase packing, gradient elution with methanol: acetonitrile = 0.1:1 to 1:0.1. In some embodiments, the solvent can be directly removed to obtain a crude product of the compound of formula (313), which can be used directly in the subsequent reaction.

[0280] In some embodiments, compound of formula (314) can be obtained by a preparation process comprising contacting compound of formula (315) with haloacetic acid in an organic solvent under deprotection reaction conditions, followed by isolation.

[0281] [ka] wherein R7 is selected from the groups shown in formula (330), (331), (332), or (333), and in some embodiments, the structure of R7 is shown in formula (330).

[0282] [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.

[0283] The haloacetic acid may be one or more selected from dichloroacetic acid, trichloroacetic acid, chloroacetic acid, and trifluoroacetic acid, and in some embodiments is dichloroacetic acid.

[0284] The deprotection reaction conditions may be a reaction temperature of 0 to 100°C and a reaction time of 0.1 to 24 hours, and in some embodiments, a reaction temperature of 10 to 40°C and a reaction time of 0.5 to 16 hours.

[0285] In some embodiments, the organic solvent is one or more of an epoxy solvent, an ether solvent, a halogenated alkyl solvent, dimethyl sulfoxide, N,N-dimethylformamide, and N,N-diisopropylethylamine. In some embodiments, the epoxy solvent is dioxane and / or tetrahydrofuran. In some embodiments, the ether solvent is ethyl ether and / or methyl tert-butyl ether. In some embodiments, the halogenated alkyl solvent is one or more of dichloromethane, trichloromethane, and 1,2-dichloroethane. In some embodiments, the organic solvent is dichloromethane. The amount of the organic solvent relative to the compound of Formula (315) is 3 to 50 L / mol, and in further embodiments, 5 to 20 L / mol.

[0286] The molar ratio of the haloacetic acid to the compound represented by formula (315) may be 5:1 to 100:1, and in some embodiments, is 10:1 to 50:1.

[0287] As described above, the compound of formula (314) can be isolated from the reaction mixture by any suitable isolation method. In some embodiments, after evaporating and removing the solvent, the compound of formula (314) can be isolated by chromatography. For example, the compound can be isolated using two chromatographic conditions: (1) normal-phase purified silica gel: 200-300 mesh silica gel packing, gradient elution with dichloromethane:methanol = 100:30 to 100:40; or (2) reverse-phase purified: C18, C8 reverse-phase packing, gradient elution with methanol:acetonitrile = 0.1:1 to 1:0.1. In some embodiments, the solvent can be directly removed to obtain a crude product of the compound of formula (314), which can be used directly in the subsequent reaction.

[0288] The compound represented by formula (315) can be obtained by a preparation method comprising contacting the compound represented by formula (317) with the compound represented by formula (316) in an organic solvent in the presence of an amidation reaction condensing agent and a tertiary amine organic base under condensation reaction conditions, followed by isolation.

[0289] [ka] In the formula, n1, n3, m1, m2, m3, R7, 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.

[0290] The compound of formula (316) may be, for example, a compound disclosed in J. Am. Chem. Soc. 2014, 136, 16958-16961. Alternatively, the compound of formula (316) can be prepared by a person skilled in the art by various methods. For example, some compounds of formula (316) can be prepared by referring to the method disclosed in Example 1 of U.S. Patent No. 8,106,022B2, the entire contents of which are incorporated herein by reference.

[0291] In some embodiments, the condensation reaction conditions include a reaction temperature of 0 to 100°C and a reaction time of 0.1 to 24 hours, and in some embodiments, a reaction temperature of 10 to 40°C and a reaction time of 0.5 to 16 hours.

[0292] The molar ratio of the compound represented by formula (316) to the compound represented by formula (317) may be 2:1 to 10:1, and in some embodiments, is 2.5:1 to 5:1.

[0293] In some embodiments, the organic solvent is one or more of acetonitrile, epoxy solvent, ether solvent, halogenated alkyl solvent, dimethyl sulfoxide, N,N-dimethylformamide, and N,N-diisopropylethylamine. In some embodiments, the epoxy solvent is dioxane and / or tetrahydrofuran. In some embodiments, the ether solvent is ethyl ether and / or methyl tert-butyl ether. In some embodiments, the halogenated alkyl solvent is one or more of dichloromethane, trichloromethane, and 1,2-dichloroethane. In some embodiments, the organic solvent is acetonitrile. The dosage of the organic solvent relative to the compound of Formula (317) is 3 to 50 L / mol, and in some embodiments, 5 to 20 L / mol.

[0294] In some embodiments, the amidation reaction condensing agent may be (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate, 3-diethoxyphosphoryl-1,2,3-benzoxazol-4(3H)-one (DEPBT), O-benzotriazole-tetramethyluronium hexafluorophosphate, or 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride, or in further embodiments, 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride. The molar ratio of the amidation reaction condensing agent to the compound represented by Formula (317) may be 2:1 to 10:1, and in some embodiments, 2.5:1 to 5:1.

[0295] The tertiary amine organic base may be N-methylmorpholine, triethylamine, or N,N-diisopropylethylamine, and in some embodiments, is N-methylmorpholine. The molar ratio of the tertiary amine organic base to the compound represented by Formula (317) may be 3:1 to 20:1, and in some embodiments, is 5:1 to 10:1.

[0296] As described above, the compound of formula (315) can be isolated from the reaction mixture by any suitable isolation method. In some embodiments, after evaporating and removing the solvent, the compound of formula (315) can be isolated by chromatography. For example, the isolation can be performed using two chromatographic conditions: (1) normal-phase purification silica gel: 200-300 mesh silica gel packing, gradient elution with dichloromethane:methanol = 100:5 to 100:7; or (2) reverse-phase purification: C18, C8 reverse-phase packing, gradient elution with methanol:acetonitrile = 0.1:1 to 1:0.1. In some embodiments, the solvent can be directly removed to obtain a crude product of the compound of formula (315), which can be used directly in the subsequent reaction.

[0297] In some embodiments, a compound of Formula (317) is reacted in a single step with a sufficient amount of a compound of Formula (316) to produce the desired compound of Formula (315), where each S1-L1 moiety is the same. In some embodiments, if desired, the compound of Formula (317) can be batch-reacted with different compounds of Formula (316), i.e., compounds of Formula (316) with different L1 and / or S1, so that the resulting compound of Formula (315) contains more than one type of S1 and / or L1. For example, 1 eq of a compound of formula (317) can be contacted with 2 eq of a first compound of formula (316) to attach a first S1-L1 moiety to the two terminal primary amine groups of the compound of formula (317), followed by contact with (n3+n1-1) eq of a second compound of formula (316) (the definitions and ranges of values ​​of n3 and n1 are as described above) to attach a second S1-L1 moiety to the (n3+n1-1) secondary amine groups of the compound of formula (317).

[0298] In some embodiments, compound of formula (317) can be obtained by a preparation process comprising contacting compound of formula (318) with aqueous methylamine in the presence of an organic solvent and under deprotection reaction conditions, followed by isolation.

[0299] [ka] In the formula, n1, n3, m1, m2, m3, R7, R 10 , R 11 , R 12 , R 13 , R 14 , R 15 The definitions and selectable ranges of each are as described above.

[0300] The deprotection reaction conditions may be a reaction temperature of 0 to 150°C and a reaction time of 5 to 72 hours, and in some embodiments, a reaction temperature of 20 to 80°C and a reaction time of 10 to 30 hours.

[0301] The organic solvent may be selected from an alcohol, and in some embodiments, is one of methanol, ethanol, and isopropanol, and in some embodiments, is methanol, and the amount of the organic solvent relative to the compound of Formula (318) is 1 to 20 L / mol, and in some embodiments, 1.5 to 10 L / mol.

[0302] The concentration of the methylamine aqueous solution may be 30 to 40% by mass, and the molar ratio of methylamine to the compound represented by formula (318) may be 10:1 to 500:1, and in some embodiments, is 50:1 to 200:1.

[0303] As described above, the compound of formula (317) can be isolated from the reaction mixture by any suitable isolation method. In some embodiments, after evaporating the solvent, the compound of formula (317) can be isolated by chromatography. For example, the following two chromatographic conditions can be used: (1) normal-phase purified silica gel: 200-300 mesh silica gel packing, gradient elution with dichloromethane:methanol:ammonia water (25 wt%) = 1:1:0.05 to 1:1:0.25; (2) reverse-phase purified: C18, C8 reverse-phase packing, gradient elution with methanol:acetonitrile = 0.1:1 to 1:0.1. In some embodiments, the solvent can be directly removed to obtain a crude product of the compound of formula (317), which can be used directly in the subsequent reaction.

[0304] In some embodiments, the compound of formula (318) can be obtained by a preparation process that includes contacting a compound of formula (319) with triphenylchloromethane (TrCl), diphenylethylphenylchloromethane, phenyldiethylphenylchloromethane, or triethylphenylchloromethane, in some embodiments, triphenylchloromethane (TrCl), in the presence of an organic solvent and under displacement reaction conditions, followed by isolation.

[0305] [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.

[0306] The substitution reaction conditions may include a reaction temperature of 0 to 100°C and a reaction time of 5 to 72 hours, and in some embodiments, the reaction conditions include a reaction temperature of 10 to 40°C and a reaction time of 10 to 30 hours.

[0307] Triphenylchloromethane (TrCl), diphenylethylphenylchloromethane, phenyldiethylphenylchloromethane, or triethylphenylchloromethane can be purchased commercially, and the molar ratio of triphenylchloromethane (TrCl), diphenylethylphenylchloromethane, phenyldiethylphenylchloromethane, or triethylphenylchloromethane to the compound represented by Formula (319) can be 1:1 to 10:1, and in some embodiments, is 1:1 to 3:1.

[0308] The organic solvent may be one or more of an epoxy solvent, an ether solvent, a halogenated alkyl solvent, dimethyl sulfoxide, N,N-dimethylformamide, and N,N-diisopropylethylamine. In some embodiments, the epoxy solvent may be dioxane and / or tetrahydrofuran. In some embodiments, the ether solvent may be ethyl ether and / or methyl tert-butyl ether. In some embodiments, the halogenated alkyl solvent may be one or more of dichloromethane, trichloromethane, and 1,2-dichloroethane. In some embodiments, the organic solvent is dichloromethane. The amount of the organic solvent relative to the compound of Formula (319) may be 3 to 50 L / mol, and in some embodiments, 5 to 20 L / mol.

[0309] As described above, the compound of Formula (318) can be isolated from the reaction mixture by any suitable isolation method. In some embodiments, after evaporating the solvent, the compound of Formula (318) can be isolated by chromatography. For example, the following two chromatographic conditions can be used: (1) normal-phase purification: 200-300 mesh silica gel packing, gradient elution with methanol:dichloromethane = 0.01:1 to 0.5:1, or methanol:dichloromethane:ethyl acetate:petroleum ether = 0.1:1:1:1 to 1:1:1:1; (2) reverse-phase purification: C18 or C8 reverse-phase packing, gradient elution with methanol:acetonitrile = 0.1:1 to 1:0.1. In some embodiments, the solvent can be directly removed to obtain a crude product of the compound of Formula (318), which can be used directly in the subsequent reaction.

[0310] In some embodiments, the compound of formula (319) can be obtained by a preparation process comprising contacting a compound of formula (320) with ethyl trifluoroacetate in an organic solvent under displacement reaction conditions, followed by isolation.

[0311] [ka] In the formula, the definitions and selectable ranges of n1, n3, m1, m2, m3, R10, R11, R12, R13, R14, and R15 are as described above.

[0312] In some embodiments, the organic solvent is one or more of acetonitrile, epoxy solvent, ether solvent, halogenated alkyl solvent, dimethyl sulfoxide, N,N-dimethylformamide, and N,N-diisopropylethylamine. In some embodiments, the epoxy solvent is dioxane and / or tetrahydrofuran. In some embodiments, the ether solvent is ethyl ether and / or methyl tert-butyl ether. In some embodiments, the halogenated alkyl solvent is one or more of dichloromethane, trichloromethane, and 1,2-dichloroethane. In some embodiments, the organic solvent is acetonitrile. The amount of the organic solvent relative to the compound of Formula (320) may be 1 to 50 L / mol, and in some embodiments, 1 to 20 L / mol.

[0313] The substitution reaction conditions may include a reaction temperature of 0 to 100°C and a reaction time of 5 to 72 hours, and in some embodiments, the substitution reaction conditions include a reaction temperature of 10 to 40°C and a reaction time of 10 to 30 hours.

[0314] 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 compound of formula (320) can be purchased commercially from Alfa Aesar.

[0315] The molar ratio of ethyl trifluoroacetate to the compound represented by Formula (320) is 2:1 to 10:1, and in some embodiments, 3:1 to 5:1.

[0316] As described above, the compound of formula (319) can be isolated from the reaction mixture by any suitable isolation method. In some embodiments, after evaporating the solvent, the compound of formula (319) can be isolated by chromatography. For example, the following two chromatographic conditions can be used: (1) normal-phase purification: 200-300 mesh silica gel packing, gradient elution with methanol:dichloromethane = 0.01:1 to 0.5:1, or methanol:dichloromethane:ethyl acetate:petroleum ether = 0.1:1:1:1 to 1:1:1:1; (2) reverse-phase purification: C18 or C8 reverse-phase packing, gradient elution with methanol:acetonitrile = 0.1:1 to 1:0.1. In some embodiments, the solvent can be directly removed to obtain a crude product of the compound of formula (319), which can be used directly in the subsequent reaction.

[0317] The oligonucleotide conjugates of the present disclosure may be used in combination with other pharmaceutically acceptable additives, which 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.

[0318] Uses of the Double-Stranded Oligonucleotides, Drug Compositions, and Oligonucleotide Conjugates of the Present Disclosure In some embodiments, the present disclosure provides use of a double-stranded oligonucleotide, drug composition, and / or oligonucleotide conjugate provided by the present disclosure in preparing a drug for treating and / or preventing a pathological condition or disease caused by the expression of a specific gene in cells. In some embodiments, the specific gene is a gene abnormally expressed in hepatocytes. In some embodiments, the specific gene is an endogenous gene expressed in the liver. In some embodiments, the specific gene is a gene of a pathogen that reproduces in the liver. In some embodiments, the specific gene is selected from genes such as ApoB, ApoC, ANGPTL3, PCSK9, SCD1, TIMP-1, Col1A1, FVII, STAT3, p53, HBV, and HCV. In some embodiments, the specific gene is selected from hepatitis B virus genes, angiopoietin-like protein 3 genes, and apolipoprotein C3 genes. Accordingly, the disease is selected from chronic liver disease, hepatitis, hepatic fibrotic disease, hepatic hyperplastic disease, and dyslipidemia. In some embodiments, the dyslipidemia is hypercholesterolemia, hypertriglyceridemia, or atherosclerosis.

[0319] In some embodiments, the present disclosure provides a method for treating a pathological condition or disease caused by the abnormal expression of a specific gene, comprising administering to a subject in need thereof an effective amount of a double-stranded oligonucleotide, pharmaceutical composition, and / or oligonucleotide conjugate provided by the present disclosure. In some embodiments, the specific gene is selected from genes such as ApoB, ApoC, ANGPTL3, PCSK9, SCD1, TIMP-1, Col1A1, FVII, STAT3, p53, HBV, and HCV. In some embodiments, the specific gene is selected from hepatitis B virus genes, angiopoietin-like protein 3 genes, and apolipoprotein C3 genes. Accordingly, the disease is selected from chronic liver disease, hepatitis, liver fibrosis, hepatic hyperplasia, and dyslipidemia. In some embodiments, the dyslipidemia is hypercholesterolemia, hypertriglyceridemia, or atherosclerosis. In some embodiments, the conjugate provided by the present disclosure can also be used to treat other liver diseases, including diseases characterized by unwanted cell proliferation, hematological disorders, metabolic disorders, and diseases characterized by inflammation. The proliferative disease of the liver may be a benign or malignant disease, such as cancer, hepatocellular carcinoma (HCC), liver metastasis, or hepatoblastoma. The hepatic hematological or inflammatory disease may be a disease related to blood coagulation factors, complement-mediated inflammation, or fibrosis. The metabolic disease of the liver includes lipid abnormalities and irregular glucose regulation. In one embodiment, the disease is treated by using one or more double-stranded oligonucleotides having a high degree of sequence identity to a gene sequence involved in the disease.

[0320] In some embodiments, the present disclosure provides a method for inhibiting expression of a specific gene in a cell, comprising contacting the cell with an effective amount of a double-stranded oligonucleotide, drug composition and / or oligonucleotide conjugate provided by the present disclosure.

[0321] By administering the double-stranded oligonucleotide, drug composition, and / or oligonucleotide complex of the present disclosure to a subject in need thereof, the goal of preventing and / or treating a pathological condition or disease caused by the expression of a specific gene in cells via a gene expression regulation mechanism can be achieved. Thus, the double-stranded oligonucleotide, drug composition, and / or oligonucleotide complex of the present disclosure can be used for the prevention and / or treatment of the pathological condition or disease, or can be used to prepare a drug for the prevention and / or treatment of the pathological condition or disease described herein.

[0322] As used herein, the term "drug administration" refers to the introduction of a double-stranded oligonucleotide, drug composition, and / or oligonucleotide conjugate into a subject's body by a method or route that localizes at least a portion of the double-stranded oligonucleotide, drug composition, and / or oligonucleotide conjugate at a desired site, thereby producing a desired effect. Suitable administration routes for the methods of the present disclosure include local administration and systemic administration. Generally, local administration delivers more of the double-stranded oligonucleotide, drug composition, and / or oligonucleotide conjugate to a specific site than to the entire body of the subject, whereas systemic administration delivers the double-stranded oligonucleotide, drug composition, and / or oligonucleotide conjugate to nearly the entire body of the subject. Given that the present disclosure is intended to provide a means for preventing and / or treating pathological conditions or diseases caused by the expression of specific genes in hepatocytes, in some embodiments, the administration method can deliver a drug to the liver.

[0323] Administration to a subject can be by any suitable route known in the art, including, but not limited to, oral or non-gastrointestinal (parenteral) routes, such as intravenous, intramuscular, subcutaneous, transdermal, intratracheal (aerosol), pulmonary, nasal, rectal, and topical (including buccal and sublingual) administration. The frequency of administration can be once or more daily, weekly, biweekly, triweekly, monthly, or yearly.

[0324] The dose of the double-stranded oligonucleotide, drug composition, and / or oligonucleotide conjugate described in this disclosure may be a dose conventional in the art, and the dose may be determined based on various parameters, particularly the age, weight, and sex of the subject. Toxicity and therapeutic efficacy may be measured in cell cultures or experimental animals using standard pharmaceutical procedures, for example, to determine the LD50 (the dose that causes death in 50% of the colony) and ED50 (the dose that produces a 50% maximum response in a quantitative response, or the dose at which a positive response occurs in 50% of experimental subjects in a qualitative response). A range of human dosages can be derived based on data obtained from cell culture assays and animal studies.

[0325] When administering the double-stranded oligonucleotide, drug composition, and / or oligonucleotide complex described in the present disclosure to, for example, male or female, 6-12 week old, 18-25 g C57BL / 6J or C3H / HeNCrlVr mice, the amount of double-stranded oligonucleotide in the double-stranded oligonucleotide, drug composition, and / or oligonucleotide complex may be 0.001-100 mg / kg body weight, in some embodiments 0.01-50 mg / kg body weight, in further embodiments 0.05-20 mg / kg body weight, in still further embodiments 0.1-15 mg / kg body weight, and in still further embodiments 0.1-10 mg / kg body weight. The above dosages are preferred when administering the double-stranded oligonucleotide, drug composition, and / or oligonucleotide complex described in the present disclosure to, for example, male or female, 6-12 week old, 18-25 g C57BL / 6J or C3H / HeNCrlVr mice.

[0326] Alternatively, the double-stranded oligonucleotide, pharmaceutical composition, and / or oligonucleotide conjugate of the present disclosure can be introduced into hepatocytes in which a specific gene is abnormally expressed, thereby achieving the goal of suppressing the expression of the specific gene in the hepatocytes through a gene expression regulation mechanism. In some embodiments, the hepatocytes are hepatitis cells, and in some embodiments, HepG2.2.15 cells. In some embodiments, the hepatocytes may be selected from hepatoma cell lines such as Hep3B, HepG2, and Huh7, or isolated primary hepatocytes, and in some embodiments, are Huh7 hepatoma cells.

[0327] The method provided by the present disclosure suppresses the expression of a specific gene in liver cells. The dose of the double-stranded oligonucleotide in the provided double-stranded oligonucleotide, drug composition, and / or oligonucleotide complex can be easily determined by one of ordinary skill in the art based on the desired effect. For example, in some embodiments, the double-stranded oligonucleotide, drug composition, and / or oligonucleotide complex is an siRNA complex, and the siRNA dose in the provided siRNA complex is an amount that can reduce target gene expression and achieve an extracellular concentration of 1 pM to 1 μM, 0.01 nM to 100 nM, 0.05 nM to 50 nM, or 0.05 nM to approximately 5 nM on the surface of the target cell. The amount required to achieve this local concentration varies depending on various factors, including the delivery method, 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 may be significantly higher than the concentration at the surface of the target cell or tissue.

[0328] <Kit> The present disclosure provides kits comprising the above-described double-stranded oligonucleotide, the above-described pharmaceutical composition, and / or the above-described oligonucleotide conjugate.

[0329] In some embodiments, the kits described herein may provide the double-stranded oligonucleotide in a container. In some embodiments, the kits described herein may include a container providing a pharmaceutically acceptable excipient. In some embodiments, the kits may include other components, such as stabilizers or preservatives. In some embodiments, the kits described herein may include at least one other therapeutic agent in a container separate from the container providing the double-stranded oligonucleotide described herein. In some embodiments, the kits may include instructions for mixing the double-stranded oligonucleotide with a pharmaceutically acceptable carrier and / or additives or other components, if present.

[0330] In the kits of the present disclosure, the double-stranded oligonucleotide and pharmaceutically acceptable carrier and / or additive, and the double-stranded oligonucleotide composition and / or complex and / or pharmaceutically acceptable additive may be provided in any format, for example, liquid format, dry format, or lyophilized format. In some embodiments, the double-stranded oligonucleotide and pharmaceutically acceptable carrier and / or additive, and the double-stranded oligonucleotide composition and / or complex and any pharmaceutically acceptable additive are essentially clean and / or sterile. In some embodiments, sterile water may be provided in the kits of the present disclosure.

[0331] The present disclosure will be further described below with reference to examples, but the present disclosure is not limited thereto in any way.

[0332] Without wishing to be limited thereto, the present invention will be further described in the following embodiments and examples relating to exemplary embodiments in which the double-stranded oligonucleotide in the composition and / or oligonucleotide complex of the present disclosure is a small interfering RNA (siRNA). In such cases, the double-stranded oligonucleotide, composition, and oligonucleotide complex of the present disclosure are siRNA, a composition containing siRNA, and an siRNA complex, respectively. In the context of the present disclosure, for convenience of explanation, the siRNA, the composition containing siRNA, and the siRNA complex in these embodiments are also referred to as the siRNA of the present disclosure, the siRNA composition of the present disclosure, and the siRNA complex of the present disclosure. This does not mean that the double-stranded oligonucleotide of the present disclosure is only siRNA; on the contrary, the double-stranded oligonucleotide may be other variants disclosed herein or known to those skilled in the art, such as small activating RNA (saRNA). Based on the detailed description of siRNA, siRNA-containing compositions, and siRNA complexes, it is conceivable that other functional double-stranded oligonucleotides will function similarly when used alone or in forming the compositions and / or complexes described in the present disclosure. (Effects of the Invention)

[0333] In some embodiments, the double-stranded oligonucleotides, compositions, or oligonucleotide complexes provided by the present disclosure may have higher stability, lower toxicity, and / or higher activity in vivo. In some embodiments, the double-stranded oligonucleotides provided by the present disclosure are saRNAs. In some embodiments, the saRNAs, saRNA compositions, or saRNA complexes provided by the present disclosure exhibit an increase in target gene expression in vivo of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%. In some embodiments, the double-stranded oligonucleotides provided by the present disclosure are siRNAs. In some embodiments, the siRNAs, siRNA compositions, or siRNA complexes provided by the present disclosure exhibit an inhibition of target gene expression in vivo of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%. In some embodiments, the siRNA, siRNA composition, or siRNA complex provided by the present disclosure exhibits at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% HBV gene expression suppression in vivo. In some embodiments, the siRNA, siRNA composition, or siRNA complex provided by the present disclosure exhibits at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% intrahepatic HBV gene expression suppression in vivo. In some embodiments, the siRNA, siRNA composition, or siRNA complex provided by the present disclosure exhibits at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% intrahepatic HBV gene expression suppression in vivo in an animal model. In some embodiments, the siRNA, siRNA composition, or siRNA complex provided by the present disclosure exhibits at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% suppression of HBV surface antigen expression in vivo. In some embodiments, the siRNA, siRNA composition, or siRNA complex provided by the present disclosure exhibits at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% suppression of ANGPTL3 gene expression in vivo.In some embodiments, the siRNA, siRNA composition, or siRNA complex provided by the present disclosure exhibits at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% in vivo suppression of intrahepatic ANGPTL3 gene expression. In some embodiments, the siRNA, siRNA composition, or siRNA complex provided by the present disclosure exhibits at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% in vivo suppression of intrahepatic ANGPTL3 gene expression in an animal model. In some embodiments, the siRNA, siRNA composition, or siRNA complex provided by the present disclosure exhibits at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% in vivo suppression of intrahepatic ANGPTL3 gene expression in human subjects. In some embodiments, the siRNA, siRNA composition, or siRNA complex provided by the present disclosure exhibits at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% APOC3 gene expression suppression in vivo. In some embodiments, the siRNA, siRNA composition, or siRNA complex provided by the present disclosure exhibits at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% intrahepatic APOC3 gene expression suppression in vivo in an animal model. In some embodiments, the siRNA, siRNA composition, or siRNA complex provided by the present disclosure exhibits at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% suppression of intrahepatic APOC3 gene expression in human subjects. In some embodiments, the double-stranded oligonucleotide, composition, or oligonucleotide complex provided by the present disclosure does not exhibit significant off-target effects. The off-target effects may be, for example, suppression of normal expression of a gene other than the target gene.An off-target effect is considered to be insignificant if the binding / inhibition of off-target gene expression is less than 50%, 40%, 30%, 20%, or 10% of the on-target gene effect.

[0334] According to some embodiments of the present disclosure, the siRNA, siRNA composition, and siRNA complex of the present disclosure exhibit excellent inhibitory effects. For example, according to one embodiment of the present disclosure, the siRNA complex provided by the present disclosure has low off-target effects and exhibits excellent HBV gene expression inhibitory properties, suppressing HBV gene expression by 66.9% to 90.9% in the liver of a hepatitis B mouse model at a dose of 1 mg / kg. At the same time, the siRNA complex of the present disclosure can also effectively reduce HBV surface antigen expression and HBV DNA in a hepatitis B mouse model. In particular, compared to those provided by the prior art, the specific siRNA complex formed by a specific modified siRNA and a specific conjugate molecule provided by the present disclosure can continue to exhibit excellent HBV expression inhibitory effects at low doses over a long experimental period of 140 days.

[0335] According to one embodiment of the present disclosure, the siRNA complex provided by the present disclosure has low off-target effects and exhibits excellent HBV gene expression suppression properties, suppressing HBV gene expression by 81.7 to 89.2% in the liver of a hepatitis B mouse model at a dose of 1 mg / kg. At the same time, the siRNA complex of the present disclosure can also effectively reduce HBV surface antigen expression and HBV DNA in a hepatitis B mouse model. In particular, compared to complexes formed with conjugate molecules provided by conventional techniques, a specific siRNA complex formed with a specific modified siRNA and a specific conjugate molecule provided by the present disclosure can maintain excellent HBV expression suppression activity at a low dose over a long experimental period of 84 days.

[0336] According to one embodiment of the present disclosure, the siRNA complex provided by the present disclosure exhibits excellent HBV gene expression suppression properties, with low off-target effects and a high HBV gene expression suppression rate of 93.8% in the liver of a hepatitis B mouse model at a dose of 1 mg / kg. At the same time, the siRNA complex provided by the present disclosure can also effectively reduce HBV surface antigen expression in a hepatitis B mouse model, achieving an HBV surface antigen expression suppression rate of 90% or more at a dose of 3 mg / kg and effectively suppressing HBV DNA. In particular, compared to a reference complex, a specific siRNA complex formed by a specific modified siRNA provided by the present disclosure and a specific conjugate molecule can maintain a high HBV expression suppression effect at a low dose over a long experimental period of 21 days.

[0337] According to one embodiment of the present disclosure, the siRNA complex provided by the present disclosure exhibits excellent HBV gene expression suppression properties, with low off-target effects and a high HBV X gene region gene suppression rate of 93.63% in the liver of a hepatitis B mouse model at a dose of 1 mg / kg. At the same time, the siRNA complex provided by the present disclosure can also effectively reduce HBV surface antigen expression in a hepatitis B mouse model, achieving an HBV surface antigen expression suppression rate of 95% or more at a dose of 3 mg / kg, thereby effectively suppressing HBV DNA. In particular, compared with complexes formed using conjugate molecules provided by the prior art, a specific siRNA complex formed using a specific modified siRNA and a specific conjugate molecule provided by the present disclosure continues to exhibit excellent HBV expression suppression effects at low doses over a long experimental period of 56 days, achieving an HBV X mRNA suppression rate of 90% or more.

[0338] In some embodiments, the siRNA complexes provided by the present disclosure exhibit excellent ANGPTL3 mRNA suppression efficiency and significantly reduce lipid levels. For example, in some embodiments, 14 days after a single subcutaneous administration, the ANGPTL3 mRNA suppression rate in mice is as high as 95% or higher. In some embodiments, after a single subcutaneous administration, the maximum triglyceride (TG) suppression rate is 93% and the maximum total cholesterol (CHO) suppression rate is 83%. At 154 days after drug administration, the TG suppression rate is maintained at 55% or higher and the CHO suppression rate is maintained at 40% or higher. In particular, compared to complexes formed with conjugate molecules provided by the prior art, the siRNA complexes provided by the present disclosure exhibit superior gene suppression rates and stronger lipid-lowering capabilities. Furthermore, the siRNA complexes provided by the present disclosure can maintain excellent lipid-lowering activity over a long experimental period of 189 days at low doses and with low administration frequency.

[0339] In some embodiments, the siRNA complexes provided by the present disclosure exhibit excellent APOC3 gene expression suppression properties, suppressing APOC3 gene expression by at least 88% in the livers of hyperlipidemia model mice at a dose of 1 mg / kg. In particular, compared to complexes formed using conjugated molecules provided by conventional techniques, the modified siRNA and siRNA complexes provided by the present disclosure exhibit excellent gene suppression rates and low off-target effects. Furthermore, the siRNA complexes provided by the present disclosure can maintain excellent lipid-suppressing activity over a long experimental period of 189 days, even at low doses and with low administration frequency.

[0340] In certain embodiments, the siRNA complexes described herein further exhibit low toxicity and good safety in animals. For example, in some embodiments, the complexes of the present disclosure did not show any obvious toxic reactions even when administered to C57BL / 6J mice at 100 times the effective concentration (assuming the effective concentration is 3 mg / kg).

[0341] From the above, it can be seen that the siRNAs, siRNA compositions and siRNA complexes provided herein efficiently reduce gene expression in target cells and exhibit excellent delivery potential. [Example]

[0342] The present disclosure will be described in detail below with reference to examples. Unless otherwise specified, all reagents and media used in the following examples are commercially available products, and all procedures such as nucleic acid electrophoresis and real-time PCR used are performed with reference to the methods described in Molecular Cloning (Cold Spring Harbor Laboratory Press (1989)).

[0343] HEK293A cells were provided by the Nucleic Acid Technology Laboratory, Institute of Molecular Medicine, Peking University. The cells were cultured in DMEM complete medium (Hyclone) containing 20% ​​fetal bovine serum (FBS, Hyclone) and 0.2% penicillin-streptomycin (Gibco, Invitrogen) at 37°C in a 5% CO2 / 95% air incubator.

[0344] HepG2.2.15 cells were purchased from ATCC and cultured in DMEM complete medium (Gibco) containing 10% fetal bovine serum (FBS, Gibco), 2 mM L-glutamine (Gibco), and 380 μg / ml G418 at 37°C in a 5% CO2 / 95% air incubator.

[0345] Huh7 cells were purchased from ATCC and cultured in DMEM complete medium (Gibco) containing 10% fetal bovine serum (FBS, Gibco), 2 mM L-glutamine (Gibco), and 380 μg / ml G418 at 37°C in a 5% CO2 / 95% air incubator.

[0346] Unless otherwise specified, when cells were transfected with the various siRNAs or siRNA complexes synthesized below, Lipofectamine was used as the transfection reagent. TM 2000 (Invitrogen), and for specific procedures, refer to the instructions provided by the manufacturer.

[0347] Unless otherwise stated, all reagent proportions provided below are calculated as volume ratios (v / v).

[0348] 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 referred to simply as C57 mice. SD rats: provided by Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd. HBV transgenic mice C57BL / 6-HBV: Breed name: B6-Tg HBV / Vst (1.28 copies, genotype A), purchased from Beijing Weitongda Biotechnology Co., Ltd. Before the experiment, COI > 10 4 Mice (hereinafter simply referred to as 1.28 copy mice) were selected. HBV transgenic mice C57BL / 6J-Tg(Alb1HBV)44Bri / J: Purchase from the Department of Laboratory Animal Science, School of Medicine, Peking University. HBV transgenic mice: designated M-TgHBV, were purchased from the Animal Department of Shanghai Municipal Public Health Center. The method for producing transgenic mice is described in Ren J. et al., J. Medical Virology. 2006, 78:551-560. AAV-HBV transgenic mice: AAV-HBV models were generated according to the published method (Dong Xiaoyan et al., Chin J Biotech 2010, May 25, 26(5):679-686). rAAV8-1.3HBV, type D (ayw), was purchased from Beijing Wujiahe Molecular Medicine Research Institute Co., Ltd., and 1 × 10 12 Viral genome (vg) / mL, Lot No. 2016123011. 5 × 10 11200 μL was injected per mouse, i.e., 1 × 10 per mouse. 11 On day 28 after virus injection, all mice were subjected to retro-orbital bleeding (approximately 100 μL), and serum was collected and used to detect HBsAg and HBV DNA. Low-concentration AAV-HBV transgenic mice: Virus was added to 1 x 10 in sterile PBS before the experiment. 11 The virus was diluted to 100 μL / mL and 100 μL of virus was injected per mouse, i.e., 1 × 10 per mouse. 10 Except for the VG injection, the modeling method was basically the same as above. 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)3707Bres / J, purchased from Jackson Laboratory, USA. Metabolic syndrome monkeys: provided by the Non-human Primate Research Center, Institute of Molecular Medicine, Peking University.

[0349] Unless otherwise stated, the following in vivo / in vitro effect experimental data are Data analysis was performed using Graphpad Prism 5.0 statistical analysis software. Data were first tested for normal distribution and homogeneity of variance. If the data met the normal distribution (p>0.20) and the variances were equal (p>0.10), multiple comparisons between groups were performed using the LSD method with one-way analysis of variance. A p<0.05 result was considered statistically significant. If the data did not meet the normal distribution or the variances were unequal, the nonparametric Kruskal-Wallis H test was used. If the Kruskal-Wallis H test result was significant (p<0.05), the data were rank-transformed and pairwise comparisons between groups were performed. A p<0.05 result was considered statistically significant.

[0350] (Preparation Example 1) Preparation of siRNA of the present disclosure In this preparation example, the siRNAs in Table 2 were synthesized according to the following method.

[0351] [Table 2] * S: sense strand, AS: antisense strand Note: Capital letters C, G, U, and A represent the base sequence of nucleotides, dT represents a deoxythymine nucleotide, lowercase m represents a 2'-methoxy-modified nucleotide on the left side of the letter m, lowercase f represents a 2'-fluoro-modified nucleotide on the left side of the letter f, lowercase s represents a bond between two adjacent nucleotides on the left and right sides of the letter s via a phosphorothioate group, VP represents a vinyl phosphate-modified nucleotide on the right side of the letter VP, P represents a phosphate-modified nucleotide on the right side of the letter P, and Ps represents a phosphorothioate-modified nucleotide on the right side of the letter Ps.

[0352] (1-1) Synthesis of Sense Strand of siRNA General-purpose solid support (UnyLinker TM Starting with NittoPhase® HL Solid Supports (Kinovate Life Sciences), the nucleoside monomers were bound one by one in the 3'-5' direction according to the above sequence order. Each binding of a nucleoside monomer involves four reactions: deprotection, coupling, capping, and oxidation. The synthesis conditions were as follows:

[0353] The nucleoside monomers were provided in a 0.1 M acetonitrile solution, and the conditions for each deprotection reaction were the same: the temperature was 25°C, the reaction time was 70 seconds, the deprotection reagent was a dichloroacetic acid solution in dichloromethane (3% v / v), and the molar ratio of dichloroacetic acid to the 4,4'-dimethoxytrityl protecting group on the solid support was 5:1.

[0354] The coupling reaction conditions were the same for all reactions: temperature 25°C, molar ratio of the nucleic acid sequence to be bound to the solid support to the nucleoside monomer 1:10, molar ratio of the nucleic acid sequence to be bound to the solid support to the coupling reagent 1:65, reaction time 600 seconds, and coupling reagent 0.5 M acetonitrile solution of 5-ethylthio-1H-tetrazole.

[0355] The capping conditions were the same for all experiments: temperature 25°C, reaction time 15 seconds. The capping reagent solution was a mixed solution of Cap1 and Cap2 in a molar ratio of 1:1, and the molar ratio of the capping reagent to the nucleic acid sequence to be bound to the solid phase carrier was acetic anhydride:N-methylimidazole:nucleic acid sequence to be bound to the solid phase carrier = 1:1:1.

[0356] The oxidation reaction conditions were the same for each experiment: the temperature was 25°C, the reaction time was 15 seconds, and the oxidation reagent was 0.05M iodine water. The molar ratio of iodine to the nucleic acid sequence bound to the solid support in the coupling step was 30:1. The reaction was carried out in a mixed solvent of tetrahydrofuran:water:pyridine = 3:1:1.

[0357] When two nucleotides in the target sequence are linked by a thiophosphate ester, the following sulfurization reaction step was used instead of the oxidation reaction step for the linkage of the latter nucleotide. The sulfurization reaction conditions for each step were the same, including a temperature of 25°C, a reaction time of 300 seconds, and xanthan hydride as the sulfurization reagent. The molar ratio of the sulfurization reagent to the nucleic acid sequence to be bound to the solid support in the coupling step was 120:1. The reaction was carried out in a mixed solvent of acetonitrile:pyridine = 1:1.

[0358] The cleavage and deprotection conditions were as follows: The synthesized carrier-bound nucleotide sequence was added to 25 wt% aqueous ammonia at a dosage of 0.5 ml / μmol and reacted at 55°C for 16 hours. The liquid was removed and the mixture was concentrated under vacuum to dryness. After treatment with aqueous ammonia, the product was dissolved in 0.4 ml / μmol N-methylpyrrolidone per amount of single-stranded nucleic acid. Then, 0.3 ml / μmol triethylamine and 0.6 ml / μmol triethylamine trihydrofluoride were added to remove the 2'-TBDMS protection from the ribose. If the 2'-position of all nucleotides in the target sequence was a modified hydroxy group, the cleavage and deprotection conditions did not include a step for removing the 2'-TBDMS protection from the ribose.

[0359] Purification and desalting: Nucleic acid purification was achieved using a preparative ion chromatography purification column (Source 15Q) with a gradient elution of NaCl. Specifically, eluent A was 20 mM sodium phosphate (pH 8.1) with a water / acetonitrile ratio of 9:1 (volume ratio), and eluent B was 1.5 M sodium chloride, 20 mM sodium phosphate (pH 8.1) with a water / acetonitrile ratio of 9:1 (volume ratio). The eluate gradient was eluted from 100:0 to 50:50 with eluent A:eluent B. The product eluates were collected and combined, and then desalted using a reversed-phase chromatography purification column. Specifically, the desalting was performed using a dextran gel column with a Dextran Gel G25 packing and deionized water elution.

[0360] Detection: Purity was detected using ion exchange chromatography (IEX-HPLC), and the molecular weight was analyzed by liquid chromatography-mass spectrometry (LC-MS) and compared with the theoretical value.

[0361] As a result, the sense strand S of siRNA was synthesized in this step.

[0362] (1-2) Synthesis of antisense strand In this process, a general-purpose solid support (UnyLinker TM The antisense strand (AS) of siRNA was synthesized using solid-phase support loaded with NittoPhase® HL Solid Supports (Kinovate Life Sciences). The conditions for deprotection, coupling, capping, oxidation and / or sulfurization, deprotection and cleavage, and isolation in the solid-phase synthesis were the same as those for the synthesis of the sense strand.

[0363] Here, a 2'-methoxy-modified uracil nucleoside monomer (VP-Um) modified with vinyl phosphate ester was synthesized according to the following method.

[0364] [ka]

[0365] (1-2-1) Synthesis of VP-U-2 The VP-U-2 molecule was synthesized according to the following method.

[0366] [ka] 2'-Methoxy-modified uracil nucleotide (2'-OMe-U, 51.30 g, 91.6 mmol), tert-butyldiphenylchlorosilane (TBDPSCl, 50.35 g, 183.2 mmol), and imidazole (12.47 g, 183.2 mmol) were dissolved in 450 mL of N,N-dimethylformamide (DMF) and stirred at room temperature for 20 h. After distillation, the DMF was removed, and the mixture was dissolved in 600 mL of dichloromethane. The mixture was washed with 300 mL of saturated sodium bicarbonate. The aqueous phase was extracted three times with 300 mL of dichloromethane (DCM). The combined organic phases were washed with 5% oxalic acid until the aqueous phase reached a pH < 5. After evaporation to dryness, crude VP-U-1 was obtained, which was used directly in the synthesis of VP-U-2.

[0367] The crude VP-U-1 product was dissolved in 100 ml of dichloromethane and stirred in an ice bath for 10 minutes. Then, 450 ml of 2% p-toluenesulfonic acid solution (solvent: methanol-dichloromethane mixed solvent in a volume ratio of 3:7) pre-chilled in a 4°C refrigerator was added and the reaction was allowed to proceed for 10 minutes. The reaction was quenched by adding 200 ml of saturated sodium bicarbonate, and the organic phase was washed with saturated aqueous sodium bicarbonate until the pH reached 8. The aqueous phases were combined and extracted twice with 200 ml of dichloromethane. The organic phases were combined and washed once with 200 ml of saturated brine, and the solvent was evaporated to dryness. The product was purified on a 200-300 mesh normal-phase silica gel column. Petroleum ether was added to the column and eluted with a gradient of petroleum ether:ethyl acetate:dichloromethane:methanol=1:1:1:0.05 to 1:1:1:0.25. The product eluate was collected, and the solvent was evaporated to dryness under reduced pressure. The product was then foam-dried using a vacuum oil pump to obtain 40.00 g of pure VP-U-2. 1H NMR (400 MHz,DMSO-d6) δ 7.96 (d,J =7.8 Hz,1H),7.64 (dtd,J =5.1,4.0,2.2 Hz,4H),7.41-7.30 (m,6H),6.79 (d,J =4.7 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, actual value: 497.45.

[0368] (1-2-2) Synthesis of VP-U-4:

[0369] [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), and trifluoroacetic acid (6.61 g, 53.2 mmol) were mixed and dissolved in 200 mL of dimethyl sulfoxide (DMSO) and stirred at room temperature for 20 h. Separately, tetraethyl methylenediphosphonate (21.44 g, 74.4 mmol) was dissolved in 120 mL of THF and cooled in an ice bath. t-BuOK (11.36 g, 101.2 mmol) was added at ice bath temperature for 10 min, then warmed to room temperature and reacted for 0.5 h. The mixture was then added to the reaction mixture over approximately 1 h and reacted at ice bath temperature for 1 h. The mixture was then warmed to room temperature and reacted for 18 h. The reaction was quenched by the addition of water, and the aqueous phase was extracted three times with 200 ml of dichloromethane. The combined organic phases were washed once with 200 ml of saturated brine, and the solvent was evaporated to dryness. The product was purified on a 200-300 mesh normal-phase silica gel column. Petroleum ether was added to the column, and a gradient elution of petroleum ether:ethyl acetate = 1:1 to 1:4 was performed. The product eluate was collected, the solvent evaporated to dryness under reduced pressure, and the mixture was foam-dried using a vacuum oil pump to obtain a total of 14.00 g of pure VP-U-4. 1H NMR (400 MHz,DMSO-d6) δ 7.96 (d,J =7.8 Hz,1H),7.64 (dtd,J =5.1,4.0,2.2 Hz,4H),7.41 - 7.30 (m,6H),6.82 - 6.71 (m,2H),5.90 (ddd,J =25.9, 15.0,1.0 Hz,1H),5.73 (d,J =7.6 Hz,1H),4.36 - 4.21 (m,3H),4.18 (t,J =4.9 Hz,1H),4.05 (ddq,J =9.7,8.5,6.9 Hz,2H),3.87 (t,J =4.8 Hz,1H),3.39 (s,3H),1.32 (td,J =6.9,0.7 Hz,6H),1.05 (s,8H). MS m / z:C 31 H 42 N2O8PSi, [M+H] + , theoretical value: 629.24, measured value: 629.51.

[0370] (1-2-3) Synthesis of VP-U-5:

[0371] [ka] VP-U-4 (14.00 g, 22.29 mmol) was dissolved in 100 mL of tetrahydrofuran, triethylamine trihydrofluoride (17.96 g, 111.45 mmol) was added, and the mixture was stirred at room temperature for 20 h to complete the reaction. The solvent was evaporated to dryness, and the crude product was obtained by dissolving in 50 mL of dichloromethane and evaporating to dryness twice. The crude product was purified on a 200-300 mesh normal-phase silica gel column. Petroleum ether was applied to the column and eluted with a gradient of petroleum ether:ethyl acetate:dichloromethane:methanol = 1:1:1:0.05 to 1:1:1:0.25. The product eluate was collected, the solvent was evaporated to dryness under reduced pressure, and the mixture was dried under a vacuum oil pump to obtain 6.70 g of pure VP-U-5. 1H NMR (400 MHz,DMSO-d6) δ 7.96 (d,J =7.8 Hz,1H),6.77 (dd,J =15.0,6.2 Hz,1H),5.99 - 5.82 (m,2H),5.73 (d,J =7.6 Hz,1H),5.27 (d,J =5.1 Hz,1H),5.10 (dd,J =5.3,4.7 Hz,1H),4.29 (ddq,J =9.8,8.6,7.0 Hz,2H),4.17 (ddd,J =6.2,5.2,1.0 Hz,1H),4.12 - 3.98 (m,3H),3.39 (s,2H),1.32 (td,J =6.9,0.6 Hz,6H).MS m / z:C 15 H 24 N2O8P, [M+H] + , theoretical value: 391.13, measured value: 391.38.

[0372] (1-2-4) Synthesis of VP-U-6:

[0373] [ka] VP-U-5 (391 mg, 1.0 mmol), pyridine trifluoroacetate (0.232 g, 1.2 mmol), N-methylimidazole (0.099 g, 1.2 mmol), and bis(diisopropylamino)(2-cyanoethoxy)phosphine (0.452 g, 1.5 mmol) were added to 10 mL of anhydrous dichloromethane under argon protection and stirred at room temperature for 5 h. The solvent was evaporated to dryness, and the product was purified by column chromatography (200-300 mesh normal phase silica gel, gradient elution with dichloromethane:acetonitrile (containing 0.5 wt% triethylamine) = 3:1 to 1:3). The product eluate was collected and the solvent was removed by concentration to give 508 mg of the desired product, VP-U-6. 31P NMR (161 MHz,DMSO-d6) δ 150.34,150.29,17.07,15.50. MS m / z:C 24 H 41 N4O9P2, [M+H] + , theoretical value: 591.23, actual value: 591.55. This indicates that VP-U-6 is the target product VP-Um, which is involved in the synthesis of RNA chains as a nucleoside monomer.

[0374] A 5'-phosphate modification was attached to the 5' end of the antisense strand using the following method.

[0375] The raw material is a phosphorylated structural monomer having the following structure: CPR-I, provided by Suzhou Jima, with the number Cat#13~2601-XX.

[0376] [ka] After all nucleoside monomers of the antisense strand were attached, the CPR-I monomer was attached to the 5' end of the antisense strand by phosphoramidite solid-phase nucleic acid synthesis through four reactions: deprotection, coupling, capping, and oxidation. The antisense strand was then obtained by cleavage and deprotection under the following conditions.

[0377] The synthesized carrier-bound nucleotide sequence was added to 25 wt% aqueous ammonia at a concentration of 0.5 ml / μmol and reacted at 55°C for 16 hours. The liquid was removed and the mixture was concentrated under vacuum to dryness. After treatment with aqueous ammonia, the product was dissolved in 0.4 ml / μmol N-methylpyrrolidone for each amount of single-stranded nucleic acid. Then, 0.3 ml / μmol triethylamine and 0.6 ml / μmol triethylamine trihydrofluoride were added to remove the 2'-TBDMS protection on the ribose. Purification and desalting: Nucleic acid purification was completed using a preparative ion chromatography purification column (Source 15Q) with a gradient elution using NaCl. Specifically, eluent A was 20 mM sodium phosphate (pH 8.1), with a solvent of water / acetonitrile = 9:1 (volume ratio), and eluent B was 1.5 M sodium chloride, 20 mM sodium phosphate (pH 8.1), with a solvent of water / acetonitrile = 9:1 (volume ratio). The elution gradient was eluted with eluent A:eluent B = 100:0 to 50:50. The product eluates were collected and combined, and desalted using a reversed-phase chromatography purification column. Specific conditions included desalting using a dextran gel column, with a packing material of dextran gel G25, and elution with deionized water.

[0378] When the target product had a 5'-thiophosphate modification, the same procedure as above was used, except that the sulfurization reaction was carried out using sulfurization reaction conditions instead of the oxidation reaction conditions described above during conjugation.

[0379] The antisense strand of the siRNA was analyzed and detected in the same manner, using the same instruments and methods as the sense strand, and finally, it was confirmed that the corresponding antisense strand of the siRNA was obtained.

[0380] (1-3) Synthesis of siRNA The S chain and AS chain were mixed in an equimolar ratio, dissolved in water for injection, heated to 95°C, and cooled to room temperature, after which they were allowed to form a double-stranded structure through hydrogen bonding.

[0381] The purity of the synthesized sense and antisense strands was detected by ion exchange chromatography (IEX-HPLC), and the molecular weight was analyzed by liquid chromatography tandem mass spectrometry (LC-MS), confirming that the synthesized nucleic acid sequences corresponded to each siRNA in Table 2.

[0382] (Preparation Example 2) Preparation of Complex 1 In this preparation example, an siRNA complex numbered Complex A1 in Table 4A was synthesized according to the following method.

[0383] (2-1) Preparation of L-10 Compound Compound L-10 was synthesized according to the following method.

[0384] [ka]

[0385] (2-1-1) Synthesis of GAL-5 Complex Terminal Segments

[0386] [ka]

[0387] (2-1-1a) 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) was dissolved in 1000 ml of anhydrous pyridine, and 540 ml of acetic anhydride (purchased from Enox, 5565.6 mmol) was added in an ice-water bath. The mixture was stirred at room temperature for 1.5 hours. The reaction mixture was poured into 10 L of ice water and filtered under reduced pressure. The cake was washed with 2 L of ice water. After complete dissolution, an acetonitrile / toluene mixed solvent (acetonitrile:toluene volume ratio = 1:1) was added. The solvent was evaporated to dryness, yielding 130.0 g of a white solid product, GAL-2.

[0388] (2-1-1b) Synthesis of GAL-3 GAL-2 (35.1 g, 90.0 mmol) obtained in step (1-1a) was dissolved in 213 ml of anhydrous 1,2-dichloroethane, and 24.0 g of TMSOTf (CAS No.: 27607-77-8, purchased from Macklin Chemical, 108.0 mmol) was added under nitrogen protection in an ice-water bath, and the mixture was allowed to react at room temperature overnight.

[0389] The reaction mixture was diluted with 400 ml of dichloromethane, filtered through diatomaceous earth, washed with 1 L of saturated aqueous sodium bicarbonate solution, and the organic phase was separated. The aqueous phase was extracted twice with 300 ml of dichloroethane. The combined organic phases were washed with 300 ml of saturated aqueous sodium bicarbonate solution and 300 ml of saturated saline solution, and the organic phase was separated and dried over anhydrous sodium sulfate. The solvent was evaporated to dryness under reduced pressure to obtain 26.9 g of a pale yellow, viscous starch syrup-like product, GAL-3.

[0390] (2-1-1c) Synthesis of GAL-4 GAL-3 (26.9 g, 81.7 mmol) obtained in step (1-1b) was dissolved in 136 ml of anhydrous 1,2-dichloroethane, and 30 g of dried 4 Å molecular sieve powder was added. 9.0 g of 5-hexen-1-ol (CAS number: 821-41-0, purchased from Adamas-beta, 89.9 mmol) was added and stirred at room temperature for 30 minutes. 9.08 g of TMSOTf (40.9 mmol) was added under nitrogen protection in an ice bath, and the reaction was stirred at room temperature overnight. The 4Å molecular sieve powder was removed by filtration, and the filtrate was diluted with 300 ml of dichloromethane and filtered through diatomaceous earth. 500 ml of saturated aqueous sodium bicarbonate solution was added and washed with stirring for 10 minutes. The organic phase was separated, and the aqueous phase was extracted once with 300 ml of dichloroethane. The combined organic phases were washed with 300 ml of saturated aqueous sodium bicarbonate solution and 300 ml of saturated brine, respectively. The organic phase was separated and dried over anhydrous sodium sulfate, and the solvent was evaporated to dryness under reduced pressure to obtain 41.3 g of a yellow starch syrup-like product, GAL-4, which was directly used in the next oxidation reaction without further purification.

[0391] (2-1-1d) Synthesis of GAL-5 GAL-4 (14.9 g, 34.7 mmol) obtained by the method described in step (1-1c) was dissolved in a mixed solvent of 77 mL of dichloromethane and 77 mL of acetonitrile, and 103 mL of deionized water and 29.7 g of sodium periodate (CAS No.: 7790-28-5, purchased from Aladdin, 138.8 mmol) were added. The mixture was stirred in an ice-water bath for 10 minutes, and ruthenium(III) chloride (CAS No.: 14898-67-0, purchased from Energy, 238 mg, 1.145 mmol) was added. The system temperature was controlled below 30 °C, and the mixture was allowed to react at room temperature overnight. 300 mL of water was added to the reaction mixture, followed by stirring. The pH was adjusted to approximately 7.5 with saturated sodium bicarbonate. The organic phase was separated and discarded. The aqueous phase was extracted three times with dichloromethane, each time using 200 mL of dichloromethane. The organic phase was discarded. The aqueous phase was adjusted to pH 3 with solid citric acid and extracted three times with dichloromethane, each time with 200 ml of dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was evaporated to dryness under reduced pressure to obtain 6.5 g of a white foamy solid product, GAL-5. 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),4.49 (d,J =8.4 Hz,1H),4.07 - 3.95 (m,3H),3.92 - 3.85 (m,1H),3.74 - 3.67 (m,1H),3.48 - 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).

[0392] (2-1-2) Synthesis of M-11-T3:

[0393] [ka] J-0 (1.883 g, 10 mmol, purchased from Alfa Aesar) was dissolved in 25 mL of acetonitrile, triethylamine (4.048 g, 40 mmol) was added, and the mixture was cooled to 0 °C in an ice-water bath. Ethyl trifluoroacetate (5.683 g, 40 mmol) was added, and the mixture was reacted at room temperature for 22 h. The solvent was evaporated to dryness under reduced pressure, and the mixture was foam-dried in a vacuum oil pump for 18 h to obtain 5.342 g of solid crude product M-11-T3, which was used directly in the subsequent reaction without further purification. MS m / z: C 15 H 22 F9N4O3, [M+H] + , theoretical value: 477.35, measured value: 477.65.

[0394] (2-1-3) Synthesis of M-11-T3-Tr:

[0395] [ka] The crude M-11-T3 product (5.342 g, 10 mmol) was dissolved in 50 ml of dichloromethane. TrCl (3.345 g, 12 mmol) and triethylamine (1.518 g, 15 mmol) were added to the reaction mixture and stirred at room temperature for 20 hours. The reaction mixture was washed twice with 20 ml of saturated sodium bicarbonate solution, and once with 20 ml of saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and the organic solvent was evaporated to dryness under reduced pressure. The mixture was then foam-dried overnight using a vacuum oil pump to obtain 7.763 g of the solid crude product M-11-T3-Tr. MS m / z: C 34 H 36 F9N4O3, [M+Na] + Theoretical value: 741.25, Found value: 741.53. The solid crude product M-11-T3-Tr was used for the subsequent synthesis of M-18-Tr without further purification.

[0396] (2-1-4) Synthesis of M-18-Tr:

[0397] [ka] The crude M-11-T3-Tr product (7.763 g, 10 mmol) obtained in step (2-1-3) was dissolved in 100 ml of methanol, 100 ml of aqueous methylamine solution (40% by mass) 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. 200 ml of a 1:1 volume ratio DCM:methanol mixed solvent was added, and the mixture was washed with 50 ml of saturated sodium bicarbonate. The aqueous phase was extracted three times with 50 ml of dichloromethane. The organic phases were combined and washed with anhydrous sodium sulfate. After drying over a sieve and filtration, the solvent was evaporated to dryness under reduced pressure and then foam-dried overnight using a vacuum oil pump. The product was then purified on a 200-300 mesh normal-phase silica gel column. Petroleum ether was added to the column, and the acidity of the silica gel was neutralized with 1 wt% triethylamine. Gradient elution was performed using dichloromethane:methanol:ammonia water (25 wt%) = 1:1:0.05 to 1:1:0.25. The product eluate was collected, and the solvent was evaporated to dryness under reduced pressure. The product was then foam-dried using a vacuum oil pump to obtain 2.887 g of pure M-18-Tr. 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,2H). MS m / z:C 28 H 39 N4, [M+H] + , theoretical value: 431.65, measured value: 432.61.

[0398] Synthesis of (2-1-5)L-5-Tr:

[0399] [ka] M-18-Tr (2.02 g, 4.69 mmol) obtained in step (2-1-4) and GAL-5 (6.93 g, 15.48 mmol) obtained in step (2-1-1) were mixed and dissolved in 47 mL of acetonitrile. N-methylmorpholine (3.13 g, 30.96 mmol) and 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride (DMTMM, 4.28 g, 15.48 mmol) were added and the mixture was stirred at room temperature for 2 h. The reaction mixture was diluted with 200 mL of dichloromethane, and the organic phase was washed with 100 mL of saturated sodium bicarbonate solution and 100 mL of saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated to dryness under reduced pressure to obtain the crude product. The product was purified on a 200-300 mesh normal-phase silica gel column. Petroleum ether was added to the column, and the acidity of the silica gel was neutralized with 1 wt% triethylamine. Gradient elution was performed with dichloromethane:methanol = 100:5 to 100:7. The product eluate was collected and evaporated to dryness under reduced pressure to obtain 7.49 g of pure L-5-Tr. 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 - 3.64 (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 119 N7O 30 , [M+H] + , theoretical value: 1718.81, actual value: 1718.03.

[0400] Synthesis of (2-1-6)L-8:

[0401] [ka] L-5-Tr (5.94 g, 3.456 mmol) obtained in step (2-1-5) was dissolved in 69 ml of dichloromethane, dichloroacetic acid (13.367 g, 103.67 mmol) 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 solution, and the mixture was washed with saturated sodium bicarbonate solution and adjusted to pH = 7-8. The aqueous phase was extracted six times with 30 ml of dichloromethane each time. The organic phases were combined, dried over anhydrous sodium sulfate, and filtered. The solvent was evaporated to dryness under reduced pressure to obtain a crude product. For purification, 200-300 mesh normal phase silica gel was used. The acidity of the silica gel was neutralized with 10 wt% triethylamine, and the column was equilibrated with 1 wt% triethylamine. Gradient elution was performed with dichloromethane:methanol = 100:30 to 100:40. The product eluate was collected, and the solvent was evaporated to dryness under reduced pressure to obtain 4.26 g of pure L-8. 1 H NMR (400 MHz,DMSO) δ 7.84 (d,J =9.0 Hz,3H),7.27 - 7.23 (m,1H),7.13 - 7.18 (m,1H),5.22 (d,J =3.1 Hz,3H),4.97 (dd,J =11.3,3.1 Hz,3H),4.48 (d,J =8.4 Hz,3H),4.09 - 3.98 (m,9H),3.88 (dd,J =19.3,9.3 Hz,3H),3.75 - 3.66 (m,3H),3.44 - 3.38 (m,3H),3.17 - 3.30 (m,4H),3.10 - 2.97 (m,4H),2.35 - 2.20 (m,6H),2.15-2.08 (m,9H),2.07-1.98 (m,13H),1.94-1.87 (m,9H),1.81-1.74 (m,9H),1.65-1.42 (m,18H). 85 H 119 N7O 30 , [M+H] + , theoretical value: 1477.59, measured value: 1477.23.

[0402] (2-1-7a) Synthesis of A-1

[0403] [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 (12.88 g, 45.0 mmol) was added. The mixture was reacted at 45°C for 22 hours. The reaction mixture was filtered, the cake was rinsed with 200 ml of DCM, and the filtrate was concentrated under reduced pressure to dryness. The residue was redissolved in 500 ml of dichloromethane and washed twice with 0.5 M triethylamine phosphate (pH = 7-8), each time with 200 ml. The aqueous phase was extracted twice with dichloromethane, each time with 200 ml. The organic phases were combined and dried over anhydrous sodium sulfate. The mixture was filtered, the solvent was evaporated to dryness under reduced pressure, and the mixture was purified on a 200-300 mesh normal phase silica gel column. Gradient elution was performed using petroleum ether: ethyl acetate: dichloromethane: methanol = 1:1:1:0.35 to 1:1:1:0.55. The product eluate was collected, the solvent was evaporated to dryness under reduced pressure, and the mixture was redissolved in 500 ml of dichloromethane and washed once with 200 ml of 0.5 M triethylamine phosphate. The aqueous phase was extracted twice with 200 ml of dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, the solvent was evaporated to dryness under reduced pressure, and the mixture was subjected to vacuum oil pump overnight to obtain 20.7 g of white solid product A-1. 1 H NMR (400 MHz,DMSO-d6) δ 7.46 (ddd,J =6.5,2.3,1.1 Hz,1H),7.40 - 7.28 (m,7H),6.89 - 6.81 (m,4H),4.84 (d,J =5.0 Hz,1H),4.36 - 4.24 MS m / z:C 24 H 23 O6, [MH] - , theoretical value: 407.15, measured value: 406.92.

[0404] (2-1-7b) Synthesis of L-7:

[0405] [ka] L-8 (2.262 g, 1.532 mmol) obtained in step (2-1-6) and A-1 (2.342 g, 4.596 mmol) obtained in step (2-1-7a) 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, 9.191 mmol) was further added. The organic phase was washed with 10 ml of saturated sodium bicarbonate, the aqueous phase was extracted with dichloromethane three times, each time with 10 ml, the organic phase was washed with 10 ml of saturated brine, the aqueous phase was extracted with dichloromethane twice, each time with 10 ml, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, the solvent was evaporated to dryness under reduced pressure, and the mixture was foam-dried overnight in a vacuum oil pump to obtain 4.900 g of 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 20 ml of triethylamine. The column was equilibrated with petroleum ether containing 1 wt% triethylamine. Gradient elution was performed with petroleum ether: ethyl acetate: dichloromethane: N,N-dimethylformamide = 1:1:1:0.5 to 1:1:1:0.6. The product eluate was collected, and the solvent was evaporated to dryness under reduced pressure to obtain 2.336 g of pure L-7. 1H NMR (400 MHz,DMSO) δ7.90 - 7.78 (m,4H),7.75 - 7.64 (m,1H),7.38 - 7.18 (m,9H),6.91 - 6.83 (m,4H),5.25 - 5.10 (m,4H),4.97 (dd,J =11.2,3.2 Hz,3H),4.48 - 4.30 (m,4H),4.02 (s,9H),3.93 - 3.84 (m,3H),3.76 - 3.66 (m,9H),3.45 - 3.35 (m,3H),3.24 - 2.98 (m,10H),2.30 - 2.20 (m,2H),2.11 - 1.88 (m,31H),1.80 - 1.40 (m, 28H). MS m / z: C 90 H 128 N7O 35 , [M-DMTr] + , theoretical value: 1564.65, measured value: 1564.88.

[0406] Synthesis of (2-1-8)L-9:

[0407] [ka] L-7 (2.300 g, 1.26 mmol) obtained in step (2-1-7b), succinic anhydride (0.378 g, 3.78 mmol), and 4-dimethylaminopyridine (DMAP, 0.462 g, 3.78 mmol) were mixed and dissolved in 13 ml of dichloromethane, and DIPEA (0.814 g, 6.30 mmol) was added and stirred at 25 °C for 24 hours. The reaction solution was washed with 5 ml of 0.5 M triethylamine phosphate, and the aqueous phase was extracted three times with 5 ml of dichloromethane each time. The organic phases were combined and evaporated to dryness under reduced pressure to obtain 2.774 g of a crude product. For column purification, 60 g of 200-300 mesh normal-phase silica gel was used. The acidity of the silica gel was neutralized with 1 wt% triethylamine, the column was equilibrated with dichloromethane, and gradient elution was performed with dichloromethane:methanol = 100:18 to 100:20 containing 1 wt% triethylamine. The product eluate was collected, and the solvent was evaporated to dryness under reduced pressure to obtain 1.874 g of pure L-9 complex molecule (compound 1). 1H 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 =19.4,9.4 Hz,3H),3.77 - 3.65 (m,9H),3.50 - 3.39 (m,6H),3.11 - 2.90 (m,5H),2.61 - 2.54 (m,4H),2.47 - 2.41 (m,2H),2.26 - 2.17 (m,2H),2.15 - 1.95 (m,22H),1.92 - 1.84 (m,9H),1.80 - 1.70 (m,10H),1.65 - 1.35 (m,17H), 1.31 - 1.19 (m,4H),0.96 (t,J =7.1 Hz,9H). MS m / z:C 94 H 132 N7O 38 , [M-DMTr] + , theoretical value: 1664.72, measured value: 1665.03.

[0408] Synthesis of (2-1-9)L-10 compound:

[0409] [ka] In this process, the L-10 compound was prepared by binding the L-9 conjugated molecule to a solid support.

[0410] The L-9 conjugate molecule (0.233 g, 0.1126 mmol) obtained in step (1-1-8), O-benzotriazole-tetramethyluronium hexafluorophosphate (HBTU, 0.064 g, 0.1689 mmol), and diisopropylethylamine (DIEA, 0.029 g, 0.2252 mmol) were mixed and dissolved in 19 ml of acetonitrile. The mixture was stirred at room temperature for 5 minutes, and the reaction solution was added with aminomethyl resin (HNResin, 0.901 g, 100-200 mesh, amino group loading capacity 40 The capping reaction was carried out by adding CapA, CapB, 4-dimethylaminopyridine (DMAP), and acetonitrile (CapA, CapB, 4-dimethylaminopyridine (DMAP), and acetonitrile) in the proportions shown in Table 3. The reaction was carried out at 25°C on a shaker at 200 rpm for 5 hours. The reaction mixture was then filtered. The cake was rinsed with acetonitrile three times at 30 mL each, filtered under suction, and dried overnight under reduced pressure on a vacuum oil pump to obtain 1.100 g of the L-10 compound (i.e., the L-9 conjugate molecule bound to the solid support) with a loading of 90.8 μmol / g.

[0411] [Table 3] Here, CapA and CapB were capping reagent solutions, CapA was a 20% by volume solution of N-methylimidazole in a pyridine / acetonitrile mixture, with the volume ratio of pyridine to acetonitrile being 3:5, and CapB was a 20% by volume solution of acetic anhydride in acetonitrile.

[0412] In the following synthesis, the sequences of the sense and antisense strands corresponded to the S and AS sequences of Complex 1 in Table 4, respectively.

[0413] (2-2) Synthesis of the sense strand 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 order of nucleotides in the sense strand. Each time a nucleoside monomer is linked, four reactions are involved: deprotection, coupling, capping, oxidation, or sulfurization. When two nucleotides are linked via a phosphate ester, the linking of the next nucleoside monomer involves four reactions: deprotection, coupling, capping, and oxidation. When two nucleotides are linked via a thiophosphate ester, the linking of the next nucleoside monomer involves four reactions: deprotection, coupling, capping, and oxidation. Escape Four reactions were carried out: protection, coupling, capping, and sulfurization. The reaction conditions were the same as those used to synthesize the sense strand in Preparation Example 1 above.

[0414] (2-3) Synthesis of antisense strand The solid-phase phosphoramidite method was used to prepare a general-purpose solid support (UnyLinker TM The antisense strand AS of Complex 1 was synthesized by cycling starting materials loaded with NittoPhase® HL Solid Supports (Kinovate Life Sciences). The conditions for deprotection, coupling, capping, oxidation or sulfurization, cleavage and deprotection, purification, and desalting in the solid-phase synthesis method were the same as those used to synthesize the antisense strand in Preparation Example 1 described above.

[0415] After the synthesis was completed, the purity of the sense and antisense strands was detected by ion exchange chromatography (IEX-HPLC), and the molecular weight was analyzed by liquid chromatography mass spectrometry (LC-MS). The measured molecular weight was compared with the theoretical value to confirm the synthesized sense and antisense strands.

[0416] (2-4) Synthesis of Complex A1 The S and AS chains were dissolved in water for injection to obtain a 40 mg / mL solution. The mixture was mixed in an equimolar ratio, heated at 50 °C for 15 min, and cooled to room temperature. The mixture was then hydrogen-bonded to form a double-stranded structure. The complex was diluted to 0.2 mg / mL with ultrapure water (homemade using a Milli-Q ultrapure water system, resistivity 18.2 MΩ*cm at 25 °C). The molecular weight was then measured using a liquid chromatography-mass spectrometry (LC-MS, Liquid Chromatography-Mass Spectrometry, purchased from Waters, model number: LCT Premier). The theoretical values ​​were S: 7516.37, AS: 7061.57, while the actual values ​​were S: 7516.6, AS: 7060.49. This confirmed the theoretical values, indicating the formation of the target complex A1, shown in formula (403).

[0417] (Preparation Example 3) Preparation of the composite of the present disclosure and comparative composite Conjugates A2 to A7, B1 to B2, C2, C12 to C13, D2, D12 to D13, E1 to E4, F1 to F3, and G1 to G3 in Tables 4A to 4G and comparative conjugates A2, C1, D1, E1, F1, and G1 were synthesized in the same manner as in Preparation Example 2, except that the siRNA sequences of the conjugates were the corresponding sequences shown in Tables 4A to 4G, respectively. It was expected that conjugates A8 to A11, B3 to B7, C1, C3, D1, D3, E5 to E9, F4 to F11, and G4 to G9 shown in Tables 4A to 4G could also be produced. After synthesis was completed, the resulting conjugates were confirmed using the same detection method as in Preparation Example 2. Among them, Theoretical values ​​for complex A2: S: 7516.37, AS: 7065.58, measured values: S: 7516.6, AS: 7064.5, Theoretical values ​​of complex A3: S: 7504.34, AS: 7139.68, measured values: S: 7515.6, AS: 7138.9, Theoretical values ​​of complex A4: S: 7516.37, AS: 7081.64, measured values: S: 7515.6, AS: 7080.9, Theoretical values ​​of complex A5: S: 8218.83, AS: 7703.05, measured values: S: 8218, AS: 7702.5, Theoretical values ​​for complex A6: S: 7516.37, AS: 6985.58, and measured values: S: 7516.5, AS: 6984.9. Theoretical values ​​of complex B1: S: 7407.22, AS: 7208.77, measured values: S: 7406.4, AS: 7208.1, Theoretical values ​​of complex B2: S: 7407.22, AS: 7170.72, measured values: S: 7406.5, AS: 7170.1, Theoretical values ​​for complex C2: S: 7485.3, AS: 7161.7, measured values: S: 7484.4, AS: 7160.9, Theoretical values ​​of complex D2: S: 7423.22, AS: 7207.78, measured values: S: 7422.6, AS: 7207.2, Theoretical values ​​of complex F2: S: 7649.55, AS: 6995.47, measured values: S: 7648.8, AS: 6994.8, Theoretical values ​​of complex F3: S: 7649.55, AS: 7011.53, measured values: S: 7648.8, AS: 7010.9, Theoretical values ​​of complex E1: S: 7584.5, AS: 7007.46, measured values: S: 7584, AS: 7006.2, Theoretical values ​​for complex E2: S: 7584.5, AS: 7011.47; measured values: S: 7584, AS: 7011.3; The theoretical values ​​for complex E4 were S: 7572.47, AS: 6907.41, and the measured values ​​were S: 7571.8, AS: 6906.9.

[0418] The measured molecular weights were consistent with the theoretical values, indicating that the desired complexes were obtained. All of these complexes have the structure shown in formula (403).

[0419] [Table 4] siRNA complex [Table 4A] TIFF0007727308000078.tif245170TIFF0007727308000079.tif108170

[0420] [Table 4B] TIFF0007727308000081.tif180170

[0421] [Table 4C] TIFF0007727308000083.tif123170

[0422] [Table 4D] TIFF0007727308000085.tif60170

[0423] [Table 4E] TIFF0007727308000087.tif213170

[0424] [Table 4F] TIFF0007727308000089.tif195170

[0425] [Table 4G] TIFF0007727308000091.tif125170 * S: sense strand, AS: antisense strand Note: Capital letters C, G, U, and A represent the base sequence of nucleotides, lowercase letter m indicates that one nucleotide adjacent to the left of the letter m is a 2'-methoxy-modified nucleotide, lowercase letter f indicates that one nucleotide adjacent to the left of the letter f is a 2'-fluoro-modified nucleotide, lowercase letter s indicates that two nucleotides adjacent to the left and right of the letter s are linked by a thiophosphate group, VP indicates that one nucleotide to the right of the letter VP is a vinyl phosphate-modified nucleotide, P indicates that one nucleotide to the right of the letter P is a phosphate-modified nucleotide, and Ps indicates that one nucleotide to the right of the letter Ps is a thiophosphate-modified nucleotide.

[0426] In the following Preparation Examples 4 to 12, various conjugate molecules were synthesized, and it was expected that by using these conjugate molecules instead of the L-10 compound in Preparation Example 2, conjugates A12 to A19, B8 to B15, C4 to C11, D4 to D11, E10 to E17, F12 to F19, and G10 to G17 in Tables 4A to 4G could be obtained according to the corresponding sequences shown in Tables 4A to 4G.

[0427] (Preparation Example 4) Preparation of P10 complex In this preparation example, it was expected that conjugates A12, B8, C4, D4, E10, F12 and G10 (hereinafter also referred to as P-10 conjugates) could be synthesized according to the following method.

[0428] (4-1) Synthesis of P-10 Compound Compound P-10 was synthesized according to the following method.

[0429] [ka]

[0430] (4-1-1) Synthesis of GAL5-C4-1 GAL-5 (13.43 g, 30.0 mmol), prepared by the method described in step (2-1-1) above, 4-amino acid tert-butyl ester hydrochloride (5.87 g, 30.0 mmol), O-benzotriazole-tetramethyluronium hexafluorophosphate (13.65 g, 36.0 mmol), and diisopropylethylamine (11.63 g, 90.0 mmol) were added to 40 mL of N,N-dimethylformamide, dissolved uniformly, and then stirred at room temperature for 5 hours. 300 mL of saturated aqueous sodium bicarbonate was added to the reaction solution, which was then extracted three times with ethyl acetate (200 mL each time). The combined organic phases were washed once with 200 mL of saturated brine, separated, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to give 30.3 g of crude oil, GAL5-C4-1, which was then directly used in the next reaction.

[0431] (4-1-2) Synthesis of GAL5-C4-2 The crude GAL5-C4-1 product (30.3 g, 30 mmol) obtained in step (4-1-1) was dissolved in 180 mL of formic acid and stirred at room temperature for 16 hours. The solvent was evaporated to dryness, and the product was purified by column chromatography (200-300 mesh normal phase silica gel, gradient elution with dichloromethane:methanol = 100:18 to 100:20). The reaction eluate was collected and the solvent was removed by concentration to obtain 14.84 g of the target product, GAL5-C4-2.

[0432] Synthesis of (4-1-3)P-6: M-18-Tr (2.02 g, 4.69 mmol) obtained by the method described in step (2-1-4) and GAL5-C4-2 (8.24 g, 15.48 mmol, a combination of two batches of product) obtained in step (4-1-2) were mixed and dissolved in 47 ml of acetonitrile, and N-methylmorpholine (3.13 g, 30.96 mmol) was added, followed by 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride (DMTMM, 4.28 g, 15.48 mmol). The mixture was stirred at room temperature for 2 hours. The reaction mixture was diluted with 20 ml of dichloromethane, the organic phase was washed with 10 ml of saturated sodium bicarbonate solution, and then with 10 ml of saturated brine. The organic phases were combined, dried over anhydrous sodium sulfate, and filtered. The solvent was evaporated to dryness under reduced pressure to obtain the crude product, which was then purified on a 200-300 mesh normal phase silica gel column. Petroleum ether was added to the column, and the acidity of the silica gel was neutralized with 1 wt% triethylamine. Gradient elution was performed with dichloromethane:methanol = 100:5-100:7, and the product eluate was collected and evaporated to dryness under reduced pressure to obtain a total of 8.27 g of pure P-6.

[0433] Synthesis of (4-1-4)P-7: P-6 (6.82 g, 3.456 mmol) obtained in (4-1-3) above was dissolved in 69 ml of dichloromethane, and dichloroacetic acid (13.367 g, 103.67 mmol) was added. The mixture was allowed to react at room temperature for 2 hours. 100 ml of dichloromethane was added to dilute the reaction mixture, and the mixture was washed with saturated sodium bicarbonate solution to adjust the pH to 7-8. The aqueous phase was extracted six times with 30 ml of dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, and filtered. The solvent was evaporated to dryness under reduced pressure to obtain the crude product. The product was purified using 200-300 mesh normal phase silica gel. The acidity of the silica gel was neutralized with 10 wt% triethylamine, and the column was equilibrated with 1 wt% triethylamine. Gradient elution was performed using dichloromethane:methanol = 100:30 to 100:40. The product eluate was collected, and the solvent was evaporated to dryness under reduced pressure to give a total of 4.82 g of P-7. MS m / z: C 78 H 127 N 10 O 33 , [M+H]+ , theoretical value: 1732.91, actual value: 1735.73.

[0434] Synthesis of (4-1-5)P-8:

[0435] [ka] (A-1) P-7 (2.653 g, 1.532 mmol) and A-1 (2.342 g, 4.596 mmol) were mixed and dissolved in 16 mL of dichloromethane. 3-Diethoxyphosphoryl-1,2,3-benzoxazol-4(3H)-one (DEPBT) (1.375 g, 4.596 mmol) and diisopropylethylamine (1.188 g, 9.191 mmol) were added and the mixture was stirred at 25 °C for 2 h. The organic phase was washed with 10 mL of saturated sodium bicarbonate, and the aqueous phase was extracted with dichloromethane three times, each time with 10 mL of sodium chloride. The organic phase was washed with 10 mL of saturated brine, and the aqueous phase was extracted twice with dichloromethane, each time with 10 mL of dichloromethane. The combined organic phases were 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 a vacuum oil pump. For column purification, 120 g of 200-300 mesh normal phase silica gel was used. The acidity of the silica gel was neutralized with 20 ml of triethylamine, and the column was equilibrated with petroleum ether containing 1 wt% triethylamine. Gradient elution was performed with petroleum ether: ethyl acetate: dichloromethane: N,N-dimethylformamide = 1:1:1:0.5 to 1:1:1:0.6. The product eluate was collected, and the solvent was evaporated to dryness under reduced pressure to obtain a total of 2.793 g of pure P-8.

[0436] Synthesis of (4-1-6)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. Diisopropylethylamine (DIEA, 149 mg, 1.155 mmol) was added and the mixture was stirred at 25°C for 21 hours. The reaction mixture was diluted with 50 mL of dichloromethane, and 100 mL of 0.5 M triethylamine phosphate was added to wash the mixture. The aqueous phase was extracted three times with 10 mL of dichloromethane. The combined organic phases were evaporated to dryness under reduced pressure to obtain the crude product. For column purification, 80 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 equilibrated with dichloromethane. Gradient elution was performed with dichloromethane:methanol = 100:18 to 100:20 containing 1 wt% triethylamine. The product 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. MS m / z:C 106 H 153 N 10 O 41 , [M-DMTr] + , theoretical value: 1921.05, actual value: 1920.97.

[0437] Synthesis of (4-1-7)P-10: P-10 was prepared by the same method as in step (2-1-9) in Preparation Example 2, except that the P-9 conjugated molecule was used instead of the L-9 conjugated molecule to obtain a P-9 conjugated molecule bound to a solid support.

[0438] (4-2) Synthesis of P10 complex Conjugates were prepared in the same manner as in steps (2-2), (2-3A), and (2-4) in Preparation Example 2, except that the sense strand was synthesized using the P-10 compound instead of the L-10 compound as the starting material. It was expected that conjugates A12, B8, C4, D4, E10, F12, and G10, whose structures are shown in formula (404), could be obtained.

[0439] (Preparation Example 5) Preparation of R5 complex In this preparation example, it was expected that conjugates A13, B9, C5, D5, E11, F13 and G11 (hereinafter also referred to as R5 conjugate) could be synthesized according to the following method.

[0440] (5-1) Synthesis of R-5 Compound The R-5 compound was synthesized according to the following method.

[0441] [ka]

[0442] (5-1-1) Synthesis of GAL-C7-1 GAL-3 (26.4 g, 80.2 mmol) obtained by the method described in step (2-1-1b) was dissolved in 134 ml of anhydrous 1,2-dichloroethane, and 60 g of 4 Å molecular sieve powder was added. 7-octen-1-ol (11.3 g, 88.2 mmol) was then added and the mixture was stirred at room temperature for 10 minutes. Trimethylsilyl trifluoromethanesulfonate (8.9 g, 40.1 mmol) was then added under nitrogen protection in an ice bath, and the mixture was stirred at room temperature for 24 hours. The 4Å molecular sieve powder was removed by filtration, and the filtrate was washed with 500 ml of saturated aqueous sodium bicarbonate solution. The organic phase was separated, and the aqueous phase was extracted once with 100 ml of dichloromethane. The combined organic phases were washed once with 250 ml of saturated brine. The organic phase was separated and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure until dry, yielding 33.3 g of a yellow starch syrup-like product, GAL-C7-1, which was directly used in the next oxidation reaction without further purification.

[0443] (5-1-2) Synthesis of GAL-C7-2 GAL-C7-1 (33.3 g, 72.8 mmol) obtained in step (5-1-1) was dissolved in a mixed solvent of 160 ml of dichloromethane and 160 ml of acetonitrile, and 216 ml of water and solid sodium periodate (62.3 g, 291.2 mmol) were added, followed by stirring in an ice-water bath for 10 minutes. Ruthenium (III) chloride (498 mg, 2.4 mmol) was added as a catalyst, and the mixture was allowed to warm to room temperature and reacted with stirring for 23 hours. The reaction mixture was diluted with 200 ml of water and stirred. Saturated sodium bicarbonate was added to adjust the pH to 7.5, the organic phase was separated, the aqueous phase was extracted three times with dichloromethane, the organic phase was discarded, the aqueous phase was adjusted to pH 3 with solid citric acid, and extracted three times with dichloromethane, each time at 200 ml. The organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The mixture was then purified by column chromatography (200-300 mesh normal phase silica gel, gradient elution with dichloromethane:methanol = 100:18-100:20) to obtain 22.4 g of white foamy solid product GAL-C7-2. MS m / z: C 21 H 32 NO 11 , [M+H] + , theoretical value: 476.50, measured value: 475.94.

[0444] (5-1-3) Synthesis of R-1: M-18-Tr (2.02 g, 4.69 mmol) obtained by the method described in step (2-1-4) and GAL-C7-2 (7.36 g, 15.48 mmol) were mixed and dissolved in 47 ml of acetonitrile, and N-methylmorpholine (3.13 g, 30.96 mmol) was added, followed by the addition of 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride (DMTMM, 4.28 g, 15.48 mmol). The mixture was stirred at room temperature for 2 hours. The reaction mixture was diluted with 200 ml of dichloromethane, the organic phase was washed with 100 ml of saturated sodium bicarbonate solution, and then with 100 ml of saturated brine. The organic phases were combined, dried over anhydrous sodium sulfate, and filtered. The solvent was evaporated to dryness under reduced pressure to obtain the crude product, which was then purified on a 200-300 mesh normal phase silica gel column. Petroleum ether was added to the column, and the acidity of the silica gel was neutralized with 1 wt% triethylamine. Gradient elution was performed with dichloromethane:methanol = 100:5-100:7, and the product eluate was collected and evaporated to dryness under reduced pressure to obtain 7.82 g of pure R-1.

[0445] (5-1-4) Synthesis of R-2: R-1 (6.23 g, 3.456 mmol) was dissolved in 69 mL of dichloromethane, and dichloroacetic acid (13.367 g, 103.67 mmol) was added. The mixture was allowed to react at room temperature for 2 h. The reaction mixture was diluted with 100 mL of dichloromethane and washed with saturated sodium bicarbonate solution to adjust the pH to 7-8. The aqueous phase was extracted six times with 30 mL of dichloromethane. The combined organic phases were dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated to dryness under reduced pressure to obtain the crude product. 200-300 mesh normal-phase silica gel was used. The acidity of the silica gel was neutralized with 10 wt% triethylamine. The column was equilibrated with 1 wt% triethylamine. Gradient elution was performed with dichloromethane:methanol = 100:30-100:40. The solvent was evaporated to dryness under reduced pressure to obtain 4.49 g of pure R-2.

[0446] (5-1-5) Synthesis of R-3: R-2 (2.391 g, 1.532 mmol) and A-1 (2.342 g, 4.596 mmol) were mixed and dissolved in 16 mL of dichloromethane. 3-Diethoxyphosphoryl-1,2,3-benzoxazol-4(3H)-one (DEPBT) (1.375 g, 4.596 mmol) and diisopropylethylamine (1.188 g, 9.191 mmol) were added and the mixture was stirred at 25 °C for 2 h. The organic phase was washed with 10 mL of saturated sodium bicarbonate, and the aqueous phase was extracted with dichloromethane three times, each time with 10 mL of sodium chloride. The organic phase was washed with 10 mL of saturated brine, and the aqueous phase was extracted twice with dichloromethane, each time with 10 mL of dichloromethane. The combined organic phases were 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 a vacuum oil pump. For column purification, 120 g of 200-300 mesh normal phase silica gel was used. The acidity of the silica gel was neutralized with 20 ml of triethylamine, and the column was equilibrated with petroleum ether containing 1 wt% triethylamine. Gradient elution was performed with petroleum ether: ethyl acetate: dichloromethane: N,N-dimethylformamide = 1:1:1:0.5 to 1:1:1:0.6, and the solvent was evaporated to dryness under reduced pressure to obtain 2.642 g of pure R-3.

[0447] (5-1-6) Synthesis of R-4: R-3 (795 mg, 0.4074 mmol), succinic anhydride (82 mg, 0.8148 mmol), and 4-dimethylaminopyridine (DMAP, 100 mg, 0.8148 mmol) were mixed and dissolved in 4 mL of dichloromethane. Diisopropylethylamine (DIEA, 100 mg, 0.8148 mmol) was added and the mixture was stirred at 25°C for 18 hours. The reaction mixture was washed with 5 mL of 0.5 M triethylamine phosphate, and the aqueous phase was extracted three times with 5 mL of dichloromethane. The combined organic phases were 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. The acidity of the silica gel was neutralized with 1 wt% triethylamine, and the column was equilibrated with dichloromethane. Gradient elution was performed with dichloromethane:methanol = 100:18 to 100:20 containing 1 wt% triethylamine. The product eluate was collected, and the solvent was evaporated to dryness under reduced pressure to obtain 505 mg of pure R-4 conjugate molecule.

[0448] (5-1-7) Synthesis of R-5 conjugate molecules: R-5 was prepared in the same manner as in step (2-1-9) in Preparation Example 2, except that the R-4 conjugate molecule was used instead of the L-9 conjugate molecule to obtain the R-4 conjugate molecule bound to a solid support.

[0449] (5-2) Synthesis of R5 complex The R5 conjugates were prepared in the same manner as in steps (2-2), (2-3A), and (2-4) in Preparation Example 2, except that the sense strand was synthesized using the R-5 compound instead of the L-10 compound as the starting compound. It was expected that the conjugates A13, B9, C5, D5, E11, F13, and G11, whose structures are shown in formula (407), could be obtained.

[0450] (Preparation Example 6) Preparation of LA5 complex In this preparation example, it was expected that conjugates A14, B10, C6, D6, E12, F14 and G12 (hereinafter also referred to as LA5 conjugate) could be synthesized according to the following method.

[0451] It was anticipated that the LA-5 compound could be synthesized by the following process route.

[0452] [ka] LA conjugates were prepared in the same manner as in steps (2-2), (2-3A), and (2-4) in Preparation Example 2, except that the sense strand was synthesized using the LA-5 compound instead of the L-10 compound as the starting compound. It was expected that conjugates A14, B10, C6, D6, E12, F14, and G12, whose structures are shown in formula (412), could be obtained.

[0453] (Preparation Example 7) Preparation of LB5 complex In this preparation example, it was expected that conjugates A15, B11, C7, D7, E13, F15 and G13 (hereinafter also referred to as LB5 conjugates) could be synthesized according to the following method.

[0454] (7-1) Synthesis of LB-5 Compound The LB-5 compound was synthesized according to the following method.

[0455] [ka]

[0456] Synthesis of (7-1-1)LB-1: L-8 (5.0 g, 3.386 mmol), obtained by the method described in step (2-1-6), adipic anhydride (870 mg, 6.772 mmol), and 4-dimethylaminopyridine (DMAP, 827 mg, 6.772 mmol) were mixed and dissolved in 130 mL of dichloromethane. Diisopropylethylamine (DIEA, 2.2 g, 16.931 mmol) was added and the mixture was stirred at 25 °C for 4 hours. The reaction solution was diluted with 70 mL of dichloromethane, washed with 0.5 M triethylamine phosphate, and the aqueous phase was extracted four times with 10 mL of dichloromethane. The organic phases were combined and evaporated to dryness under reduced pressure to obtain the 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 equilibrated with dichloromethane. Gradient elution was performed with petroleum ether: ethyl acetate: dichloromethane: methanol = 1:1:1:0.2 to 1:1:1:1. The solvent was evaporated to dryness under reduced pressure to obtain 4.267 g of pure LB-1.

[0457] Synthesis of (7-1-2)LB-2: LB-1 (4.697 g, 2.753 mmol, two batches combined) obtained by the method described in step (7-1-1), 3-amino-1,2-propanediol (313 mg, 3.442 mmol), 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride (DMTMM, 953 mg, 3.442 mmol), and N-methylmorpholine (700 mg, 6.884 mmol) were added sequentially to a mixture of 30 mL of acetonitrile and 3 mL of methanol and stirred overnight at room temperature. The solvent was evaporated to dryness and the mixture was purified by column chromatography (200-300 mesh normal phase silica gel with a gradient elution of 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 desired product, LB-2.

[0458] Synthesis of (7-1-3)LB-3: LB-2 (2.27 g, 1.353 mmol) was dissolved in 14 mL of anhydrous pyridine. 4,4'-bismethoxytrityl chloride (688 mg, 2.03 mmol) was added and the reaction was stirred overnight at room temperature. 150 mL of methanol was added to quench the reaction, and the solvent was evaporated to dryness. The product was purified by column chromatography (200-300 mesh normal phase silica gel, gradient elution with 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, LB-3.

[0459] Synthesis of (7-1-4)LB-4: LB-3 (822 mg, 0.415 mmol), succinic anhydride (83 g, 0.83 mmol), and 4-dimethylaminopyridine (DMAP, 102 mg, 0.83 mmol) were mixed and dissolved in 4 mL of dichloromethane. DIEA (270 mg, 2.075 mmol) was added and the mixture was stirred overnight at 25°C. The reaction mixture was washed three times with 0.5 M triethylamine phosphate, and the aqueous phase was extracted three times with 2 mL of dichloromethane. The combined organic phases were evaporated to dryness under reduced pressure to obtain the crude product. For column purification, 200-300 mesh normal phase silica gel was used. The acidity of the silica gel was neutralized with 5 wt% triethylamine, and the column was equilibrated with petroleum ether. Gradient elution was performed with dichloromethane:methanol = 100:5 to 100:20 containing 1 wt% triethylamine. The solvent was evaporated to dryness under reduced pressure to obtain 787 mg of pure LB-4 complex molecule.

[0460] Synthesis of (7-1-5)LB-5: LB-5 was prepared in the same manner as in step (2-1-9) in Preparation Example 2, except that the LB-4 conjugate molecule was used instead of the L-9 conjugate molecule to obtain the LB-4 conjugate molecule bound to a solid support.

[0461] (7-2) Synthesis of LB5 complex The LB5 conjugates were prepared in the same manner as in steps (2-2), (2-3A), and (2-4) in Preparation Example 2, except that the sense strand was synthesized using the LB-5 compound instead of the L-10 compound as the starting compound. It was expected that the conjugates A15, B11, C7, D7, E13, F15, and G13, whose structures are shown in formula (413), could be obtained.

[0462] (Preparation Example 8) Synthesis of V8 complex In this preparation example, it was expected that conjugates A16, B12, C8, D8, E14, F16 and G14 (hereinafter also referred to as V8 conjugate) could be synthesized according to the following method.

[0463] It was anticipated that compound V-8 could be synthesized via the following process route.

[0464] [ka] The V8 conjugates were prepared in the same manner as in steps (2-2), (2-3A), and (2-4) in Preparation Example 2, except that the sense strand was synthesized using the V-8 compound instead of the L-10 compound as the starting compound. It was expected that conjugates A15, B12, C8, D8, E14, F16, and G14 (hereinafter also referred to as V8 conjugates) whose structures are shown in formula (414) could be obtained.

[0465] (Preparation Example 9) Preparation of W8 complex In this preparation example, it was expected that conjugates A17, B13, C9, D9, E15, F17 and G15 (hereinafter also referred to as W8 conjugate) could be synthesized according to the following method.

[0466] (9-1) Synthesis of W-8 Compound Compound W-8 was synthesized according to the following method.

[0467] [ka]

[0468] Synthesis of (9-1-1)W-1: W-0 (2.024 g, 10 mmol) was dissolved in 25 mL of acetonitrile, triethylamine (4.048 g, 40 mmol) was added, the mixture was cooled to approximately 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 h. The solvent was evaporated to dryness under reduced pressure, and the mixture was foam-dried using a vacuum oil pump for 18 h to obtain 5.835 g of solid crude product W-1.

[0469] (9-1-2) Synthesis of W-2: Crude product W-1 (5.835 g, 10 mmol) was dissolved in 50 mL of dichloromethane. TrCl (3.345 g, 12 mmol) and triethylamine (1.518 g, 15 mmol) were added to the reaction mixture, and the mixture was stirred at room temperature for 20 hours. The reaction mixture was washed twice with 20 mL of saturated sodium bicarbonate and once with 20 mL of saturated brine. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the organic solvent was evaporated to dryness under reduced pressure. The mixture was then foam-dried overnight using a vacuum oil pump to obtain 8.012 g of solid crude product W-2. This product was then used for the subsequent deprotection reaction without further treatment.

[0470] Synthesis of (9-1-3)W-3: The crude product W-2 (8.012 g, 10 mmol) was dissolved in 100 ml of methanol, and 100 ml of an aqueous methylamine solution (40 wt %) was added thereto, followed by stirring at 50° C. for 23 hours. The insoluble particles were removed by filtration, and the solvent was evaporated to dryness under reduced pressure. 200 ml of a 1:1 volumetric ratio DCM-methanol mixed solvent was added, and the organic phase was washed with 50 ml of saturated sodium bicarbonate. The aqueous phase was extracted with dichloromethane three times, each time with 50 ml of dichloromethane. The combined organic phases were dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated to dryness under reduced pressure. The extract was then foam-dried overnight using a vacuum oil pump. The extract was then purified on a 200-300 mesh normal-phase silica gel column. Petroleum ether was added to the column, and the acidity of the silica gel was neutralized with 1 wt% triethylamine. Gradient elution was performed using dichloromethane:methanol:ammonia water (25 wt%) = 1:1:0.05 to 1:1:0.25. The product eluate was collected, and the solvent was evaporated to dryness under reduced pressure. The extract was then foam-dried using a vacuum oil pump to obtain 3.062 g of pure W-3.

[0471] (9-1-4) 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. Diisopropylethylamine (1.57 g, 12.14 mmol) was added, and finally 3-diethoxyphosphoryl-1,2,3-benzoxazol-4(3H)-one (DEPBT, 1.816 g, 6.04 mmol) was added, and the mixture was stirred at room temperature for 2.5 h. The reaction mixture was diluted with 100 ml of dichloromethane, the organic phase was washed with 80 ml of saturated sodium bicarbonate solution, and then with 80 ml of saturated brine. The organic phases were combined, dried over anhydrous sodium sulfate, and filtered. The solvent was evaporated to dryness under reduced pressure to obtain the crude product, which was then purified on a 200-300 mesh normal phase silica gel column. Petroleum ether was added to the column, and the acidity of the silica gel was neutralized with 1 wt% triethylamine. Gradient elution was performed with dichloromethane:methanol = 100:5-100:7, and the product eluate was collected and evaporated to dryness under reduced pressure to obtain 1.610 g of pure W-4.

[0472] Synthesis of (9-1-5)W-5: W-4 (1.61 g, 0.886 mmol) was dissolved in 125 mL of dichloromethane, and dichloroacetic acid (3.5 mL, 42.43 mmol) was added. The mixture was allowed to react at room temperature for 1 hour. 150 mL of pyridine was added to neutralize the reaction mixture, and the solvent was evaporated to dryness under reduced pressure to obtain the crude product. Using 200-300 mesh normal-phase silica gel, the acidity of the silica gel was neutralized with 10 wt% triethylamine. The column was equilibrated with 1 wt% triethylamine and subjected to gradient elution with dichloromethane:methanol = 100:30 to 100:40. The product eluate was collected, and the solvent was evaporated to dryness under reduced pressure to obtain 1.26 g of pure W-5.

[0473] Synthesis of (9-1-6)W-6: W-5 (1.25 g, 0.793 mmol) and A-1 (1.21 g, 2.38 mmol) obtained by the method described in step (2-1-7a) were mixed and dissolved in 12 mL of dichloromethane. 3-Diethoxyphosphoryl-1,2,3-benzoxazol-4(3H)-one (DEPBT, 0.712 g, 2.38 mmol) and diisopropylethylamine (0.615 g, 4.76 mmol) were added and the mixture was stirred at 25 °C for 3 h. The organic phase was washed with 80 mL of saturated sodium bicarbonate, and the aqueous phase was extracted with dichloromethane three times, each time with 10 mL of dichloromethane. The combined organic phases were washed with 10 mL of saturated brine, 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 in a vacuum oil pump. For column purification, 185 g of 200-300 mesh normal phase silica gel was used. The acidity of the silica gel was neutralized with 20 ml of triethylamine. The column was equilibrated with petroleum ether containing 1 wt% triethylamine. Gradient elution was performed with petroleum ether: ethyl acetate: dichloromethane: N,N-dimethylformamide = 1:1:1:0.1 to 1:1:0.7. The product eluate was collected, and the solvent was evaporated to dryness under reduced pressure to obtain 1.57 g of pure W-6.

[0474] Synthesis of (9-1-7)W-7: W-6 (1.238 g, 0.63 mmol), succinic anhydride (0.189 g, 1.89 mmol), and 4-dimethylaminopyridine (DMAP, 0.231 g, 1.89 mmol) were mixed and dissolved in 7 mL of dichloromethane. 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 5 mL of 0.5 M triethylamine phosphate, and the aqueous phase was extracted three times with 5 mL of dichloromethane. The combined organic phases were 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. The acidity of the silica gel was neutralized with 1 wt% triethylamine, and the column was equilibrated with dichloromethane. Gradient elution was performed with dichloromethane:methanol = 100:18 to 100:20 containing 1 wt% triethylamine. The product eluate was collected, and the solvent was evaporated to dryness under reduced pressure to obtain 1.033 g of pure W-7 complex. MS m / z:C 101 H 146 N7O 38 , [M-DMTr] + , theoretical value: 1763.92, actual value: 1763.21.

[0475] Synthesis of (9-1-8)W-8: W-8 was prepared in the same manner as in step (2-1-9) in Preparation Example 2, except that the W-7 conjugated molecule was used instead of the L-9 conjugated molecule to obtain a W-7 conjugated molecule bound to a solid support.

[0476] (9-2) Synthesis of W8 complex The W8 conjugate was prepared in the same manner as in steps (2-2), (2-3A), and (2-4) in Preparation Example 2, except that the sense strand was synthesized using the W-8 compound instead of the L-10 compound as the starting compound. It was expected that the conjugates A17, B13, C9, D9, E15, F17, and G15, whose structures are shown in formula (415), could be obtained.

[0477] (Preparation Example 10) Preparation of X8 complex In this preparation example, it was expected that conjugates A18, B14, C10, D10, E16, F18 and G16 (hereinafter also referred to as X8 conjugates) could be synthesized according to the following method.

[0478] It was anticipated that compound X-8 could be synthesized by the following process route.

[0479] [ka] Except for synthesizing the sense strand using the X-8 compound instead of the L-10 compound as the starting material, the X8 conjugates were prepared by the same methods as steps (2-2), (2-3A), and (2-4) in Preparation Example 2. In this preparation example, it was expected that conjugates A18, B14, C10, D10, E16, F18, and G16, whose structures are represented by formula (421), could be synthesized according to the following method.

[0480] (Preparation Example 11) Preparation of Z5 complex In this preparation example, it was expected that conjugates A19, B15, C11, D11, E12, F14 and G12 (hereinafter also referred to as Z5 conjugate) could be synthesized according to the following method.

[0481] (11-1) Synthesis of Z-5 Compound Compound Z-5 was synthesized according to the following method.

[0482] [ka]

[0483] Synthesis of (11-1-1)Z-1: W-3 (1.50 g, 3.37 mmol) obtained by the method described in step (9-1-3) and GAL5-C4-2 (7.18 g, 13.48 mmol) obtained by the method described in step (4-1-2) were mixed and dissolved in 34 ml of dichloromethane, and diisopropylethylamine (3.48 g, 26.96 mmol) was added, and finally 3-diethoxyphosphoryl-1,2,3-benzoxazol-4(3H)-one (DEPBT, 4.04 g, 13.48 mmol) was added, and the mixture was stirred at room temperature for 4.5 hours. The reaction mixture was diluted with 100 ml of dichloromethane, the organic phase was washed with 80 ml of saturated sodium bicarbonate solution, and then with 80 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 to obtain the crude product. The crude product was purified on a 200-300 mesh normal phase silica gel column. Petroleum ether was added to the column, and the acidity of the silica gel was neutralized with 1 wt% triethylamine. Gradient elution was performed with dichloromethane:methanol = 30:1 to 15:1. The product eluate was collected and evaporated to dryness under reduced pressure to obtain 3.97 g of pure Z-1. MS m / z: C 98 H 143 N 10 O 33 , [M+H] + , theoretical value: 1987.98, measured value: 1987.90.

[0484] (11-1-2) Synthesis of Z-2: Z-1 (3.97 g, 2.00 mmol) was dissolved in 250 ml of dichloromethane, and dichloroacetic acid (10.941 g, 84.85 mmol) was added. The mixture was allowed to react at room temperature for 1 hour. Pyridine was added to neutralize the reaction mixture, and the solvent was evaporated to dryness under reduced pressure to obtain the crude product. 220 g of 200-300 mesh normal phase silica gel was placed in a column. The acidity of the silica gel was neutralized with 10% pyridine, and the column was equilibrated with 1% pyridine. Gradient elution was performed with dichloromethane:methanol = 10:1 to 2:1. The product eluate was collected, and the solvent was evaporated to dryness under reduced pressure to obtain 3.49 g of pure Z-2. MS m / z: C 79 H 129 N 10 O 33 , [M+H] +, theoretical value: 1746.94, actual value: 1746.90.

[0485] Synthesis of (11-1-3)Z-3: Z-2 (3.49 g, 2.0 mmol) and A-1 (3.06 g, 6.0 mmol) obtained by the method described in step (2-1-7a) were mixed and dissolved in 30 mL of dichloromethane. 3-diethoxyphosphoryl-1,2,3-benzoxazol-4(3H)-one (DEPBT, 1.80 g, 6.0 mmol) was added, followed by diisopropylethylamine (1.55 g, 12.0 mmol), and the mixture was stirred at 25 °C for 3 h. The reaction mixture was diluted with 100 mL of dichloromethane, and the organic phase was washed twice with saturated sodium bicarbonate, each time with 30 mL of water. The aqueous phase was extracted with 10 mL of dichloromethane. The combined organic phases were washed with 50 mL of saturated brine, 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 in a vacuum oil pump. For column purification, 200 g of 200-300 mesh normal phase silica gel was used. The acidity of the silica gel was neutralized with 20 ml of triethylamine. The column was equilibrated with petroleum ether containing 1 wt% triethylamine. Gradient elution was performed with dichloromethane:methanol = 25:1 to 15:1. The product eluate was collected, and the solvent was evaporated to dryness under reduced pressure to obtain 2.2 g of pure Z-3. MS m / z:C 103 H 151 N 10 O 38 , [M+H] + , Theoretical value: 2136.02, Measured value: 2136.20.

[0486] Synthesis of (11-1-4)Z-4: Z-3 (2.10 g, 0.983 mmol) was dissolved in 14.8 mL of dichloromethane containing DIEA (0.635 g, 4.915 mmol). 4-Dimethylaminopyridine (DMAP, 240 mg, 1.966 mmol) was added and the mixture was stirred to clarify. Succinic anhydride (197 mg, 1.966 mmol) was then added and the mixture was stirred at 25°C for 18 hours. The reaction mixture was diluted with 50 mL of dichloromethane, and the organic phase was washed with 80 mL of 0.5 M triethylamine phosphate. The aqueous phase was extracted twice with 50 mL of dichloromethane. The combined organic phases were evaporated to dryness under reduced pressure to obtain the crude product. For column purification, 188 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 equilibrated with dichloromethane. Gradient elution was performed with dichloromethane containing 1 wt% triethylamine:methanol = 10:1 to 3:1. The product eluate was collected, and the solvent was evaporated to dryness under reduced pressure to obtain 1.95 g of pure Z-4 ​​complex. MS m / z:C 107 H 155 N 10 O 41 , [M+H] + , theoretical value: 1935.07, actual value: 1935.29.

[0487] Synthesis of (11-1-5)Z-5 Z-5 was prepared by the same method as in step (2-1-9) in Preparation Example 2, except that the Z-4 ​​conjugate molecule was used instead of the L-9 conjugate molecule to obtain the Z-4 ​​conjugate molecule bound to a solid support.

[0488] (11-2) Synthesis of Z5 complex The Z5 conjugate was prepared in the same manner as in steps (2-2), (2-3A), and (2-4) in Preparation Example 2, except that the sense strand was synthesized using the Z-5 compound instead of the L-10 compound as the starting compound. It was expected that the conjugates A19, B15, C11, D11, E17, F19, and G17, whose structures are shown in formula (422), could be obtained.

[0489] (Preparation Example 12) Preparation of FIN complex In this preparation example, conjugates A20 to A23, B16 to B17, C14, D14, E18 to E20, and F20 shown in Tables 4A to 4G, and comparative conjugates A1 and B1 (hereinafter also referred to as FIN conjugates) were synthesized. The sequences of the siRNA conjugated to these conjugates are shown in the corresponding sequences in Tables 4A to 4G.

[0490] (12-1) Synthesis of FIN-2 conjugate molecules The FIN-2 conjugate molecule was synthesized by the following process route, with reference to the preparation method described in Rajeev et al., ChemBioChem 2015, 16, 903-908.

[0491] Synthesis of (12-1-1)PRO-10

[0492] [ka]

[0493] (12-1-1a) Synthesis of PRO-7 2.93 g of PRO-6 (L-hydroxyproline, CAS number: 51-35-4, purchased from Energy, 22.4 mmol) was dissolved in 22.5 ml of 1,4-dioxane (1,4-dioxane, CAS number: 123-91-1), and 34 ml of 10% (w / w) aqueous Na2CO3 solution was added to form a suspension. 6.95 g of Fmoc-Cl (9-fluorenylmethyl chloroformate, CAS number: 28920-43-6, purchased from Energy, 26.8 mmol) was dissolved in 34 ml of 1,4-dioxane and added to the suspension in an ice bath. The mixture was allowed to warm to room temperature and react overnight. The reaction mixture was poured into 150 ml of ice water and extracted three times with 100 ml of methyl tert-butyl ether. The organic phase was discarded, and the aqueous phase was adjusted to pH 5 or less with concentrated HCl. The mixture was extracted twice with 100 ml of ethyl acetate. The combined organic phases were dried over anhydrous sodium sulfate, and the solvent was evaporated to dryness under reduced pressure to obtain 7.83 g of a white foamy solid product, PRO-7. 1H NMR (400 MHz,DMSO-d6) δ 7.91 (t,J =7.2 Hz,2H),7.67 (d,J =7.5 Hz,2H),7.48 - 7.39 (m,2H),7.38 - 7.27 (m,2H),5.17 (s,1H),4.27 (s,2H),4.23 - 4.11 (m,2H),3.55 - 3.41 (m,3H),2.31 - 2.10 (m,1H),2.08 - 1.88 (m,1H). HRMS (ESI) m / z theoretical value (calcd for) C 20 H 19 NO5[MH] - 352.1190, Actual value: 352.1033.

[0494] (12-1-1b) Synthesis of PRO-8 7.83 g of PRO-7 (22.2 mmol) was dissolved in 80 ml of THF (CAS No.: 109-99-9) and heated to 65°C in an oil bath. 36.6 ml of a 2 mol / L THF solution of BH3-Me2S (CAS No. 13292-87-0, purchased from J&K Scientific, 73.2 mmol) was added under reflux and the reaction was continued at reflux for 3 hours. The reaction mixture was drained and the remaining solid was dissolved with methanol. Methanol was added under stirring until the reaction mixture was free of gas, and the mixture was stirred for 30 minutes. The solvent was removed under reduced pressure and then purified three times with petroleum ether to yield 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 HRMS (ESI) m / z Theoretical value C 20 H 21 NO4[M+Na] + 362.1368, Actual value: 362.1012.

[0495] (12-1-1c) Synthesis of PRO-9 7.1 g of PRO-8 (21 mmol) was dissolved in 100 ml of pyridine, and 14.2 g of DMTr-Cl (4,4'-bismethoxytrityl chloride, 42 mmol) was added. The mixture was stirred at room temperature for 5 hours. The solvent was removed under reduced pressure, and the crude product was dissolved in ethyl acetate. Salt impurities were removed by filtration. The solvent was then removed under reduced pressure and the product was purified on a silica gel column. The silica gel column was pre-basified with pyridine, and the crude product was dissolved in DCM and loaded. DMTr-Cl was eluted with DCM containing 1% (v / v) pyridine, and the product was eluted with ethyl acetate. The product eluate was collected, and the solvent was evaporated to dryness under reduced pressure to give 8.2 g of a white solid product, PRO-9. HRMS (ESI) m / z theoretical value C 41 H 39 NO6[M+Na] + 664.2675, actual measurement 664.2348, C18 RP-HPLC (lot number JJS160324-1) purity 94.20%.

[0496] (12-1-1d) Synthesis of PRO-10 8.2 g of PRO-9 (12.8 mmol) was dissolved in 64 ml of N,N-dimethylformamide (DMF), 40 ml of piperidine (384 mmol) was added, and the mixture was stirred at room temperature for 30 minutes. The reaction mixture was poured into 300 ml of ice water and extracted three times with 150 ml of ethyl acetate. The combined organic phases were washed with 200 ml of saturated brine, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The product was then purified on a silica gel column. The silica gel column was pre-basified with pyridine and then loaded with DCM. The Fmoc group was eluted with DCM containing 1% (v / v) pyridine, followed by ethyl acetate. The product eluate was collected, and the solvent was evaporated to dryness under reduced pressure to give 4.65 g of a white solid product, PRO-10. 1H NMR (400 MHz, DMSO-d6) δ 7.40 (d,J =7.2 Hz,2H),7.35 - 7.18 (m,7H),6.93 - 6.84 (m,4H),4.56 (d,J =3.9 Hz,1H),4.12 (s,1H),3.74 (s,6H),3.46 - 3.37 (m,1H),2.88 (ddd,J =18.5,10.0,5.5 Hz,2H),2.75 (dd,J =8.7,5.8 Hz,1H),2.62 (dd,J =11.0,2.7 Hz,1H),1.74 - 1.65 (m,1H),1.40 (ddd,J =12.9,8.5,5.9 Hz,1H),HRMS (ESI) m / z theoretical value C 26 H 29 NO4[M+Na] + 442.1994, measured 442.1999, C18 RP-HPLC (Lottery No. JJS160329-1), purity 97.07%.

[0497] (12-1-2) FIN-1 synthesis

[0498]

change

[0499] (12-1-3) FIN-2 synthesis

[0500]

change

[0501] (12-2) Binding of FIN-2 conjugate molecules to solid supports The FIN-2 conjugated molecule obtained in step (12-1-3) was circulated three times by the solid-phase nucleic acid synthesis method to form a nucleic acid on a general-purpose solid support (UnyLinker TM The RNA was bound to NittoPhase® HL Solid Supports (loaded NittoPhase® HL Solid Supports) to achieve binding of a composite group (FIN_FIN_FIN) to the 3' end of the RNA sense strand.

[0502] The conjugation was carried out in accordance with the preparation method described in Rajeev et al., ChemBioChem 2015, 16, 903-908. Specifically, starting with the above-mentioned general-purpose solid support, the hydroxy-protecting group on the solid support was removed, and the solid support was contacted with a FIN-2 conjugated molecule under coupling reaction conditions and in the presence of a coupling reagent, followed by capping and oxidation to obtain a FIN conjugated molecule. The hydroxy-protecting group DMTr on the FIN conjugated molecule was removed, and the solid support was contacted with a FIN-2 conjugated molecule, followed by capping and oxidation. The above deprotection-coupling-capping-oxidation process was repeated once more to conjugate a third FIN-2 conjugated molecule, yielding a conjugate group (FIN_FIN_FIN) bound to the solid support.

[0503] In the above reaction, the reaction conditions, solvents, and reagent amounts for the above-mentioned deprotection, coupling, capping, and oxidation were the same as those for the solid phase nucleic acid synthesis method described in Preparation Example 1 above.

[0504] (12-3) Synthesis of Complexes F1 to F5 The title complexes were prepared in the same manner as in steps (2-2), (2-3A), and (2-4) in Preparation Example 2, except that: 1) a sense strand was synthesized starting from the compound obtained in step (12-2); and 2) the complexed siRNA had sequences corresponding to complexes A20 to A23, B16 to B17, C14, D14, E18 to E20, and F20 and comparative complexes A1 and B1 shown in Tables 4A to 4G.

[0505] The molecular weight was detected using a liquid chromatography mass spectrometer (LC-MS, Liquid Chromatography-Mass Spectrometry, purchased from Waters, model number: LCT Premier). As a result, the measured value agreed with the theoretical value, and the synthesized complex was confirmed to be the intended compound whose structure is shown in formula (307).

[0506] (Preparation Example 13) Preparation of Comparative Complexes A3, E2, and F2 In this preparation example, comparative complexes A3, E2, and F2 were synthesized, and the sequences of the siRNAs conjugated to these complexes are shown in Tables 4A, 4E, and 4F.

[0507] (13-1) Synthesis of (GalNAc)3 conjugated molecules Compound 30, i.e., the above-described linker-(L A )3-trihydroxymethylaminomethane-L B - and a targeting group, N-acetylgalactosamine molecule (wherein each L A A conjugated molecule (also called a (GalNAc)3 conjugated molecule) containing (one N-acetylgalactosamine molecule can be bound to one linker, so that three N-acetylgalactosamine molecules can be bound to one linker) was synthesized, and the structure of compound 30 is shown in the following formula.

[0508] [ka]

[0509] (13-2)(GalNAc)3 conjugation to solid support The (GalNAc)3 conjugated molecule was bound to a solid support by the same method as in step (2-1-9) in Preparation Example 2 to obtain a (GalNAc)3 conjugated molecule bound to a solid support.

[0510] (13-3) Synthesis of comparative complexes A3, E2, and F2 Comparative complexes A3, E2, and F2 were prepared in the same manner as steps (2-2), (2-3A), and (2-4) in Preparation Example 2, except that 1) a sense strand was synthesized starting from the compound obtained in step (13-2), and 2) the complex siRNAs had the sequences shown in Tables 4A, 4E, and 4F as numbers A3, E2, and F2.

[0511] The molecular weight was detected using a liquid chromatography mass spectrometer (LC-MS, Liquid Chromatography-Mass Spectrometry, purchased from Waters, model number: LCT Premier). As a result, the measured value agreed with the theoretical value, and the synthesized complex was confirmed to be the intended compound whose structure is shown in formula (305).

[0512] After the preparation of the complex of the present disclosure is completed, it is freeze-dried as a solid powder by standard means and stored for use, and when in use, it may be reconstituted with, for example, water for injection to prepare a solution of the desired concentration.

[0513] The properties of the siRNA and siRNA complexes of the present disclosure prepared as described above were investigated through the following experimental examples.

[0514] The following describes an experiment on the effect of the siRNA complexes in Table 4A.

[0515] (Experimental Example A1) This experiment demonstrates the toxicity of the siRNA complex of the present disclosure. C57BL / 6J mice were subcutaneously administered a single dose of 100 mg / kg or 200 mg / kg (calculated as siRNA) of complex A1 per mouse (0.9% sodium chloride aqueous solution, concentrations of 10 mg / mL and 20 mg / mL, respectively, in a volume of 10 mL / kg, administered to three male and three female mice for each concentration). Clinical observations were conducted during the administration period, and no animal deaths or clinical symptoms associated with drug side effects were observed. 24 hours after administration, blood samples were collected for clinical pathological examination, and the animals were dissected. No abnormalities were observed in either clinical pathological examination or gross dissection. As is clear from the above results, the complexes of the present disclosure have low toxicity at the animal level.

[0516] (Experimental Example A2) This experiment demonstrates the stability of the siRNA complexes of the present disclosure. (Experimental Example 2-1) Stability of siRNA complex in in vitro lysosomal lysate Preparation of lysosomal lysis solution-treated test samples: Comparative Complex A1 and Complex A21 (each provided as a 0.9% sodium chloride solution with an siRNA concentration of 20 μM, 6 μL per group) were uniformly mixed with 27.2 μL of sodium citrate solution (pH 5.0), 4.08 μL of deionized water, and 2.72 μL of Tritosome (purchased from Xenotech, product code R0610LT, lot number 1610069). The mixture was incubated at 37°C. Five μL of the sample was removed at 0, 1, 2, 4, 6, 8, 24, and 48 h. Each sample was denatured with 15 μL of 9 M urea, followed by the addition of 4 μL of 6x sample loading buffer (Solarbio, product code 20160830). The reaction was then immediately frozen in a -80°C refrigerator to terminate the reaction. Time 0 represents the time when the test sample and the lysosomal lysate were mixed uniformly and then immediately removed.

[0517] Preparation of reference samples not treated with lysosomal lysate: 1.5 μl of each of the above complexes (20 μM) was homogenously mixed with 7.5 μL of aqueous sodium citrate solution (pH 5.0) and 1 μL of deionized water. 30 μL of 9 M urea solution was added for denaturation, followed by the addition of 8 μL of 6x sample loading buffer, mixing homogenously, and immediately freezing in a refrigerator at -80°C to terminate the reaction. The reference sample for each complex is marked "Con" on the electropherogram.

[0518] A 16% wt% non-denaturing polyacrylamide gel was formulated. 20 μl of each of the test and reference samples was loaded onto the gel and electrophoresed at a constant current of 20 mA for 10 minutes, followed by 30 minutes at a constant current of 40 mA. After electrophoresis, the gel was placed on a shaker and stained with GelRed dye (BioTium, product code 13G1203) for 10 minutes. The gel was then imaged and photographed, and the results are shown in Figure 1.

[0519] 1 shows the results of semi-quantitative detection of the stability of the test siRNA complex in in vitro tritosomes. The results show that the complex of the present disclosure remains undegraded in tritosomes for a long period of time, demonstrating excellent stability.

[0520] (Experimental Example A2-2) Stability of siRNA complexes in in vitro lysosomal lysate The same method as in Experimental Example 2-1 was used, except that the test samples were complexes A1, A6, and comparative siRNA1, and the incubation times with tritozoan were 0 h, 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, and 8 h, respectively.

[0521] The results of non-denaturing polyacrylamide gel electrophoresis are shown in FIG.

[0522] 2 shows the results of semi-quantitative detection of the stability of the test siRNA complex in in vitro tritosomes. The results show that the complex of the present disclosure remains undegraded in tritosomes for a long period of time, demonstrating excellent stability.

[0523] The results shown in Figures 1 and 2 above demonstrate that siRNAs having specific modifications according to the present disclosure exhibit satisfactory stability in lysosomal lysates.

[0524] (Experimental Example A2-3) Stability in human plasma Complexes A1, A6, and comparative siRNA2 (each provided as a 20 μM siRNA in 0.9% sodium chloride solution, 12 μL per group) were homogenously mixed with 108 μL of 90% human plasma (diluted with PBS) and incubated at 37°C. At 0, 2, 4, 6, 8, 24, 48, and 72 hours, 10 μL samples were withdrawn and immediately flash-frozen in liquid nitrogen and stored in a refrigerator at -80°C. After sampling at each time point, the frozen samples were diluted 5-fold with 1x PBS (pH 7.4), and 10 μL of each sample was removed for use. At the same time, an equimolar amount of the test sample (2 μM, 2 μL) was homogenously mixed with 8 μL of 1x PBS (pH 7.4) to prepare a 10 μL sample without human plasma treatment, designated Con. A 20% (wt%) non-denaturing polyacrylamide gel was prepared. All samples from each group in the preliminary sample were mixed with 4 μL of sample loading buffer (20 mM EDTA, 36% (wt%) glycerin, and 0.06% (wt%) bromphenol blue in water. The gel was then loaded and electrophoresed at a constant current of 80 mA for 60 minutes. After electrophoresis, the samples were stained with 1x Sybr Gold dye (Invitrogen, Cat. 11494) for 15 minutes and then imaged. The results are shown in Figure 3.

[0525] FIG. 3 shows the results of semi-quantitative detection of the stability of the test conjugate in human plasma in vitro.

[0526] As can be seen from the results in FIG. 3, the conjugate of the present disclosure was not decomposed even after 72 hours in human plasma, demonstrating excellent stability in human plasma.

[0527] (Experimental Example A2-4) Stability of the complex in monkey plasma Complexes A1, A6, and comparative siRNA2 (each provided as a 20 μM siRNA in 0.9% sodium chloride solution, 12 μL per group) were homogenously mixed with 108 μL of 90% cynomolgus monkey plasma (monkey plasma, purchased from Kosen Biosciences, HQ70082, diluted with PBS) and incubated at 37°C. At 0, 2, 4, 6, 8, 24, 48, and 72 hours, 10 μL samples were withdrawn, immediately flash-frozen in liquid nitrogen, and stored in a refrigerator at -80°C. After sampling at each time point, the frozen samples were diluted 5-fold with 1x PBS (pH 7.4), and 10 μL of each sample was removed for use. At the same time, an equimolar amount of the test sample (2 μM, 2 μL) was homogenously mixed with 8 μL of 1x PBS (pH 7.4) to prepare a 10 μL untreated monkey plasma sample, designated Con. A 20% (wt%) non-denaturing polyacrylamide gel was prepared. All samples from each group in the preliminary sample were mixed with 4 μL of sample loading buffer (20 mM EDTA, 36% (wt%) glycerin, and 0.06% (wt%) bromphenol blue in water. The gel was then loaded and electrophoresed at a constant current of 80 mA for 60 minutes. After electrophoresis, the samples were stained with 1x Sybr Gold dye (Invitrogen, Cat. 11494) for 15 minutes and then imaged. The results are shown in Figure 4.

[0528] FIG. 4 shows the results of semi-quantitative detection of the stability of test siRNAs in monkey plasma in vitro.

[0529] As can be seen from the results in FIG. 4, the siRNA complex of the present disclosure was not decomposed in cynomolgus monkey plasma for up to 72 hours, demonstrating excellent stability in monkey plasma.

[0530] (A2-5) This experiment demonstrates the stability of the disclosed siRNA complex in an in vitro lysosomal lysate. 1) Detection of stability in mouse lysosomal lysates Preparation of lysosomal lysate-treated test samples: 6 μl of Complex A2 and comparative siRNA2 (20 μM) were mixed uniformly with 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 Tritosomes, Xenotech, No. R0610.LT, Lot No. 1610069), resulting in a final acid phosphatase concentration of 0.2 mU / μL. The mixture was incubated at 37°C. At 0, 1, 2, 4, 6, and 24 hours, 5 μl of the mixture was removed and denatured with 15 μL of 9 M urea solution. Then, 4 μl of 6x sample loading buffer (Solarbio, No. 20160830) was added and the mixture was immediately frozen in a -80°C refrigerator to terminate the reaction. Time 0 represents the time when the test sample and the lysosomal lysate were mixed uniformly and then immediately removed.

[0531] Preparation of a reference sample not treated with lysosomal lysate: 1.5 μl each of equimolar amounts of Complex A2 and comparative siRNA2 (20 μM) was mixed uniformly with 7.5 μL of aqueous sodium citrate solution (pH 5.0) and 1 μL of deionized water. 30 μL of 9 M urea solution was added for denaturation, followed by 8 μL of 6× sample loading buffer, mixing uniformly, and immediately freezing in a -80°C refrigerator to stop the reaction. The reference sample for each sample was designated M and used for comparison with the electrophoresis results of the sample.

[0532] A 16% wt% non-denaturing polyacrylamide gel was prepared. 20 μl of each of the test and reference samples was loaded onto the gel. Electrophoresis was performed at a constant current of 20 mA for 10 minutes, followed by 30 minutes at a constant current of 40 mA. After electrophoresis, the gel was placed on a shaker and stained with GelRed dye (BioTium, product code 13G1203) for 10 minutes. The gel was then imaged and photographed, and the results are shown in Figure 5.

[0533] 2) Stability in human lysosomal lysates The stability of comparative siRNA2 and complex A2 in human lysosomal lysate was determined using the same method as in 1), except that mouse lysosomal lysate was replaced with human lysosomal lysate (Human Liver Lysosomes, Xenotech, product code H0610.L, lot number 1610316). The results are shown in Figure 6.

[0534] As is clear from the results, the siRNA complex of the present disclosure exhibits satisfactory stability in both human-derived lysosomal lysate and mouse-derived lysosomal lysate, and can be maintained without degradation for at least 24 hours.

[0535] (Experimental Example A3) This experiment shows the results of a pharmacokinetic study of Complex A1 in rats. In this experiment, rats in each experimental group (10 rats per group, half of which were male and half of which were female) were given a single subcutaneous injection of Complex A1 at doses of 10 mg / kg and 50 mg / kg, and then the drug concentrations in the rat plasma, liver, and kidney tissues were measured at each time point.

[0536] The SD rats used in this experiment were provided by Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.

[0537] First, SD rats were randomly divided by sex according to their body weight using the PRISTIMA 7.2.0 data system, and then administered the conjugates in each group at the designed dose. The doses for all animals were calculated according to their body weight and administered subcutaneously in a single dose of 10 and 50 mg / kg. The conjugates were administered in 0.9% sodium chloride solution at 1 mg / ml and 5 mg / ml, respectively, in a volume of 10 ml / kg. Whole blood samples were collected from the jugular vein of rats at 5 minutes (±30 seconds), 30 minutes (±1 minute), 1 hour (±2 minutes), 2 hours (±2 minutes), 6 hours (±5 minutes), 24 hours (±10 minutes), 48 hours (±20 minutes), 72 hours (±20 minutes), 120 hours (±30 minutes), and 168 hours (±30 minutes) before and after drug administration. Plasma was isolated by centrifugation at 1800 × g for 10 minutes at 2-8°C. Approximately 70 μL of the plasma sample was placed in a tube, and the remaining sample was placed in another tube and frozen at -70 to -86°C until detection. Approximately 24, 48, 72, 120, and 168 hours after drug administration, rats were anesthetized with sodium pentobarbital (60 mg / kg intraperitoneally) according to their body weight, euthanized by blood collection from the abdominal aorta, and liver and kidney tissues were collected by gross dissection. Liver and kidney samples were collected from each rat, stored in 1 mL cryopreservation tubes, and kept at -68°C or below until detection and analysis.

[0538] The concentrations of complex A1 in rat plasma, liver, and kidney tissues were quantitatively detected by HPLC-FLD (high performance liquid chromatography with fluorescence detection). Specifically, the following steps were performed.

[0539] (1) The tissue was ground to a mass of 80 mg or less, and then a tissue and cell lysis solution (Tissue and Cell Lysis Solution, supplier: epicentre, product code: MTC096H) was added to prepare a 66.7 mg / mL tissue homogenate. (2) The tissue homogenate was sonicated (150 W, 30 s) to disrupt the cells. (3) For tissue samples, 75 μL of tissue sample was added to a 96-well PCR plate, followed by 5 μL of protease K (supplier: Invitrogen, number: 25530-015), 10 μL of a mixed aqueous solution of 10 wt% acetonitrile and 0.01 wt% Tween 20; for plasma samples, 20 μL of plasma was added to a 96-well PCR plate, followed by 45 μL of tissue and cell lysate, 5 μL of protease K, 20 μL of a mixed aqueous solution of 10 wt% acetonitrile and 0.01 wt% Tween 20. (4) The plate was sealed and placed in a PCR machine (supplier: Applied Biosystems, model number: GeneAmp (registered trademark) PCR system 9700) and incubated at 65°C for 45 minutes. (5) After the incubation, 10 μL of a 3M KCl aqueous solution (supplier: Sigma-Aldrich, product code: 60135-250ML) was added, the mixture was shaken to homogenize, and the mixture was centrifuged at 4° C. and 3200 rcf for 15 minutes. (6) For tissue samples, add 80 μL of the supernatant to 120 μL of hybridization mixture (preparation of hybridization mixture: 0.5 mL of 6 μM PNA probe (supplier: Hangzhou Taihe Biotechnology Co., Ltd.), 1 mL of 200 mM Trizma / pH = 8, 5 mL of 8 M urea solution, 3.5 mL of H2O, and 2 mL of acetonitrile). For plasma samples, 40 μL of the supernatant was added to 160 μL of hybridization mixture (preparation of hybridization mixture: 0.5 mL of 6 μM PNA probe, 1 mL of 200 mM Trizma / pH=8, 5 mL of 8 M urea aqueous solution, 7.5 mL of H2O, and 2 mL of acetonitrile). (7) The plate was sealed, placed in a PCR machine, incubated at 95°C for 15 minutes, and immediately placed on ice for 5 minutes. (8) The mixture was transferred to another 96-well conical plate, shaken to homogenize, and centrifuged at 3200 rcf for 1 minute. (9) The sample was loaded and detected, and then analyzed and quantified by HPLC-FLD (liquid phase system supplier: Thermo Fisher, chromatograph model number: ultimate 3000).

[0540] The analysis results are shown in Figures 7 to 10. Figures 7 to 10 show the time-dependent metabolic curves of the PK / TK plasma concentration in rat plasma and the time-dependent metabolic curves of the PK / TK tissue concentration in rat liver and kidney for complex A1 at a dose of 10 mg / kg or 50 mg / kg, respectively. Specifically, FIG. 7 shows the time course metabolism curve of PK / TK plasma concentration of conjugate A1 in rat plasma at a dose of 10 mg / kg.

[0541] FIG. 8 shows the time course metabolism curves of PK / TK tissue concentrations of complex A1 in rat liver and kidney at a dose of 10 mg / kg.

[0542] FIG. 9 shows the time course metabolism curve of PK / TK plasma concentration of conjugate A1 in rat plasma at a dose of 50 mg / kg.

[0543] FIG. 10 shows the time course metabolism curves of PK / TK tissue concentrations of complex A1 in rat liver and kidney at a dose of 50 mg / kg.

[0544] As can be seen from the results in Figures 7 to 10, the concentration of complex A1 in rat plasma rapidly decreased to below the detection limit within several hours, regardless of whether it was administered at a low dose (10 mg / kg) or a relatively high dose (50 mg / kg), but in liver tissue, a high and stable tissue concentration was maintained for at least 168 hours. This indicates that the siRNA complex of the present disclosure can be significantly enriched and stabilized specifically in the liver, and has high targeting properties.

[0545] (Experimental Example A4) This experiment demonstrates the inhibitory effect of the RNA complex of the present disclosure on HBV mRNA expression in vivo. In this experiment, the inhibitory efficiency of complexes A5 and A7 on HBV mRNA expression levels was examined in HBV transgenic mice C57BL / 6J-Tg(Alb1HBV)44Bri / J.

[0546] A hepatitis B virus surface antigen diagnostic kit (enzyme-linked immunosorbent assay)...

Claims

1. A double-stranded oligonucleotide comprising a sense strand and an antisense strand, Each nucleotide in the sense strand and the antisense strand is a modified nucleotide, the sense strand comprises nucleotide sequence 1, the antisense strand comprises nucleotide sequence 2, the length of nucleotide sequence 1 and the length of nucleotide sequence 2 are both 19 nucleotides ... a double-stranded oligonucleotide, wherein each nucleotide is independently one of a non-fluoro-modified nucleotide, wherein each non-fluoro-modified nucleotide is a 2'-alkoxy-modified nucleotide, a 2'-substituted alkoxy-modified nucleotide, a 2'-alkyl-modified nucleotide, a 2'-substituted alkyl-modified nucleotide, a 2'-amino-modified nucleotide, a 2'-substituted amino-modified nucleotide, or a 2'-deoxynucleotide, wherein the first nucleotide at the 5' end of nucleotide sequence 2 is the first nucleotide at the 5' end of the antisense strand, the nucleotides at positions 2, 6, 14, and 16 of nucleotide sequence 2 are fluoro-modified nucleotides, and each nucleotide at any other position of nucleotide sequence 2 is independently one of a non-fluoro-modified nucleotide.

2. At least nucleotides 2 to 19 of said nucleotide sequence 2 from the 5' to the 3' end are complementary to the first nucleotide sequence fragment; or 2. The double-stranded oligonucleotide of claim 1, wherein the first nucleotide of said nucleotide sequence 2 from the 5' end to the 3' end is A or U.

3. 3. The double-stranded oligonucleotide of claim 1, wherein the sense strand further comprises nucleotide sequence 3 and the antisense strand further comprises nucleotide sequence 4, each nucleotide of nucleotide sequence 3 and nucleotide sequence 4 being independently one of non-fluoro-modified nucleotides, each of nucleotide sequence 3 and nucleotide sequence 4 being 1 to 4 nucleotides in length, nucleotide sequence 3 and nucleotide sequence 4 being equal in length and essentially perfectly reverse complementary or completely reverse complementary, nucleotide sequence 3 being linked to the 5' end of nucleotide sequence 1 and nucleotide sequence 4 being linked to the 3' end of nucleotide sequence 2, and nucleotide sequence 4 being essentially perfectly reverse complementary or completely reverse complementary to a second nucleotide sequence fragment, the second nucleotide sequence fragment referring to a nucleotide sequence adjacent to the first nucleotide sequence fragment in the target mRNA and having the same length as nucleotide sequence 4, and wherein "essentially perfectly reverse complementary" refers to the presence of one or less base mismatches between the two nucleotide sequences, and "completely reverse complementary" refers to the absence of mismatches between the two nucleotide sequences.

4. The double-stranded oligonucleotide according to any one of claims 1 to 3, further comprising a nucleotide sequence 5, wherein each nucleotide of the nucleotide sequence 5 is independently one of non-fluoro-modified nucleotides, the nucleotide sequence 5 is 1 to 3 nucleotides in length, and is attached to the 3' end of the antisense strand, constituting a 3' overhang end of the antisense strand.

5. 5. The double-stranded oligonucleotide according to claim 4, wherein the length of nucleotide sequence 5 is two nucleotides, and from the 5' end to the 3' end, nucleotide sequence 5 is two consecutive thymine deoxyribonucleotides, two consecutive uracil ribonucleotides, or is completely reverse-complementary to a third nucleotide sequence fragment, wherein the third sequence fragment refers to a nucleotide sequence that is adjacent to the first nucleotide sequence fragment or the second nucleotide sequence fragment in the target mRNA and has a length equal to that of nucleotide sequence 5.

6. The double-stranded oligonucleotide according to any one of claims 1 to 5, wherein 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 a methoxy group.

7. In the double-stranded oligonucleotide, at least one phosphate group is a thiophosphate, and the thiophosphate is 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 double-stranded oligonucleotide according to any one of claims 1 to 6, 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.

8. The double-stranded oligonucleotide according to any one of claims 1 to 7, wherein the 5'-terminal nucleotide of the antisense strand is a 5'-phosphate nucleotide or a 5'-phosphate analog-modified nucleotide.

9. The double-stranded oligonucleotide according to any one of claims 1 to 8, which is saRNA or siRNA.

10. The double-stranded oligonucleotide according to any one of claims 1 to 9, wherein the target mRNA is one selected from the mRNAs corresponding to the genes ApoB, ApoC, ANGPTL3, PCSK9, SCD1, TIMP-1, Col1A1, FVII, STAT3, p53, HBV, and HCV.

11. said nucleotide sequence 1 is the sequence shown in SEQ ID NO: 1 and said nucleotide sequence 2 is the sequence shown in SEQ ID NO: 2; or the nucleotide sequence 1 is the sequence shown in SEQ ID NO:3, and the nucleotide sequence 2 is the sequence shown in SEQ ID NO:4; or the nucleotide sequence 1 is the sequence shown in SEQ ID NO:5, and the nucleotide sequence 2 is the sequence shown in SEQ ID NO:6; or said nucleotide sequence 1 is the sequence shown in SEQ ID NO: 7 and said nucleotide sequence 2 is the sequence shown in SEQ ID NO: 8; or said nucleotide sequence 1 is the sequence shown in SEQ ID NO: 9 and said nucleotide sequence 2 is the sequence shown in SEQ ID NO: 10; or the nucleotide sequence 1 is the sequence shown in SEQ ID NO: 11, and the nucleotide sequence 2 is the sequence shown in SEQ ID NO: 12; or The double-stranded oligonucleotide of claim 1, wherein the nucleotide sequence 1 is the sequence shown in SEQ ID NO: 13, and the nucleotide sequence 2 is the sequence shown in SEQ ID NO: 14: 5'-CmCmUmUmGmAmGfGfCfAmUmAmCmUmUmCmAmAmAm-3' (SEQ ID NO: 1) 5'-UmUfUmGmAmAfGmUmAmUmGmCmCmUfCmAfAmGmGm-3' (SEQ ID NO: 2) 5'-UmGmCmUmAmUmGfCfCfUmCmAmUmCmUmUmCmUmAm-3' (SEQ ID NO: 3) 5'-UmAfGmAmAmGfAmUmGmAmGmGmCmAfUmAfGmCmAm-3' (SEQ ID NO: 4) 5'-UmCmUmGmUmGmCfCfUfUmCmUmCmAmUmCmUmGmAm-3' (SEQ ID NO: 5) 5'-UmCfAmGmAmUfGmAmGmAmAmGmGmCfAmCfAmGmAm-3' (SEQ ID NO: 6) 5'-CmGmUmGmUmGmCfAfCfUmUmCmGmCmUmUmCmAmAm-3' (SEQ ID NO: 7) 5'-UmUfGmAmAmGfCmGmAmAmGmUmGmCfAmCfAmCmGm-3' (SEQ ID NO: 8) 5'-GmAmAmAmGmUmAfUfGfUmCmAmAmCmGmAmAmUmAm-3' (SEQ ID NO: 9) 5'-UmAfUmUmCmGfUmUmGmAmCmAmUmAfCmUfUmUmCm-3' (SEQ ID NO: 10) 5'-CmCmAmAmGmAmGfCfAfCmCmAmAmGmAmAmCmUmAm-3' (SEQ ID NO: 11) 5'-UmAfGmUmUmCfUmUmGmGmUmGmCmUfCmUfUmGmGm-3' (SEQ ID NO: 12) 5'-CmAmAmUmAmAmAmAfGfCfUmGmGmAmCmAmAmGmAmAm-3' (SEQ ID NO: 13) 5'-UmUfCmUmUmGfUmCmCmAmGmCmUmUfUmAfUmUmGm-3' (SEQ ID NO: 14) Here, the capital letters C, G, U, and A represent the base sequence of nucleotides, the lower case letter m represents that one nucleotide adjacent to the left side of the letter m is a 2'-methoxy-modified nucleotide, and the lower case letter f represents that one nucleotide adjacent to the left side of the letter f is a 2'-fluoro-modified nucleotide.

12. A pharmaceutical composition comprising the double-stranded oligonucleotide according to any one of claims 1 to 11 and a pharmaceutically acceptable carrier.

13. An oligonucleotide conjugate comprising the double-stranded oligonucleotide according to any one of claims 1 to 11 and a conjugated group conjugated to the double-stranded oligonucleotide.

14. having a structure shown in formula (308), 【Chemical 1】 In the formula, 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 C １ -C １０ Alkyl group, C １ -C １０ Halogenated alkyl group and C １ -C １０ alkoxy groups; R ３ is a group having the structure shown in formula A59, 【Chemistry 2】 In the formula, E １ is OH, SH or BH ２ and Nu is a double-stranded oligonucleotide; R ２ is a straight chain alkylene group of 1 to 20 carbon atoms in length, one or more of which carbon atoms are selected from C(O), NH, O, S, CH═N, S(O) ２ , C ２ -C １０ Alkenylene group, C ２ -C １０ Alkynylene group, C ６ -C １０ Arylene group, C ３ -C １８ Heterocyclylene group and C ５ -C １０ heteroarylene groups, R ２ is C １ -C １０ Alkyl group, C ６ -C １０ Aryl group, C ５ -C １０ Heteroaryl group, C １ -C １０ Halogenated alkyl group, —OC １ -C １０ Alkyl group, —OC １ -C １０ alkylphenyl group, —C １ -C １０ Alkyl-OH, —OC １ -C １０ Halogenated alkyl group, -SC １ -C １０ Alkyl group, —SC １ -C １０ alkylphenyl group, —C １ -C １０ Alkyl-SH, -SC １ -C １０ Halogenated alkyl groups, halogen substituents, -OH, -SH, -NH ２ , -C １ -C １０ Alkyl-NH ２ , -N(C １ -C １０ alkyl group) (C １ -C １０ alkyl group), -NH(C １ -C １０ alkyl group), 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 groups, —OC(O)C １ -C １０ Alkyl group, —SO ２ (C １ -C １０ alkyl group), -SO ２ (phenyl group), —SO ２ (C １ -C １０ halogenated alkyl group), —SO ２ NH ２ , -SO ２ NH (C １ -C １０ alkyl group), -SO ２ NH (phenyl group), -NHSO ２ (C １ -C １０ alkyl group), -NHSO ２ (phenyl group) and -NHSO ２ (C １ -C １０ halogenated alkyl groups), Each L １ is a straight chain alkylene group of 1 to 70 carbon atoms in length, one or more of which carbon atoms are selected from C(O), NH, O, S, CH═N, S(O) ２ , C ２ -C １０ Alkenylene group, C ２ -C １０ Alkynylene group, C ６ -C １０ Arylene group, C ３ -C １８ Heterocyclylene group and C ５ -C １０ heteroarylene groups; １ is C １ -C １０ Alkyl group, C ６ -C １０ Aryl group, C ５ -C １０ Heteroaryl group, C １ -C １０ Halogenated alkyl group, —OC １ -C １０ Alkyl group, —OC １ -C １０ alkylphenyl group, —C １ -C １０ Alkyl-OH, —OC １ -C １０ Halogenated alkyl group, -SC １ -C １０ Alkyl group, —SC １ -C １０ alkylphenyl group, —C １ -C １０ Alkyl-SH, -SC １ -C １０ Halogenated alkyl groups, halogen substituents, -OH, -SH, -NH ２ , -C １ -C １０ Alkyl-NH ２ , -N(C １ -C １０ alkyl group) (C １ -C １０ alkyl group), -NH(C １ -C １０ alkyl group), 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 groups, —OC(O)C １ -C １０ Alkyl group, —SO ２ (C １ -C １０ alkyl group), -SO ２ (phenyl group), —SO ２ (C １ -C １０ halogenated alkyl group), —SO ２ NH ２ , -SO ２ NH (C １ -C １０ alkyl group), -SO ２ NH (phenyl group), -NHSO ２ (C １ -C １０ alkyl group), -NHSO ２ (phenyl group) and -NHSO ２ (C １ -C １０ halogenated alkyl groups), 【Chemistry 3】 represents the site at which the group is attached to the rest of the molecule, and M １ The oligonucleotide conjugate of claim 13 , wherein represents a targeting group.

15. Each L １ is a combination of one or more bonds independently selected from groups of formulae A1 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 A45 or 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 is a combination of at least two bonds selected from A1, A4, A8, A10 and A11, or L １ The oligonucleotide complex of claim 15, wherein is a combination of at least two bonds selected from A1, A8, and A10.

17. L １ is 3 to 25 atoms in length, or L １ 17. The oligonucleotide conjugate of any one of claims 14 to 16, wherein the length of

18. n １ and n3 are each independently 1 or 2, or n1+n3=2 to 3. The oligonucleotide complex of any one of claims 14 to 17.

19. each m １ , each m ２ and each m ３ are each independently selected from integers of 2 to 5, or m1 = m2 = m3. The oligonucleotide complex of any one of claims 14 to 18.

20. each said targeting group is selected from a ligand capable of binding to a cell surface receptor; or each said targeting group is selected from ligands that have affinity for the asialoglycoprotein receptor on the surface of mammalian liver cells; or 20. The siRNA conjugate of any one of claims 14 to 19, wherein at least one or each of the targeting groups is galactose or N-acetylgalactosamine, GalNAc.

21. Each R １０ , R １１ , R １２ , R １３ , R １４ and R １５ The oligonucleotide conjugate of any one of claims 14 to 20, wherein are independently H, a methyl group, or an ethyl group.

22. R ２ forms an amide bond with an N on the nitrogen-containing backbone, or R ２ is selected from B5, B6, B5' or B6'; 【Chemistry 6】 During the ceremony, 【Chemistry 7】 represents the site at which the group is attached to the rest of the molecule, and q ２ The oligonucleotide complex of any one of claims 14 to 21, wherein is an integer from 1 to 10.

23. The oligonucleotide complex according to any one of claims 14 to 22, 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 8】 【change】 【change】 【change】 【change】 【change】

24. Use of the double-stranded oligonucleotide according to any one of claims 1 to 11, the pharmaceutical composition according to claim 12 and / or the oligonucleotide conjugate according to any one of claims 13 to 23 in the preparation of a drug for the treatment and / or prevention of a pathological condition or disease caused by abnormal expression of a gene.

25. 25. The use according to claim 24, wherein the gene is selected from the group consisting of a hepatitis B virus gene, an angiopoietin-like protein 3 gene, and an apolipoprotein C3 gene.

26. 26. The use according to claim 24 or 25, wherein the disease is selected from a chronic disease, an inflammation, a fibrotic disease, a hyperplastic disease or a lipid disorder.

27. 27. The use of claim 26, wherein the dyslipidemia is hypercholesterolemia, hypertriglyceridemia or atherosclerosis.

28. A method for inhibiting gene expression, comprising contacting an effective amount of the double-stranded oligonucleotide according to any one of claims 1 to 11, the pharmaceutical composition according to claim 12, and / or the oligonucleotide complex according to any one of claims 13 to 23 with a cell expressing the gene in vitro.

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