Double-stranded oligonucleotides, compositions and conjugates containing double-stranded oligonucleotides, and methods of making and uses

Abstract

A double-stranded oligonucleotide containing a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, each nucleotide in the double-stranded oligonucleotide is independently a modified or unmodified nucleotide, a portion of the sense strand and the antisense strand reverse complement to form a double-stranded region, wherein the 5' terminal nucleotide of the antisense strand is a ribose 5' modified nucleotide having a structure as shown in formula (101). The double-stranded oligonucleotide of the present disclosure, the pharmaceutical composition containing the double-stranded oligonucleotide of the present disclosure and / or the oligonucleotide conjugate have better activity and lower toxicity.

Description

Technical Field

[0001] This disclosure relates to a modified double-stranded oligonucleotide and pharmaceutical compositions and oligonucleotide conjugates containing the double-stranded oligonucleotide. This disclosure also relates to methods for preparing and using these double-stranded oligonucleotides, pharmaceutical compositions, and oligonucleotide conjugates. Background Technology

[0002] Double-stranded oligonucleotides are known to the public as active pharmaceutical ingredients. In recent years, considerable progress has been made in the development of double-stranded oligonucleotide drugs.

[0003] In the drug development of double-stranded oligonucleotides, the safety of the oligonucleotides themselves remains a top priority. For example, prior art WO2019 / 105437A1 discloses an siRNA or a pharmaceutical composition or siRNA conjugate containing that siRNA. This siRNA, pharmaceutical composition, or siRNA conjugate exhibits good inhibitory activity against target gene expression in mice, but subsequent studies have revealed some hepatotoxicity, thus hindering its further application in drug development. Therefore, how to obtain double-stranded oligonucleotides with both good pharmaceutical activity and low toxicity requires further in-depth exploration in this field, and related unresolved needs still exist in actual research and development. Summary of the Invention

[0004] The inventors unexpectedly discovered that by chemically modifying the first nucleotide at the 5' end of the antisense strand of a double-stranded oligonucleotide with a specific ribose 5', the resulting modified double-stranded oligonucleotide, as well as pharmaceutical compositions and oligonucleotide conjugates containing this double-stranded oligonucleotide, exhibit high stability, good activity, and low toxicity in vivo. Therefore, the inventors made the following invention.

[0005] In a first aspect, this disclosure provides a double-stranded oligonucleotide containing a sense strand and an antisense strand, each strand having 14 to 30 nucleotides. Each nucleotide in the double-stranded oligonucleotide is independently modified or unmodified. A portion of the sense strand and the antisense strand are inversely complementary to form a double-stranded region. The 5' terminal nucleotide of the antisense strand is a nucleotide with a ribose 5' modification, which has the structure shown in formula (101). The group R0 constitutes the 5' overhang of the antisense strand, and R0 has the structure shown in formula (102).

[0006]

[0007] in:

[0008] R 201 Hydroxyl or phosphate group:

[0009] G1 is OH, O- Or OJ1, wherein J1 is a C1-C6 alkyl, a substituted C1-C6 alkyl, a C3-C6 cycloalkyl, or a substituted C3-C6 cycloalkyl;

[0010] Bx1 is a hydrogen, a heterocyclic base, or a base substitution group, and if the 3' end of the positive chain includes a protruding end, Bx1 does not pair with the base at that protruding end, wherein the base substitution group is a phenyl or a substituted phenyl group; Bx2 is a heterocyclic base;

[0011] Z is a group having one of the divalent linking groups represented by the structures shown in formulas (Z1) to (Z5), or Z is a 1,2-cycloalkylene or heterocyclic group having 3-6 carbon atoms, or a substituted 1,2-cycloalkylene or heterocyclic group having 3-6 carbon atoms:

[0012]

[0013]

[0014] Wherein, P1 and P2 are each independently H, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C2-C6 alkenyl or substituted C2-C6 alkenyl.

[0015] P3 is either O or S;

[0016] X1 is H or hydroxyl, and X2 is selected from H, halogen, hydroxyl, C1-C6 alkoxy or substituted C1-C6 alkoxy.

[0017] T2 is a phosphate ester subunit or a thiophosphate ester subunit; Y1, Y2, Y3, Y4, Y5, Y6, Y7 and Y8 are each independently H, halogen, hydroxyl, methyl, ethyl, n-propyl or isopropyl;

[0018] This indicates the site where the group is covalently linked.

[0019] Secondly, this disclosure also provides a pharmaceutical composition comprising the double-stranded oligonucleotide provided in this disclosure and a pharmaceutically acceptable carrier.

[0020] Thirdly, this disclosure also provides an oligonucleotide conjugate containing a double-stranded oligonucleotide provided in this disclosure and a conjugating group conjugated to the double-stranded oligonucleotide. The conjugating group includes a linker and a pharmaceutically acceptable targeting group and / or a delivery aid group. The double-stranded oligonucleotide, the linker, and the targeting group or the delivery aid group are sequentially covalently or non-covalently linked. Each targeting group is selected from ligands capable of binding to cell surface receptors, and each delivery aid group is selected from groups capable of increasing the biocompatibility of the oligonucleotide conjugate in the target organ or tissue.

[0021] Fourthly, this disclosure also provides the use of the double-stranded oligonucleotides, pharmaceutical compositions and / or oligonucleotide conjugates of this disclosure in the preparation of medicaments for treating and / or preventing diseases or symptoms associated with the level of mRNA expression of the target group.

[0022] Fifthly, this disclosure also provides a method for treating and / or preventing diseases or symptoms associated with mRNA levels of a target gene expression, the method comprising administering to a subject in need the double-stranded oligonucleotide, pharmaceutical composition, and / or oligonucleotide conjugate of this disclosure.

[0023] In a sixth aspect, this disclosure also provides a method for regulating the expression level of a target gene in a cell, the method comprising contacting the cell with an effective amount of the double-stranded oligonucleotide, pharmaceutical composition and / or oligonucleotide conjugate of this disclosure.

[0024] In a seventh aspect, this disclosure also provides a kit comprising the double-stranded oligonucleotides, pharmaceutical compositions, and / or oligonucleotide conjugates of this disclosure.

[0025] Incorporate by reference

[0026] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference to the same extent that each individual publication, patent or patent application is specifically and individually incorporated herein by reference.

[0027] Beneficial effects

[0028] The double-stranded oligonucleotides, pharmaceutical compositions, and / or oligonucleotide conjugates disclosed herein exhibit significantly low toxicity while maintaining good stability and good target gene expression regulatory activity. Specific details are as follows:

[0029] First, the double-stranded oligonucleotides, pharmaceutical compositions, and / or oligonucleotide conjugates disclosed herein exhibit low toxicity in vivo. For example, when the double-stranded oligonucleotide of this disclosure is siRNA, the siRNA conjugate provided by this disclosure was administered subcutaneously to each rat at a single dose of 30 mg / kg for 14 consecutive days. The rats were then dissected, and liver tissue pathological sections were observed under an optical microscope. The results showed that the rats administered the conjugate of this disclosure had clear hepatic cord structures, tightly packed hepatocytes with clear boundaries, abundant and uniformly stained cytoplasm, round and normal-sized nuclei, intact and normal venous endothelium, and no obvious tissue abnormalities. In contrast, the rats administered the reference conjugate showed extensive edema and degeneration of hepatocytes, cell swelling, loose and pale stained cytoplasm, and numerous hepatocytes with fatty degeneration. Circular vacuoles of varying numbers were visible in the cytoplasm, and multiple focal infiltrations of inflammatory cells were observed within the lobules. This indicates that the hepatotoxicity of the siRNA conjugate of this disclosure is significantly reduced compared to the reference siRNA conjugate.

[0030] Second, the double-stranded oligonucleotides, pharmaceutical compositions, and / or oligonucleotide conjugates disclosed herein exhibit excellent target gene expression regulatory activity in in vitro cell experiments. For example, when the double-stranded oligonucleotides disclosed herein are siRNAs, the siRNA conjugates provided herein exhibit excellent target gene inhibitory activity in in vitro cell experiments. In some embodiments, at a siRNA concentration of 10 nM, the siRNA conjugates provided herein showed an 81.63%-84.04% inhibition rate of target mRNA expression in primary hepatocytes of HBV transgenic mice.

[0031] Furthermore, the double-stranded oligonucleotides, pharmaceutical compositions, and / or oligonucleotide conjugates disclosed herein exhibit higher stability and / or higher activity in vivo. For example, when the double-stranded oligonucleotides disclosed herein are siRNAs, the siRNA conjugates provided herein can exhibit higher target gene inhibitory activity in vivo. For instance, the siRNA conjugates provided herein showed a high HBV mRNA inhibition rate of 60.85%-69.73% in HBV transgenic mice at a dose of 0.1 mg / kg. As another example, the siRNA conjugates disclosed herein showed a high inhibition rate of FXI mRNA in mouse liver tissue on days 8, 15, and 29 at a dose of 3 mg / kg siRNA, with an inhibition rate reaching up to 82%, and the FXI mRNA inhibition rate did not show a decreasing trend during the experiment, suggesting that it may stably and efficiently inhibit FXI mRNA for a longer period. For example, the siRNA conjugate of this disclosure showed a high inhibition rate of ANGPTL3 mRNA in mouse liver tissue on days 8, 15 and 29 at a dose of 3 mg / kg siRNA, with an inhibition rate of up to 92%, and still showed an inhibition rate of at least 63% of ANGPTL3 mRNA on day 29.

[0032] This demonstrates that the double-stranded oligonucleotides and oligonucleotide conjugates provided in this disclosure can effectively regulate the expression of target genes in vivo and in vitro for extended periods, while exhibiting low toxicity. Therefore, they can effectively treat and / or prevent diseases and / or symptoms related to the mRNA level of target gene expression with significantly higher safety, showing promising application prospects. Attached Figure Description

[0033] The accompanying drawings are provided to further illustrate the present disclosure and form part of the specification. They are used together with the following detailed description to explain the present disclosure, but do not constitute a limitation thereof. In the drawings:

[0034] Figure 1 This is a line graph showing the relative expression levels of FXI mRNA in mouse liver tissue on days 8, 15, and 29 after administration of different siRNA conjugates at 3 mg / kg, compared to the blank control.

[0035] Figure 2 This is a line graph showing the relative expression levels of ANGPTL3 mRNA in mouse liver tissue on days 8, 15, and 29 after administration of different siRNA conjugates at 3 mg / kg, compared to the blank control. Detailed Implementation

[0036] The following provides a detailed description of specific embodiments of this disclosure. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit this disclosure.

[0037] Unless otherwise specified, the nomenclature, procedures, and techniques used in the analytical chemistry, synthetic organic chemistry, and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard calculations are applicable to chemical synthesis and chemical analysis.

[0038] definition

[0039] Unless otherwise specified, in the preceding and following text, uppercase letters C, G, U, T, and A represent the base composition of nucleotides; lowercase letter m indicates that the nucleotide adjacent to the left of letter m is a methoxy-modified nucleotide; lowercase letter f indicates that the nucleotide adjacent to the left of letter f is a fluorinated nucleotide; lowercase letter s indicates that the two nucleotides adjacent to the left and right of letter s are linked by thiophosphate groups; and the letter combination VP indicates that the nucleotide adjacent to the right of letter combination VP is a vinyl phosphate (5'-(E)-vinylphosphonate, E-VP) modified nucleotide.

[0040] In the preceding and following text, "fluorinated nucleotides" refers to nucleotides formed by replacing the 2'-hydroxyl group of the ribosome with fluorine, and "non-fluorinated nucleotides" refers to nucleotides or nucleotide analogs formed by replacing the 2'-hydroxyl group of the ribosome with a non-fluorinated group. "Nucleotide analogs" refer to groups that can replace nucleotides in nucleic acids but whose structure differs from adenine ribonucleotides, guanine ribonucleotides, cytosine ribonucleotides, uracil ribonucleotides, or thymine deoxyribonucleotides. Examples include isonucleotides, bridged nucleic acids (BNAs), or acyclic nucleotides. "Methoxylated nucleotides" refers to nucleotides formed by replacing the 2'-hydroxyl group of the ribosome with a methoxy group.

[0041] In the context of this document, the terms "complementary" and "reverse complementary" are used interchangeably and have the meaning known to those skilled in the art: in a double-stranded nucleic acid molecule, the bases of one strand are paired complementaryly with the bases of the other strand. In DNA, the purine base adenine (A) always pairs with the pyrimidine base thymine (T) (or uracil (U) in RNA); the purine base guanine (C) always pairs with the pyrimidine base cytosine (G). Each base pair consists of 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 two strands are considered complementary, and the sequence of the complementary strand can be inferred from its sequence. Correspondingly, "mismatch" in the art means, in a double-stranded nucleic acid, that the bases at corresponding positions are not paired complementaryly.

[0042] Unless otherwise specified above and below, "substantially anticomplementary" means that there are no more than 3 base mismatches between the two nucleotide sequences involved; "substantially anticomplementary" means that there are no more than 1 base mismatch between the two nucleotide sequences; and "completely anticomplementary" means that there are no base mismatches between the two nucleotide sequences.

[0043] In the foregoing and hereinafter, particularly in the description of methods for preparing double-stranded oligonucleotides, pharmaceutical compositions, or oligonucleotide conjugates of this disclosure, unless otherwise specified, the nucleoside monomer refers to the modified or unmodified RNA phosphoramidites (sometimes also called nucleoside phosphoramidites) used in phosphoramidite solid-phase synthesis, depending on the type and sequence of nucleotides in the desired double-stranded oligonucleotide or oligonucleotide conjugate. Phosphoramidite solid-phase synthesis is a method known to those skilled in the art for RNA synthesis. All nucleoside monomers used in this disclosure are commercially available.

[0044] Unless otherwise stated above or below, a "substituted" group refers to a group formed by replacing one or more hydrogen atoms in the group with a substituent. For example, "substituted C1-C5 hydrocarbon group" refers to a group formed by replacing one or more hydrogen atoms in a C1-C5 hydrocarbon group with a substituent. Those skilled in the art will understand that compounds applicable to this disclosure may contain various substituents, as long as the introduction of such substituents does not affect the function of this disclosure and achieves its purpose. In some embodiments, the substituents are selected from the group consisting of C1-C5 hydrocarbon groups. 10Alkyl, C6-C 10 Aryl, C5-C 10 heteroaryl, C1-C 10 Halogenated alkyl, -OC1-C 10 Alkyl, -OC1-C 10 Alkylphenyl, -C1-C 10 Alkyl-OH, -OC1-C 10 Halogenated alkyl, -SC1-C 10 Alkyl, -SC1-C 10 Alkylphenyl, -C1-C 10 Alkyl-SH, -SC1-C 10 Halogenated alkyl groups, halogenated substituents, -OH, -SH, -NH2, -C1-C 10 Alkyl-NH2,-N(C1-C) 10 Alkyl) (C1-C 10 alkyl), -NH(C1-C 10 Alkyl), -N(C1-C 10 Alkyl) (C1-C 10 alkylphenyl), -NH(C1-C 10 Alkylphenyl), cyano, nitro, -CO2H, -C(O)O(C1-C 10 Alkyl), -CON (C1-C) 10 Alkyl) (C1-C 10 Alkyl), -CONH (C1-C) 10 Alkyl group), -CONH2, -NHC(O) (C1-C 10 alkyl), -NHC(O)(phenyl), -N(C1-C 10 Alkyl)C(O)(C1-C 10 Alkyl), -N(C1-C 10 alkyl)C(O)(phenyl), -C(O)C1-C 10 Alkyl, -C(O)C1-C 10 Alkylphenyl, -C(O)C1-C 10 Haloalkyl, -OC(O)Cl-C 10 Alkyl group, -SO2 (C1-C) 10 alkyl), -SO2 (phenyl), -SO2 (C1-C 10 Halogenated alkyl groups), -SO2NH2, -SO2NH (C1-C 10 alkyl), -SO2NH (phenyl), -NHSO2 (C1-C 10 Alkyl), -NHSO2 (phenyl) and -NHSO2 (C1-C) 10(Haloalkyl). In some embodiments, the substituent is one of C1-C3 alkyl, C6-C8 aryl, -OC1-C3 alkyl, -OC1-C3 alkylphenyl, halogen substituent, cyano, or nitro. Those skilled in the art will understand that for any group containing one or more substituents, these groups are not intended to introduce any substitution or substitution pattern that is spatially impractical, synthetically infeasible, and / or inherently unstable.

[0045] As used herein, “halogen” refers to F, Cl, Br, or I. As used herein, “alkyl” refers to a straight-chain and branched saturated hydrocarbon group having a specified number of carbon atoms, typically 1 to 20 carbon atoms, such as 1 to 10 carbon atoms, or 1 to 8 or 1 to 3 carbon atoms. For example, C1-C6 alkyl groups comprise straight-chain and branched alkyl groups with 1 to 6 carbon atoms. When referring to residues having a specific number of alkyl groups, the intention is to encompass all branched and straight-chain forms having that number of carbon atoms; thus, for example, “butyl” means including n-butyl, sec-butyl, isobutyl, and tert-butyl; “propyl” includes n-propyl and isopropyl. Alkylenes are subsets of alkyl groups, referring to residues that are identical to alkyl groups but have two connection points.

[0046] 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 a hydrogen molecule from an adjacent carbon atom of the parent alkyl group. The group can be in either a cis or trans configuration of the double bond. Typical alkenyl groups include, but are not limited to: vinyl; propenyl, such as propyl-1-en-1-yl, propyl-1-en-2-yl, propyl-2-en-1-yl (allyl), propyl-2-en-2-yl; butenyl, such as buten-1-en-1-yl, buten-1-en-2-yl, 2-methylpropen-1-en-1-yl, buten-2-en-1-yl, buten-2-en-2-yl, buten-1,3-dien-1-yl, buten-1,3-dien-2-yl, etc. In some embodiments, the alkenyl group has 2 to 20 carbon atoms, while in other embodiments, it has 2 to 10, 2 to 8, or 2 to 6 carbon atoms. Cycloalkenyl groups are monocyclic hydrocarbon groups having a specific number of carbon atoms and at least one carbon-carbon double bond.

[0047] As used herein, "alkynyl" refers to an unsaturated branched or straight-chain alkyl group having at least one carbon-carbon triple bond obtained by removing two hydrogen molecules from adjacent carbon atoms of the parent alkyl group. Typical alkynyl groups include, but are not limited to: ethynyl; propynyl, such as prop-1-yn-1-yl, prop-2-yn-1-yl; butynyl, such as but-1-yn-1-yl, but-1-yn-3-yl, but-3-yn-1-yl, etc. In some embodiments, the alkynyl group has 2 to 20 carbon atoms, while in other embodiments, it has 2 to 10, 2 to 8, or 2 to 6 carbon atoms. A subset of alkynyl groups refers to residues that are identical to the alkynyl group but have two attachment sites.

[0048] As used herein, “cycloalkyl” refers to a saturated or unsaturated non-aromatic hydrocarbon cyclic group having 3 to 14 carbon atoms, unless otherwise specified. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, methylcyclopropyl, 2,2-dimethylcyclobutyl, 2-ethylcyclopentyl, or cyclohexyl. Cycloalkyl groups may comprise multiple spirocyclic or fused rings. Cycloalkyl groups may be optionally mono-, di-, tri-, tetra-, or penta-substituted at any position, subject to conventional valence conditions.

[0049] As used herein, "alkoxy" refers to an alkyl group with a specified number of carbon atoms attached by an oxygen bridge, such as methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, pentooxy, 2-pentoxy, isopentoxy, neopentoxy, hexoxy, 2-hexoxy, 3-hexoxy, 3-methylpentoxy, etc. Alkoxy groups typically have 1 to 10, 1 to 8, 1 to 6, or 1 to 4 carbon atoms attached by an oxygen bridge.

[0050] As used herein, "aryl" refers to a group derived from an aromatic monocyclic or polycyclic hydrocarbon ring system by removing a hydrogen atom from a ring carbon atom. This aromatic monocyclic or polycyclic hydrocarbon ring system contains only hydrogen and carbon atoms of 6 to 18, wherein at least one ring in the ring system is fully unsaturated, i.e., it comprises a cyclic, delocalized (4n+2)π-electron system according to Hückel's theory. Aryl groups include, but are not limited to, groups such as phenyl, fluorenyl, and naphthyl.

[0051] "Heteroaryl" refers to a group derived from a 3- to 18-membered aromatic ring radical, comprising 2 to 17 carbon atoms and 1 to 6 heteroatoms selected from nitrogen, oxygen, and sulfur. As used herein, a heteroaryl can be a monocyclic, bicyclic, tricyclic, or tetracyclic system, wherein at least one ring in the ring system is fully unsaturated, i.e., it comprises a cyclic delocalized (4n+2) π-electron system according to Hückel's theory. Heteroaryls include fused ring or bridged ring systems. Alternatively, the heteroatoms in a heteroaryl are oxidized heteroatoms. Alternatively, one or more nitrogen atoms (if present) are quaternized. The heteroaryl is attached to the rest of the molecule via any ring atom. Examples of heteroaryl groups include, but are not limited to: aziridinetrienyl, acridine, benzimidazolyl, benzoindolyl, 1,3-benzodioxazolyl, benzofuranyl, benzooxazolyl, benzo[d]thiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxepinyl, benzo[b][1,4]oxazinyl, 1,4-benzodioxanyl, benzonaphthofuranyl, and benzooxazolyl. Benzadioxolyl, benzodioxinyl, benzopyranyl, benzopyranone, benzofuranyl, benzofuranone, benzothiophenyl, benzothiophene[3,2-d]pyrimidinyl, benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridyl, carbazole, cinnolinyl, cyclopentano[d]pyrimidinyl, 6,7-dihydro-5H-cyclopentano[4,5]thiophene[2,3-d]pyrimidinyl, 5,6-dihydrobenzo[ [h]quinazolinyl, 5,6-dihydrobenzo[h]quinazolinyl, 5,6-dihydrobenzo[h]cinnolinyl, 6,7-dihydro-5H-benzo[6,7]cycloheptano[1,2-c]pyridazinyl, dibenzofuranyl, dibenzothiophenyl, furanyl, furanone, furano[3,2-c]pyridyl, 5,6,7,8,9,10-hexahydrocyclooctano[d]pyrimidinyl, 5,6,7,8,9,10-hexahydrocyclooctano[d]pyrimidinyl, 5,6,7,8,9,10-hexahydrocyclooctano[d]pyrimidinyl Alkyl[d]pyridazinyl, 5,6,7,8,9,10-hexahydrocyclooctyl[d]pyridinyl, isothiazolyl, imidazolyl, indazolyl, indole, isoyindolyl, dihydroindolyl, isodihydroindolyl, isoquinolinyl, indolizinyl, isoxazolyl, 5,8-methano-5,6,7,8-tetrahydroquinazolinyl, naphthyridinyl, 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, phenotoxazinyl, phthalazinyl, pteridinyl, purine, pyrrolyl, pyrazolyl, pyrazolo[3,4-d]pyrimidinyl, pyridinyl, pyrido[3,2-d]pyrimidinyl, pyrido[3,4-d]pyrimidinyl, pyrazinyl, pyridinyl, pyridazinyl, quinazolinyl, Quinoxalinyl, quinolinyl, tetrahydroquinolinyl, 5,6,7,8-tetrahydroquinazolinyl, 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidinyl, 6,7,8,9-tetrahydro-5H-cycloheptane[4,5]thieno[2,3-d]pyrimidinyl, 5,6,7,8-tetrahydropyridin[4,5-c]pyridazinyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl, triazinyl, thieno[2,3-d]pyrimidinyl, thieno[3,2-d]pyrimidinyl, thieno[2,3-c]pyridinyl, and thiophenyl / thienyl.

[0052] A "heterocyclic group" refers to a stable 3- to 18-membered non-aromatic cyclic group containing 2-12 carbon atoms and 1-6 heteroatoms selected from nitrogen, oxygen, and sulfur. Unless otherwise specified in the specification, a heterocyclic group is a monocyclic, bicyclic, tricyclic, or tetracyclic system, and may include fused or bridged ring systems. Alternatively, the heteroatoms in the heterocyclic group are oxidized heteroatoms. Alternatively, one or more nitrogen atoms (if present) are quaternized. The heterocyclic group is partially or fully saturated. The heterocyclic group can be attached to the rest of the molecule via any ring atom. Examples of such heterocyclic groups include, but are not limited to: dioxane, thienyl[1,3]dithianyl, decahydroisoquinolinyl, imidazolinyl, imidazoalkyl, isothiazolyl, isoxazolyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxaperazinyl, 2-oxaperidinyl, 2-oxaperidinyl, 2-oxaperpyrrolyl, oxazolyl, piperidinyl, piperazine, 4-piperidinoneyl, pyrrolyl, pyrazolyl, quininecycloyl, thiazoalkyl, tetrahydrofuranyl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl, and 1,1-dioxo-thiomorpholinyl.

[0053] As used herein, "heterocyclic base" refers to a nucleic acid base or a modified nucleic acid base. In some embodiments, the heterocyclic base is a pyrimidine, a substituted pyrimidine, a purine, or a substituted purine. In some embodiments, the heterocyclic base is a naturally occurring purine. In some embodiments, the heterocyclic base is a non-naturally occurring purine or a substituted purine. In some embodiments, the heterocyclic base is a naturally occurring pyrimidine. In some embodiments, the heterocyclic base is a non-naturally occurring pyrimidine or a substituted pyrimidine.

[0054] Various protecting groups, such as hydroxyl protecting groups, may be used in this disclosure. Generally, protecting groups insensitize chemical functional groups to specific reaction conditions and can be added to and removed from the functional group in a molecule without substantially impairing the rest of the molecule. Representative hydroxyl 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, all of which are incorporated herein by reference in their entirety. 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 hydroxyl protecting groups that may be used herein include dimethoxytriphenylmethyl (DMT), monomethoxytriphenylmethyl, 9-phenyloxanthracene-9-yl (Pixyl), and 9-(p-methoxyphenyl)oxanthracene-9-yl (Mox). In some embodiments, non-exclusive examples of hydroxyl protecting groups that may be used herein include Tr (triphenylmethyl), MMTr (4-methoxytriphenylmethyl), DMTr (4,4'-dimethoxytriphenylmethyl), and TMTr (4,4',4”-trimethoxytriphenylmethyl).

[0055] The term “subject,” as used herein, refers to any animal, such as a mammal or marsupial. Subjects in this disclosure include, but are not limited to, humans, non-human primates (e.g., rhesus monkeys or other types of macaques), mice, pigs, horses, donkeys, cattle, rabbits, sheep, rats, and any kind of poultry. As used herein, “treatment” refers to a method of obtaining a beneficial or desired outcome, including but not limited to therapeutic benefits. A “therapeutic benefit” means the eradication or improvement of the underlying disorder being treated. Furthermore, a therapeutic benefit is obtained by eradicating or improving one or more physiological symptoms associated with the underlying disorder, thereby observing improvement in the subject, although the subject may still suffer from the underlying disorder.

[0056] As used herein, “prevention” refers to methods for obtaining a beneficial or desired outcome, including but not limited to preventive benefits. To obtain a “preventive benefit,” a double-stranded oligonucleotide, pharmaceutical composition, or oligonucleotide conjugate may be given to a subject at risk of developing a specific disease, or to a subject who reports one or more physiological symptoms of a disease, even if a diagnosis of the disease may not have been made.

[0057] Double-stranded oligonucleotides

[0058] In a first aspect, this disclosure provides a double-stranded oligonucleotide comprising a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, wherein each nucleotide in the double-stranded oligonucleotide is independently modified or unmodified, portions of the sense strand and the antisense strand are anticomplementary to form a double-stranded region, wherein the 5' terminal nucleotide of the antisense strand is a nucleotide with a ribose 5' modification having the structure shown in formula (101), and a group R0 constitutes the 5' overhang of the antisense strand, R0 having the structure shown in formula (102):

[0059]

[0060] in:

[0061] R 201 It can be a hydroxyl or phosphate group;

[0062] G1 is OH, O - Or OJ1, wherein J1 is a C1-C6 alkyl, a substituted C1-C6 alkyl, a C3-C6 cycloalkyl, or a substituted C3-C6 cycloalkyl;

[0063] Bx1 is a hydrogen, a heterocyclic base, or a base substitution group, and if the 3' end of the positive chain includes a protruding end, Bx1 does not pair with the base at that protruding end, wherein the base substitution group is a phenyl or a substituted phenyl group; Bx2 is a heterocyclic base;

[0064] Z is a group having one of the divalent linking groups represented by the structures shown in formulas (Z1) to (Z5), or Z is a 1,2-cycloalkylene or heterocyclic group having 3-6 carbon atoms, or a substituted 1,2-cycloalkylene or heterocyclic group having 3-6 carbon atoms:

[0065]

[0066]

[0067] P1 and P2 are each independently H, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C2-C6 alkenyl or substituted C2-C6 alkenyl.

[0068] P3 is either O or S;

[0069] X1 is H or hydroxyl, and X2 is selected from H, halogen, hydroxyl, C1-C6 alkoxy or substituted C1-C6 alkoxy.

[0070] T2 is a phosphate ester subunit or a thiophosphate ester subunit; Y1, Y2, Y3, Y4, Y5, Y6, Y7 and Y8 are each independently H, halogen, hydroxyl, methyl, ethyl, n-propyl or isopropyl;

[0071] This indicates the site where the group is covalently linked.

[0072] Those skilled in the art will understand that Bx1 is a hydrogen, a heterocyclic base, or a base substitution group, and if the 3' end of the positive chain includes a protruding end, Bx1 does not pair with the base at that protruding end, wherein the base substitution group is a phenyl or a substituted phenyl group. x2 The use of heterocyclic bases does not alter the properties of the siRNA disclosed herein, and the objectives of the invention can still be achieved. In the context of this disclosure, a heterocyclic base refers to a nucleobase or a modified nucleobase. In some embodiments, the heterocyclic base is a pyrimidine, a substituted pyrimidine, a purine, or a substituted purine. For ease of synthesis, in some embodiments, the heterocyclic base is uracil, thymine, cytosine, 5-methylcytosine, adenine, or guanine.

[0073] In some embodiments, the base substitution group is a phenyl or substituted phenyl group, wherein the substituted phenyl group refers to a phenyl group in which one or more hydrogen atoms on the phenyl group are each independently replaced by F, Cl, CH3, CH2F or CF3; in some embodiments, the substituted phenyl group refers to a group formed by replacing one or more hydrogen atoms on the phenyl group with F, Cl or CH3.

[0074] In some embodiments, Z is a divalent linker represented by any one of formulas (Z1) to (Z5) capable of linking two nucleotide molecules in formula (101). In some embodiments, Z has the structure shown in formula (Z1) or (Z2), and P1 and P2 in formulas (Z1) and (Z2) are each independently H.

[0075] According to the present invention, Z can also be a 1,2-cycloalkylene or heterocyclic group having 3-6 carbon atoms, or a substituted 1,2-cycloalkylene or heterocyclic group having 3-6 carbon atoms. This structure does not change the properties of the double-stranded oligonucleotide of the present invention and can also achieve the inventive purpose of the present invention. Considering factors such as ease of synthesis, structural / process cost, etc., in some embodiments, Z is 1,2-cyclopropylene.

[0076] According to the present invention, the selection of X1 and X2 may affect the structure and properties of the double-stranded oligonucleotide. In some embodiments, X1 is independently selected from H or hydroxyl groups, and X2 is selected from hydroxyl groups or OCH3. In some embodiments, X1 is H and X2 is OCH3.

[0077] For ease of synthesis, in some embodiments, Y1, Y2, Y3, Y4, Y5, Y6, Y7 and Y8 are all H.

[0078] In some implementations, formula (101) is selected from one of the following structures:

[0079]

[0080]

[0081]

[0082] Wherein, Base refers to C, G, U, T or A. In the antisense strand, the 5' end of nucleotide sequence II contains a ribose 5' modified nucleotide as shown in one of formulas (501) to (507), thereby the double-stranded oligonucleotide with the overhang of the antisense strand 5' end can have a good balance of further target gene expression regulation activity and low toxicity.

[0083] As previously stated, each nucleotide in the double-stranded oligonucleotides disclosed herein is either modified or unmodified. In the context of this disclosure, the term "modified nucleotide" refers to a nucleotide or nucleotide analog formed by replacing the 2' hydroxyl group of the ribosyl group with another group, or a nucleotide whose bases are modified. The modified nucleotides do not result in a significant reduction or loss of the function of the double-stranded oligonucleotide in regulating gene expression. For example, the modified nucleotides disclosed in JKWatts, GGFDeleavey, and MJDamha, Chemically modified siRNA: tools and applications. Drug Discov Today, 2008, 13(19-20):842-55 may be selected.

[0084] In some embodiments, the double-stranded oligonucleotide comprises a sense strand and an antisense strand, wherein the sense strand comprises nucleotide sequence I, the antisense strand comprises nucleotide sequence II, the 5' terminal nucleotide of nucleotide sequence II is a ribose 5' modified nucleotide having the structure shown in formula (101); nucleotide sequence I consists of 19 nucleotides, with the group shown in formula (101) counted as 2 nucleotides, nucleotide sequence II consists of 20 nucleotides, the group R0 constitutes the overhang at the 5' end of the antisense strand, and the remaining portion of nucleotide sequence II forms a double-stranded region with nucleotide sequence I, nucleotide sequence II is at least partially anticomplementary to a first nucleotide sequence, the first nucleotide sequence being a nucleotide sequence of 19 nucleotides in length in the mRNA of the target gene expression; nucleotides 7-9 of nucleotide sequence I are fluorinated nucleotides in the direction from 5' end to 3' end; nucleotides 3, 7, 15, and 17 of nucleotide sequence II are fluorinated nucleotides in the direction from 5' end to 3' end.

[0085] In some embodiments, nucleotides 7-9 of nucleotide sequence I, in the direction from 5' end to 3' end, are fluorinated nucleotides, and each nucleotide at other positions of nucleotide sequence I is independently one of non-fluorinated nucleotides; nucleotides 3, 7, 15, and 17 of nucleotide sequence II, in the direction from 5' end to 3' end, are fluorinated nucleotides, and each nucleotide at other positions of nucleotide sequence II is independently one of non-fluorinated nucleotides.

[0086] In some embodiments, the 2nd to 20th nucleotides of the nucleotide sequence II are substantially anticomplementary, substantially anticomplementary, or completely anticomplementary to the first nucleotide sequence in the direction from the 5' end to the 3' end.

[0087] In some embodiments, at least the nucleotides at positions 3-20 of the nucleotide sequence II are completely reverse complementary to the nucleotides at positions 1-18 of the first nucleotide sequence, in the direction from the 5' end to the 3' end.

[0088] In some embodiments, the 2nd to 20th nucleotides of nucleotide sequence II are substantially anticomplementary, substantially anticomplementary, or completely anticomplementary to nucleotide sequence I, in the direction from the 5' end to the 3' end.

[0089] In some embodiments, the 2nd to 20th nucleotides of nucleotide sequence II are completely anticomplementary to nucleotide sequence I in the 5' to 3' direction, or there is a base mismatch between the 3rd nucleotide of nucleotide sequence II and the 2nd nucleotide of nucleotide sequence I in the 3' to 5' direction.

[0090] In some embodiments, the sense strand further contains nucleotide sequence III, and the antisense strand further contains nucleotide sequence IV. Each nucleotide of nucleotide sequence III and nucleotide sequence IV is independently one of non-fluorinated modified nucleotides. The length of nucleotide sequence III is 1, 2, 3, or 4 nucleotides. The length of nucleotide sequence IV is equal to that of nucleotide sequence III, and nucleotide sequences IV and III are substantially anticomplementary or completely anticomplementary. Nucleotide sequence III is attached to the 5' end of nucleotide sequence I, and nucleotide sequence IV is attached to the 3' end of nucleotide sequence II. Nucleotide sequence IV is substantially anticomplementary or completely anticomplementary to a second nucleotide sequence, which is a nucleotide sequence in the mRNA expressed by the target gene that is adjacent to the first nucleotide sequence and has the same length as nucleotide sequence IV. Thus, the double-stranded oligonucleotide of this disclosure can have a double-stranded complementary region of 19-23 nucleotides in length.

[0091] In some embodiments, the double-stranded oligonucleotide further comprises a nucleotide sequence V, each nucleotide of which is independently one of a non-fluorinated modified nucleotide, the length of which is 1 to 3 nucleotides, and is attached to the 3' end of the antisense strand to form a 3' overhang of the antisense strand. Thus, the length ratio of the sense strand to the antisense strand of the double-stranded oligonucleotide provided in this disclosure can 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.

[0092] In some embodiments, the nucleotide sequence V is 2 nucleotides in length and, from the 5' end to the 3' end, consists of two consecutive thymine deoxyribonucleotides, two consecutive uracil ribonucleotides, or is completely anticomplementary to the third nucleotide sequence. The third nucleotide sequence refers to a nucleotide sequence in the mRNA expressing the target gene that is adjacent to the 5' end of the first or second nucleotide sequence and has the same length as the nucleotide sequence V. Therefore, in some embodiments, the sense and antisense strands of the double-stranded oligonucleotide of this disclosure each have a length of 19 / 21 or 21 / 23 nucleotides, in which case the double-stranded oligonucleotide of this disclosure has better target gene expression regulatory activity.

[0093] In some embodiments, each non-fluorinated modified nucleotide is independently selected from one of the nucleotides or nucleotide analogs formed by replacing the hydroxyl group at the 2' position of the ribosyl group of the nucleotide with a non-fluorinated group.

[0094] In some embodiments, each of the non-fluorinated nucleotides is a methoxylated nucleotide, meaning a nucleotide formed by replacing the 2'-hydroxyl group of the ribosyl group with a methoxy group. In the context of this disclosure, a "fluorinated nucleotide" refers to a nucleotide formed by replacing the 2'-hydroxyl group of the ribosyl group with a fluorine group, having the structure shown in formula (7). A "non-fluorinated nucleotide" refers to a nucleotide or nucleotide analog formed by replacing the 2'-hydroxyl group of the ribosyl group with a non-fluorinated group. In some embodiments, each non-fluorinated nucleotide is independently selected from one of the nucleotides or nucleotide analogs formed by replacing the 2'-hydroxyl group of the ribosyl group with a non-fluorinated group.

[0095] The nucleotides formed by replacing the hydroxyl group at the 2' position of the ribosyl group with a non-fluorinated group are well known to those skilled in the art, and these nucleotides may be selected from one of the following: 2'-alkoxy modified nucleotides, 2'-alkyl modified nucleotides, 2'-substituted alkyl modified nucleotides, 2'-amino modified nucleotides, 2'-substituted amino modified nucleotides, and 2'-deoxynucleotides.

[0096] In some embodiments, the 2'-alkoxy-modified nucleotide is a methoxy-modified nucleotide (2'-OMe), as shown in formula (8). In some embodiments, the 2'-amino-modified nucleotide (2'-NH2) is shown in formula (9). In some embodiments, the 2'-deoxynucleotide (DNA) is shown in formula (10).

[0097]

[0098] Nucleotide analogs are groups that can replace nucleotides in nucleic acids, but whose structure differs from that of adenine ribonucleotides, guanine ribonucleotides, cytosine ribonucleotides, uracil ribonucleotides, or thymine deoxyribonucleotides. In some embodiments, nucleotide analogs can be isonucleotides, bridged nucleic acids (BNAs), or acyclic nucleotides.

[0099] BNA refers to a restricted or inaccessible nucleotide. BNA can contain a bridging structure with a "fixed" C3'-endoglucan condensation, consisting of a five-membered, six-membered, or seven-membered ring. This bridge is typically incorporated into the 2'-, 4'-position of the ribose to provide a 2', 4'-BNA nucleotide. In some embodiments, BNA can be LNA, ENA, cET BNA, etc., where LNA is shown in formula (12), ENA in formula (13), and cET BNA in formula (14).

[0100]

[0101] Acyclic nucleotides are a class of nucleotides formed by opening the sugar ring of a nucleotide. In some embodiments, acyclic nucleotides can be unblocking nucleic acids (UNA) or glycerol nucleic acids (GNA), wherein UNA is shown in formula (15) and GNA is shown in formula (16):

[0102]

[0103] In formulas (15) and (16) above, R is selected from H, OH or alkoxy (O-alkyl).

[0104] Isonucleotides are compounds formed by altering the position of a base on the ribose ring in a nucleotide. In some embodiments, an isonucleotide can be a compound formed by moving a base from the 1'-position to the 2'-position or 3'-position on the ribose ring, as shown in formula (17) or (18).

[0105] In the compounds of formulas (17)-(18) above, Base represents a nucleic acid base, such as A, U, G, C or T; R is selected from H, OH, F or non-fluorine groups as described above.

[0106]

[0107]

[0108] In some embodiments, the nucleotide analogue is selected from one of the following: isonucleotides, LNA, ENA, cET, UNA, and GNA. In some embodiments, each non-fluorinated nucleotide is a methoxylated nucleotide, and in the preceding and following text, the methoxylated nucleotide refers to a nucleotide formed by replacing the 2'-hydroxyl group of the ribosome with a methoxy group.

[0109] In the preceding and following text, “fluorinated nucleotides,” “2’-fluorinated nucleotides,” “nucleotides in which the 2’-hydroxyl group of the ribose group is replaced by fluorine,” and “nucleotides with a 2’-fluorinated ribose group” have the same meaning, all referring to compounds in which the 2’-hydroxyl group of the nucleotide is replaced by fluorine, resulting in compounds with the structure shown in formula (7); “methoxylated nucleotides,” “2’-methoxylated nucleotides,” “nucleotides in which the 2’-hydroxyl group of the ribose group is replaced by methoxyl,” and “nucleotides with a 2’-methoxy ribose group” have the same meaning, all referring to compounds in which the 2’-hydroxyl group of the ribose group of the nucleotide is replaced by methoxyl, resulting in compounds with the structure shown in formula (8).

[0110] The modified double-stranded oligonucleotides are not only low in cost, but also less susceptible to cleavage by ribonucleases in the blood, thereby increasing their stability and making them more resistant to nuclease hydrolysis. Simultaneously, these modified double-stranded oligonucleotides exhibit higher activity in regulating target gene expression.

[0111] In some embodiments, at least one of the phosphate ester groups in the phosphate-sugar backbone of at least one single chain of the sense chain and the antisense chain is a phosphate ester group with a modifying group. In some embodiments, the phosphate ester group with the modifying group is a thiophosphate ester group formed by replacing at least one oxygen atom in the phosphodiester bond of the phosphate ester group with a sulfur atom; in some embodiments, the phosphate ester group with the modifying group is a thiophosphate ester group having the structure shown in formula (121):

[0112]

[0113] This modification can stabilize the double-stranded structure of double-stranded oligonucleotides, maintaining high specificity and high affinity of base pairing.

[0114] In some embodiments, in the double-stranded oligonucleotide, the phosphate ester group having the modifying group is present at at least one of the following positions:

[0115] Between the first and second nucleotides at the 5' end of the positive strand;

[0116] Between the second and third nucleotides at the 5' end of the positive strand;

[0117] Between the first and second nucleotides at the 3' end of the positive strand;

[0118] Between the second and third nucleotides at the 3' end of the positive strand;

[0119] Between the third and fourth nucleotides at the 5' end of the antisense strand;

[0120] Between the first and second nucleotides at the 3' end of the antisense strand; and

[0121] Between the second and third nucleotides at the 3' end of the antisense strand.

[0122] The double-stranded oligonucleotides disclosed herein can be various double-stranded oligonucleotides that regulate gene expression. In some embodiments, they can be double-stranded oligonucleotides that inhibit or downregulate gene expression, such as siRNA; in other embodiments, they can be double-stranded oligonucleotides that activate or upregulate gene expression, such as saRNA.

[0123] The double-stranded oligonucleotides disclosed herein exhibited excellent target gene expression regulatory activity while unexpectedly reducing toxicity.

[0124] The modified double-stranded oligonucleotides, pharmaceutical compositions, and oligonucleotide conjugates disclosed herein can be used to regulate various abnormal gene expression and treat various pathological conditions or diseases caused by abnormal gene expression. Alternatively, the double-stranded oligonucleotides, pharmaceutical compositions, and nucleotide conjugates disclosed herein can be used to treat or improve pathological conditions or diseases related to gene expression by regulating gene expression. These genes can be various endogenous genes in the human or animal body, or pathogen genes that proliferate in the human or animal body. Double-stranded oligonucleotides with specific nucleotide sequences and the modification schemes described herein can be designed and prepared based on the mRNA expressing the target gene. In some embodiments, the mRNA expressed by the target gene is selected from the mRNA transcribed from the following genes: ACE2, ANGPTL3, ApoA, ApoB, ApoC, AR, ASK1, C5, Col1A1, CTGF, Ebola, FOXO1, FTO, FVII, FXI, FXII, GCGR, HBV, HCV, HSD17B13, p53, PCSK9, PNP, PLG, PKK, KNG, SARS-CoV-2, SCD1, SCNN1A, SOD1, STAT3, TIMP-1, TMPRSS6, XO, INSR, SREBF1, HDV, RPTOR, TLK2, LPA, C3, AGT.

[0125] In some embodiments, the double-stranded oligonucleotide is siRNA, and the mRNA expressing the target gene is selected from the mRNA expressed by the hepatitis B virus (HBV) gene. In some embodiments, the double-stranded oligonucleotide is siRNA, and the mRNA expressing the target gene is selected from the mRNA expressed by plasma coagulation factor XI (FXI). In some embodiments, the double-stranded oligonucleotide is siRNA, and the mRNA expressing the target gene is selected from the mRNA expressed by angiopoietin-like protein 3 (ANGPTL3).

[0126] In some embodiments, the siRNA has the siRNA sequence shown in Table 1 below. Specifically, for siRNAs 1-7, the mRNA expressing the target gene is selected from the mRNA expressing the hepatitis B virus gene (HBV); for siRNAs 8-14, the mRNA expressing the target gene is selected from the mRNA expressing plasma coagulation factor XI (FXI); and for siRNAs 15-21, the mRNA expressing the target gene is selected from the mRNA expressing angiopoietin-like protein 3 (ANGPTL3).

[0127] Table 1 siRNA sequences

[0128]

[0129]

[0130] In this context, uppercase letters C, G, U, and A represent the base composition of a nucleotide; lowercase letter m indicates that the nucleotide adjacent to the left of m is methoxylated; lowercase letter f indicates that the nucleotide adjacent to the left of f is fluorinated; and lowercase letter s indicates that the two nucleotides to its left and right are linked by a phosphate thioester group. (The last part, "siRNA1-7," appears to be a typographical error and is left untranslated.) VPD These refer to the structures shown in formulas (501) to (507), respectively, in siRNA8-14. VPD These refer to the structures shown in formulas (501) to (507), respectively, in siRNA15-21. VPD These refer to the structures shown in equations (501) to (507) respectively.

[0131] The method for preparing double-stranded oligonucleotides disclosed herein

[0132] The method for preparing double-stranded oligonucleotides disclosed herein includes synthesizing the sense and antisense strands of a double-stranded oligonucleotide separately using a solid-phase phosphoramide method according to the desired double-stranded oligonucleotide sequence, and annealing the sense and antisense strands to form an oligonucleotide duplex. The difference lies in that, when linking the last nucleotide at the 5' end of the antisense strand, a 5' ribose-modified phosphoramide monomer is used as the nucleoside phosphoramide monomer for linking, followed by a sulfidation reaction. The 5' ribose-modified phosphoramide monomer is described later. In some embodiments, the preparation method further includes separating and purifying the double-stranded oligonucleotide.

[0133] In some embodiments, when the double-stranded oligonucleotide is siRNA, the method for preparing the siRNA disclosed herein includes:

[0134] (1) The positive strand of siRNA was synthesized by phosphoramide solid-phase synthesis method according to the 3'-5' direction;

[0135] (2) The antisense strand of siRNA was synthesized by phosphoramide solid-phase synthesis method according to the 3'-5' direction;

[0136] (3) The sense and antisense strands of the siRNA are separated and annealed to obtain the siRNA described in this disclosure.

[0137] It will be readily understood by those skilled in the art that the reaction conditions for solid-phase synthesis described in steps (1) and (2), including nucleoside monomer deprotection conditions, the type and amount of deprotection reagent, coupling reaction conditions, the type and amount of coupling reagent, capping reaction conditions, the type and amount of capping reagent, oxidation reaction conditions, the type and amount of oxidizing reagent, and sulfidation reaction conditions, the type and amount of sulfidation reagent, can be any reasonable process route and method conditions, reagent type, and amount. For example, in some embodiments, the solid-phase synthesis described in steps (1) and (2) can use the following conditions:

[0138] The deprotection conditions for nucleoside monomers include a temperature of 0-50°C, and in some embodiments 15-35°C; a reaction time of 30-300 seconds, and in some embodiments 50-150 seconds; and a deprotection reagent selected from one or more of trifluoroacetic acid, trichloroacetic acid, dichloroacetic acid, and monochloroacetic acid, and in some embodiments dichloroacetic acid. The molar ratio of the deprotection reagent to the 4,4'-dimethoxytriphenylmethyl protecting group on the solid support can be 2:1-100:1, and in some embodiments 3:1-50:1.

[0139] The coupling reaction conditions include a temperature of 0-50℃, and in some embodiments 15-35℃; the molar ratio of the nucleic acid sequence to the nucleoside monomer linked on the solid-phase support can be 1:1-1:50, and in some embodiments 1:5-1:15; the molar ratio of the nucleic acid sequence to the coupling reagent linked on the solid-phase support can be 1:1-1:100, and in some embodiments 1:50-1:80; and the reaction time and the selection of the coupling reagent are the same as described above.

[0140] The capping reaction conditions include a temperature of 0-50°C, and in some embodiments 15-35°C; a reaction time of 5-500 seconds, and in some embodiments 10-100 seconds; and the selection of the capping reagent is the same as described above. The molar ratio of the total amount of capping reagent to the nucleic acid sequence linked on the solid-phase support can be 1:100-100:1, and in some embodiments 1:10-10:1. When using equimolar amounts of acetic anhydride and N-methylimidazole as capping reagents, the molar ratio of acetic anhydride, N-methylimidazole, and the nucleic acid sequence linked on the solid-phase support can be 1:1:10-10:10:1, and in some embodiments 1:1:2-2:2:1.

[0141] The oxidation reaction conditions include a temperature of 0-50°C, in some embodiments 15-35°C, a reaction time of 1-100 seconds, in some embodiments 5-50 seconds, and an oxidizing agent of iodine in some embodiments (provided in the form of iodine solution in a further embodiment). The molar ratio of the oxidizing agent to the nucleic acid sequence linked on the solid-phase support in the coupling step can be 1:1-100:1, in some embodiments 5:1-50:1. In some embodiments, the oxidation reaction is carried out in a mixed solvent of tetrahydrofuran:water:pyridine = 3:1:1-1:1:3. The sulfidation reaction conditions include a temperature of 0-50°C, in some embodiments 15-35°C, a reaction time of 50-2000 seconds, in some embodiments 100-1000 seconds, and a sulfidation agent of hydroflavin in some embodiments. The molar ratio of the sulfidation agent to the nucleic acid sequence linked on the solid-phase support in the coupling step can be 10:1–1000:1, in some embodiments 10:1-500:1. In some embodiments, the sulfidation reaction is carried out in a mixed solvent of acetonitrile:pyridine = 1:3-3:1.

[0142] The annealing method in step (3) is also well known to those skilled in the art. For example, the synthesized sense and antisense strands can be simply mixed in equimolar ratio in water for injection and heated to 70-95°C, followed by cooling to room temperature, to allow them to form a double-stranded structure through hydrogen bonds. This yields the siRNA disclosed herein.

[0143] During the synthesis process, the purity and molecular weight of the nucleic acid sequence can be detected at any time, thereby better controlling the synthesis quality. The detection methods are well known to those skilled in the art. For example, the purity of nucleic acid can be detected by ion exchange chromatography, and the molecular weight can be determined by liquid chromatography-mass spectrometry (LC-MS).

[0144] After obtaining the siRNA disclosed herein, in some embodiments, the synthesized siRNA can be characterized by methods such as liquid chromatography-mass spectrometry (LC-MS) and molecular weight detection to determine that the synthesized siRNA is the target-designed siRNA, for example, matching one of the sequences listed in Table 1 above.

[0145] The aforementioned 5' modified phosphorus amide monomer has the structure shown in formula (301):

[0146]

[0147] The definitions and selection ranges of X1, X2, Z, Bx1, Bx2, Y1, Y2, Y3, Y4, Y5, Y6, Y7, and Y8 are as described above. In some embodiments, the Bx1 group and Bx2 group in the compound shown in formula (301) are the active functional groups (e.g., amino groups) of the Bx1 group and Bx2 group in the group shown in formula (101), and if present, all of them are protected by a protecting group. When Bx1 is a heterocyclic base, the commonly used protecting groups for active functional groups in nucleoside heterocyclic bases are known to those skilled in the art and can be used in the double-stranded oligonucleotides of this disclosure. In some embodiments, the X1 group and X2 group in the compound shown in formula (301) are the active functional groups (e.g., hydroxyl groups) of the X1 group and X2 group in the group shown in formula (101), and if present, all of them are protected by a protecting group. In some embodiments, the X1 group in formula (101) is a hydroxyl group. In this case, X1 in the compound of formula (301) is a protected hydroxyl group, such as a hydroxyl group protected by a silane protecting group (such as TBDMS or TBDPS). These protecting groups can be removed during the subsequent synthesis of the double-stranded oligonucleotide, releasing the active functional group, thereby enabling the prepared double-stranded oligonucleotide to have target sequence expression regulatory activity.

[0148] R k To provide R 201 The group. In some embodiments, R k It is a group R 201 A group formed where all active groups are protected by protecting groups. In some embodiments, R 201 For hydroxyl group, R kThe hydroxyl protecting group is a hydroxyl protecting group. The hydroxyl protecting group can be one or more of Tr (triphenylmethyl), MMTr (4-methoxytriphenylmethyl), DMTr (4,4'-bismethoxytriphenylmethyl), and TMTr (4,4',4'-trimethoxybenzyl). In some embodiments, R... k It could be DMTr, which stands for 4,4'-dimethoxytrityl.

[0149] Each B1 is independently selected from substituted or unsubstituted C1-C5 hydrocarbon groups; each B2 is independently selected from one of C1-C5 alkyl, cyanoethyl, cyanopropyl, and cyanobutyl groups. In some embodiments, each B1 is isopropyl or tert-butyl, and each B2 is 2-cyanoethyl or 3-cyanopropyl.

[0150] The compound shown in formula (301) is commercially available, or, if any reasonable synthetic route is used by a person skilled in the art, the compound shown in formula (301) can be prepared.

[0151] For example, the compound of formula (301) can be obtained by a method comprising the following steps: in an organic solvent, under condensation reaction conditions and in the presence of a condensation reaction accelerant, contacting the compound of formula (302) with the compound of formula (303) to separate the compound of formula (301):

[0152]

[0153] Among them, R k The definitions and selectable ranges of X1, X2, Z, Bx1, Bx2, Y1, Y2, Y3, Y4, Y5, Y6, Y7, Y8, B1, and B2 are as described above.

[0154] B3 is N(B1)2 or a halogen. In some embodiments, B3 is diisopropylamino (N(iPr)2) or chlorine.

[0155] The condensation reaction conditions include a reaction temperature of 0-50°C and a reaction time of 0.5-5 hours. In some embodiments, the reaction temperature is 15-35°C and the reaction time is 1-3 hours. The reaction pressure can be atmospheric pressure.

[0156] The organic solvent is one or more selected from epoxy solvents, ether solvents, haloalkane 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 diethyl ether and / or methyl tert-butyl ether, and the haloalkane solvent is one or more selected from dichloromethane, trichloromethane, and 1,2-dichloroethane. In some embodiments, the organic solvent is N,N-diisopropylethylamine. The amount of the organic solvent used relative to the compound shown in formula (302) is 1-50 L / mol, and in some embodiments it is 3-20 L / mol.

[0157] The condensation reaction accelerator can be any substance capable of promoting the condensation reaction between the compound shown in formula (302) and the compound shown in formula (303) to generate the compound shown in formula (301). In some embodiments, B3 is diisopropylamino, and the condensation reaction accelerator is a mixture of N-methylimidazolium and tetrazolium. In some embodiments, B3 is chlorine, and the reaction accelerator is N,N-diisopropylethylamine. The molar ratio of the total amount of the condensation reaction accelerator to the compound of formula (302) is 1:1 to 10:1, and in some embodiments, it is 1:1 to 5:1.

[0158] The molar ratio of the compound of formula (303) to the compound of formula (302) is 1:1 to 10:1, and in some embodiments, it is 1:1 to 5:1. The compound of formula (303) is commercially available or can be synthesized by those skilled in the art using known methods. In some embodiments, the compound of formula (303) is a readily available commercially available bis(diisopropylamino)(2-cyanoethoxy)phosphine or 3-((chloro(diisopropylamino)phosphino)oxy)propionitrile.

[0159] The compound of formula (301) can be separated from the reaction mixture using any suitable separation method. In some embodiments, the solvent can be removed by evaporation, and the compound of formula (301) produced by the reaction can be separated using, for example, column chromatography, with separation conditions such as normal-phase silica gel packing and elution using a mixed eluent of dichloromethane:methanol = 15:1, or a gradient elution of dichloromethane:methanol = 50:1-(20-30):1.

[0160] The compound represented by formula (302) is commercially available or can be prepared by a person skilled in the art using a reasonable synthetic route. In some embodiments, Z in formula (302) represents any one of formulas (Z1)-(Z5), and the compound represented by formula (302) can be obtained by the following preparation method: the method includes contacting the compound of formula (304) with the compound of formula (305) in an organic solvent under condensation reaction conditions in the presence of a condensation reaction accelerant, and separating the compound of formula (302):

[0161]

[0162]

[0163] Among them, X1, X2, R k The definitions and selectable ranges of Bx1, Bx2, Y1, Y2, Y3, Y4, Y5, Y6, Y7, Y8 and B2 are as described above; Z is the structure shown in any of the terms (Z1)-(Z5), and W is Cl, Br or I.

[0164] The condensation reaction conditions include a reaction temperature of 20-90°C and a reaction time of 0.5-5 hours. In some embodiments, the reaction temperature is 40-80°C and the reaction time is 1-8 hours. The reaction pressure can be atmospheric pressure. In some embodiments, the reaction can be carried out under microwave heating, for example, under microwave heating at 50-70°C for 1-5 hours.

[0165] The organic solvent is one or more selected from epoxy solvents, ether solvents, haloalkane 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 diethyl ether and / or methyl tert-butyl ether, and the haloalkane solvent is one or more selected from dichloromethane, trichloromethane, and 1,2-dichloroethane. In some embodiments, the organic solvent is tetrahydrofuran. The amount of the organic solvent used relative to the compound shown in formula (304) is 1-50 L / mol, and in some embodiments it is 3-20 L / mol.

[0166] The molar ratio of the compound shown in formula (305) to the compound shown in formula (304) is 1:1 to 10:1, and in some embodiments it is 1:1 to 5:1.

[0167] The condensation reaction accelerator is a mixture of a ligand, propylene oxide, and a palladium-containing compound, or a mixture of 1,1'-bis(diphenylphosphine)ferrocene palladium dichloride and propylene oxide. The ligand is selected from 1,1'-bis(diphenylphosphine)ferrocene, 4,5-bis(diphenylphosphine)-9,9-dimethyloxanthracene, 1,1'-bi-naphthol, triphenylphosphine, and 2-biscyclohexylphosphine-2',4',6'-triisopropylbiphenyl. The palladium compound is selected from palladium acetate, palladium chloride, tetra(triphenylphosphine) palladium, bis(triphenylphosphine) palladium dichloride, and palladium acetylacetonate, and in some embodiments, palladium acetate. In the condensation reaction accelerator, the molar ratio of the ligand, propylene oxide, and palladium-containing compound is (1-10):(40-80):(0.1-10), or the molar ratio of 1,1'-bis(diphenylphosphine)ferrocene palladium dichloride and propylene oxide in the catalyst is 1:1-1:10, and in some embodiments, 1:1-5:1. The total amount of the condensation reaction accelerator is in a molar ratio of 1:1-10:1 to the compound of formula (304), and in some embodiments, 1:1-5:1. In some embodiments, the condensation reaction accelerator further comprises a tertiary amine, and in some embodiments, triethylamine. The molar ratio of the triethylamine to the compound of formula (304) is 1:1-10:1, and in some embodiments, 1:1-5:1.

[0168] The compound of formula (302) can be separated from the reaction mixture using any suitable separation method. In some embodiments, the solvent can be removed by evaporation, and the compound of formula (302) produced by the reaction can be separated using, for example, column chromatography, with separation conditions such as normal-phase silica gel packing and elution using a gradient of dichloromethane:methanol = (50-80):1-(20-40):1. In some embodiments, the solvent can be removed directly to obtain a crude product of compound (302), which can be used directly in subsequent reactions.

[0169] Formula (304) can be prepared by synthetic methods commonly used in the art or obtained commercially. For example, when R in formula (304) k For DMTr, Y5, Y6, Y7, and Y8 are all H, B2 is 2-cyanoethyl, B x1When X1 is thymine and H is H, the compound represented by formula (304) can be prepared according to the method described in Sahar Abbas et al., ORGANIC LETTERS, 2001, Vol.3, No.21, 3365-3367, Commercially Availabel 5'-DMT Phosphoramidites as Reagents for the Synthesis of Vinylphosphonate-Linked Oligonucleic Acids, Scheme 2.

[0170] The compound shown in formula (305) is commercially available or can be synthesized by those skilled in the art using known methods. In some embodiments, the compound of formula (305) is commercially available. In some embodiments, formula (305) can be obtained by preparation methods known in the art. For example, when Z in formula (305) is the linking group shown in (Z1), W is Br, Y1, Y2, Y3, and Y4 are all H, B x2 When uracil is used and X2 is OCH3, the compound shown in formula (305) can be prepared according to the method described in the literature Collis, Alana EC (2008) The synthesis of vinylphosphonate-linked RNA. PhDthesis, University of Nottingham, Scheme 81, except that the corresponding nucleoside modified with 2' methoxy group is used instead of deoxyribonucleoside for preparation.

[0171] In some embodiments, the nucleoside phosphoramidide compound with ribose 5' modification shown in formula (301) has one of the structures shown in formulas (3011)-(3016):

[0172]

[0173]

[0174]

[0175] Pharmaceutical Composition

[0176] In a second aspect, this disclosure provides a pharmaceutical composition comprising the double-stranded oligonucleotide provided in this disclosure, and a pharmaceutically acceptable carrier.

[0177] The pharmaceutically acceptable carrier can be a carrier conventionally used in the field of double-stranded oligonucleotide drug delivery, such as, but not limited to, magnetic nanoparticles (e.g., Fe3O4 or Fe2O3-based nanoparticles), 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), and poly(2-aminoethyl ethylene phosphate). One or more of the following: phosphate), PPEEA, and poly(2-dimethylaminoethylmethacrylate), PDMAEMA, and their derivatives.

[0178] In some embodiments, there are no particular requirements for the content of double-stranded oligonucleotides and pharmaceutically acceptable carriers in the pharmaceutical composition. In some embodiments, the weight ratio of double-stranded oligonucleotides to pharmaceutically acceptable carriers can be 1:(1-500), and in some embodiments, the weight ratio is 1:(1-50).

[0179] In some embodiments, the pharmaceutical composition may also contain other pharmaceutically acceptable excipients, which may be one or more of a variety of formulations or compounds conventionally used in the art. For example, the other pharmaceutically acceptable excipients may include at least one of pH buffers, protectants, and osmotic pressure regulators.

[0180] The pH buffer solution can be a tris(hydroxymethyl)aminomethane hydrochloride buffer with a pH of 7.5-8.5 and / or a phosphate buffer with a pH of 5.5-8.5, for example, a phosphate buffer with a pH of 5.5-8.5.

[0181] The protective agent may be at least one selected from inositol, sorbitol, sucrose, trehalose, mannose, maltose, lactose, and glucose. Based on the total weight of the pharmaceutical composition, the content of the protective agent may be 0.01-30% by weight.

[0182] The osmotic pressure regulator may be sodium chloride and / or potassium chloride. The content of the osmotic pressure regulator results in an osmotic pressure of 200-700 milliosm / kg (mOsm / kg) for the pharmaceutical composition. The content of the osmotic pressure regulator can be readily determined by those skilled in the art based on the desired osmotic pressure. In some embodiments, the dosage of the formulation made from the pharmaceutical composition may be adjusted during administration depending on the route of administration.

[0183] In some embodiments, the pharmaceutical composition may be a liquid formulation, such as an injection; or it may be a lyophilized powder for injection, which is mixed with liquid excipients to form a liquid formulation for administration. The liquid formulation may be used, but is not limited to, for subcutaneous, intramuscular, or intravenous administration, or may be delivered via, but is not limited to, aerosol administration to the lungs, aerosol administration to other organs (such as the liver), or oral inhalation, or nasal administration. In some embodiments, the pharmaceutical composition is used for aerosol administration.

[0184] In some embodiments, the pharmaceutical composition may be in the form of a liposomal formulation. In some embodiments, the pharmaceutically acceptable carrier used in the liposomal formulation comprises an amine-containing transfection compound (hereinafter also referred to as an organic amine), a cofactor lipid, and / or a polyethylene glycol-modified lipid. The organic amine, cofactor lipid, and polyethylene glycol-modified lipid may be selected from one or more of the amine-containing transfection compounds or their pharmaceutically acceptable salts or derivatives, cofactor lipids, and polyethylene glycol-modified lipids described in Chinese patent application CN103380113A (which is incorporated herein by reference in its entirety).

[0185] In some embodiments, the organic amine may be a compound of formula (601) as described in Chinese patent application CN103380113A, or a pharmaceutically acceptable salt thereof:

[0186]

[0187] in:

[0188] X 101 and X 102 Each can be independently O, S, NA, or CA, where A is hydrogen or C1-C. 20 hydrocarbon chain;

[0189] Y 101 and Z 101Each can be independently C=O, C=S, S=O, CH-OH, or SO2;

[0190] R 101 R 102 R 103 R 104 R 105 R 106 and R 107 Each is independently hydrogen, cyclic or acyclic, substituted or unsubstituted, branched or straight aliphatic group, cyclic or acyclic, substituted or unsubstituted, branched or straight heteroaliphatic group, substituted or unsubstituted, branched or straight acyl group, substituted or unsubstituted, branched or straight aryl group, substituted or unsubstituted, branched or straight heteroaryl group;

[0191] x is an integer from 1 to 10;

[0192] n is an integer from 1 to 3, m is an integer from 0 to 20, and p is 0 or 1; where, if m = p = 0, then R 102 It is hydrogen;

[0193] Furthermore, if at least one of n or m is 2, then R 103 The nitrogen in formula (601) forms a structure as shown in formula (602) or formula (603):

[0194]

[0195] In this context, g, e, and f are each an integer from 1 to 6, "HCC" represents a hydrocarbon chain, and each *N represents a nitrogen atom in formula (601).

[0196] In some implementations, R 103 It is a polyamine. In other embodiments, R 103 It is a ketal. In some embodiments, R in formula (601) 101 and R 102 Each of them is independently an arbitrary substituted or unsubstituted, branched or straight-chain alkyl or alkenyl group having 3 to 20 carbon atoms, such as 8 to 18 carbon atoms, and 0 to 4 double bonds, such as 0 to 2 double bonds.

[0197] In some implementations, if each of n and m independently has a value of 1 or 3, then R 103 It can be any one of the following equations (604)-(613):

[0198]

[0199]

[0200] In equations (604)-(613), g, e, and f are each independent integers from 1 to 6, each "HCC" represents a hydrocarbon chain, and each * indicates R. 103 Possible connection points with nitrogen atoms in formula (601), wherein each H at any * position can be replaced to achieve connection with nitrogen atoms in formula (601).

[0201] Those skilled in the art can obtain the compound represented by formula (601) by any reasonable method. In some embodiments, the compound represented by formula (601) can be prepared according to the description in Chinese patent application CN103380113A.

[0202] In some embodiments, the organic amine is an organic amine as shown in formula (614) and / or an organic amine as shown in formula (615):

[0203]

[0204]

[0205] The auxiliary lipid is cholesterol, cholesterol analogues and / or cholesterol derivatives;

[0206] The PEGylated lipid is 1,2-dipalmitoyl-sn-glycerol-3-phosphatidylethanolamine-N-[methoxy(polyethylene glycol)]-2000.

[0207] In some embodiments, the molar ratio of the organic amine, the auxiliary lipid, and the polyethylene glycol-modified lipid in the pharmaceutical composition is (19.7-80):(19.7-80):(0.3-50), for example, (50-70):(20-40):(3-20).

[0208] In some embodiments, the pharmaceutical composition particles formed from the double-stranded oligonucleotide of the present disclosure and the above-mentioned amine-containing transfection reagent have an average diameter of about 30 nm to about 200 nm, typically about 40 nm to about 135 nm, and 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.

[0209] In some embodiments, in the pharmaceutical composition formed from the double-stranded oligonucleotide of this disclosure and the above-mentioned amine-containing transfection reagent, the weight ratio (weight / weight ratio) of the double-stranded oligonucleotide to all lipids (e.g., organic amines, auxiliary lipids and / or polyethylene glycol-modified lipids) is in 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 this disclosure to all 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.

[0210] In some embodiments, the components of the pharmaceutical composition may exist independently when sold, and may be in liquid form when used. In some embodiments, the pharmaceutical composition formed by the double-stranded oligonucleotide provided in this disclosure and the above-described pharmaceutically acceptable carrier can be prepared according to various known methods, simply by replacing existing double-stranded oligonucleotides with the double-stranded oligonucleotide provided in this disclosure; in some embodiments, it can be prepared according to the following method:

[0211] An organic amine, auxiliary lipid, and polyethylene glycol-modified lipid are suspended in an alcohol at the above molar ratio and mixed to obtain a lipid solution. The amount of alcohol used is such that the total mass concentration of the resulting lipid solution is 2-25 mg / mL, for example, 8-18 mg / mL. The alcohol is selected from pharmaceutically acceptable alcohols, such as alcohols that are liquid near room temperature, for example, one or more of ethanol, propylene glycol, benzyl alcohol, glycerol, polyethylene glycol 200, polyethylene glycol 300, and polyethylene glycol 400, for example, ethanol.

[0212] The double-stranded oligonucleotide provided in this disclosure is dissolved in a buffer salt solution to obtain an aqueous solution of the double-stranded oligonucleotide. The concentration of the buffer salt solution is 0.05-0.5M, for example, 0.1-0.2M. The pH of the buffer salt solution is adjusted to 4.0-5.5, for example, 5.0-5.2. The amount of buffer salt solution used is such that the concentration of the double-stranded oligonucleotide does not exceed 0.6 mg / mL, for example, 0.2-0.4 mg / mL. The buffer salt is selected from one or more of soluble acetate and soluble citrate, for example, sodium acetate and / or potassium acetate.

[0213] The lipid solution and the aqueous solution of the double-stranded oligonucleotide are mixed, and the resulting product is incubated at 40-60°C for at least 2 minutes, for example, 5-30 minutes, to obtain the incubated liposome formulation. The volume ratio of the lipid solution to the aqueous solution of the double-stranded oligonucleotide is 1:(2-5), for example, 1:4.

[0214] The incubated liposome formulation is concentrated or diluted, impurities are removed, and sterilization is performed to obtain the pharmaceutical composition provided in this disclosure. Its physicochemical parameters are: pH value of 6.5-8, encapsulation efficiency of not less than 80%, particle size of 40-200 nm, polydispersity index of not more than 0.30, and osmotic pressure of 250-400 mOsm / kg; for example, the physicochemical parameters can be: pH value of 7.2-7.6, encapsulation efficiency of not less than 90%, particle size of 60-100 nm, polydispersity index of not more than 0.20, and osmotic pressure of 300-400 mOsm / kg.

[0215] Concentration or dilution can be performed before, after, or simultaneously with impurity removal. Impurity removal can be achieved using various existing methods, such as ultrafiltration at 100 kDa using a tangential flow system or hollow fiber column, with the ultrafiltration exchange solution being phosphate-buffered saline (PBS) at pH 7.4. Sterilization can be achieved using various existing methods, such as filtration sterilization through a 0.22 μm filter.

[0216] Oligonucleotide conjugates

[0217] In a third aspect, this disclosure provides an oligonucleotide conjugate comprising the double-stranded oligonucleotide provided in this disclosure, and a conjugating group conjugated to the double-stranded oligonucleotide. In some embodiments, the conjugating group comprises a linker and a pharmaceutically acceptable targeting group and / or a delivery aid group, and the double-stranded oligonucleotide, the linker, and the targeting group or the delivery aid group are sequentially covalently or non-covalently linked, each targeting group being selected from ligands capable of binding to cell surface receptors, and each delivery aid group being selected from groups capable of increasing the biocompatibility of the oligonucleotide conjugate in a target organ or tissue.

[0218] In the context of this disclosure, unless otherwise stated, "conjugation" refers to the covalent connection between two or more chemical moieties, each having a specific function; correspondingly, "conjugated compound" refers to a compound formed by the covalent connection of these chemical moieties. Further, "oligonucleotide conjugated compound" refers to a compound formed by the covalent attachment of one or more chemical moieties having a specific function to an oligonucleotide. Oligonucleotide conjugated compounds should be understood, depending on the context, as a collective term for multiple oligonucleotide conjugated compounds or an oligonucleotide conjugated compound represented by a particular chemical formula. In the context of this disclosure, "conjugated molecule" should be understood as a specific compound that can be reactively conjugated to an oligonucleotide to ultimately form the oligonucleotide conjugated compounds of this disclosure.

[0219] Generally, the conjugation group comprises at least one pharmaceutically acceptable target group and an optional linker, and the double-stranded oligonucleotide, the linker, and the target group are sequentially linked. In one embodiment, there are 1-6 target groups. In another embodiment, there are 2-4 target groups. The double-stranded oligonucleotide molecule can be non-covalently or covalently conjugated to the conjugation group, for example, it can be covalently conjugated to the conjugation group. The conjugation site of the double-stranded oligonucleotide to the conjugation group can be at the 3' or 5' end of the sense strand of the double-stranded oligonucleotide, at the 5' end of the antisense strand, or within the internal sequence of the double-stranded oligonucleotide. In some specific embodiments, the conjugation site of the double-stranded oligonucleotide to the conjugation group is at the 3' end of the sense strand of the double-stranded oligonucleotide.

[0220] In some embodiments, the conjugation group may be attached to a phosphate group, a 2'-hydroxyl group, or a base of the nucleotide. In some embodiments, the conjugation group may be attached to a 3'-hydroxyl group, in which case the nucleotides are linked by a 2'-5' phosphodiester bond. When the conjugation group is attached to the end of a double-stranded oligonucleotide chain, the conjugation group is usually attached to a phosphate group of the nucleotide; when the conjugation group is attached to the inner sequence of a double-stranded oligonucleotide, the conjugation group is usually attached to a ribose ring or a base. Various connection methods can be found in: Muthiah Manoharan et al. siRNA conjugates carrying sequentially assembled trivalent N-acetylgalactosamine linked through nucleosides elicit robust gene silencing in vivo inhepatocytes. ACS Chemical biology, 2015, 10(5): 1181-7.

[0221] In some embodiments, the double-stranded oligonucleotide and the conjugation group are linked by acid-labile or reducible chemical bonds that can degrade in the acidic environment of the endosome, thus freeing the double-stranded oligonucleotide. For non-degradable conjugations, the conjugation group can be attached to the positive and negative strands of the double-stranded oligonucleotide to minimize the impact of the conjugation on the activity of the double-stranded oligonucleotide.

[0222] The targeting group can be linked to the double-stranded oligonucleotide molecule via a suitable adapter. Those skilled in the art can select a suitable adapter based on the specific type of the targeting group. For details on these adapters, types of targeting groups, and the methods of linking them to the double-stranded oligonucleotide, please refer to the disclosure of WO2015006740A2, the entire contents of which are incorporated herein by reference.

[0223] In some embodiments, the targeting group may be a ligand commonly used in the field of double-stranded oligonucleotide drug delivery, such as the various ligands described in WO2009082607A2, the entire disclosure of which is incorporated herein by reference.

[0224] In some embodiments, at least one or each of the target groups is selected from ligands capable of binding to cell surface receptors expressing the target gene.

[0225] In some embodiments, at least one or each of the target groups is selected from ligands capable of binding to receptors on the surface of mammalian hepatocytes. In some embodiments, each of the target groups is independently a ligand with affinity for desialyl glycoprotein receptors on the surface of mammalian hepatocytes. In some embodiments, each of the target groups is independently a desialyl glycoprotein or a sugar. In some embodiments, each of the target groups is independently selected from D-mannose pyranoyl ... α-D-Furfural, β-D-Furfural, α-D-Fructose, α-D-Galactopyranose, α-D-Galactopyranose, β-D-Galactopyranose, α-D-Galactopyranose, β-D-Galactopyranose, Glucosamine, Sialic acid, Galactosamine, N-acetylgalactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-butyrylgalactosamine, N-isobutyrylgalactosamine, 2 -Amino-3-O-[(R)-1-carboxyethyl]-2-deoxy-β-D-glucopyranose, 2-deoxy-2-methylamino-L-glucopyranose, 4,6-dideoxy-4-carboxamido-2,3-di-O-methyl-D-mannopyranose, 2-deoxy-2-sulfonamido-D-glucopyranose, N-ethanolyl-α-neuraminic acid, 5-thio-β-D-glucopyranose, 2,3,4 One of the following: tri-O-acetyl-1-thio-6-O-triphenylmethyl-α-D-glucopyranoside methyl ester, 4-thio-β-D-galactopyranose, 3,4,6,7-tetra-O-acetyl-2-deoxy-1,5-dithio-α-D-glucopyranoside ethyl ester, 2,5-dehydrated-D-alosulfonyl, ribose, D-ribose, D-4-thioribose, L-ribose, and L-4-thioribose. In some embodiments, at least one or each of the target groups is galactose or N-acetylgalactosamine.

[0226] In some embodiments, at least one or each of the target groups is selected from ligands capable of binding to receptors on the surface of lung epithelial cells. In some embodiments, each of the target groups is selected from groups targeting integrin αvβ6 or groups targeting integrin αvβ3. In some embodiments, each of the target groups is independently a peptide or a small molecule ligand.

[0227] In some embodiments, at least one or each of the delivery aid groups is selected from groups capable of increasing the biocompatibility of the oligonucleotide conjugate in the central nervous system. In some embodiments, at least one or each of the delivery aid groups is selected from lipophilic molecules. In some embodiments, each of the delivery aid groups is C5-C. 18 Straight-chain hydrocarbon groups or steroidal compounds.

[0228] In some embodiments, the linkers in the oligonucleotide conjugates of this disclosure have a structure as shown in formula (701):

[0229]

[0230] Where k is an integer from 1 to 3;

[0231] L A Having a structure containing amide bonds as shown in formula (702), L B Having a structure containing N-acylpyrrolidine as shown in formula (303), containing a carbonyl group and an oxygen atom, L C It is a linking group based on hydroxymethylaminomethane, dihydroxymethylaminomethane, or trihydroxymethylaminomethane;

[0232]

[0233] Where, n 302 q 302 and p 302 Each is an independent integer from 2 to 6; optionally, n 302 q 302 and p 302 Each is independently 2 or 3; n 303 n is an integer between 4 and 16, optionally n 303 For integers between 8 and 12, This indicates the site where the group is covalently linked.

[0234] In the aforementioned connector, each L A Each of the target groups is connected via an ether bond and via L C The oxygen atom of the hydroxyl group in some of the middle groups is related to L. C Partially linked by ether bonds; L B Through the carbonyl group in formula (703) and L C The nitrogen atom of the amino group in some of the amino groups forms an amide bond and is linked to the double-stranded oligonucleotide through the oxygen atom in formula (703) by forming a phosphate ester bond or a thiophosphate ester bond.

[0235] In some embodiments, the oligonucleotide conjugates provided in this disclosure have a structure as shown in formula (705):

[0236]

[0237] Wherein, Nu represents the double-stranded oligonucleotide provided in this disclosure, or the double-stranded oligonucleotide obtained according to the method of this disclosure.

[0238] In some embodiments, the linkers in the oligonucleotide conjugates of this disclosure have the structure shown in formula (706):

[0239]

[0240] Where, n 306 For each p, the integer is between 0 and 3. 306 Independently, integers from 1 to 6. The site indicates a covalently linked group; the linking group is connected to the target group by an ether bond formed by an oxygen atom marked with *; the linking group is connected to the double-stranded oligonucleotide by at least one of the oxygen atoms marked with # forming a phosphate ester bond or a thiophosphate ester bond, and the remaining oxygen atoms marked with # are connected to hydrogen atoms to form hydroxyl groups, or connected to C1-C3 alkyl groups to form C1-C3 alkoxy groups;

[0241] In some embodiments, the oligonucleotide conjugates of this disclosure have a structure as shown in formula (707):

[0242]

[0243] Wherein, Nu represents the double-stranded oligonucleotide provided in this disclosure, or the double-stranded oligonucleotide obtained according to the method of this disclosure.

[0244] In some embodiments, the oligonucleotide conjugates of this disclosure have the structure shown in formula (708):

[0245]

[0246] in,

[0247] n1 is an integer selected from 1 to 3, and n3 is an integer selected from 0 to 4;

[0248] Each m1, m2, or m3 is an independent integer selected from 2 to 10;

[0249] R 10 R 11 R 12 R 13 R 14 Or R 15 Each is independently H, or selected from the group consisting of C1-C. 10 Alkyl, C1-C 10 Halogenated alkyl groups and C1-C10 Alkoxy;

[0250] R3 has the structure shown in Equation A59:

[0251]

[0252] Wherein, E1 is OH, SH or BH2, and Nu represents the double-stranded oligonucleotide provided in this disclosure;

[0253] R2 is a straight-chain alkylene group with a length of 1-20 carbon atoms, wherein one or more carbon atoms are optionally replaced by any one or more groups selected from the group consisting of: C(O), NH, O, S, CH=N, S(O)2, C2-C 10 alkenyl, C2-C 10 Ethyne group, C6-C 10 Aromatic, C3-C 18 Heterocyclic groups and C5-C 10 Heteroaryl; and wherein R2 may optionally have any one or more substituents from the group consisting of: C1-C 10 Alkyl, C6-C 10 Aryl, C5-C 10 heteroaryl, C1-C 10 Halogenated alkyl, -OC1-C 10 Alkyl, -OC1-C 10 Alkylphenyl, -C1-C 10 Alkyl-OH, -OC1-C 10 Halogenated alkyl, -SC1-C 10 Alkyl, -SC1-C 10 Alkylphenyl, -C1-C 10 Alkyl-SH, -SC1-C 10 Halogenated alkyl groups, halogenated substituents, -OH, -SH, -NH2, -C1-C 10 Alkyl-NH2,-N(C1-C) 10 Alkyl) (C1-C 10 alkyl), -NH(C1-C 10 Alkyl), -N(C1-C 10 Alkyl) (C1-C 10 alkylphenyl), -NH(C1-C 10 Alkylphenyl), cyano, nitro, -CO2H, -C(O)O(C1-C 10 Alkyl), -CON (C1-C) 10 Alkyl) (C1-C 10 Alkyl), -CONH (C1-C) 10 Alkyl groups, -CONH2, -NHC(O) (C1-C)10 alkyl), -NHC(O)(phenyl), -N(C1-C 10 Alkyl)C(O)(C1-C 10 Alkyl), -N(C1-C 10 alkyl)C(O)(phenyl), -C(O)C1-C 10 Alkyl, -C(O)C1-C 10 Alkylphenyl, -C(O)C1-C 10 Haloalkyl, -OC(O)Cl-C 10 Alkyl group, -SO2 (C1-C) 10 alkyl), -SO2 (phenyl), -SO2 (C1-C 10 Halogenated alkyl groups), -SO2NH2, -SO2NH (C1-C 10 alkyl), -SO2NH (phenyl), -NHSO2 (C1-C 10 Alkyl), -NHSO2 (phenyl) and -NHSO2 (C1-C) 10 (Halogenated alkyl);

[0254] Each L1 is independently a straight-chain alkylene group with a length of 1-70 carbon atoms, wherein one or more carbon atoms are optionally replaced by any one or more groups selected from the group consisting of: C(O), NH, O, S, CH=N, S(O)2, C2-C 10 alkenyl, C2-C 10 Ethyne group, C6-C 10 Aromatic, C3-C 18 Heterocyclic groups and C5-C 10 Heteroaryl; and wherein L1 may optionally have any one or more substituents from the group consisting of: C1-C 10 Alkyl, C6-C 10 Aryl, C5-C 10 heteroaryl, C1-C 10 Halogenated alkyl, -OC1-C 10 Alkyl, -OC1-C 10 Alkylphenyl, -C1-C 10 Alkyl-OH, -OC1-C 10 Halogenated alkyl, -SC1-C 10 Alkyl, -SC1-C 10 Alkylphenyl, -C1-C 10 Alkyl-SH, -SC1-C 10 Halogenated alkyl groups, halogenated substituents, -OH, -SH, -NH2, -C1-C 10 Alkyl-NH2,-N(C1-C) 10 Alkyl) (C1-C10 alkyl), -NH(C1-C 10 Alkyl), -N(C1-C 10 Alkyl) (C1-C 10 alkylphenyl), -NH(C1-C 10 Alkylphenyl), cyano, nitro, -CO2H, -C(O)O(C1-C 10 Alkyl), -CON (C1-C) 10 Alkyl) (C1-C 10 Alkyl), -CONH (C1-C) 10 Alkyl group), -CONH2, -NHC(O) (C1-C 10 alkyl), -NHC(O)(phenyl), -N(C1-C 10 Alkyl)C(O)(C1-C 10 Alkyl), -N(C1-C 10 alkyl)C(O)(phenyl), -C(O)C1-C 10 Alkyl, -C(O)C1-C 10 Alkylphenyl, -C(O)C1-C 10 Haloalkyl, -OC(O)Cl-C 10 Alkyl group, -SO2 (C1-C) 10 alkyl), -SO2 (phenyl), -SO2 (C1-C 10 Halogenated alkyl groups), -SO2NH2, -SO2NH (C1-C 10 alkyl), -SO2NH (phenyl), -NHSO2 (C1-C 10 Alkyl), -NHSO2 (phenyl) and -NHSO2 (C1-C) 10 (Halogenated alkyl);

[0255] Indicates the site where groups are covalently linked;

[0256] M1 represents a targeting group, the definition of which and the range of possible selections are the same as described above. In some embodiments, each M1 is independently selected from a group of ligands that have affinity for desialylate glycoprotein receptors on the surface of mammalian liver cells.

[0257] Those skilled in the art will understand that although L1 is defined as a linear alkyl group for convenience, it may not be a linear group or may have a different name, such as an amine or alkenyl group resulting from the above substitutions and / or replacements. For the purposes of this disclosure, the length of L1 is the number of atoms in the chain connecting the two attachment points. For this purpose, the ring (such as a heterocyclic or heteroaryl group) obtained by replacing the carbon atom of the linear alkylene group is counted as one atom. In some embodiments, each L1 is independently selected from one or more combinations of groups of formulas A1-A26:

[0258]

[0259]

[0260] Wherein, j1 is an integer from 1 to 20; R' is a C1-C10 alkyl group; Ra is selected from one of the groups of formula A27-A45:

[0261]

[0262]

[0263] Rb is a C1-C10 alkyl group;

[0264] When M1 is a ligand with affinity for the desialyl glycoprotein receptor on the surface of mammalian liver cells, in some embodiments, n1 can be an integer from 1 to 3, and n3 can 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, which ensures that the number of M1 ligands is at least 3, making it easier for the M1 ligand to bind to the desialyl glycoprotein receptor on the liver surface, thereby promoting the conjugate's entry into the cell via endocytosis. Experiments show that when the number of M1 ligands is greater than 3, the ease of binding of the M1 ligand to the desialyl glycoprotein receptor on the liver surface does not increase significantly. Therefore, considering factors such as ease of synthesis, structural / process 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 - 3.

[0265] In some embodiments, when m1, m2, and m3 are independently selected from integers of 2 to 10, the spatial positions between the multiple M1 ligands can be adapted to the binding of the M1 ligand to the liver surface desialylate glycoprotein receptor. In order to make the conjugates provided in this disclosure simpler, easier to synthesize, and / or reduce costs, in some embodiments, m1, m2, and m3 are each independently an integer of 2 to 5, and in some embodiments, m1 = m2 = m3.

[0266] Those skilled in the art will understand that when R10 R 11 R 12 R 13 R 14 and R 15 Each is independently selected from H, C1-C 10 Alkyl, C1-C 10 Halogenated alkyl groups and C1-C 10 Using one of the alkoxy groups will not change the properties of the conjugates disclosed herein, and the objectives of this disclosure can still be achieved. In some embodiments, R 10 R 11 R 12 R 13 R 14 and R 15 Each is independently selected from H, methyl, and ethyl. In some embodiments, R 10 R 11 R 12 R 13 R 14 and R 15 All are H.

[0267] According to the oligonucleotide conjugates provided in this disclosure, R3 is a group with the structure shown in Formula A59, wherein E1 is OH, SH or BH2. In some embodiments, E1 is OH or SH, based on the consideration of the availability of raw materials.

[0268] In some implementations, R2 is chosen to enable the connection between N and A59 on the nitrogen-containing framework. In the context of this disclosure, "nitrogen-containing framework" refers to a framework connected to R. 10 R 11 R 12 R 13 R 14 and R 15 The chain structure in which carbon atoms are interconnected with nitrogen (N) is used. Therefore, R2 can be any linking group capable of connecting the A59 group to the N on the nitrogen-containing backbone in a suitable manner. In some embodiments, when the oligonucleotide conjugates of this disclosure are prepared by solid-phase synthesis, the R2 group needs to contain both a linking site for the N on the nitrogen-containing backbone and a linking site for the P in R3. In some embodiments, the site in R2 that links to the N on the nitrogen-containing backbone forms an amide bond with N, and the site that links to the P on R3 forms a phosphate ester bond with P. In some embodiments, R2 is B5, B6, B5', or B6'.

[0269]

[0270]

[0271] in, This indicates the site where a group is covalently bonded.

[0272] The value of q2 can be an integer from 1 to 10. In some implementations, q2 is an integer from 1 to 5.

[0273] The function of L1 is to link the M1 ligand to the N on the nitrogen-containing backbone, providing targeting functionality for the oligonucleotide conjugates of this disclosure. In some embodiments, L1 is selected from one or more linkage combinations of groups of formulas A1-A26. In some embodiments, L1 is selected from one or more linkage combinations of A1, A4, A5, A6, A8, A10, A11, and A13; in some embodiments, L1 is selected from a linkage combination of at least two of A1, A4, A8, A10, and A11; in some embodiments, L1 is selected from a linkage combination of at least two of A1, A8, and A10.

[0274] In some embodiments, the length of L1 can be 3-25 atoms, 3-20 atoms, 4-15 atoms, or 5-12 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.

[0275] 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 formulas A1-A26 are selected respectively to achieve N-linking of the M1 ligand to the nitrogen-containing backbone and to make the spatial positions between the M1 ligands more suitable for the binding of the M1 ligand to the liver surface desialyl glycoprotein receptor.

[0276] In some embodiments, the oligonucleotide conjugates of this disclosure have the structures shown in formulas (403), (404), (405), (406), (407), (408), (409), (410), (411), (412), (413), (414), (415), (416), (417), (418), (419), (420), (421), or (422):

[0277]

[0278]

[0279]

[0280]

[0281]

[0282]

[0283]

[0284] In some embodiments, P in Formula A59 can be attached to any possible position in the double-stranded oligonucleotide sequence; for example, P in Formula A59 can be attached to any nucleotide of the sense or antisense strand of the double-stranded oligonucleotide. In some embodiments, P in Formula A59 is attached to any nucleotide of the sense strand of the double-stranded oligonucleotide. In some embodiments, P in Formula A59 is attached to the end of the sense 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 first four nucleotides of the sense or antisense strand counting from one end. In some embodiments, P in Formula A59 is attached to the end of the sense or 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 attached to the above-described positions on the sense strand of the double-stranded oligonucleotide, the conjugate provided in this disclosure, upon entering the cell, can release the individual antisense strand of the double-stranded oligonucleotide upon unwinding to regulate target gene expression.

[0285] The P in Formula A59 can be attached to any possible position on the nucleotide in the double-stranded oligonucleotide, such as the 5' position, 2' position, 3' position, or base of the nucleotide. In some embodiments, the P in Formula A59 can be attached to the 2', 3', or 5' position of the nucleotide in the double-stranded oligonucleotide by forming a phosphodiester bond. In some embodiments, the P in Formula A59 is attached to the oxygen atom formed by the dehydrogenation of the 3'-hydroxyl group of the 3'-terminal nucleotide of the positive strand of the double-stranded oligonucleotide, or the P in Formula A59 is attached to the nucleotide by substituting a hydrogen atom in the 2'-hydroxyl group of one nucleotide in the positive strand of the double-stranded oligonucleotide, or the P in Formula A59 is attached to the nucleotide by substituting a hydrogen atom in the 5'-hydroxyl group of the 5'-terminal nucleotide of the positive strand of the double-stranded oligonucleotide.

[0286] In some embodiments, the double-stranded oligonucleotides contained in the oligonucleotide conjugates of this disclosure may be siRNAs, in which case the oligonucleotide conjugates of this disclosure are also referred to as siRNA conjugates. In some embodiments, the double-stranded oligonucleotides contained in the oligonucleotide conjugates of this disclosure may be, for example, siRNAs listed in Table 1. Oligonucleotide conjugates containing these siRNAs exhibit low toxicity and high mRNA repressive activity against the expression of the target gene.

[0287] This disclosure relates to the preparation of oligonucleotide conjugates.

[0288] The aforementioned oligonucleotide conjugates can be synthesized using methods already described in detail in the prior art. For example, WO2015006740A2 describes in detail various methods for preparing siRNA conjugates. When the double-stranded oligonucleotide is siRNA, the oligonucleotide conjugates disclosed herein can also be obtained using methods well known to those skilled in the art. For instance, WO2014025805A1 describes a method for preparing the structure shown in formula (705), and Rajeev et al. describe a method for preparing the structure shown in formula (707) in ChemBioChem 2015, 16, 903-908. Chinese patent application CN110959011A also discloses in detail a method for preparing the oligonucleotide conjugate shown in formula (708). The entire contents of the above-mentioned documents are incorporated herein by reference.

[0289] The oligonucleotide conjugates disclosed herein may also be used in combination with other pharmaceutically acceptable excipients, which may be one or more of a variety of formulations or compounds conventionally used in the art, as detailed in the above description of the pharmaceutical compositions of this disclosure.

[0290] Applications of the double-stranded oligonucleotides, pharmaceutical compositions, and oligonucleotide conjugates disclosed herein

[0291] In some embodiments, this disclosure provides the use of the double-stranded oligonucleotides provided herein, double-stranded oligonucleotides obtained according to the methods of this disclosure, pharmaceutical compositions, and / or oligonucleotide conjugates in medicaments for treating and / or preventing diseases or symptoms associated with the mRNA level of a target gene expression. 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 pathogen gene that proliferates in the liver. In some embodiments, the specific gene is a gene expressed in lung epithelial cells. In some embodiments, the specific gene is a gene expressed in the central nervous system. In some embodiments, the specific gene is a gene expressed in tumor cells. In some embodiments, the mRNA expressing the target gene is selected from one of the following mRNAs transcribed from genes: ACE2, ANGPTL3, ApoA, ApoB, ApoC, AR, ASK1, C5, Col1A1, CTGF, Ebola, FOXO1, FTO, FVII, FXI, FXII, GCGR, HBV, HCV, HSD17B13, p53, PCSK9, PNP, PLG, PKK, KNG, SARS-CoV-2, SCD1, SCNN1A, SOD1, STAT3, TIMP-1, TMPRSS6, XO, INSR, SREBF1, HDV, RPTOR, TLK2, LPA, C3, AGT. In some embodiments, the mRNA expressing the target gene is selected from mRNA expressed by the hepatitis B virus (HBV) gene, mRNA expressed by the angiopoietin-like protein 3 (ANGPTL3) gene, or mRNA expressed by the apolipoprotein C3 (ApoC3) gene. In some embodiments, the disease or symptom associated with the mRNA level of the target gene expression is chronic liver disease, hepatitis, liver fibrosis, hepatoproliferative disorders, and / or dyslipidemia. In some embodiments, the disease or symptom associated with the mRNA level of the target gene expression is hepatitis B.

[0292] In some embodiments, this disclosure provides a method for treating and / or preventing diseases or symptoms associated with the level of mRNA expression of a target gene, the method comprising administering to a subject in need an effective amount of the double-stranded oligonucleotide, pharmaceutical composition, and / or oligonucleotide conjugate provided in this disclosure. In some embodiments, the mRNA expressing the target gene is selected from mRNAs transcribed from the following genes: ACE2, ANGPTL3, ApoA, ApoB, ApoC, AR, ASK1, C5, Col1A1, CTGF, Ebola, FOXO1, FTO, FVII, FXI, FXII, GCGR, HBV, HCVHSD17B13, p53, PCSK9, PNP, PLG, PKK, KNG, SARS-CoV-2, SCD1, SCNN1A, SOD1, STAT3, TIMP-1, TMPRSS6, XO, INSR, SREBF1, HDV, RPTOR, TLK2, LPA, C3, AGT. In some embodiments, the mRNA expressed by the target gene is selected from the mRNA expressed by the hepatitis B virus (HBV) gene, the mRNA expressed by the angiopoietin-like protein 3 (ANGPTL3) gene, or the mRNA expressed by the apolipoprotein C3 (ApoC3) gene. In some embodiments, the disease or symptom associated with the level of the target gene's expressed mRNA is chronic liver disease, hepatitis, liver fibrosis, hepatoproliferative disorders, and / or dyslipidemia. In some embodiments, the disease or symptom associated with the level of the target gene's expressed mRNA is hepatitis B.

[0293] In some embodiments, the conjugates provided in this disclosure can also be used to treat other liver diseases, including diseases characterized by unwanted cell proliferation, hematologic disorders, metabolic disorders, and diseases characterized by inflammation. Proliferative liver diseases can be benign or malignant, such as cancer, hepatocellular carcinoma (HCC), liver metastases, or hepatoblastoma. Hematologic or inflammatory liver diseases can involve clotting factors, complement-mediated inflammation, or fibrosis. Metabolic liver diseases include dyslipidemia and irregularities in glucose regulation. In one embodiment, the disease is treated by administering one or more double-stranded oligonucleotides having a sequence highly homologous to the gene sequence involved in the disease.

[0294] In some embodiments, this disclosure provides a method for regulating the expression level of a target gene in cells, the method comprising contacting the cells with an effective amount of the double-stranded oligonucleotide, pharmaceutical composition, and / or oligonucleotide conjugate provided in this disclosure. In some embodiments, the mRNA expressing the target gene is selected from the mRNAs transcribed from the following genes: ACE2, ANGPTL3, ApoA, ApoB, ApoC, AR, ASK1, C5, Col1A1, CTGF, Ebola, FOXO1, FTO, FVII, FXI, FXII, GCGR, HBV, HCV, HSD17B13, p53, PCSK9, PNP, PLG, PKK, KNG, SARS-CoV-2, SCD1, SCNN1A, SOD1, STAT3, TIMP-1, TMPRSS6, XO, INSR, SREBF1, HDV, RPTOR, TLK2, LPA, C3, AGT. In some embodiments, the regulation refers to inhibiting the expression of a target gene in the cell, wherein the mRNA expressing the target gene is selected from the mRNA of hepatitis B virus (HBV) gene expression, the mRNA of angiopoietin-like protein 3 (ANGPTL3) gene expression, or the mRNA of apolipoprotein C3 (ApoC3) gene expression.

[0295] By administering the double-stranded oligonucleotides, pharmaceutical compositions, and / or oligonucleotide conjugates provided in this disclosure to subjects in need, the prevention and / or treatment of pathological conditions or diseases caused by the expression of specific genes in cells can be achieved through mechanisms that regulate gene expression. Therefore, the double-stranded oligonucleotides provided in this disclosure, the double-stranded oligonucleotides obtained according to the methods of this disclosure, the pharmaceutical compositions, and / or oligonucleotide conjugates can be used for the prevention and / or treatment of said pathological conditions or diseases, or for the preparation of medicaments for the prevention and / or treatment of the pathological conditions or diseases described herein.

[0296] As used herein, the term "administration" refers to the placement of a double-stranded oligonucleotide, pharmaceutical composition, and / or oligonucleotide conjugate into a subject by means of a method or route that at least partially targets a desired site to produce a desired effect. Routes of administration suitable for the methods of this disclosure include local administration and systemic administration. Generally, local administration results in the delivery of more double-stranded oligonucleotides, pharmaceutical compositions, and / or oligonucleotide conjugates to a specific site compared to the entire body of the subject; while systemic administration results in the delivery of said double-stranded oligonucleotides, pharmaceutical compositions, and / or oligonucleotide conjugates to substantially the entire body of the subject.

[0297] The medication may be administered to the subject via any suitable route known in the art, including but not limited to: oral or parenteral routes, such as intravenous administration, intramuscular administration, subcutaneous administration, transdermal administration, airway administration (aerosol), pulmonary administration, nasal administration, rectal administration, and local administration (including oral and sublingual administration). Administration frequency may be once or more daily, weekly, bi-weekly, bi-weekly, monthly, or annually.

[0298] The dosages of the double-stranded oligonucleotides, pharmaceutical compositions, and / or oligonucleotide conjugates described in this disclosure are conventional dosages in the art, which can be determined based on various parameters, particularly the age, weight, and sex of the subject. Toxicity and efficacy can be determined in cell culture or laboratory animals using standard pharmaceutical procedures, such as determining the LD50 (the dose that causes 50% of the population to die) and ED50 (the dose that elicits 50% of the maximum response intensity in a quantitative response, and the dose that elicits a positive response in 50% of the subjects in a qualitative response). Ranges of human dosages can be derived based on data obtained from cell culture analysis and animal studies.

[0299] When administering the double-stranded oligonucleotides, pharmaceutical compositions, and / or oligonucleotide conjugates described in this disclosure, for example, to male or female C57BL / 6J or C3H / HeNCrlVr mice, 6-12 weeks old, weighing 18-25 g, the amount of double-stranded oligonucleotides in the double-stranded oligonucleotides, pharmaceutical compositions, and / or oligonucleotide conjugates 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 even further embodiments 0.1-15 mg / kg body weight, and in still further embodiments 0.1-10 mg / kg body weight. The above amounts are preferred when administering the double-stranded oligonucleotides, pharmaceutical compositions, and / or oligonucleotide conjugates described in this disclosure.

[0300] Furthermore, by introducing the double-stranded oligonucleotides, pharmaceutical compositions, and / or oligonucleotide conjugates disclosed herein into cells with abnormal gene expression, the expression of that specific gene in the cells can also be inhibited through gene expression regulation mechanisms. In some embodiments, the cells are hepatocytes. In some embodiments, the hepatocytes may be cells selected from hepatocellular carcinoma cell lines such as Hep3B, HepG2, and Huh7, or isolated primary hepatocytes; in some embodiments, they are primary hepatocytes.

[0301] The methods provided in this disclosure for inhibiting the expression of specific genes in cells involve double-stranded oligonucleotides, pharmaceutical compositions, and / or oligonucleotide conjugates, the amount of which is readily determined by those skilled in the art based on the desired effect. For example, in some embodiments, the double-stranded oligonucleotides, pharmaceutical compositions, and / or oligonucleotide conjugates are siRNA conjugates, and the amount of siRNA in the provided siRNA conjugate is sufficient to reduce the expression of the target gene and result in an extracellular concentration of 1 pM to 1 μM, or 0.01 nM to 100 nM, or 0.05 nM to 50 nM, or about 5 nM at the surface of the target cells. The amount required to achieve this local concentration will vary depending on various factors, including the delivery method, delivery site, the number of cell layers between the delivery site and the target cells or tissue, and whether the delivery is local or systemic. The concentration at the delivery site can be significantly higher than the concentration at the surface of the target cells or tissue.

[0302] Reagent test kit

[0303] This disclosure provides a kit comprising the double-stranded oligonucleotide, pharmaceutical composition and / or oligonucleotide conjugate provided in this disclosure.

[0304] In some embodiments, the kit described herein may provide double-stranded oligonucleotides, pharmaceutical compositions, and / or conjugates in a single container. In some embodiments, the kit described herein may include a container providing pharmaceutically acceptable excipients. In some embodiments, the kit may also contain other components, such as stabilizers or preservatives. In some embodiments, the kit described herein may contain at least one other therapeutic agent in a container other than the one providing the double-stranded oligonucleotides, pharmaceutical compositions, and / or conjugates described herein. In some embodiments, the kit may include instructions for mixing the double-stranded oligonucleotides, pharmaceutical compositions, and / or conjugates with pharmaceutically acceptable carriers and / or excipients or other components (if any).

[0305] In the kits disclosed herein, the double-stranded oligonucleotides and pharmaceutically acceptable carriers and / or excipients, as well as the pharmaceutical compositions and / or conjugates, and / or pharmaceutically acceptable excipients, may be provided in any form, such as liquid, dry, or lyophilized. In some embodiments, the double-stranded oligonucleotides and pharmaceutically acceptable carriers and / or excipients, as well as the pharmaceutical compositions and / or conjugates and optional pharmaceutically acceptable excipients, are substantially pure and / or sterile. In some embodiments, sterile water may be provided in the kits disclosed herein.

[0306] The present disclosure will be further illustrated by the following examples, but the present disclosure is not limited thereto.

[0307] Without intending to be limiting, the invention is further described in detail below in the embodiments and in exemplary embodiments relating to small interfering RNA (siRNA) in pharmaceutical compositions and / or oligonucleotide conjugates of this disclosure. In this context, the double-stranded oligonucleotides, pharmaceutical compositions, and oligonucleotide conjugates of this disclosure are siRNA, siRNA-containing pharmaceutical compositions, and siRNA conjugates, respectively. In the context of this disclosure, for ease of description, the siRNA, siRNA-containing pharmaceutical compositions, and siRNA conjugates in these embodiments are also referred to as the siRNA, pharmaceutical compositions, and siRNA conjugates of this disclosure. This does not mean that the double-stranded oligonucleotides of this disclosure can only be siRNA; rather, the double-stranded oligonucleotides can be other variants disclosed herein or known to those skilled in the art, such as small activating RNA (saRNA), etc. It is contemplated that, based on the detailed description of siRNA, siRNA-containing pharmaceutical compositions, and siRNA conjugates, other double-stranded oligonucleotides will similarly function when used alone or in the formation of the pharmaceutical compositions and / or oligonucleotide conjugates described in this disclosure.

[0308] Example

[0309] Unless otherwise specified, all reagents used in the following examples are commercially available products.

[0310] Example 1: Preparation of siRNA1

[0311] (1-1) Synthesis of compound Z2

[0312]

[0313] At 25°C, 25 g of compound Z1 was dissolved in 100 mL of anhydrous pyridine and protected under an argon atmosphere. Then, 32.8 g of 4,4'-dimethoxytriphenylchloromethane was added to the reaction mixture, and the reaction was allowed to proceed for 3 h. The anhydrous pyridine was removed by vacuum distillation. The remaining mixture was dissolved in 500 mL of dichloromethane, washed twice with 300 mL of saturated NaHCO3 each time, and washed once with 350 mL of saturated brine. The mixture was dried over anhydrous Na2SO4, filtered, and the organic phase filtrate was concentrated. The resulting residue was subjected to silica gel column chromatography [V(petroleum ether):V(ethyl acetate) = 5:1-1:1] gradient elution. The eluent containing the reaction products was collected, and the solvent was evaporated to obtain 43.4 g of compound Z2. m / z (ES+): theoretical, [M+H]+, 561.61; detection, [M+H]+, 561.23.

[0314] (1-2) Synthesis of compound Z3

[0315]

[0316] 30 g of compound Z2 and 7.7 g of imidazole were added to 150 mL of N,N-dimethylformamide, followed by 29.5 g of tert-butyldiphenylchlorosilane (TBDPSCl). The reaction was allowed to proceed for 5 h. Then, 50 mL of saturated sodium bicarbonate solution and 500 mL of ethyl acetate were added to the reaction solution. The resulting organic phase was washed once with 300 mL of saturated brine and once with 300 mL of 5 wt% citric acid aqueous solution. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain 42.75 g of compound Z3, which was used directly in subsequent reactions without further purification. m / z (ES+): theoretical, [M+H]+, 800.02; detection, [M+H]+, 799.64

[0317] (1-3) Synthesis of compound Z4

[0318]

[0319] 42.75 g of compound Z3 was added to 100 mL of dichloromethane. The reaction solution was cooled to -5 °C in an ice bath. Then, 300 mL of buffer B (a mixed solution of 2% p-toluenesulfonic acid hydrate, 28% methanol, and 70% dichloromethane) was added to the reaction solution. After reacting for 30 min, 300 mL of dichloromethane and 150 mL of saturated sodium bicarbonate solution were added to the reaction solution. The mixture was separated, and the organic phase was washed with 30 mL of saturated brine and concentrated. Then, it was dissolved in 80 mL of ethyl acetate. 200 mL of petroleum ether was added to the solution at room temperature, and the mixture was stirred for 30 min. The filtrate was removed by suction filtration, and the residual solid was dried to give 14.5 g of compound Z4. m / z(ES+): theoretical, [M+H] + 497.64, detection, [M+H]+, 497.80

[0320] (1-4) Synthesis of compound Z5

[0321]

[0322] Under argon protection, 10.4 g of compound Z4 was added to 120 mL of dichloromethane, followed by 34 g of Des Martin periodane (DMP). The reaction was allowed to proceed for 30 min. Then, 300 mL of saturated sodium thiosulfate solution and 500 mL of ethyl acetate were added to the reaction mixture. After stirring for 30 min, the mixture was allowed to stand and extracted separately. The organic phase was washed once with 100 mL of saturated sodium bicarbonate solution and once with 100 mL of brine. After drying with anhydrous Na2SO4 at room temperature and filtering, the solvent was removed by vacuum distillation to obtain 9.8 g of compound Z5. m / z (ES+): theoretical, [M+NH4]+, 512.66; detection, [M+NH4]+, 512.80

[0323] (1-5) Synthesis of compound Z6

[0324]

[0325] Under argon protection at 0℃, 13.9 g of triphenylphosphine and 38 g of carbon tetrabromide were added to 100 mL of dichloromethane, respectively. After stirring for 30 min, 9.8 g of compound Z5 was added, and the reaction was allowed to proceed for 2 h. Then, 5 mL of saturated ammonium chloride solution and 100 mL of dichloromethane were added to the reaction mixture, and the mixture was stirred for 30 min. The mixture was separated, and the resulting organic phase was concentrated under reduced pressure to remove the solvent. The solution was then purified by column chromatography [V(petroleum ether):V(ethyl acetate) = 20:1]. The eluent containing the reaction product was collected, and the solvent was evaporated to obtain 4.68 g of compound Z6. m / z(ES+): theoretical, [M+H] + 651.44 detection, [M+H] + 651.50

[0326] (1-6) Synthesis of compound Z7

[0327]

[0328] 1.8 g of dimethyl phosphate, 1.1 g of triethylamine, and 4.68 g of compound Z6 were added separately to 50 mL of N,N-dimethylformamide solution and reacted at room temperature for 2 h. 150 mL of ethyl acetate was added to the reaction solution, followed by washing three times with 300 mL of 5 wt% sodium bicarbonate solution each time, and twice with 100 mL of saturated brine each time. The organic phase was dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography [V(petroleum ether):V(ethyl acetate) = 1:1]. The eluent containing the reaction product was collected, and the solvent was evaporated to give 2.4 g of compound Z7. m / z(ES+): theoretical, [M+H] + 572.54, Detection, [M+H] + 572.7

[0329] (1-7) Synthesis of compound Z8

[0330]

[0331] Under argon protection, 2.4 g of compound Z7 and 4.6 mL of tetrabutylammonium fluoride (TBAF) were added to 25 mL of tetrahydrofuran, and the reaction was carried out at room temperature for 2 h. The reaction solution was concentrated and purified by column chromatography [V(petroleum ether):V(ethyl acetate) = 1:1] to remove the solvent, yielding 1.0 g of compound Z8. m / z (ES+): theoretical, [M+H] + 334.14, Detection, [M+H] + 334.80

[0332] (1-8) Synthesis of compound S2

[0333]

[0334] Under argon protection, 5.0 g of compound S1 and 0.8 g of tetrazolium were added to 50 mL of anhydrous acetonitrile and reacted at room temperature for 10 min. Then, 8.5 mL of water was added, and the reaction continued for 1.0 h. 500 mL of dichloromethane and 300 mL of saturated sodium bicarbonate aqueous solution were added to the reaction solution, and the mixture was stirred for 5 min and allowed to stand for phase separation. The resulting organic phase was washed once with 100 mL of saturated saline solution, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and dried under vacuum to obtain 4.6 g of compound S2. m / z (ES+): theoretical, [M+H]+: 778.30, detection, [M+H]+: 778.89.

[0335] (1-9) Synthesis of compound Z9

[0336]

[0337] 0.32 g of compound Z8, 0.1 g of compound S2, 0.068 g of 1,1'-bis(diphenylphosphine)ferrocene, 800 μL of propylene oxide, and 0.017 g of palladium acetate were added to 10 mL of tetrahydrofuran and reacted under microwave at 70 °C for 1.5 h. Then, 300 mL of saturated sodium bicarbonate aqueous solution and 200 mL of ethyl acetate were added, stirred for 5 min, and allowed to stand for phase separation. The aqueous phase was extracted with 300 mL of ethyl acetate, and the organic phases were combined. The resulting organic phase was washed once with 300 mL of saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by gradient elution on a silica gel column (V(dichloromethane):V(methanol) = 50:1-40:1), and dried under vacuum to obtain 92 mg of compound Z9. m / z (ES+): theoretical, [M+H] + 1030.37, Detection, [M+H] +:1031.12.

[0338] (1-10) Synthesis of compound Z10

[0339]

[0340] 0.65 g of compound Z9, 0.326 g of N,N-diisopropylethylamine, and 0.224 g of 3-((chloro(diisopropylamino)phosphono)oxy)propionitrile) were added to 5 mL of dichloromethane and reacted at room temperature for 3.0 h. 300 mL of saturated sodium bicarbonate aqueous solution was added to the reaction solution and stirred for 1 h. After standing and separating the layers, the aqueous phase was extracted with 300 mL of dichloromethane, and the organic phases were combined. The resulting organic phase was washed once with 100 mL of saturated brine, dried with anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by silica gel column chromatography using a mixed eluent [V(dichloromethane):V(methanol) = 50:1-30:1] as the mobile phase. The eluent containing the reaction product was collected, and the solvent was evaporated to obtain 300 mg of compound Z10. The structure of the obtained compound Z10 is shown in formula (3011). 1 H NMR(300MHz,DMSO)δ11.46(s,2H),7.65(dd,J=13.0,8.1Hz,3H),7.42–7.16(m,9H),6.89(d,J=8.9Hz,4H) ,6.15–5.99(m,1H),5.79(dd,J=12.3,6.3Hz,2H),5.68–5.60(m,1H),5.43–5.36(m,1H),4.81(d,J=4.5Hz ,1H),4.51(d,J=5.1Hz,4H),4.25–4.04(m,5H),3.78(s,3H),3.74(s,5H),3.58(d,J=6.0Hz,3H),3.41–3. 35(m,6H),3.33(s,6H),2.84(dt,J=26.0,5.8Hz,4H),1.27–1.08(m,12H),0.82(s,9H),0.09–0.01(m,6H). 1 P NMR (122MHz, DMSO) δ150.4,,150.3,140.0,139.7,18.9,18.9,18.5,18.5.

[0341] (1-11) Synthesis of the positive strand of siRNA1

[0342] Nucleoside monomers were sequentially ligated from 3' to 5' along the positive strand nucleotide arrangement of siRNA1 in Table 1 using a solid-phase phosphoramide method. Each ligation of a nucleoside monomer involved four steps: deprotection, coupling, capping, and oxidation or sulfidation. Specifically, when two nucleotides were linked using a phosphate ester, the ligation of the subsequent nucleoside monomer involved deprotection, coupling, capping, and oxidation. When two nucleotides were linked using a thiophosphate ester, the ligation of the subsequent nucleoside monomer involved protection, coupling, capping, and sulfidation. The synthetic conditions are given below:

[0343] The nucleoside monomer was provided in a 0.1 M acetonitrile solution. The deprotection reaction conditions were the same for each step: 25 °C, 70 seconds, and the deprotection reagent was a dichloromethane solution of dichloroacetic acid (3% v / v). The molar ratio of dichloroacetic acid to the 4,4'-dimethoxytriphenylmethyl protecting group on the solid support was 5:1.

[0344] The coupling reaction conditions were identical for each step, including a temperature of 25°C, a molar ratio of nucleic acid sequence to nucleoside monomer linked on the solid-phase support of 1:10, a molar ratio of nucleic acid sequence to coupling reagent linked on the solid-phase support of 1:65, a reaction time of 600 seconds, and a 0.5 M acetonitrile solution of 5-ethylthio-1H-tetrazole.

[0345] Each capping step was performed under identical conditions, including a temperature of 25°C and a reaction time of 15 seconds. The capping reagent solution was a 1:1 molar mixture of CapA and CapB, and the molar ratio of the capping reagent to the nucleic acid sequence linked on the solid-phase support was acetic anhydride:N-methylimidazole:nucleic acid sequence linked on the solid-phase support = 1:1:1.

[0346] Each oxidation step was performed under identical conditions, including a temperature of 25°C, a reaction time of 15 seconds, and 0.05M iodine solution as the oxidizing agent. The molar ratio of iodine to the nucleic acid sequence linked on the solid-phase support in the coupling step was 30:1. The reaction was carried out in a mixed solvent of tetrahydrofuran:water:pyridine = 3:1:1.

[0347] The conditions for each sulfidation reaction were identical, including a temperature of 25°C, a reaction time of 300 seconds, and the use of hydroflavin as the sulfidation reagent. The molar ratio of the sulfidation reagent to the nucleic acid sequence linked on the solid-phase support in the coupling step was 120:1. The reaction was carried out in a mixed solvent of acetonitrile and pyridine in a ratio of 1:1.

[0348] The cleavage and deprotection conditions are as follows: The synthesized nucleotide sequence linked to the vector is added to ammonia water with a concentration of 25 wt% (0.5 ml / μmol), and reacted at 55 °C for 16 h. The liquid is then removed and the mixture is concentrated to dryness under vacuum.

[0349] Purification and Desalting: Nucleic acid purification was achieved using a preparative ion chromatography column (Source 15Q) with gradient elution using NaCl. Specifically: Eluent A: 20 mM sodium phosphate (pH 8.1), solvent: water / acetonitrile = 9:1 (v / v); Eluent B: 1.5 M sodium chloride, 20 mM sodium phosphate (pH 8.1), solvent: water / acetonitrile = 9:1 (v / v); Elution gradient: eluent A: eluent B = 100:0-50:50. The product eluates were collected and combined, then desalted using a reversed-phase chromatography column. Specific conditions included using a dextran gel column (g25 packing material) and elution with deionized water.

[0350] Detection: Purity was determined using ion exchange chromatography (IEX-HPLC), and molecular weight was analyzed using liquid chromatography-mass spectrometry (LC-MS).

[0351] (1-12) Synthesis of the antisense strand of siRNA1

[0352] Using the solid-phase phosphorus amide method, a universal solid support (UnyLinker) was employed. TM loaded Starting with HLSolid Supports (Kinovate Life Sciences), the antisense strand of siRNA1 was synthesized according to the antisense strand composition in Table 1. Nucleoside monomers were linked one by one from the 3'-5' direction. When linking the last nucleoside monomer, the compound of formula Z10 prepared in steps (1-10) above was used. The deprotection, coupling, capping, oxidation or sulfidation reaction conditions, cleavage and deprotection, purification and desalting conditions in the solid-phase synthesis method were the same as those for the synthesis of the sense strand.

[0353] (1-13) Annealing

[0354] The sense and antisense strands were mixed in an equimolar ratio, dissolved in water for injection, and heated to 95°C. After slowly cooling to room temperature, the two single strands formed a double-stranded structure through hydrogen bonding. Following the above synthesis, purity was determined using ion-exchange chromatography (IEX-HPLC), and molecular weight was determined using liquid chromatography-mass spectrometry (LC-MS) to confirm that the synthesized siRNA was siRNA1 as listed in Table 1.

[0355] Example 2 Preparation of siRNA2

[0356] (2-1) Synthesis of compound P2

[0357]

[0358] Under argon protection, 11.9 g of compound P1 and 2.26 g of tetrazolium were dissolved in 110 mL of anhydrous acetonitrile and reacted at room temperature for 10 min. Then, 21.4 mL of water was added, and the reaction was continued for 1.0 h. 500 mL of dichloromethane and 300 mL of saturated sodium bicarbonate aqueous solution were added to the reaction solution, and the mixture was stirred and allowed to stand for separation. The resulting organic phase was washed once with 100 mL of saturated saline solution, dried over anhydrous ammonium sulfate, filtered, concentrated, and then dried under vacuum to obtain 9.3 g of compound P2.

[0359] (2-2) Synthesis of compound P3

[0360]

[0361] Under argon protection, 2.0 g of compound Z8, 5.96 g of compound P2, 1.33 g of 1,1'-bis(diphenylphosphine)ferrocene (DPPF), and 0.37 g of palladium acetate, obtained according to steps 1-7 of Example 1, were added to 20 mL of tetrahydrofuran, followed by 8 mL of propylene oxide. The mixture was then heated to 70 °C and reacted for 5.0 h. 300 mL of saturated sodium bicarbonate aqueous solution and 200 mL of ethyl acetate were added to the reaction solution, and the mixture was stirred briefly and allowed to stand for separation. The aqueous phase was extracted with 200 mL of ethyl acetate, and the combined organic phases were washed once with 100 mL of saturated saline solution, dried with anhydrous sodium sulfate, filtered, concentrated, purified by silica gel column chromatography [V(dichloromethane):V(methanol) = 80:1-20:1], and the eluent containing the reaction products was collected. The solvent was evaporated and the eluent was dried under vacuum to obtain 4.2 g of compound P3. m / z(ES+): Theoretical, [M+H] + 914.30, Detection, [M+H] + 914.88.

[0362] (2-3) Synthesis of compound P4

[0363]

[0364] Under argon protection, 4 g of compound P3 and 0.3 g of tetrazolium were added to 50 mL of N,N-dimethylformamide. Then, 0.36 g of N-methylimidazole and 1.98 g of bis(diisopropylamino)(2-cyanoethoxy)phosphine were added to the reaction solution, and the reaction was carried out at room temperature for 2.0 h. 300 mL of saturated sodium bicarbonate aqueous solution was added to the reaction solution, and after stirring, the mixture was allowed to stand for separation. The aqueous phase was extracted with 200 mL of dichloromethane, and the organic phases were combined. The organic phase was washed once with 100 mL of saturated saline solution, dried with anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by silica gel column chromatography [V(dichloromethane):V(methanol) = 50:1-30:1]. The eluent containing the reaction product was collected, the solvent was evaporated, and the residue was dried under vacuum to obtain 1.4 g of compound P4. The structure of the obtained compound P4 is shown in formula (3012). 1 H NMR (500MHz, DMSO) δ11.43(d,J=21.1Hz,2H),7.72–7.59(m,1H),7.49(s,1H),7.38(d,J=7.4Hz,2H),7.31(t,J=7.5Hz,2H),7. 28–7.20(m,5H),6.89(dd,J=7.9,4.5Hz,4H),6.29–6.04(m,2H),5.81(dd,J=6.8,3.7Hz,1H),5.66(d,J=8.0Hz,1H),5.12(s,1H ),4.42(dddd,J=60.7,55.6,44.3,31.3Hz,2H),4.22–3.99(m,4H),3.86–3.74(m,2H),3.71(d,J=23.9Hz,6H),3.67–3.52(m,2 H),3.41(s,1H),3.35(s,3H),3.31–3.15(m,2H),2.97–2.87(m,1H),2.85–2.75(m,2H),1.52–1.38(m,3H),1.25–1.05(m,12H). 1 P NMR (202MHz, DMSO) δ149.7,149.6,149.4,149.3,17.6,17.5,17.3,17.2.

[0365] (2-4) Synthesis of the positive strand of siRNA2

[0366] Nucleoside monomers were sequentially ligated from 3' to 5' along the positive strand nucleotide arrangement of siRNA2 in Table 1 using a solid-phase phosphoramide method. Each ligation of a nucleoside monomer involved four steps: deprotection, coupling, capping, and oxidation or sulfidation. Specifically, when two nucleotides were linked using a phosphate ester, the ligation of the subsequent nucleoside monomer involved deprotection, coupling, capping, and oxidation. When two nucleotides were linked using a thiophosphate ester, the ligation of the subsequent nucleoside monomer involved protection, coupling, capping, and sulfidation. The synthetic conditions are given below:

[0367] The nucleoside monomer was provided in a 0.1 M acetonitrile solution. The deprotection reaction conditions were the same for each step: 25 °C, 70 seconds, and the deprotection reagent was a dichloromethane solution of dichloroacetic acid (3% v / v). The molar ratio of dichloroacetic acid to the 4,4'-dimethoxytriphenylmethyl protecting group on the solid support was 5:1.

[0368] The coupling reaction conditions were identical for each step, including a temperature of 25°C, a molar ratio of nucleic acid sequence to nucleoside monomer linked on the solid-phase support of 1:10, a molar ratio of nucleic acid sequence to coupling reagent linked on the solid-phase support of 1:65, a reaction time of 600 seconds, and a 0.5 M acetonitrile solution of 5-ethylthio-1H-tetrazole.

[0369] Each capping step was performed under identical conditions, including a temperature of 25°C and a reaction time of 15 seconds. The capping reagent solution was a 1:1 molar mixture of CapA and CapB, and the molar ratio of the capping reagent to the nucleic acid sequence linked on the solid-phase support was acetic anhydride:N-methylimidazole:nucleic acid sequence linked on the solid-phase support = 1:1:1.

[0370] Each oxidation step was performed under identical conditions, including a temperature of 25°C, a reaction time of 15 seconds, and 0.05M iodine solution as the oxidizing agent. The molar ratio of iodine to the nucleic acid sequence linked on the solid-phase support in the coupling step was 30:1. The reaction was carried out in a mixed solvent of tetrahydrofuran:water:pyridine = 3:1:1.

[0371] The conditions for each sulfidation reaction were identical, including a temperature of 25°C, a reaction time of 300 seconds, and the use of hydroflavin as the sulfidation reagent. The molar ratio of the sulfidation reagent to the nucleic acid sequence linked on the solid-phase support in the coupling step was 120:1. The reaction was carried out in a mixed solvent of acetonitrile and pyridine in a ratio of 1:1.

[0372] The cleavage and deprotection conditions are as follows: The synthesized nucleotide sequence linked to the vector is added to ammonia water with a concentration of 25 wt% (0.5 ml / μmol), and reacted at 55 °C for 16 h. The liquid is then removed and the mixture is concentrated to dryness under vacuum.

[0373] Purification and Desalting: Nucleic acid purification was achieved using a preparative ion chromatography column (Source 15Q) with gradient elution using NaCl. Specifically: Eluent A: 20 mM sodium phosphate (pH 8.1), solvent: water / acetonitrile = 9:1 (v / v); Eluent B: 1.5 M sodium chloride, 20 mM sodium phosphate (pH 8.1), solvent: water / acetonitrile = 9:1 (v / v); Elution gradient: eluent A: eluent B = 100:0-50:50. The product eluates were collected and combined, then desalted using a reversed-phase chromatography column. Specific conditions included using a dextran gel column (g25 packing material) and elution with deionized water.

[0374] Detection: Purity was determined using ion exchange chromatography (IEX-HPLC), and molecular weight was analyzed using liquid chromatography-mass spectrometry (LC-MS).

[0375] (2-5) Synthesize the antisense strand of siRNA2

[0376] Using the solid-phase phosphorus amide method, a universal solid support (UnyLinker) was employed. TM loaded Starting with HLSolid Supports (Kinovate Life Sciences), the antisense strand of siRNA2 was synthesized according to the composition of the antisense strand in Table 1. Nucleoside monomers were linked one by one from the 3'-5' direction. When linking the last nucleoside monomer, the compound of formula P4 prepared in steps (2-3) above was used. The deprotection, coupling, capping, oxidation or sulfidation reaction conditions, cleavage and deprotection, purification and desalting conditions in the solid-phase synthesis method were the same as those for the synthesis of the sense strand.

[0377] (2-6) Annealing

[0378] The sense and antisense strands were mixed in an equimolar ratio, dissolved in water for injection, and heated to 95°C. After slowly cooling to room temperature, the two single strands formed a double-stranded structure through hydrogen bonding. Following the above synthesis, purity was determined using ion-exchange chromatography (IEX-HPLC), and molecular weight was determined using liquid chromatography-mass spectrometry (LC-MS) to confirm that the synthesized siRNA was siRNA2 as listed in Table 1.

[0379] Example 3 Preparation of siRNA3

[0380] (3-1) Synthesis of compound L2

[0381]

[0382] Under argon protection, 3.0 g of compound L1 and 343 mg of tetrazolium were dissolved in 30 mL of anhydrous acetonitrile and reacted at room temperature for 10 min. Then, 5.2 mL of water was added, and the reaction was continued for 1.0 h. 300 mL of dichloromethane and 200 mL of saturated sodium bicarbonate aqueous solution were added to the reaction solution, and the mixture was stirred for 5 min and allowed to stand for separation. The organic phase was washed once with 100 mL of saturated saline solution, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and dried under vacuum to obtain 2.5 g of compound L2. m / z(ES+): theoretical, [M+H] + 538.56, Detection, [M+H] + 538.62.

[0383] (3-2) Synthesis of compound L3

[0384]

[0385] 2.7 g of compound Z8, 1.24 g of compound L2, 827 mg of 1,1'-bis(diphenylphosphine)ferrocene, 154 mg of palladium acetate, and 4.4 g of propylene oxide, obtained according to steps 1-7 of Example 1, were added to 10 mL of tetrahydrofuran and reacted under an argon atmosphere for 3.0 h. Then, 300 mL of saturated sodium bicarbonate aqueous solution and 200 mL of ethyl acetate were added to the reaction solution, stirred for 5 min, and allowed to stand for phase separation. The aqueous phase was extracted with ethyl acetate, and the organic phases were combined and washed once with 100 mL of saturated brine. The solution was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by gradient elution on a silica gel column [V(dichloromethane):V(methanol) = 30:1-20:1]. The eluent containing the reaction product was collected, the solvent was evaporated, and the solution was dried under vacuum to obtain 840 mg of compound L3. m / z (ES+): theoretical, [M+H] + 790.78, Detection, [M+H] + 790.84.

[0386] (3-3) Synthesis of compound L4

[0387]

[0388] At room temperature, 0.84 g of compound L3, 0.416 g of bis(diisopropylamino)(2-cyanoethoxy)phosphine, 0.056 g of tetrazolium, and 0.022 g of azirmonidazole were added to 5 mL of N,N-dimethylformamide, and the reaction was carried out for 1.0 h. Then, 300 mL of saturated sodium bicarbonate aqueous solution and 200 mL of ethyl acetate were added to the reaction solution, stirred briefly, and allowed to stand for separation. The aqueous phase was extracted with ethyl acetate, and the organic phases were combined. The resulting organic phase was washed once with 100 mL of saturated brine, dried with anhydrous sodium sulfate, filtered, concentrated, and the residue was subjected to silica gel column chromatography [V(dichloromethane):V(methanol) = 50:1-30:1] gradient elution. The eluent containing the reaction product was collected, and the solvent was evaporated to obtain 400 mg of compound L4. The structure of the obtained compound L4 is shown in formula (3013). 1 H NMR(500MHz,DMSO)δ11.45(s,1H),7.76–7.59(m,1H),7.46–7.23(m,12H),6.90 (m,5H),6.17-6.06(m,1H),5.82–5.80(m,1H),5.66–5.63(m,1H),4.98–4.81(m, 2H),4.56–4.34(m,2H),4.13-3.99(m,7H),3.88–3.70(m,11H),3.62-3.59(m,3H ),3.41-3.27(m,3H),3.08-2.80(m,6H),2.25-2.03(m,3H),1.18–1.12(m,12H). 1 P NMR (202MHz, DMSO) δ149.5,149.4,149.3,149.2,17.2,17.1,16.9,16.7.

[0389] (3-4) Synthesis of the positive strand of siRNA3

[0390] Nucleoside monomers were sequentially ligated from 3' to 5' along the positive strand nucleotide arrangement of siRNA3 in Table 1 using a solid-phase phosphoramide method. Each ligation of a nucleoside monomer involved four steps: deprotection, coupling, capping, and oxidation or sulfidation. When two nucleotides were linked using a phosphate ester, the ligation of the subsequent nucleoside monomer involved deprotection, coupling, capping, and oxidation. When two nucleotides were linked using a thiophosphate ester, the ligation of the subsequent nucleoside monomer involved protection, coupling, capping, and sulfidation. The synthetic conditions are given below:

[0391] The nucleoside monomer was provided in a 0.1 M acetonitrile solution. The deprotection reaction conditions were the same for each step: 25 °C, 70 seconds, and the deprotection reagent was a dichloromethane solution of dichloroacetic acid (3% v / v). The molar ratio of dichloroacetic acid to the 4,4'-dimethoxytriphenylmethyl protecting group on the solid support was 5:1.

[0392] The coupling reaction conditions were identical for each step, including a temperature of 25°C, a molar ratio of nucleic acid sequence to nucleoside monomer linked on the solid-phase support of 1:10, a molar ratio of nucleic acid sequence to coupling reagent linked on the solid-phase support of 1:65, a reaction time of 600 seconds, and a 0.5 M acetonitrile solution of 5-ethylthio-1H-tetrazole.

[0393] Each capping step was performed under identical conditions, including a temperature of 25°C and a reaction time of 15 seconds. The capping reagent solution was a 1:1 molar mixture of CapA and CapB, and the molar ratio of the capping reagent to the nucleic acid sequence linked on the solid-phase support was acetic anhydride:N-methylimidazole:nucleic acid sequence linked on the solid-phase support = 1:1:1.

[0394] Each oxidation step was performed under identical conditions, including a temperature of 25°C, a reaction time of 15 seconds, and 0.05M iodine solution as the oxidizing agent. The molar ratio of iodine to the nucleic acid sequence linked on the solid-phase support in the coupling step was 30:1. The reaction was carried out in a mixed solvent of tetrahydrofuran:water:pyridine = 3:1:1.

[0395] The conditions for each sulfidation reaction were identical, including a temperature of 25°C, a reaction time of 300 seconds, and the use of hydroflavin as the sulfidation reagent. The molar ratio of the sulfidation reagent to the nucleic acid sequence linked on the solid-phase support in the coupling step was 120:1. The reaction was carried out in a mixed solvent of acetonitrile and pyridine in a ratio of 1:1.

[0396] The cleavage and deprotection conditions are as follows: The synthesized nucleotide sequence linked to the vector is added to ammonia water with a concentration of 25 wt% (0.5 ml / μmol), and reacted at 55 °C for 16 h. The liquid is then removed and the mixture is concentrated to dryness under vacuum.

[0397] Purification and Desalting: Nucleic acid purification was achieved using a preparative ion chromatography column (Source 15Q) with gradient elution using NaCl. Specifically: Eluent A: 20 mM sodium phosphate (pH 8.1), solvent: water / acetonitrile = 9:1 (v / v); Eluent B: 1.5 M sodium chloride, 20 mM sodium phosphate (pH 8.1), solvent: water / acetonitrile = 9:1 (v / v); Elution gradient: eluent A: eluent B = 100:0-50:50. The product eluates were collected and combined, then desalted using a reversed-phase chromatography column. Specific conditions included using a dextran gel column (g25 packing material) and elution with deionized water.

[0398] Detection: Purity was determined using ion exchange chromatography (IEX-HPLC), and molecular weight was analyzed using liquid chromatography-mass spectrometry (LC-MS).

[0399] (3-5) Synthesize the antisense strand of siRNA3

[0400] Using the solid-phase phosphorus amide method, a universal solid support (UnyLinker) was employed. TM loaded Starting with HLSolid Supports (Kinovate Life Sciences), the antisense strand of siRNA1 was synthesized according to the antisense strand composition of siRNA3 in Table 1. Nucleoside monomers were linked one by one from the 3'-5' direction. When linking the last nucleoside monomer, the compound of formula L4 prepared in step (3-3) above was used. The deprotection, coupling, capping, oxidation or sulfidation reaction conditions, cleavage and deprotection, purification and desalting conditions in the solid-phase synthesis method were the same as those for the synthesis of the sense strand.

[0401] (3-6) Annealing

[0402] The sense and antisense strands were mixed in an equimolar ratio, dissolved in water for injection, and heated to 95°C. After slowly cooling to room temperature, the two single strands formed a double-stranded structure through hydrogen bonding. Following the synthesis, purity was determined using ion-exchange chromatography (IEX-HPLC), and molecular weight was analyzed by liquid chromatography-mass spectrometry (LC-MS) to confirm that the synthesized siRNA was siRNA3 as listed in Table 1.

[0403] Example 4: Preparation of siRNA4

[0404] (4-1) Synthesis of compound Y2

[0405]

[0406] Under argon protection, 2.61 g of compound Y1 and 490 mg of tetrazolium were dissolved in 25 mL of anhydrous acetonitrile and reacted at room temperature for 10 min. Then, 4.5 mL of water was added, and the reaction continued for 1.5 h. 200 mL of ethyl acetate and 200 mL of saturated sodium bicarbonate aqueous solution were added to the reaction solution, and the mixture was stirred for 5 min and allowed to stand for separation. The organic phase was washed once with 100 mL of saturated saline solution, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and dried under vacuum to obtain 2.95 g of compound Y2. m / z (ES+): theoretical, [M+H] + 775.58, Detection, [M+H] + 775.64.

[0407] (4-2) Synthesis of compound Y3

[0408]

[0409] At room temperature, 0.84 g of compound Z8, 2.9 g of compound Y2, 560 mg of 1,1'-bis(diphenylphosphine)ferrocene, 2.9 g of propylene oxide, and 113 mg of palladium acetate, obtained according to steps 1-7 of Example 1, were added to 45 mL of tetrahydrofuran and reacted at 70 °C for 5.0 h. 200 mL of saturated sodium bicarbonate aqueous solution and 200 mL of ethyl acetate were added to the reaction solution, stirred for 5 minutes, and allowed to stand for phase separation. The aqueous phase was extracted once with 100 mL of ethyl acetate, and the organic phases were combined. The resulting organic phase was washed once with 100 mL of saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was subjected to silica gel column chromatography [V(dichloromethane):V(methanol) = 30:1-20:1] gradient elution. The eluent containing the reaction products was collected, and the solvent was evaporated to obtain 1.72 g of compound Y3. m / z (ES+): theoretical, [M+H] + 1028.00, Detection, [M+H] + 1028.06.

[0410] (4-3) Synthesis of compound Y4

[0411]

[0412] Under argon protection, 1.36 g of compound Y3, 0.6 g of tetrazolium, 27 mg of N-methylimidazole, and 600 mg of bis(diisopropylamino)(2-cyanoethoxy)phosphine were sequentially added to 15 mL of dimethylformamide. The reaction was carried out at room temperature for 3.0 h. 50 mL of saturated sodium bicarbonate aqueous solution and 70 mL of ethyl acetate were added to the reaction solution, and the mixture was stirred for 5 minutes. After standing and separating the layers, the aqueous phase was extracted with 70 mL of ethyl acetate. The organic phases were combined and washed once with 100 mL of saturated brine. After drying with anhydrous sodium sulfate, the mixture was filtered, concentrated, and the residue was purified by silica gel column chromatography [V(dichloromethane):V(methanol) = 40:1-20:1]. The eluent containing the reaction products was collected, and the solvent was evaporated to obtain 800 mg of compound Y4. The structure of the obtained compound Y4 is shown in formula (3015). 1 H NMR(500MHz,DMSO)δ11.46(d,J=7.6Hz,1H),11.22(s,1H),8.64–8.51(m,2H),8.05(d,J =7.5Hz,2H),7.67(dt,J=14.6,7.2Hz,2H),7.56(t,J=7.7Hz,2H),7.34(t,J=6.5Hz,2H), 7.28–7.10(m,8H),6.95(ddd,J=22.6,19.8,5.7Hz,1H),6.82(dt,J=7.8,5.2Hz,5H),6.5 4(t,J=6.8Hz,1H),6.19(ddd,J=22.2,17.8,8.7Hz,1H),5.89–5.76(m,1H),5.66(t,J=8. 1Hz,1H),5.30(d,J=3.8Hz,1H),4.52(ddd,J=27.3,9.7,4.3Hz,1H),4.43–4.24(m,2H),4 .17(dt,J=8.9,5.7Hz,3H),3.87–3.62(m,9H),3.61(dt,J=12.0,6.8Hz,2H),3.41(d,J=5 .7Hz,1H),3.36(d,J=5.8Hz,2H),3.34(s,3H),3.32–3.21(m,3H),2.93(dd,J=11.1,5.7H z,1H),2.81(t,J=5.3Hz,2H),2.77–2.64(m,1H),1.29–1.21(m,3H),1.18–1.10(m,12H). 1 P NMR (202MHz, DMSO) δ149.6,149.6,149.3,149.3,17.6,17.5,17.3,17.1.

[0413] (4-4) Synthesis of the positive strand of siRNA4

[0414] Nucleoside monomers were sequentially ligated from 3' to 5' along the positive strand nucleotide arrangement of siRNA4 in Table 1 using a solid-phase phosphoramide method. Each ligation of a nucleoside monomer involved four steps: deprotection, coupling, capping, and oxidation or sulfidation. When two nucleotides were linked using a phosphate ester, the ligation of the subsequent nucleoside monomer involved deprotection, coupling, capping, and oxidation. When two nucleotides were linked using a thiophosphate ester, the ligation of the subsequent nucleoside monomer involved protection, coupling, capping, and sulfidation. The synthetic conditions are given below:

[0415] The nucleoside monomer was provided in a 0.1 M acetonitrile solution. The deprotection reaction conditions were the same for each step: 25 °C, 70 seconds, and the deprotection reagent was a dichloromethane solution of dichloroacetic acid (3% v / v). The molar ratio of dichloroacetic acid to the 4,4'-dimethoxytriphenylmethyl protecting group on the solid support was 5:1.

[0416] The coupling reaction conditions were identical for each step, including a temperature of 25°C, a molar ratio of nucleic acid sequence to nucleoside monomer linked on the solid-phase support of 1:10, a molar ratio of nucleic acid sequence to coupling reagent linked on the solid-phase support of 1:65, a reaction time of 600 seconds, and a 0.5 M acetonitrile solution of 5-ethylthio-1H-tetrazole.

[0417] Each capping step was performed under identical conditions, including a temperature of 25°C and a reaction time of 15 seconds. The capping reagent solution was a 1:1 molar mixture of CapA and CapB, and the molar ratio of the capping reagent to the nucleic acid sequence linked on the solid-phase support was acetic anhydride:N-methylimidazole:nucleic acid sequence linked on the solid-phase support = 1:1:1.

[0418] Each oxidation step was performed under identical conditions, including a temperature of 25°C, a reaction time of 15 seconds, and 0.05M iodine solution as the oxidizing agent. The molar ratio of iodine to the nucleic acid sequence linked on the solid-phase support in the coupling step was 30:1. The reaction was carried out in a mixed solvent of tetrahydrofuran:water:pyridine = 3:1:1.

[0419] The conditions for each sulfidation reaction were identical, including a temperature of 25°C, a reaction time of 300 seconds, and the use of hydroflavin as the sulfidation reagent. The molar ratio of the sulfidation reagent to the nucleic acid sequence linked on the solid-phase support in the coupling step was 120:1. The reaction was carried out in a mixed solvent of acetonitrile and pyridine in a ratio of 1:1.

[0420] The cleavage and deprotection conditions are as follows: The synthesized nucleotide sequence linked to the vector is added to ammonia water with a concentration of 25 wt% (0.5 ml / μmol), and reacted at 55 °C for 16 h. The liquid is then removed and the mixture is concentrated to dryness under vacuum.

[0421] Purification and Desalting: Nucleic acid purification was achieved using a preparative ion chromatography column (Source 15Q) with gradient elution using NaCl. Specifically: Eluent A: 20 mM sodium phosphate (pH 8.1), solvent: water / acetonitrile = 9:1 (v / v); Eluent B: 1.5 M sodium chloride, 20 mM sodium phosphate (pH 8.1), solvent: water / acetonitrile = 9:1 (v / v); Elution gradient: eluent A: eluent B = 100:0-50:50. The product eluates were collected and combined, then desalted using a reversed-phase chromatography column. Specific conditions included using a dextran gel column (g25 packing material) and elution with deionized water.

[0422] Detection: Purity was determined using ion exchange chromatography (IEX-HPLC), and molecular weight was analyzed using liquid chromatography-mass spectrometry (LC-MS).

[0423] (4-5) Synthesize the antisense strand of siRNA4

[0424] Using the solid-phase phosphorus amide method, a universal solid support (UnyLinker) was employed. TM loaded Starting with HLSolid Supports (Kinovate Life Sciences), the antisense strand of siRNA1 was synthesized according to the antisense strand composition of siRNA4 in Table 1. Nucleoside monomers were linked one by one from the 3'-5' direction. When linking the last nucleoside monomer, the compound of formula Y4 prepared in step (5-3) above was used. The deprotection, coupling, capping, oxidation or sulfidation reaction conditions, cleavage and deprotection, purification and desalting conditions in the solid-phase synthesis method were the same as those for the synthesis of the sense strand.

[0425] (4-6) Annealing

[0426] The sense and antisense strands were mixed in an equimolar ratio, dissolved in water for injection, and heated to 95°C. After slowly cooling to room temperature, the two single strands formed a double-stranded structure through hydrogen bonding. Following the above synthesis, purity was determined using ion-exchange chromatography (IEX-HPLC), and molecular weight was determined using liquid chromatography-mass spectrometry (LC-MS) to confirm that the synthesized siRNA was siRNA4 as listed in Table 1.

[0427] Example 5: Preparation of siRNA5

[0428] (5-1) Synthesis of compound K2

[0429]

[0430] Under argon protection, 3.75 g of compound K1 and 0.63 g of tetrazolium were added to 28 mL of anhydrous acetonitrile and reacted at room temperature for 10 min. Then, 5 mL of water was added, and the reaction continued for 2 h. 200 mL of ethyl acetate and 200 mL of saturated sodium bicarbonate aqueous solution were added to the reaction mixture, stirred for 5 min, and allowed to stand for separation. The aqueous phase was extracted with ethyl acetate, and the organic phases were combined. The resulting organic phase was washed once with 100 mL of saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain 3.45 g of compound K2. m / z(ES+): theoretical, [M+H] + 751.75, Detection, [M+H] + 751.82.

[0431] (5-2) Synthesis of compound K3

[0432]

[0433] Under argon protection, 964 mg of compound Z8, 3.26 g of compound K2, 641 mg of 1,1'-bis(diphenylphosphine)ferrocene, 3.36 g of propylene oxide, and 129 mg of palladium acetate, obtained according to steps 1-7 of Example 1, were added to 45 mL of tetrahydrofuran. The reaction was carried out at 70 °C for 5.0 h. 200 mL of saturated sodium bicarbonate aqueous solution and 200 mL of ethyl acetate were added to the reaction solution, and the mixture was stirred for 5 minutes and allowed to stand for separation. The aqueous phase was extracted with 100 mL of ethyl acetate, and the organic phases were combined. The obtained organic phase was washed once with 100 mL of saturated brine, dried with anhydrous sodium sulfate, filtered, concentrated, and the residue was subjected to silica gel column chromatography [V(dichloromethane):V(methanol) = 50:1-20:1] gradient elution. The eluent containing the reaction products was collected, and the solvent was evaporated to obtain 1.47 g of compound K3. m / z(ES+): Theoretical, [M+H] + :1003.98, Detection, [M+H] + 1004.04.

[0434] (5-3) Synthesis of compound K4

[0435]

[0436] Under an argon atmosphere, 1.45 g of compound K3, 78.6 mg of tetrazolium, 30 mg of N-methylimidazole, and 655 mg of bis(diisopropylamino)(2-cyanoethoxy)phosphine were added to 15 mL of methylformamide solution and reacted at room temperature for 3.0 h. Then, 50 mL of saturated sodium bicarbonate aqueous solution and 70 mL of ethyl acetate were added to the reaction solution, and the mixture was stirred for 5 minutes and allowed to stand for phase separation. The aqueous phase was extracted with 70 mL of ethyl acetate, and the organic phases were combined. The resulting organic phase was washed once with 100 mL of saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was subjected to silica gel column chromatography [V(dichloromethane):V(methanol) = 50:1-20:1] gradient elution. The eluent containing the reaction products was collected, and the solvent was evaporated to obtain 700 mg of compound K4. The structure of the obtained K4 compound is shown in formula (3014). 1 H NMR (500MHz, DMSO) δ11.44(s,1H),11.29(s,1H),8.16(d,J=7.3Hz,1H),8.00(d,J=7.6Hz,2H),7.72–7.59(m,2H),7.52(t,J=7.7Hz,2H),7.42– 7.18(m,11H),6.89(t,J=8.0Hz,5H),6.23–6.04(m,2H),5.82(t,J=3.6H z,1H),5.71–5.64(m,1H),5.06(s,1H),4.64–4.44(m,1H),4.41(ddd,J= 36.5,11.5,6.3Hz,1H),4.25(d,J=3.7Hz,1H),4.15(dd,J=11.9,5.8Hz,2H),4.08(d,J=6.1Hz,1H),3.85–3.74(m,2H),3.73(s,6H),3.59-3.55 (m,2H),3.40(d,J=1.7Hz,1H),3.36(d,J=1.7Hz,2H),3.32(s,3H),2.91(dd,J=11.0,5.7Hz,1H),2.69(s,1H),2.44(s,1H),1.17–1.09(m,12H). 1 P NMR (202MHz, DMSO) δ149.7,149.6,149.3,149.2,17.6,17.5,17.3,17.3.

[0437] (5-4) Synthesis of the positive strand of siRNA5

[0438] Nucleoside monomers were sequentially ligated from 3' to 5' along the positive strand nucleotide arrangement of siRNA5 in Table 1 using a solid-phase phosphoramide method. Each ligation of a nucleoside monomer involved four steps: deprotection, coupling, capping, and oxidation or sulfidation. Specifically, when two nucleotides were linked using a phosphate ester, the ligation of the subsequent nucleoside monomer involved deprotection, coupling, capping, and oxidation. When two nucleotides were linked using a thiophosphate ester, the ligation of the subsequent nucleoside monomer involved protection, coupling, capping, and sulfidation. The synthetic conditions are given below:

[0439] The nucleoside monomer was provided in a 0.1 M acetonitrile solution. The deprotection reaction conditions were the same for each step: 25 °C, 70 seconds, and the deprotection reagent was a dichloromethane solution of dichloroacetic acid (3% v / v). The molar ratio of dichloroacetic acid to the 4,4'-dimethoxytriphenylmethyl protecting group on the solid support was 5:1.

[0440] The coupling reaction conditions were identical for each step, including a temperature of 25°C, a molar ratio of nucleic acid sequence to nucleoside monomer linked on the solid-phase support of 1:10, a molar ratio of nucleic acid sequence to coupling reagent linked on the solid-phase support of 1:65, a reaction time of 600 seconds, and a 0.5 M acetonitrile solution of 5-ethylthio-1H-tetrazole.

[0441] Each capping step was performed under identical conditions, including a temperature of 25°C and a reaction time of 15 seconds. The capping reagent solution was a 1:1 molar mixture of CapA and CapB, and the molar ratio of the capping reagent to the nucleic acid sequence linked on the solid-phase support was acetic anhydride:N-methylimidazole:nucleic acid sequence linked on the solid-phase support = 1:1:1.

[0442] Each oxidation step was performed under identical conditions, including a temperature of 25°C, a reaction time of 15 seconds, and 0.05M iodine solution as the oxidizing agent. The molar ratio of iodine to the nucleic acid sequence linked on the solid-phase support in the coupling step was 30:1. The reaction was carried out in a mixed solvent of tetrahydrofuran:water:pyridine = 3:1:1.

[0443] The conditions for each sulfidation reaction were identical, including a temperature of 25°C, a reaction time of 300 seconds, and the use of hydroflavin as the sulfidation reagent. The molar ratio of the sulfidation reagent to the nucleic acid sequence linked on the solid-phase support in the coupling step was 120:1. The reaction was carried out in a mixed solvent of acetonitrile and pyridine in a ratio of 1:1.

[0444] The cleavage and deprotection conditions are as follows: The synthesized nucleotide sequence linked to the vector is added to ammonia water with a concentration of 25 wt% (0.5 ml / μmol), and reacted at 55 °C for 16 h. The liquid is then removed and the mixture is concentrated to dryness under vacuum.

[0445] Purification and Desalting: Nucleic acid purification was achieved using a preparative ion chromatography column (Source 15Q) with gradient elution using NaCl. Specifically: Eluent A: 20 mM sodium phosphate (pH 8.1), solvent: water / acetonitrile = 9:1 (v / v); Eluent B: 1.5 M sodium chloride, 20 mM sodium phosphate (pH 8.1), solvent: water / acetonitrile = 9:1 (v / v); Elution gradient: eluent A: eluent B = 100:0-50:50. The product eluates were collected and combined, then desalted using a reversed-phase chromatography column. Specific conditions included using a dextran gel column (g25 packing material) and elution with deionized water.

[0446] Detection: Purity was determined using ion exchange chromatography (IEX-HPLC), and molecular weight was analyzed using liquid chromatography-mass spectrometry (LC-MS).

[0447] (5-5) Synthesis of the antisense strand of siRNA5

[0448] Using the solid-phase phosphorus amide method, a universal solid support (UnyLinker) was employed. TM loaded Starting with HLSolid Supports (Kinovate Life Sciences), the antisense strand of siRNA1 was synthesized according to the antisense strand composition of siRNA3 in Table 1. Nucleoside monomers were linked one by one from the 3'-5' direction. When linking the last nucleoside monomer, the compound of formula K4 prepared in step (4-3) above was used. The deprotection, coupling, capping, oxidation or sulfidation reaction conditions, cleavage and deprotection, purification and desalting conditions in the solid-phase synthesis method were the same as those for synthesizing the sense strand.

[0449] (5-6) Annealing

[0450] The sense and antisense strands were mixed in an equimolar ratio, dissolved in water for injection, and heated to 95°C. After slowly cooling to room temperature, the two single strands formed a double-stranded structure through hydrogen bonding. Following the above synthesis, purity was determined using ion-exchange chromatography (IEX-HPLC), and molecular weight was determined using liquid chromatography-mass spectrometry (LC-MS) to confirm that the synthesized siRNA was siRNA5 as listed in Table 1.

[0451] Example 6 Preparation of siRNA6

[0452] (6-1) Synthesis of compound N2

[0453]

[0454] Under argon protection, 2.08 g of compound N1 was added to 30 mL of N,N-dimethylformamide, followed by 3.88 g of di-tert-butylsilylbis(trifluoromethanesulfonic acid), and the reaction was carried out at 0 °C for 40 min. The resulting reaction mixture was used directly in the next reaction without purification.

[0455] (6-2) Synthesis of compound N3

[0456]

[0457] Under argon protection and in an ice bath, the reaction mixture obtained in step (6-1) was added to 2.4 g of imidazole, and the reaction was allowed to proceed to room temperature for 30 min. Then, 3.26 g of tert-butyldimethylchlorosilane was added, and the reaction was continued for 2 h. 50 mL of sodium bicarbonate solution was added to the reaction mixture, and the aqueous phase was extracted with 500 mL of ethyl acetate. The organic phases were combined, washed once with 100 mL of saturated brine, dried over anhydrous Na₂SO₄, filtered, and concentrated to obtain 4.3 g of compound N₃. m / z(ES⁺): theoretical, [M⁺H] + 515.87, detection, [M+H] + 515.89

[0458] (6-3) Synthesis of compound N4

[0459]

[0460] Under argon protection and ice bath temperature, 10 mL of pyridine was added to 1.5 mL of pyridine hydrogen fluoride solution to form mixture A. 7.5 g of compound N3, prepared according to the method described in (6-2), was dissolved in 70 mL of dichloromethane to form mixture B. Mixture A was added to mixture B and reacted for 2 h. Then, 50 mL of sodium bicarbonate solution was added to the reaction mixture, and the aqueous phase was extracted with 50 mL of ethyl acetate. The organic phases were combined, and the resulting organic phase was washed five times with 50 mL of 5 wt% citric acid aqueous solution, followed by washing once with 100 mL of saturated sodium bicarbonate solution. The organic phase was dried with anhydrous Na2SO4, filtered, and concentrated to obtain 5.77 g of compound N4. m / z(ES+): theoretical, [M+Na] + 397.49, Detection, [M+H] + 397.50.

[0461] (6-4) Synthesis of compound N5

[0462]

[0463] At 25°C, 5.55 g of compound N4 was added to 50 mL of anhydrous pyridine. 4.84 g of 4,4'-dimethoxytriphenylchloromethane was added to the reaction solution, and the reaction was allowed to proceed for 3 h. The solvent was removed by vacuum evaporation. The residue was dissolved in 100 mL of ethyl acetate and 50 mL of 5 wt% citric acid aqueous solution. The resulting organic phase was washed five times with 100 mL of 5 wt% citric acid aqueous solution each time. Finally, the organic phase was washed with 200 mL of saturated NaHCO3 and 100 mL of brine. The organic phase was dried over anhydrous Na2SO4, filtered, and concentrated. The residue was purified by silica gel column chromatography [V(petroleum ether):V(ethyl acetate) = 10:1-7:1] gradient elution. The eluent containing the reaction product was collected, and the solvent was evaporated to obtain 6.23 g of compound N5. m / z (ES+): theoretical, [M+H] + 677.88, Detection, [M+H] + 677.90.

[0464] (6-5) Synthesis of compound N6

[0465]

[0466] Under an argon atmosphere, 5.4 g of compound N5, 432 mg of tetrazolium, 164 mg of N-methylimidazole, and 3.6 g of bis(diisopropylamino)(2-cyanoethoxy)phosphine were added to 50 mL of dimethylformamide, and the reaction was carried out at room temperature for 3.0 h. 100 mL of saturated sodium bicarbonate aqueous solution and 70 mL of ethyl acetate were added to the reaction solution, and the mixture was stirred for 5 minutes and allowed to stand for separation. The aqueous phase was extracted with 200 mL of ethyl acetate, and the organic phases were combined. The resulting organic phase was washed once with 100 mL of saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by silica gel column chromatography [V(petroleum ether):V(dichloromethane):V(ethyl acetate) = 10:1-7:1] gradient elution. The eluent containing the reaction products was collected, the solvent was evaporated, and the residue was dried under vacuum to give 6.2 g of compound N6. m / z(ES+): theoretical, [M+H] + 878.10, Detection, [M+H] + 878.15.

[0467] (6-6) Synthesis of compound N7

[0468]

[0469] Under argon protection, 6.2 g of compound N6 and 996 mg of tetrazolium were added to 50 mL of anhydrous acetonitrile and reacted at room temperature for 10 minutes. Then, 9 mL of water was added, and the reaction continued for 2 hours. 200 mL of ethyl acetate and 200 mL of saturated sodium bicarbonate aqueous solution were added to the reaction mixture, and after stirring for 5 minutes, the mixture was allowed to stand for separation. The organic phase was washed once with 100 mL of saturated saline solution, dried over anhydrous ammonium sulfate, filtered, concentrated under reduced pressure, and dried under vacuum using an oil pump to obtain 5.38 g of compound N7. m / z(ES+): theoretical, [M+Na] + 816.91, detection, [M+Na] + 816.96.

[0470] (6-7) Synthesis of compound N8

[0471]

[0472] Under argon protection, 1.38 g of compound Z8, 4.94 g of compound N7, 920 mg of 1,1'-bis(diphenylphosphine)ferrocene, 4.8 g of propylene oxide, and 186 mg of palladium acetate, obtained according to steps 1-7 of Example 1, were added to 45 ml of tetrahydrofuran and reacted at 70 °C for 5.0 h. Then, 200 ml of saturated sodium bicarbonate aqueous solution and 200 ml of ethyl acetate were added, stirred for 5 minutes, and allowed to stand for phase separation. After extraction of the aqueous phase with 100 mL of ethyl acetate, the organic phases were combined and washed once with 100 mL of saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and dried under vacuum. The residue was purified by silica gel column chromatography with a gradient elution of [V(dichloromethane):V(dioxane = 17:1-4:1]]. The eluent containing the reaction products was collected, the solvent was evaporated, and the residue was dried under vacuum to give 2.6 g of compound N8. m / z (ES+): theoretical, [M+H] + :1047.15, Detection, [M+H] + 1047.21.

[0473] (6-8) Synthesis of compound N9

[0474]

[0475] Under an argon atmosphere, 1.3 g of compound N8, 67 mg of tetrazolium, 25 mg of N-methylimidazole, and 561 mg of bis(diisopropylamino)(2-cyanoethoxy)phosphine were sequentially added to 15 mL of dimethylformamide solution, and the reaction was carried out at room temperature for 3.0 h. 50 mL of saturated sodium bicarbonate aqueous solution and 70 mL of ethyl acetate were added to the reaction solution, and the mixture was stirred for 5 minutes and allowed to stand for separation. The aqueous phase was extracted with ethyl acetate, and the organic phases were combined. The resulting organic phase was washed with 100 mL of saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was eluted using a silica gel column (V(petroleum ether):V(ethyl acetate) = 1:1-0:1). The eluent containing the reaction products was collected, the solvent was evaporated, and the residue was dried under vacuum to obtain 460 mg of compound N9. The structure of the obtained N9 compound is shown in formula (3016). 1 H NMR (500MHz, DMSO) δ11.45(s,1H),7.62(ddd,J=29.8,18.7,6.1Hz,2H),7.42(d,J=7.6Hz,2H),7.26(dt,J=27.2,9.8Hz,9H),6.93–6.82(m,5H) ,6.12–5.99(m,1H),5.81(d,J=2.7Hz,1H),5.63(dd,J=8.0,2.9Hz,1H),4.96(d,J=6.1Hz,1H),4.75(d,J=32.4Hz,1H),4.55–4.41(m,1H),4.37 –4.27(m,2H),4.11(dd,J=12.5,8.0Hz,2H),3.82–3.71(m,8H),3.62(dd,J=11.0,6.4Hz,2H),3.41(d,J=5.5Hz,1H),3.37(d,J=5.7Hz,2H),3.3 4(s,3H),2.90–2.75(m,4H),1.99(s,3H),1.14(dd,J=14.0,6.9Hz,12H),0.73(d,J=18.2Hz,9H),-0.06(s,3H),-0.21(dd,J=15.6,4.3Hz,3H). 1 P NMR (202MHz, DMSO) δ149.5,149.4,149.4,149.2,18.2,17.9,17.8,17.5. 19 F NMR (471MHz, DMSO) δ-113.8,-113.8,-113.8,-113.8,-118.9,-118.9,-119.0,-119.0.

[0476] (6-9) Synthesis of the positive strand of siRNA6

[0477] Nucleoside monomers were sequentially ligated from 3' to 5' along the positive strand nucleotide arrangement of siRNA6 in Table 1 using a solid-phase phosphoramide method. Each ligation of a nucleoside monomer involved four steps: deprotection, coupling, capping, and oxidation or sulfidation. Specifically, when two nucleotides were linked using a phosphate ester, the ligation of the subsequent nucleoside monomer involved deprotection, coupling, capping, and oxidation. When two nucleotides were linked using a thiophosphate ester, the ligation of the subsequent nucleoside monomer involved protection, coupling, capping, and sulfidation. The synthetic conditions are given below:

[0478] The nucleoside monomer was provided in a 0.1 M acetonitrile solution. The deprotection reaction conditions were the same for each step: 25 °C, 70 seconds, and the deprotection reagent was a dichloromethane solution of dichloroacetic acid (3% v / v). The molar ratio of dichloroacetic acid to the 4,4'-dimethoxytriphenylmethyl protecting group on the solid support was 5:1.

[0479] The coupling reaction conditions were identical for each step, including a temperature of 25°C, a molar ratio of nucleic acid sequence to nucleoside monomer linked on the solid-phase support of 1:10, a molar ratio of nucleic acid sequence to coupling reagent linked on the solid-phase support of 1:65, a reaction time of 600 seconds, and a 0.5 M acetonitrile solution of 5-ethylthio-1H-tetrazole.

[0480] Each capping step was performed under identical conditions, including a temperature of 25°C and a reaction time of 15 seconds. The capping reagent solution was a 1:1 molar mixture of CapA and CapB, and the molar ratio of the capping reagent to the nucleic acid sequence linked on the solid-phase support was acetic anhydride:N-methylimidazole:nucleic acid sequence linked on the solid-phase support = 1:1:1.

[0481] Each oxidation step was performed under identical conditions, including a temperature of 25°C, a reaction time of 15 seconds, and 0.05M iodine solution as the oxidizing agent. The molar ratio of iodine to the nucleic acid sequence linked on the solid-phase support in the coupling step was 30:1. The reaction was carried out in a mixed solvent of tetrahydrofuran:water:pyridine = 3:1:1.

[0482] The conditions for each sulfidation reaction were identical, including a temperature of 25°C, a reaction time of 300 seconds, and the use of hydroflavin as the sulfidation reagent. The molar ratio of the sulfidation reagent to the nucleic acid sequence linked on the solid-phase support in the coupling step was 120:1. The reaction was carried out in a mixed solvent of acetonitrile and pyridine in a ratio of 1:1.

[0483] The cleavage and deprotection conditions are as follows: The synthesized nucleotide sequence linked to the vector is added to ammonia water with a concentration of 25 wt% (0.5 ml / μmol), and reacted at 55 °C for 16 h. The liquid is then removed and the mixture is concentrated to dryness under vacuum.

[0484] Purification and Desalting: Nucleic acid purification was achieved using a preparative ion chromatography column (Source 15Q) with gradient elution using NaCl. Specifically: Eluent A: 20 mM sodium phosphate (pH 8.1), solvent: water / acetonitrile = 9:1 (v / v); Eluent B: 1.5 M sodium chloride, 20 mM sodium phosphate (pH 8.1), solvent: water / acetonitrile = 9:1 (v / v); Elution gradient: eluent A: eluent B = 100:0-50:50. The product eluates were collected and combined, then desalted using a reversed-phase chromatography column. Specific conditions included using a dextran gel column (g25 packing material) and elution with deionized water.

[0485] Detection: Purity was determined using ion exchange chromatography (IEX-HPLC), and molecular weight was analyzed using liquid chromatography-mass spectrometry (LC-MS).

[0486] (6-10) Synthesize the antisense strand of siRNA6

[0487] Using the solid-phase phosphorus amide method, a universal solid support (UnyLinker) was employed. TM loaded Starting with HLSolid Supports (Kinovate Life Sciences), the antisense strand of siRNA1 was synthesized according to the composition of the antisense strand of siRNA6 in Table 1. Nucleoside monomers were linked one by one from the 3'-5' direction. When linking the last nucleoside monomer, the compound of formula N9 prepared in steps (6-8) above was used. The deprotection, coupling, capping, oxidation or sulfidation reaction conditions, cleavage and deprotection, purification and desalting conditions in the solid-phase synthesis method were the same as those for the synthesis of the sense strand.

[0488] (6-11) Annealing

[0489] The sense and antisense strands were mixed in an equimolar ratio, dissolved in water for injection, and heated to 95°C. After slowly cooling to room temperature, the two single strands formed a double-stranded structure through hydrogen bonding. Following the above synthesis, purity was determined using ion-exchange chromatography (IEX-HPLC), and molecular weight was determined using liquid chromatography-mass spectrometry (LC-MS), confirming that the synthesized siRNA was siRNA6 as listed in Table 1.

[0490] Example 7 Preparation of siRNA8

[0491] siRNA8 was prepared according to the preparation method described in Example 1, except that nucleoside monomers were linked one by one from the 3'-5' direction according to the nucleotide arrangement sequence shown in Table 1 for the sense and antisense strands of siRNA8. When linking the last nucleoside monomer of the antisense strand, the compound of formula Z10 prepared according to steps (1-10) in Example 1 was used.

[0492] Following the method described in steps (1-13) of Example 1, the sense and antisense strands were mixed in an equimolar ratio, dissolved in water for injection, and heated to 95°C. After slowly cooling to room temperature, the two single strands formed a double-stranded structure through hydrogen bonding. After the above synthesis was completed, the purity was detected by ion exchange chromatography (IEX-HPLC), and the molecular weight was determined by liquid chromatography-mass spectrometry (LC-MS), confirming that the synthesized siRNA was siRNA8 in Table 1.

[0493] Example 8 Preparation of siRNA9

[0494] siRNA9 was prepared according to the preparation method described in Example 2, except that nucleoside monomers were linked one by one from the 3'-5' direction according to the nucleotide arrangement sequence shown in Table 1 for the sense and antisense strands of siRNA9. When linking the last nucleoside monomer of the antisense strand, the compound of formula P4 prepared according to steps (2-3) of Example 2 was used.

[0495] Following the method described in steps (2-6) of Example 2, the sense and antisense strands were mixed in an equimolar ratio, dissolved in water for injection, and heated to 95°C. After slowly cooling to room temperature, the two single strands formed a double-stranded structure through hydrogen bonding. After the above synthesis was completed, the purity was detected by ion exchange chromatography (IEX-HPLC), and the molecular weight was determined by liquid chromatography-mass spectrometry (LC-MS), confirming that the synthesized siRNA was siRNA9 as listed in Table 1.

[0496] Example 9 Preparation of siRNA15

[0497] siRNA15 was prepared according to the preparation method described in Example 1, except that nucleoside monomers were linked one by one from the 3'-5' direction according to the nucleotide arrangement sequence shown in Table 1 for the sense and antisense strands of siRNA15. When linking the last nucleoside monomer of the antisense strand, the compound of formula Z10 prepared in steps (1-10) of Example 1 was used.

[0498] Following the method described in steps (1-13) of Example 1, the sense and antisense strands were mixed in an equimolar ratio, dissolved in water for injection, and heated to 95°C. After slowly cooling to room temperature, the two single strands formed a double-stranded structure through hydrogen bonding. After the above synthesis was completed, the purity was detected by ion exchange chromatography (IEX-HPLC), and the molecular weight was determined by liquid chromatography-mass spectrometry (LC-MS), confirming that the synthesized siRNA was siRNA15 as listed in Table 1.

[0499] Example 10 Preparation of siRNA16

[0500] siRNA16 was prepared according to the preparation method described in Example 2, except that nucleoside monomers were linked one by one from the 3'-5' direction according to the nucleotide arrangement sequence shown in Table 1 for the sense and antisense strands of siRNA16. When linking the last nucleoside monomer of the antisense strand, the compound shown in formula P4 prepared in steps (2-3) of Example 2 was used.

[0501] Following the method described in steps (2-6) of Example 2, the sense and antisense strands were mixed in an equimolar ratio, dissolved in water for injection, and heated to 95°C. After slowly cooling to room temperature, the two single strands formed a double-stranded structure through hydrogen bonding. After the above synthesis was completed, the purity was detected by ion exchange chromatography (IEX-HPLC), and the molecular weight was determined by liquid chromatography-mass spectrometry (LC-MS), confirming that the synthesized siRNA was siRNA16 in Table 1.

[0502] Example 11 Synthesis of Conjugate 1, Conjugate 8 and Conjugate 15

[0503] Following the method for preparing "Conjugate 1" in Preparation Example 1 of WO2019 / 105437A1, Conjugate 1, Conjugate 8, and Conjugate 15 of this disclosure were prepared respectively, with the difference being that: nucleoside monomers were sequentially linked from 3' to 5' according to the nucleotide arrangement order of the sense and antisense strands of siRNA1, siRNA8, and siRNA15 in Table 1; when linking the last nucleotide of the antisense strand (i.e., the 5' terminal nucleotide), the nucleoside phosphoramide monomer used for linking this nucleotide was replaced with Z10 prepared according to steps (1-10) of Example 1 of this disclosure. Molecular weight was determined by liquid chromatography-mass spectrometry (LC-MS). The results showed that the theoretical value of the sense strand of Conjugate 1 was 7516.5, and the measured value was 7515.3; the theoretical value of the antisense strand was 7287.8, and the measured value was 7286.0. The measured values ​​are consistent with the theoretical values, thus confirming that the synthesized conjugate 1 is the target-designed double-stranded nucleic acid sequence containing the group shown in formula (501); the theoretical value of the sense strand of conjugate 8 is 7605.4, and the measured value of the sense strand is 7604.3; the theoretical value of the antisense strand is 7229.8, and the measured value of the antisense strand is 7228.7. The measured values ​​are consistent with the theoretical values, thus confirming that the synthesized conjugate 8 is the target-designed double-stranded nucleic acid sequence containing the group shown in formula (501); the theoretical value of the sense strand of conjugate 15 is 7584.5, and the measured value of the sense strand is 7583.5; the theoretical value of the antisense strand is 7233.7, and the measured value of the antisense strand is 7233.6. The measured values ​​are consistent with the theoretical values, thus confirming that the synthesized conjugate 15 is the target-designed double-stranded nucleic acid sequence containing the group shown in formula (501). The structures of conjugate 1, conjugate 8, and conjugate 15 are shown in formula (801):

[0504]

[0505] In formula (801), Nu is siRNA1, siRNA8 or siRNA15 in Table 1 of this publication.

[0506] Example 12 Synthesis of Conjugate 2, Conjugate 9 and Conjugate 16

[0507] Conjugate 2 of this disclosure was prepared according to the method of "Conjugate 1" in Example 1 of WO2019 / 105437A1, with the difference that: nucleoside monomers were linked one by one from the 3'-5' direction according to the nucleotide arrangement order of the sense and antisense strands of siRNA2, siRNA9, and siRNA16 in Table 1; when linking the last nucleotide of the antisense strand (i.e., the 5' terminal nucleotide), this nucleotide was replaced with compound P4 prepared according to steps (2-3) of Example 2 of this disclosure. Molecular weight was determined by liquid chromatography-mass spectrometry (LC-MS). The results showed that the theoretical value of the sense strand of conjugate 2 was 7516.5, and the measured value was 7515.3; the theoretical value of the antisense strand was 7285.8, and the measured value was 7284.6. The measured values ​​are consistent with the theoretical values, thus confirming that the synthesized conjugate 2 is the target-designed double-stranded nucleic acid sequence containing the group shown in formula (502); the theoretical value of the sense strand of conjugate 2 is 7605.4, and the measured value of the sense strand is 7604.3; the theoretical value of the antisense strand is 7227.8, and the measured value of the antisense strand is 7226.6. The measured values ​​are consistent with the theoretical values, thus confirming that the synthesized conjugate 9 is the target-designed double-stranded nucleic acid sequence containing the group shown in formula (502); the theoretical value of the sense strand of conjugate 16 is 7584.5, and the measured value of the sense strand is 7583.5; the theoretical value of the antisense strand is 7233.7, and the measured value of the antisense strand is 7232.6. The measured values ​​are consistent with the theoretical values, thus confirming that the synthesized conjugate 16 is the target-designed double-stranded nucleic acid sequence containing the group shown in formula (502). The structures of conjugate 2, conjugate 9, and conjugate 16 are shown in formula (802) below:

[0508]

[0509] In formula (802), Nu is siRNA2, siRNA9 or siRNA16 in Table 1 of this publication.

[0510] Example 13 Synthesis of conjugates 3-6

[0511] Conjugates 3 to 6 of this disclosure were obtained according to the method of "Conjugate 1" in Example 1 of WO2019 / 105437A1, with the only difference being that when connecting the last nucleotide (i.e., the 5' terminal nucleotide) of the antisense strand, the nucleotide was replaced with compound L4 prepared according to step (3-3) of Example 3 of this disclosure, compound Y4 prepared according to step (4-3) of Example 4 of this disclosure, compound K4 prepared according to step (5-3) of Example 5 of this disclosure, or compound N4 prepared according to step (6-3) of Example 6 of this disclosure. As a result, the theoretical value of the sense strand of conjugate 3 was 7516.5, and the measured value of the sense strand was 7515.3; the theoretical value of the antisense strand was 7161.7, and the measured value of the antisense strand was 7160.7. The measured values ​​are consistent with the theoretical values, thus confirming that the synthesized conjugate 3 is the target-designed double-stranded nucleic acid sequence containing the group shown in formula (503); the theoretical value of the sense strand of conjugate 4 is 7516.5, and the measured value of the sense strand is 7515.3; the theoretical value of the antisense strand is 7294.8, and the measured value of the antisense strand is 7293.8. The measured values ​​are consistent with the theoretical values, thus confirming that the synthesized conjugate 4 is the target-designed double-stranded nucleic acid sequence containing the group shown in formula (504); the theoretical value of the sense strand of conjugate 5 is 7516.5, and the measured value of the sense strand is 7515.3; the theoretical value of the antisense strand is 7270.8, and the measured value of the antisense strand is 7270.7. The measured values ​​are consistent with the theoretical values, thus confirming that the synthesized conjugate 5 is the target-designed double-stranded nucleic acid sequence containing the group shown in formula (505); the theoretical value of the sense strand of conjugate 6 is 7516.5, and the measured value of the sense strand is 7515.3; the theoretical value of the antisense strand is 7303.8, and the measured value of the antisense strand is 7302.8. The measured values ​​are consistent with the theoretical values, thus confirming that the synthesized conjugate 6 is the target-designed double-stranded nucleic acid sequence containing the group shown in formula (506). The structures of conjugates 3, 4, 5, and 6 are shown in formula (802).

[0512]

[0513] In formula (802), Nu is siRNA3, siRNA4, siRNA5 or siRNA6 in Table 1 of this publication.

[0514] Synthesis of reference conjugates 1-3 in Comparative Example 1

[0515] Reference conjugates 1, 2, and 3 were obtained according to the method described in Example 1 of WO2019 / 105437A1, with the only difference being that nucleoside monomers were sequentially linked from the 3'-5' direction according to the nucleotide arrangement sequence shown for the sense and antisense strands of siRNA22, siRNA23, and siRNA24 below. The structures of reference conjugates 1-3 are shown in formula (803) below:

[0516]

[0517] For reference conjugate 1, Nu in formula (803) is siRNA22 with the following composition:

[0518] siRNA22

[0519] Chain of Justice: CmsCmsUmUmGmAmGfGfCfAmUmAmCmUmUmCmAmAmAm(SEQ ID NO:25)

[0520] Antonym chain: VPUmsUfsUmGmAmAfGmUmAmUmGmCmCmUfCmAfAmGmGmsUmsUm(SEQ ID NO:26)

[0521] Theoretical value for the sense strand: 7516.5, measured value for the sense strand: 7515.3; theoretical value for the antisense strand: 7061.7, measured value for the antisense strand: 7060.7. The measured values ​​are consistent with the theoretical values, thus confirming that the synthesized reference conjugate 1 is the target-designed double-stranded siRNA conjugate with the structure shown in formula (803).

[0522] For reference conjugate 2, Nu in formula (803) is siRNA23 with the following composition:

[0523] siRNA23

[0524] Chain of Justice: GmsUmsAmCmGmUmGfGfAfCmUmGmGmAmUmUmCmUmGm(SEQ ID NO:27)

[0525] Antonym chain: VPCmsAfsGmAmAmUfCmCmAmGmUmCmCmAfCmGfUmAmCmsUmsUm(SEQ ID NO:28)

[0526] Theoretical value for the sense strand: 7605.4, measured value for the sense strand: 7604.3; theoretical value for the antisense strand: 7002.6, measured value for the antisense strand: 7001.5. The measured values ​​are consistent with the theoretical values, thus confirming that the synthesized reference conjugate 2 is the target-designed double-stranded siRNA conjugate with the structure shown in formula (803).

[0527] For reference conjugate 3, Nu in formula (803) is siRNA24 with the following composition:

[0528] siRNA24

[0529] Chain of Justice: CmsCmsAmAmGmAmGfCfAfCmCmAmAmGmAmAmCmUmAm(SEQ ID NO:29)

[0530] Antonym chain: VPUmsAfsGmUmUmCfUmUmGmGmUmGmCmUfCmUfUmGmGmsCmsUm(SEQ ID NO:30)

[0531] Theoretical value for the sense strand: 7584.5, measured value for the sense strand: 7583.5; theoretical value for the antisense strand: 7007.4, measured value for the antisense strand: 7006.4. The measured values ​​are consistent with the theoretical values, thus confirming that the synthesized reference conjugate 3 is the target-designed double-stranded siRNA conjugate with the structure shown in formula (803).

[0532] After the above-mentioned siRNA or siRNA conjugate is prepared, it is freeze-dried into a solid powder using standard methods and stored for later use.

[0533] Experimental Example 1 This experiment demonstrates the inhibitory efficiency of the disclosed siRNA conjugate on HBV mRNA expression in vitro.

[0534] HBV transgenic mice C57BL / 6J-Tg(A1b1HBV)44Bri / J were purchased from the Department of Laboratory Animal Science, Peking University School of Medicine. Mice with S / COV>10 were selected for the experiment before the experiment, and are referred to as 44Bri mice.

[0535] Primary hepatocytes were obtained from fresh liver tissue of 44 Bri mice and the cell density was adjusted to 2 × 10⁶ cells / mL in Opti-MEM (1X) medium (GIBCO, catalog number 31985-070). 5 Primary mouse liver cell suspension was obtained by adding the mouse primary liver cell suspension to different wells of a 12-well plate. The volume of mouse primary liver cell suspension added was 0.5 mL / well, and the number of primary mouse liver cells was 1 × 10⁶ cells / mL. 5 Cells / pores.

[0536] Prepare a 20 μM (based on siRNA) working solution for each of the following siRNA conjugates using DEPC-treated water. The siRNA conjugates used are conjugate 1, conjugate 2, or reference conjugate 1, respectively.

[0537] In different culture wells containing mouse primary liver cell suspension, siRNA conjugate working solution for each conjugate was added and mixed thoroughly at a volume of 2.5 μL / well. Each siRNA conjugate was added to three wells to obtain a transfection mixture containing siRNA (final concentration of 10 nM), designated as the test group. The mixtures in the other three wells containing mouse primary liver cell suspension were designated as the blank control group.

[0538] Each siRNA-containing transfection mixture and the blank control group were placed in an incubator containing 5% CO2 and cultured at 37°C for 24 hours.

[0539] Subsequently, total RNA was extracted from the cells in each well using TRIZOL (purchased from SIGMA, catalog number T9424) according to the method described in the instructions, and an aqueous solution of total RNA was obtained.

[0540] For each well of cells, a total RNA aqueous solution containing 1 μg of total RNA was taken and used to reverse transcriptase the cells using the Goldenstar reverse transcription kit. TM The reagents provided in the RT6 cDNA Synthesis Kit (purchased from Beijing Qingke Xinyue Biotechnology Co., Ltd., catalog number TSK301M) included Goldenstar. TM Oligo(dT) 17 As primers, a 20 μL reverse transcription reaction system was prepared according to the reverse transcription procedure in the kit instructions, and the total RNA from each well was reverse transcribed. The reverse transcription conditions were as follows: for each reverse transcription reaction system, the system was incubated at 50°C for 50 min, then at 85°C for 5 min, and finally at 4°C for 5 min. After the reaction was completed, 80 μL of DEPC water was added to the reverse transcription reaction system to obtain a solution containing cDNA.

[0541] For each reverse transcription reaction system, take 5 μL of the above solution containing cDNA as a template, and use... 15 μL of qPCR reaction system was prepared using the reagents provided by the SYBR qPCR SuperMix Plus kit (purchased from Nearshore Protein Technology Co., Ltd., catalog number E096-01B). The PCR primer sequences for amplifying the target gene HBV and the internal reference gene GAPDH are shown in Table 2, with a final concentration of 0.25 μM for each primer. Each qPCR reaction system was placed on an ABI StepOnePlus Real-Time PCR instrument, and a three-step amplification method was used. The amplification program was: 95℃ pre-denaturation for 10 min, followed by 95℃ denaturation for 30 s, 60℃ annealing for 25 s, and 72℃ extension for 25 s. This denaturation, annealing, and extension process was repeated 40 times to obtain product W containing amplified target gene HBV and internal reference gene GAPDH. Product W was then incubated sequentially at 95℃ for 1 min, 55℃ for 30 s, and 95℃ for 30 s. The melting curves of the target gene HBV and the internal reference gene GAPDH in product W were collected by real-time quantitative PCR instrument, and the Ct values ​​of the target gene HBV and the internal reference gene GAPDH were obtained.

[0542] Table 2 Sequences of the detection primers

[0543]

[0544]

[0545] The relative expression level and inhibition rate of the target gene HBV in each test group were calculated using the Ct(ΔΔCt) method. The calculation method is as follows:

[0546] ΔCt(test group) = Ct(target gene in test group) – Ct(internal reference gene in test group)

[0547] ΔCt(control group) = Ct(target gene in control group) – Ct(internal reference gene in control group)

[0548] ΔCt(test group) = ΔCt(test group) - ΔCt(control group average)

[0549] ΔCt(control group) = ΔCt(control group) - ΔCt(control group average)

[0550] Here, ΔCt (control group average) is the arithmetic mean of ΔCt (control group) for each of the three culture wells in the control group. Thus, each culture well in both the test group and the control group corresponds to a ΔCt value.

[0551] Using the control group as a baseline, the expression level of HBV mRNA in the test group was normalized, and the HBV mRNA expression level in the blank control group was defined as 100%.

[0552] The relative expression level of HBV mRNA in the test group was 2. -ΔΔCt(测试组) ×100%

[0553] HBV mRNA inhibition rate in the test group = (1 - relative expression level of HBV mRNA in the test group) × 100%

[0554] Table 3 below shows the results of detecting the inhibitory activity of the various conjugates of this disclosure on HBV mRNA expression in primary liver cells.

[0555] Table 3. In vitro activity assays of siRNA conjugates

[0556] siRNA conjugates mRNA inhibition rate (%) Conjugate 1 81.63 Conjugate 2 84.04 Reference conjugate 1 83.14

[0557] As can be seen from the results in Table 3, both conjugate 1 and conjugate 2 of this disclosure exhibit excellent HBV gene expression inhibitory activity at the cellular level, maintaining the same level as the reference conjugate 1.

[0558] Experimental Example 2 This experiment demonstrates the inhibitory efficiency of the disclosed siRNA conjugate on HBV mRNA expression on day 8 in vivo.

[0559] The serum HBsAg content of 44Bri mice was detected using a hepatitis B virus surface antigen diagnostic kit (enzyme-linked immunosorbent assay) (Shanghai Kehua Biotechnology) according to the method described in the instructions. Mice with S / COV>10 were selected and randomly divided into groups of 5 (all males). Each group was numbered and administered conjugate 1, conjugate 2, or reference conjugate 1 to each mouse subcutaneously at a dose of 0.1 mg / kg body weight (based on siRNA). The siRNA conjugates were provided in 1×PBS solution containing 0.02 mg / ml (based on siRNA). The administration volume was 5 ml / kg. These were the experimental groups. Each mouse in the other two groups was given 1×PBS at a dose of 5 ml / kg.

[0560] Using the drug administration point as day 1, animals were sacrificed on day 8, and liver tissue was collected from each mouse. The liver tissue was preserved using RNA later (Sigma Aldrich). 1 mL of Trizol (Sigma) was added to each liver tissue sample, and the tissue was homogenized three times for 30 seconds each time using a Tissuelyset II automated tissue homogenizer to obtain a liver tissue homogenate. 0.2 mL of chloroform was added, and the homogenate was allowed to stand for 3 minutes. The homogenate was then centrifuged at 12000 rpm for 10 minutes at 4°C, and 0.4 mL of the supernatant was collected. 0.5 mL of isopropanol was added to the supernatant, and the homogenate was allowed to stand at room temperature for 10 minutes. The homogenate was then centrifuged at 12000 rpm for 10 minutes at 4°C, and the supernatant was discarded. 1 mL of ethanol was added to the precipitate to wash it, and the precipitate was centrifuged at 12000 rpm for 5 minutes at 4°C, and the supernatant was discarded. 70 μL of DEPC-diluted water was added to the precipitate to obtain the extracted total RNA solution.

[0561] For total RNA from the liver tissue of each mouse, 10.5 μL of total RNA aqueous solution containing 1 μg of total RNA was prepared into a 20 μL reverse transcription reaction system using the Reverse Transcription System (Promega, catalog number A3500) according to the reverse transcription procedure in the kit instructions. The reverse transcription conditions were as follows: for each reverse transcription reaction system, the system was incubated at 42°C for 30 min, then at 95°C for 5 min, and finally at 4°C for 5 min. After the reaction, 80 μL of DEPC water was added to the reverse transcription system to obtain a solution containing cDNA.

[0562] For each reverse transcription reaction system, 5 μL of the above-mentioned cDNA-containing solution was used as a template. A 20 μL qPCR reaction system was prepared using the reagents provided by the SYBR Select Master Mix kit (Applied Biosystems). The PCR primer sequences for amplifying the target gene HBV and the internal reference gene GAPDH are shown in Table 2, with a final concentration of 0.25 μM for each primer. Each qPCR reaction system was placed on an ABI StepOnePlus Real-Time PCR instrument, and a three-step amplification method was used. The amplification program was: 95℃ pre-denaturation for 10 min, followed by 95℃ denaturation for 30 s, 60℃ annealing for 30 s, and 72℃ extension for 30 s. This denaturation, annealing, and extension process was repeated 40 times to obtain product W containing amplified target gene HBV and internal reference gene GAPDH. Product W was then incubated sequentially at 95℃ for 1 min, 55℃ for 30 s, and 95℃ for 30 s. The melting curves of the target gene HBV and the internal reference gene GAPDH in product W were collected by real-time quantitative PCR instrument, and the Ct values ​​of the target gene HBV and the internal reference gene GAPDH were obtained.

[0563] Following the method described in Experiment Example 1, the relative expression level and inhibition rate of HBV mRNA in mouse liver tissue after administration of each siRNA conjugate were calculated using Ct values.

[0564] Table 4 below shows the results of detecting the inhibitory activity of the various conjugates of this disclosure on HBV mRNA expression in mice.

[0565] Table 4. Inhibition rate of different conjugates on HBV mRNA in mouse liver

[0566]

[0567]

[0568] As can be seen from the results in Table 4, both conjugate 1 and conjugate 2 of this disclosure showed excellent HBV gene expression inhibitory activity in mice, with an HBV mRNA inhibition rate of at least 67.54% at a low dose of 0.1 mg / kg. Furthermore, compared with reference conjugate 1, conjugate 1 showed superior inhibitory activity in vivo.

[0569] Experimental Example 3 This experiment demonstrates the animal-level toxicity of the siRNA conjugate of the present invention.

[0570] In SD rats (purchased from SPAF (Beijing) Biotechnology Co., Ltd.), each rat was subcutaneously administered a single dose of 30 mg / kg (calculated as siRNA) of conjugate 1, conjugate 2, and reference conjugate 1 disclosed herein, with PBS serving as the control group. The rats were observed for 14 consecutive days, during which no deaths or behavioral abnormalities were observed. The rats were dissected, and liver tissue was harvested. After sampling, dehydration, embedding, slide preparation, and staining, pathological sections were prepared and observed under an optical microscope (microscope model: NIKON Eclipse ci, imaging system: NIKON digital sight DS-FI2, MADE IN JAPAN). As a result, in the liver pathological sections of rats administered conjugates 1 and 2, the hepatic cord structure was clear, hepatocytes were tightly arranged with clear boundaries, abundant cytoplasm, uniform staining, round and normal-sized nuclei, and intact and normal venous endothelium, with no obvious abnormalities observed in the tissue. In contrast, in the liver pathological sections of rats administered reference conjugate 1, extensive edema and degeneration of hepatocytes were observed, with swollen cells, loose and lightly stained cytoplasm, and a large number of hepatocytes also showing fatty degeneration. A varying number of round vacuoles were visible in the cytoplasm, and multiple focal infiltrations of inflammatory cells were observed within the lobules. Therefore, compared with reference conjugate 1, conjugates 1 and 2 of this disclosure have significantly improved in terms of toxicity.

[0571] Experimental Example 4 This experiment demonstrates the inhibitory efficiency of the disclosed siRNA conjugate on HBV mRNA expression on day 8 in vivo.

[0572] Following the method in Experiment 2, mice were administered conjugates 3, 4, 5, or 6, respectively. The siRNA conjugates were provided as 1×PBS solutions containing 0.02 mg / ml (calculated as siRNA), with an administration volume of 5 ml / kg, serving as the experimental group. Each mouse in the other two groups was given 1×PBS, with an administration volume of 5 ml / kg, serving as the blank control group.

[0573] Using the time of drug administration as day 1, the animals were sacrificed on day 8, and the relative expression level and inhibition rate of HBV mRNA in the liver tissue of mice were obtained according to the method in Experiment Example 2.

[0574] Table 5 below shows the results of detecting the inhibitory activity of the various conjugates of this disclosure on HBV mRNA expression in mice.

[0575] Table 5. Inhibition rate of different conjugates on HBV mRNA in mouse liver

[0576]

[0577]

[0578] As can be seen from the results in Table 5, the conjugates 3-6 disclosed herein all exhibited excellent HBV gene expression inhibitory activity in mice. Conjugate 3 achieved an HBV mRNA inhibition rate of 69.57% at a low dose of 0.1 mg / kg.

[0579] Experimental Example 5 This experiment demonstrates the inhibitory efficiency of the disclosed siRNA conjugate on HBV mRNA expression on day 15 in vivo.

[0580] Following the method in Experiment Example 2, the inhibitory efficiency of conjugate 2, conjugate 3, and reference conjugate 1 on HBV mRNA expression on day 15 in vivo was tested.

[0581] Table 6 below shows the results of the detection of the inhibitory activity of the various conjugates of this disclosure on HBV mRNA expression in mice.

[0582] Table 6. Inhibition rate of different conjugates on HBV mRNA in mouse liver

[0583]

[0584] As shown in Table 5, compared with reference conjugate 1, the siRNA conjugate 2 or conjugate 3 disclosed herein exhibited comparable or even better inhibitory activity against FXI mRNA expression on day 15 at a dose of 0.1 mg / kg siRNA.

[0585] Experimental Example 6 This experiment demonstrates the inhibitory efficiency of the disclosed siRNA conjugate on FXI mRNA expression in vivo.

[0586] C57BL / 6N mice (all female) were randomly divided into groups of five, and each group was numbered. Each group of mice was administered the test conjugate 8, conjugate 9, or reference conjugate 2 via subcutaneous injection at a dose of 3 mg / kg (based on siRNA). The siRNA conjugates were provided as 1 mg / ml siRNA conjugate in PBS solution, with an administration volume of 3 ml / kg for each conjugate.

[0587] One group of mice was given 1×PBS at a volume of 3 ml / kg as the control group.

[0588] Using the time of drug administration as day 1, animals were sacrificed on day 8, and liver tissue from each mouse was collected and preserved using RNA later (Sigma Aldrich). The liver tissue was homogenized using a tissue homogenizer, and total RNA was extracted using Trizol (ThermoFisher) according to the procedure described in the manufacturer's instructions.

[0589] C57BL / 6N mice (all female) were randomly divided into groups of five, each group was numbered, and administered the test conjugate 8, conjugate 9, reference conjugate 2, and PBS (3 ml / kg) at the same dose (3 mg / kg), concentration (1 mg / ml), and volume (3 ml / kg) as described above. Day 1 was designated as the time of administration. Animals were sacrificed on day 15, and liver tissue was collected from each mouse. The liver tissue was preserved using RNA later (Sigma Aldrich). The liver tissue was homogenized using a tissue homogenizer, and total RNA was extracted using Trizol (Thermo Fisher Scientific) according to the manufacturer's instructions.

[0590] The expression level and inhibition rate of FXI mRNA were detected and calculated using quantitative real-time PCR according to the method in Example 1. Specifically, total RNA extracted was reverse transcribed into cDNA using the ImProm-II™ reverse transcription kit (Promega) according to its instructions, resulting in a cDNA-containing solution. The expression level of FXI mRNA in liver tissue was then detected using a quantitative real-time PCR kit (Beijing Kangwei Century Biotechnology Co., Ltd.). In this quantitative real-time PCR method, the mouse GAPDH (mGAPDH) gene was used as an internal reference gene. Primers targeting FXI and mouse GAPDH were used to detect FXI and mouse GAPDH, respectively. The sequences of the detection primers are shown in Table 6. In the calculation of FXI mRNA expression level and inhibition rate, the control group consisted of mice administered PBS, and each test group consisted of mice administered different siRNA conjugates. The FXI mRNA expression level in the control group was recorded as 100%, and the corresponding FXI mRNA expression level inhibition rate was recorded as 0%. The test results were standardized with the FXI mRNA expression level in the control group, and the results are shown in Table 8.

[0591] Table 7 Sequences of the detection primers

[0592]

[0593] Table 8 below shows the results of detecting the inhibitory activity of the various conjugates of this disclosure on FXI mRNA expression in mice. The relative expression levels of FXI mRNA in the liver of each group of mice are shown in Table 8. Figure 1 .

[0594] Table 8. Inhibition rate of siRNA conjugates on FXI mRNA

[0595]

[0596] Figure 1 This is a line graph showing the relative expression levels of FXI mRNA in mouse liver tissue on days 8, 15, and 29 after administration of different siRNA conjugates at 3 mg / kg, compared to the blank control. (See Table 8 or...) Figure 1 The results showed that, compared with reference conjugate 2, the siRNA conjugate of this disclosure exhibited superior inhibitory activity against FXI mRNA expression on days 8, 15, and 29 at a dose of 3 mg / kg siRNA.

[0597] Experiment 7 This experiment demonstrates the inhibitory efficiency of the disclosed siRNA conjugate on ANGPTL3 mRNA expression in vivo.

[0598] C57BL / 6N mice were randomly divided into groups of five. Each group was administered conjugate 15, conjugate 16, a reference conjugate, and PBS, respectively. Mice in each group were administered conjugate 15, conjugate 16, or reference conjugate 3 at a dose of 3 mg / kg (based on siRNA). The siRNA conjugates were provided as 1 mg / ml siRNA conjugate solution in PBS, with a dosage volume of 3 ml / kg.

[0599] One group of mice was given 1×PBS at a volume of 3 ml / kg as the control group.

[0600] Using the time of drug administration as day 1, animals were sacrificed on day 8, and liver tissue from each mouse was collected and preserved using RNA later (Sigma Aldrich). The liver tissue was homogenized using a tissue homogenizer, and total RNA was extracted using Trizol (ThermoFisher) according to the procedure described in the manufacturer's instructions.

[0601] The expression level and inhibition rate of ANGPTL3 mRNA were detected and calculated using quantitative real-time PCR according to the method in Example 1. Specifically, cDNA was obtained by reverse transcription using a reverse transcription kit (Promega, catalog number A3500) following the manufacturer's instructions. The expression level of ANGPTL3 mRNA was detected using a 2×Ultra SYBR Mixture (with ROX) kit (Beijing Kangwei Century Biotechnology Co., Ltd., catalog number CW0956) with cDNA as a template, following the manufacturer's instructions. The PCR primers used to amplify ANGPTL3 and GAPDH as an internal reference gene are shown in Table 6.

[0602] Table 9: Primer Sequences

[0603]

[0604] In the calculation of ANGPTL3 mRNA expression levels and inhibition rates, the control group consisted of mice administered PBS, and the test groups consisted of mice administered different siRNA conjugates. The ANGPTL3 mRNA expression level in the control group was recorded as 100%, and the corresponding inhibition rate was recorded as 0%. The test results were standardized using the ANGPTL3 mRNA expression level in the control group, and the results are shown in Table 10. The relative expression levels of ANGPTL3 mRNA in the liver of each group of mice are shown below. Figure 2 .

[0605] Table 10 Inhibition rate of siRNA conjugates on ANGPTL3 mRNA

[0606]

[0607] Figure 2 This is a line graph showing the relative expression levels of ANGPTL3 mRNA in mouse liver tissue at days 8, 15, and 29 after administration of different siRNA conjugates at 3 mg / kg, compared to the blank control. (See Table 10 or...) Figure 2 The results show that the siRNA conjugate of this disclosure, at a dosage of 3 mg / kg siRNA, exhibited comparable inhibitory activity against ANGPTL3 mRNA expression on days 8, 15, and 29 to the reference conjugate 3. Some embodiments of this disclosure have been described in detail above. However, this disclosure is not limited to the specific details of the above embodiments. Within the scope of the technical concept of this disclosure, various simple modifications can be made to the technical solutions of this disclosure, and all such simple modifications fall within the protection scope of this disclosure.

[0608] It should also be noted that the various specific technical features described in the above embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, this disclosure will not describe the various possible combinations separately.

[0609] Furthermore, various different embodiments of this disclosure can be combined in any way, as long as they do not violate the spirit of this disclosure, they should also be regarded as the content disclosed in this disclosure.

Claims

1. A double-stranded oligonucleotide comprising a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, wherein each nucleotide in the double-stranded oligonucleotide is independently modified or unmodified, and portions of the sense strand and the antisense strand are anticomplementary to form a double-stranded region, wherein, The 5' terminal nucleotide of the antisense strand is a nucleotide with a ribose 5' modification, which has the structure shown in formula (101). The group R0 constitutes the 5' overhang of the antisense strand, and R0 has the structure shown in formula (102). in, R 201 It is a hydroxyl group; G1 is OH or O - ; Bx1 is a hydrogen, a heterocyclic base, or a base substitution group, and if the 3' end of the positive chain includes a protruding end, Bx1 does not pair with the base at that protruding end, wherein the base substitution group is a phenyl or a substituted phenyl group; Bx2 is a heterocyclic base; The heterocyclic base is uracil, thymine, cytosine, 5-methylcytosine, adenine, or guanine; The substituted phenyl group refers to a group formed by one or more hydrogen atoms on a phenyl group being independently replaced by F, Cl, CH3, CH2F or CF3. Z has the structure shown in formula (Z1) or (Z2), and both P1 and P2 are H; X1 is H or hydroxyl, and X2 is selected from H, halogen, hydroxyl or C1-C6 alkoxy. T2 is a phosphate ester subunit or a thiophosphate ester subunit; Y1, Y2, Y3, Y4, Y5, Y6, Y7 and Y8 are each independently H, halogen, hydroxyl, methyl, ethyl, n-propyl or isopropyl; This indicates the site where the group is covalently linked.

2. The double-stranded oligonucleotide of claim 1, wherein, G1 is OH, and T2 is a thiophosphate subunit.

3. The double-stranded oligonucleotide as described in claim 1, wherein, X2 is OCH3.

4. The double-stranded oligonucleotide of claim 1, wherein, Y1, Y2, Y3, Y4, Y5, Y6, Y7, and Y8 are all H.

5. The double-stranded oligonucleotide of claim 1, wherein, The group represented by formula (101) is selected from the group represented by one of formulas (501), (502), (503), (504), (505), or (506): Where Base is C, G, U, T or A.

6. The double-stranded oligonucleotide according to any one of claims 1-5, wherein, The sense strand comprises nucleotide sequence I, and the antisense strand comprises nucleotide sequence II. The 5' terminal nucleotide of nucleotide sequence II is a nucleotide with a ribose 5' modification, which has the structure shown in formula (101). Nucleotide sequence I consists of 19 nucleotides, with the group shown in formula (101) counted as 2 nucleotides. Nucleotide sequence II consists of 20 nucleotides, with the group R0 forming the overhang at the 5' end of the antisense strand. The remaining portion of nucleotide sequence II and nucleotide sequence I form a double-stranded region. Nucleotide sequence II is at least partially anticomplementary to a first nucleotide sequence, which is a nucleotide sequence of 19 nucleotides in length in the mRNA of the target gene expression. Nucleotides 7-9 of nucleotide sequence I are fluorinated nucleotides in the direction from 5' end to 3' end. Nucleotides 3, 7, 15, and 17 of nucleotide sequence II are fluorinated nucleotides in the direction from 5' end to 3' end.

7. The double-stranded oligonucleotide of claim 6, wherein, Following the direction from the 5' end to the 3' end, nucleotides 7-9 of nucleotide sequence I are fluorinated nucleotides, and each nucleotide at other positions of nucleotide sequence I is independently one of non-fluorinated nucleotides; following the direction from the 5' end to the 3' end, nucleotides 3, 7, 15, and 17 of nucleotide sequence II are fluorinated nucleotides, and each nucleotide at other positions of nucleotide sequence II is independently one of non-fluorinated nucleotides.

8. The double-stranded oligonucleotide of claim 7, wherein, In the direction from the 5' end to the 3' end, the 2nd to 20th nucleotides of the nucleotide sequence II are substantially anticomplementary, substantially anticomplementary, or completely anticomplementary to the first nucleotide sequence. The phrase "basically reverse complementary" means that there are no more than three base mismatches between the two nucleotide sequences involved. The term "substantially reverse complementary" refers to a base mismatch of no more than one between two nucleotide sequences. The term "completely reverse complementary" means that there are no base mismatches between the two nucleotide sequences.

9. The double-stranded oligonucleotide of claim 8, wherein, With the direction from the 5' end to the 3' end, the nucleotides at positions 3-20 of the nucleotide sequence II are completely opposite complementary to the nucleotides at positions 1-18 of the first nucleotide sequence.

10. The double-stranded oligonucleotide of claim 8, wherein, Following the direction from the 5' end to the 3' end, nucleotides 2-20 of nucleotide sequence II are substantially anticomplementary, substantially anticomplementary, or completely anticomplementary to nucleotide sequence I.

11. The double-stranded oligonucleotide of claim 10, wherein, In the direction from 5' end to 3' end, the 2nd to 20th nucleotides of nucleotide sequence II are completely anticomplementary to nucleotide sequence I, or in the direction from 5' end to 3' end, the 3rd nucleotide of nucleotide sequence II has a base mismatch with the 2nd nucleotide of nucleotide sequence I in the direction from 3' end to 5' end.

12. The double-stranded oligonucleotide of claim 6, wherein, The sense strand further contains nucleotide sequence III, and the antisense strand further contains nucleotide sequence IV. Each nucleotide of nucleotide sequence III and nucleotide sequence IV is independently one of non-fluorinated nucleotides. The length of nucleotide sequence III is 1, 2, 3, or 4 nucleotides. The length of nucleotide sequence IV is equal to that of nucleotide sequence III, and nucleotide sequence IV and nucleotide sequence III are substantially anticomplementary or completely anticomplementary. Nucleotide sequence III is attached to the 5' end of nucleotide sequence I, and nucleotide sequence IV is attached to the 3' end of nucleotide sequence II. Nucleotide sequence IV is substantially anticomplementary or completely anticomplementary to a second nucleotide sequence, which is a nucleotide sequence in the mRNA expressed by the target gene that is adjacent to the first nucleotide sequence and has the same length as nucleotide sequence IV.

13. The double-stranded oligonucleotide of claim 6, wherein, The double-stranded oligonucleotide also contains a nucleotide sequence V, each nucleotide of which is independently one of non-fluorinated modified nucleotides, the length of which is 1 to 3 nucleotides, and is attached to the 3' end of the antisense strand to form the 3' overhang of the antisense strand.

14. The double-stranded oligonucleotide of claim 13, wherein, The nucleotide sequence V is 2 nucleotides in length and, in the direction from the 5' end to the 3' end, the nucleotide sequence V is two consecutive thymine deoxyribonucleotides, two consecutive uracil ribonucleotides, or completely reverse complementary to the third nucleotide sequence. The third nucleotide sequence refers to a nucleotide sequence in the mRNA expressed by the target gene that is adjacent to the 5' end of the first or second nucleotide sequence and has the same length as the nucleotide sequence V.

15. The double-stranded oligonucleotide of claim 12, wherein, The double-stranded oligonucleotide also contains a nucleotide sequence V, each nucleotide of which is independently one of non-fluorinated modified nucleotides, the length of which is 1 to 3 nucleotides, and is attached to the 3' end of the antisense strand to form the 3' overhang of the antisense strand.

16. The double-stranded oligonucleotide of claim 15, wherein, The nucleotide sequence V is 2 nucleotides in length and, in the direction from the 5' end to the 3' end, the nucleotide sequence V is two consecutive thymine deoxyribonucleotides, two consecutive uracil ribonucleotides, or completely reverse complementary to the third nucleotide sequence. The third nucleotide sequence refers to a nucleotide sequence in the mRNA expressed by the target gene that is adjacent to the 5' end of the first or second nucleotide sequence and has the same length as the nucleotide sequence V.

17. The double-stranded oligonucleotide of claim 7, wherein, Each non-fluorinated modified nucleotide is independently selected from one of the nucleotides or nucleotide analogs formed by replacing the hydroxyl group at the 2' position of the ribosyl group of a nucleotide with a non-fluorinated group.

18. The double-stranded oligonucleotide of claim 7, wherein, Each of the non-fluorinated nucleotides is a methoxylated nucleotide, which refers to a nucleotide formed by replacing the 2'-hydroxyl group of the ribosome with a methoxy group.

19. The double-stranded oligonucleotide of claim 7, wherein, At least one of the phosphate ester groups in the phosphate-sugar backbone of at least one single strand of the sense chain and the antisense chain is a phosphate ester group with a modifying group, wherein the phosphate ester group with the modifying group is present at least at one of the following positions: Between the first and second nucleotides at the 5' end of the positive strand; Between the second and third nucleotides at the 5' end of the positive strand; Between the first and second nucleotides at the 3' end of the positive strand; Between the second and third nucleotides at the 3' end of the positive strand; Between the third and fourth nucleotides at the 5' end of the antisense strand; Between the first and second nucleotides at the 3' end of the antisense strand; and Between the second and third nucleotides at the 3' end of the antisense strand.

20. The double-stranded oligonucleotide of claim 6, wherein, The double-stranded oligonucleotide is saRNA or siRNA.

21. The double-stranded oligonucleotide of claim 20, wherein, The double-stranded oligonucleotide is siRNA1, siRNA2, siRNA3, siRNA4, siRNA5, siRNA6, siRNA7, siRNA8, siRNA9, siRNA10, siRNA11, siRNA12, siRNA13, siRNA14, siRNA15, siRNA16, siRNA17, siRNA18, siRNA19, siRNA20, or siRNA21.

22. A pharmaceutical composition comprising the double-stranded oligonucleotide as described in any one of claims 1-21 and a pharmaceutically acceptable carrier.

23. An oligonucleotide conjugate comprising a double-stranded oligonucleotide as described in any one of claims 1-21 and a conjugating group conjugated to the double-stranded oligonucleotide, the conjugating group comprising a linker and a pharmaceutically acceptable targeting group and / or a delivery aid group, wherein the double-stranded oligonucleotide, the linker, and the targeting group or the delivery aid group are covalently or non-covalently linked in sequence, each of the targeting groups being selected from ligands capable of binding to cell surface receptors, and each delivery aid group being selected from groups capable of increasing the biocompatibility of the oligonucleotide conjugate in a target organ or tissue.

24. Use of the double-stranded oligonucleotide of any one of claims 1-21, and / or the pharmaceutical composition of claim 22 and / or the oligonucleotide conjugate of claim 23 in the preparation of a medicament for treating and / or preventing diseases or symptoms associated with the mRNA level of a target gene expression.

25. The use as described in claim 24, wherein, The mRNA expressed by the target gene is selected from one of the following mRNAs transcribed from genes: ACE2, ANGPTL3, ApoA, ApoB, ApoC, AR, ASK1, C5, Col1A1, CTGF, Ebola, FOXO1, FTO, FVII, FXI, FXII, GCGR, HBV, HCV, HSD17B13, p53, PCSK9, PNP, PLG, PKK, KNG, SARS-CoV-2, SCD1, SCNN1A, SOD1, STAT3, TIMP-1, TMPRSS6, XO, INSR, SREBF1, HDV, RPTOR, TLK2, LPA, C3, AGT.

26. The use as described in claim 25, wherein, The mRNA expressed by the target gene is selected from the mRNA expressed by the hepatitis B virus gene.

27. The use as described in claim 25 or 26, wherein, The disease or symptom associated with the mRNA level of the target gene expression is hepatitis B.

28. A kit comprising the double-stranded oligonucleotide of any one of claims 1-21, and / or the pharmaceutical composition of claim 22 and / or the oligonucleotide conjugate of claim 23.

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